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Morphogen Directed Coordination of GPCR Activity Promotes Primary Cilium Function for Downstream Signaling Primary cilium dysfunction triggers catastrophic failure of signal transduction pathways that organize through cilia, thus conferring significant pressure on such signals to ensure ciliary homeostasis. Intraflagellar transport (IFT) of cargo that maintains the primary cilium is powered by high ciliary cAMP. Paradoxically, Sonic Hedgehog (SHH) signaling, for which ciliary function is crucial, triggers a reduction in ciliary cAMP that could blunt downstream signaling by slowing IFT. We investigated this paradox and mapped a novel signal relay driven by SHH-stimulated prostaglandin E2 that stabilizes ciliary cAMP flux through by activating Gs-coupled EP4 receptor. Chemical or genetic blockade of the SHH-EP4 relay cripples cAMP buffering, which leads to decreased intraciliary cAMP, short cilia, and attenuated SHH pathway induction. Accordingly, EP4-/- mice show pronounced ciliary defects and altered SHH-dependent neural tube patterning. Thus, SHH orchestrates a sophisticated ciliary GPCR-cAMP signaling network that ensures primary cilium fitness for a robust downstream signaling response. | cell biology |
Independent regulation of mtDNA quantity and quality resets the mitochondrial genome in C. elegans primordial germ cells Mitochondria contain an independent genome, called mtDNA, which contains essential metabolic genes. Although mtDNA mutations occur at high frequency, they are inherited infrequently, indicating that germline mechanisms limit their accumulation. To determine how germline mtDNA is regulated, we examined the control of mtDNA quantity and quality in C. elegans primordial germ cells (PGCs). We show that PGCs generate a bottleneck in mtDNA number by segregating mitochondria into lobe-like protrusions that are cannibalized by adjacent cells, reducing mtDNA content two-fold. As PGCs exit quiescence and divide, mtDNAs replicate to maintain a set point of [~]200 mtDNAs per germline stem cell. Whereas PGC lobe cannibalism eliminates mtDNAs stochastically, we show that the kinase PINK1, operating independently of Parkin and autophagy, preferentially reduces the fraction of mutant mtDNAs. Thus, PGCs employ parallel mechanisms to control both the quantity and quality of the founding population of germline mtDNAs. | cell biology |
New hypotheses of cell type diversity and novelty from comparative single cell and nuclei transcriptomics in echinoderms Cell types are the fundamental building blocks of metazoan biodiversity and offer a powerful perspective for inferring evolutionary phenomena. With the development of single-cell transcriptomic techniques, new definitions of cell types are emerging. This allows a conceptual reassessment of traditional definitions of novel cell types and their evolution. Research in echinoderms, particularly sea star and sea urchin embryos have contributed significantly to understanding the evolution of novel cell types, in particular the primary mesenchyme cells (PMCs) and pigment cells that are found in sea urchin but not sea star embryos. This paper outlines the development of a gene expression atlas for the bat star, Patiria miniata, using single nuclear RNA sequencing (snRNA-seq) of embryonic stages. The atlas revealed 22 cell clusters covering all expected cell types from the endoderm, mesoderm and ectoderm germ layers. In particular, four distinct neural clusters, an immune cluster, and distinct right and left coelom clusters were revealed as distinct cell states. A comparison with Strongylocentrotus purpuratus embryo single cell transcriptomes was performed using 1:1 orthologs to anchor and then compare gene expression patterns. S. purpuratus primordial germ cell equivalents were not detected in P. minata, while the left coelom of P. miniata has no equivalent cell cluster in S. purpuratus. Pigment cells of S. purpuratus map to clusters containing immune mesenchyme and neural cells of P. miniata, while the PMCs of S. purpuratus are revealed as orthologous to the right coelom cluster of P. miniata. These results suggest a new interpretation of the evolution of these well-studied cell types and a reflection on the definition of novel cell types. | evolutionary biology |
Reduced representation sequencing accurately quantifies relative abundance and reveals population-level variation in Pseudo-nitzschia spp. Certain species within the genus Pseudo-nitzschia are able to produce the neurotoxin domoic acid (DA), which can cause illness in humans, mass-mortality of marine animals, and closure of commercial and recreational shellfisheries during toxic events. Understanding and forecasting blooms of these harmful species is a primary management goal. However, accurately predicting the onset and severity of bloom events remains difficult, in part because the underlying drivers of bloom formation have not been fully resolved. Furthermore, Pseudo-nitzschia species often co-occur, and recent work suggests that the genetic composition of a Pseudo-nitzschia bloom may be a better predictor of toxicity than prevailing environmental conditions. We developed a novel next-generation sequencing assay using restriction site-associated DNA (2b-RAD) genotyping and applied it to mock Pseudo-nitzschia communities generated by mixing cultures of different species in known abundances. On average, 94% of the variance in observed species abundance was explained by the expected abundance. In addition, the false positive rate was low (0.45% on average) and unrelated to read depth, and false negatives were never observed. Application of this method to environmental DNA samples collected during natural Pseudo-nitzschia spp. bloom events in Southern California revealed that increases in DA were associated with increases in the relative abundance of P. australis. Although the absolute correlation across time-points was weak, an independent species fingerprinting assay (Automated Ribosomal Intergenic Spacer Analysis) supported this and identified other potentially toxic species. Finally, we assessed population-level genomic variation by mining SNPs from the environmental 2bRAD dataset. Consistent shifts in allele frequencies in P. pungens and P. subpacifica were detected between high and low DA years, suggesting that different intraspecific variants may be associated with prevailing environmental conditions or the presence of DA. Taken together, this method presents a potentially cost-effective and high-throughput approach for studies aiming to evaluate both population and species dynamics in mixed samples.
HighlightsO_LI2bRAD method facilitates species- and population-level analysis of the same sample
C_LIO_LIMethod accurately quantifies species relative abundance with low false positives
C_LIO_LIConsistent shifts in allele frequencies were detected between high and low DA years
C_LIO_LICertain Pseudo-nitzschia spp. populations may be more associated with DA presence
C_LI | genomics |
Proteome Remodeling of the Eye Lens at 50 Years Identified with Data-Independent Acquisition The eye lens is responsible for focusing and transmitting light to the retina. The lens does this in the absence of organelles yet maintains transparency for at least five decades before onset of age-related nuclear cataract (ARNC). It is hypothesized that oxidative stress contributes significantly to ARNC formation. It is additionally hypothesized that transparency is maintained by a microcirculation system (MCS) that delivers antioxidants to the lens nucleus and exports small molecule waste. Common data-ependent acquisition (DDA) methods are hindered by dynamic range of lens protein expression and provide limited context to age-related changes in the lens. In this study we utilized data-independent acquisition (DIA) mass spectrometry to analyze the urea insoluble, membrane protein fractions of 16 human lenses subdivided into three spatially distinct lens regions to characterize age-related changes, particularly concerning the lens MCS and oxidative stress response. In this pilot cohort, we measured 4,788 distinct protein groups, 46,681 peptides, and 7,592 deamidated sequences, more than in any previous human lens DDA approach. Our results reveal age-related changes previously known in lens biology and expand on these findings, taking advantage of the rich dataset afforded by DIA. Principally, we demonstrate that a significant proteome remodeling event occurs at approximately 50 years of age, resulting in metabolic preference for anaerobic glycolysis established with organelle degradation, decreased abundance of protein networks involved in calcium-dependent cell-cell contacts while retaining networks related to oxidative stress response. Further, we identified multiple antioxidant transporter proteins not previously detected in the human lens and describe their spatiotemporal and age-related abundance changes. Finally, we demonstrate that aquaporin-5, among other proteins, is modified with age by PTMs including deamidation and truncation. We suggest that the continued accumulation of each of these age-related outcomes in proteome remodeling contribute to decreased fiber cell permeability and result in ARNC formation. | biochemistry |
Replicative Instability Drives Cancer Progression In the past decade, defective DNA repair has been increasingly linked with cancer progression. Human tumors with markers of defective DNA repair and increased replication stress have been shown to exhibit genomic instability and poor survival rates across tumor types. Here we utilize-omics data from two independent consortia to identify the genetic underpinnings of replication stress, therapy resistance, and primary carcinoma to brain metastasis in BRCA wildtype tumors. In doing so, we have defined a new pan-cancer class of tumors characterized by replicative instability (RIN). RIN is defined by genomic evolution secondary to replicative challenge. Our data supports a model whereby defective single-strand break repair, translesion synthesis, and non-homologous end joining effectors drive RIN. Collectively, we find that RIN accelerates cancer progression by driving copy number alterations and transcriptional program rewiring that promote tumor evolution.
Statement of SignificanceDefining the genetic basis of genomic instability with wildtype BRCA repair effectors is a significant unmet need in cancer research. Here we identify and characterize a pan-cancer cohort of tumors driven by replicative instability (RIN). We find that RIN drives therapy resistance and distant metastases across multiple tumor types. | cancer biology |
Single-cell atlas of mouse limb development reveals a complex spatiotemporal dynamics of skeleton formation Limb development has long served as a model system for coordinated spatial patterning of progenitor cells. Here, we identify a population of naive limb progenitors and show that they differentiate progressively to form the skeleton in a complex nonconsecutive three-dimensional pattern.
Single-cell RNA sequencing of the developing mouse forelimb revealed three progenitor states: naive, proximal and autopodial, as well as Msx1 as a marker for the naive progenitors. In vivo lineage tracing confirmed this role and localized the naive progenitors to the outer margin of the limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that the progressive transition of Msx1+ naive progenitors into proximal and autopodial progenitors coincides with their differentiation to Sox9+ chondroprogenitors, which occurs along all the forming skeletal segments.
Indeed, tracking the spatiotemporal sequence of differentiation showed that the skeleton forms progressively in a complex pattern. These findings suggest a new model for limb skeleton development. | developmental biology |
A synthetic tear protein resolves dry eye through promoting corneal nerve regeneration. Corneal architecture is essential for vision and is greatly perturbed by the absence of tears due to the highly prevalent disorder dry eye. With no regenerative therapies available, pathological alterations of the ocular surface in response to dryness, including persistent epithelial defects and poor wound healing, result in lifelong morbidity. Here, using a mouse model of aqueous-deficient dry eye, we reveal that topical application of the synthetic tear protein lacripep reverses the pathological outcomes of dry eye through restoring the extensive network of corneal nerves that are essential for tear secretion, barrier function, epithelial homeostasis and wound healing. Intriguingly, the restorative effects of lacripep occur despite extensive immune cell infiltration, suggesting tissue reinnervation and regeneration can be achieved under chronic inflammatory conditions. In summary, our data highlight lacripep as a first-in-class regenerative therapy for returning the cornea to a near homeostatic state in individuals who suffer from dry eye.
TeaserTopical application of a synthetic tear protein repairs dry eye-mediated corneal damage through driving functional sensory reinnervation. | cell biology |
Crosstalk between age accumulated DNA-damage and the SIRT1-AKT-GSK3b axis in urine derived renal progenitor cells The aging process is manifested by a multitude of interlinked biological processes. These processes contribute to genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. Together these are recognized as of the main risk factors of the worlds most prevalent diseases, such as neurodegenerative disorders, cancer, cardiovascular disease, and metabolic disease.
The mammalian ortholog of the yeast silent information regulator (Sir2) SIRT1 is a NAD+-dependent class III histone deacetylase and has been recognized to be involved in many of the forementioned processes. Therefore, its activity is connected to aging via the regulation of apoptosis, cell differentiation, development, stress response, metabolism, and tumorigenesis. Furthermore, the physiological activity of several sirtuin family members has been connected to the regulation of life span of lower organisms (Caenorhabditis elegans and Drosophila melanogaster) as well as mammals. Aging in somatic cells of mammals is accompanied by mutations and other forms of DNA damage. These might manifest in transient cell cycle arrest associated with DNA repair, apoptosis, senescence, or cell differentiation. The activity of SIRT1 has previously been reported to be regulated by the DNA damage response pathway. On the one hand, SIRT1 is recruited from ATM to DBS and is required for DNA damage repair, but on the other hand, SIRT1 activity was also found to be negatively regulated by genotoxic stress via the interaction of ATM with Deleted in Breast Cancer 1 (DBC1). Increased levels of DBS are associated with downregulation of ATM and lower phosphorylation levels of AKT and GSK3{beta}, with significant implications for mesenchymal stem cell (MSC) maintenance and differentiation. In this proposed "stem cell checkpoint," the ATM signalling pathway initiated by DBS maintains MSCs and blocks their differentiation. Based on this, it has already been established that in senescent mesenchymal stem cells, SIRT1 expression is decreased, while its overexpression delays the onset of senescence and loss of differentiation capacity/ability.
In the present study, we provide evidence that SIX2-positive urine derived renal progenitor cells-UdRPCs isolated directly from human urine show typical hallmarks of aging when obtained from elderly donors. This includes the transcriptional downregulation of SIRT1 and its downstream targets AKT and GSK3{beta}. This transcriptional downregulation is accompanied by an increase in DNA damage and transcriptional levels several cell cycle inhibitors such as P16, reflecting possibly the ATM induced "stemness checkpoint" to maintain UdRPC stemness and differentiation capacity. We provide evidence that the renal progenitor transcription factor SIX2 binds to the coding sequence of SIRT1 and both factors mutually influence the transcription of each other. Furthermore, we show that the SIRT1 promoter region is methylation sensitive and becomes subsequently methylated in UdRPCs derived from aged donors, dividing them into SIRT1 high and low expressing UdRPCs. This downregulation might render the cells more vulnerable to endogenous noxae accelerating the accumulation of DNA damage and ultimately the accumulation of aging associated hallmarks. | cell biology |
Protrusion growth driven by myosin-generated force Actin-based protrusions are found on the surface of all eukaryotic cells, where they support diverse biological activities essential for life. Models of protrusion growth hypothesize that actin filament assembly provides the mechanical force for bending the plasma membrane outward. However, membrane-associated myosin motors are also abundant in protrusions, though their potential for contributing growth-promoting force remains unexplored. Using a novel inducible system that docks myosin motor domains to membrane binding modules with temporal control, we found that the application of myosin-generated force to the plasma membrane is sufficient for driving robust elongation of protrusions. Protrusion growth scaled with motor accumulation, required active, barbed end-directed force, and was independent of cargo delivery or the recruitment of canonical barbed end elongation factors. Application of growth-promoting force was also supported by structurally distinct myosin motor domains and membrane binding modules. We conclude that myosin-generated force can drive protrusion growth and this mechanism is likely active in diverse biological contexts. | cell biology |
MorphoFeatures: unsupervised exploration of cell types, tissues and organs in volume electron microscopy Electron microscopy (EM) provides a uniquely detailed view of cellular morphology, including organelles and fine subcellular ultrastructure. While the acquisition and (semi-)automatic segmentation of multicellular EM volumes is now becoming routine, large-scale analysis remains severely limited by the lack of generally applicable pipelines for automatic extraction of comprehensive morphological descriptors. Here, we present a novel unsupervised method for learning cellular morphology features directly from 3D EM data: a convolutional neural network delivers a representation of cells by shape and ultrastructure. Applied to the full volume of an entire three-segmented worm of the annelid Platynereis dumerilii, it yields a visually consistent grouping of cells supported by specific gene expression profiles. Integration of features across spatial neighbours can retrieve tissues and organs, revealing, for example, a detailed organization of the animal foregut. We envision that the unbiased nature of the proposed morphological descriptors will enable rapid exploration of very different biological questions in large EM volumes, greatly increasing the impact of these invaluable, but costly resources. | developmental biology |
Musashi1 and its short C-terminal variants regulate pluripotency states in embryonic stem cells Musashi1 (MSI1) is a marker for adult stem cells, but little is known for its expression and function in pluripotent stem cells (PSCs). Here we report that MSI1 is expressed in embryonic stem cells (ESCs) and is required for pluripotency maintenance. We found that there exit short c-terminal MSI1 variants (MSI1-C, hMSI1272-362 or mMSI1138-362) in naive but not primed ESCs. When overexpressed, MSI1 and MSI1-C variants facilitate primed-to-naive pluripotency transition by elevating the pluripotency of primed hESCs toward a formative-like state, enable better survival of hESCs in human-mouse interspecies cell completion, and enhance the ability of blastoid formation of hESCs after naive induction. Mechanistically, we show that the MSI1-C variants, though lacking RNA recognition motifs, bind to RNAs, enhance stress resistance and upregulate DNA damage repair genes. Thus, this study demonstrates that ESCs utilize MSI1 and the newly identified short MSI1-C proteins as double swords to regulate pluripotency states. | developmental biology |
The effect of dietary chitin on Atlantic salmon (Salmo salar) chitinase activity, gene expression, and microbial composition BackgroundChitin is a common component in the natural diet of many fish, and a range of chitinases with the potential to down chitin have been identified. Yet whether chitin is metabolized in fish is still unclear. Here we used a combination of chitinase activity assay, transcriptomics, and 16S rRNA bacterial analysis to assess the effect of chitin supplementation on Atlantic salmon gene expression and microbial community.
ResultsAtlantic salmon express multiple genes associated with chitin metabolism, and we show that the expression and activity of Atlantic salmon chitinases are not affected by the addition of dietary chitin. We do, however, demonstrate an association between gut microbial composition, chitinase activity in the gut, and host chitinase expression.
ConclusionThe findings presented here support the idea that chitin metabolism genes are linked to the maintenance of a chitin-based barrier in the teleost gut. These results contribute to a greater understanding of chitin metabolism in fish. | genetics |
Evolution towards small colony variants of pandemic multidrug resistant ST131 Escherichia coli isolates from a 10-year bone infection BackgroundChronic wounds are usually challenging to treat due to underlying medical conditions of the individual and as they readily become infected by microorganisms due to the failure of mechanical and physiological first line innate immune responses. We report here the characterization of host adaptation of five E. coli genomes including three E. coli ST131 genomes that occurred concomitantly with Enterococcus faecalis from a 10-year chronic wound infection after a foot fracture during the 2004 tsunami.
MethodsThe five E. coli strains were characterized by various microbiological and genomic approaches. Microbiological methods were antimicrobial resistance, growth in different media and biofilm formation. Genomic methods were determination of the genome sequence by PacBio RSII and Illumina sequencing. Phylogenetic analyses and genome alterations such as single nucleotide polymorphisms, deletion and rearrangements that led to pseudogenes and chromosomal inversions were documented. Relevant selected metabolic and physiological pathways were analyzed for integrity.
FindingStrains of two initially present sequence types, including the highly antimicrobial resistant ST405 clone, were subsequently replaced by isolates of the ubiquitous ST131 clone. The three E. coli ST131 strains showed a heavily host-adapted genome with a high number of pseudogenes and a large chromosomal inversion compared to ST131 reference strains. Furthermore, two of three E. coli ST131 isolates were small colony variants with its genetic basis in multiple genome alterations including pseudogenes and deletions in the pathway for heme biosynthesis. Pseudogene analysis indicated also the three ST131 strains to be mutator strains. Although enhanced capability of biofilm formation of the ST131 isolates was indicated by the agar plate assay, the a liquid culture biofilm assay did not display pronounced biofilm formation suggesting unconventional modes of biofilm formation.
InterpretationST131 clone members, which originally appeared as commensal strains can cause urinary tract and blood stream infections and are ubiquitously found in the environment including waste water and in animals. ST131 strains have presumably been already acquired from the environment on occurrence of the initial foot fracture and can persist in wounds showing an outmost genome plasticity and adaptability which might causing the chronic infection. Although co-infection with E. faecalis might have supported chronicity, these findings indicate that in individuals with underlying metabolic diseases wound infection by ST131 E. coli isolates can be a health risk.
FundingThis work was partially funded by ALF. | microbiology |
Multi-level force-dependent allosteric enhancement of αE-catenin binding to F-actin by vinculin Classical cadherins are transmembrane proteins whose extracellular domains link neighboring cells, and whose intracellular domains connect to the actin cytoskeleton via {beta}-catenin, - catenin. The cadherin-catenin complex transmits forces that drive tissue morphogenesis and wound healing. In addition, tension-dependent changes in E-catenin conformation enables it to recruit the actin-binding protein vinculin to cell-cell junctions, where it contributes to junctional strengthening. How and whether multiple cadherin-complexes cooperate to reinforce cell-cell junctions in response to load remains poorly understood. Here, we used single-molecule optical trap measurements to examine how multiple cadherin-catenin complexes interact with F-actin under load, and how this interaction is influenced by the presence of vinculin. We show that force oriented toward the (-) end of the actin filament results in mean lifetimes 3-fold longer than when force was applied towards the barbed (+) end. Further, load is distributed asymmetrically among complexes, such that only one bears the majority of applied load. We also measured force-dependent actin binding by a quaternary complex comprising the cadherin-catenin complex and the vinculin head region, which cannot itself bind actin. Binding lifetimes of this quaternary complex increased as additional complexes bound F-actin, but only when load was oriented toward the (-) end. In contrast, the cadherin-catenin complex alone did not show this form of cooperativity. These findings reveal multi-level, force-dependent regulation that enhances the strength of the association of multiple cadherin/catenin complexes with F-actin, conferring positive feedback that may strengthen the junction and polarize F-actin to facilitate the emergence of higher-order cytoskeletal organization. | biophysics |
A zebrafish embryo screen utilizing gastrulation for identification of anti-metastasis drugs Few models exist that allow for rapid and effective screening of anti-metastasis drugs. Here, we present a phenotype-based chemical screen utilizing gastrulation of zebrafish embryos for identification of anti-metastasis drugs. Based on our hypothesis that small molecules which interrupt zebrafish gastrulation might suppress metastasis progression of cancer cells, we designed a drug screening concept which uses epiboly, the first morphogenetic movement in gastrulation, as a marker. The screen only needs zebrafish embryos and enables 100 chemicals to be tested in five hours through just observing epiboly progression of a test chemical-treated embryos. In the screen, embryos at two-cell stage are firstly corrected and then developed to sphere stage. The embryos are treated with a test chemical and incubated in the presence of the chemical until vehicle-treated embryos develop to 90% epiboly stage. Finally, positive hit chemicals that interrupt epiboly progression are selected through comparing epiboly progression of a test chemical-treated embryos with that of vehicle-treated embryos under a stereoscopic microscope. Previous study subjected 1280 FDA-approved drugs into the screen and identified Adrenosterone and Pizotifen as epiboly-interrupting drugs. Mouse models of metastasis validated that the drugs could suppress metastasis of breast cancer cells. Furthermore, the screen can be diverted to a chemical genetic screening platform for identification of metastasis-promoting genes. Hydroxysteroid (11-Beta) Dehydrogenase 1 (HSD11b1) and serotonin receptor 2C (HTR2C), which are primary target of Adrenosterone and Pizotifen respectively, induces epithelial-mesenchymal transition (EMT) and promote metastasis of breast cancer cells. | cancer biology |
Herpes simplex virus 1 entry glycoproteins form stable complexes prior to and during membrane fusion Herpesviruses - ubiquitous pathogens that cause persistent infections - have some of the most complex cell entry mechanisms. Entry of the prototypical herpes simplex virus 1 (HSV-1) requires coordinated efforts of 4 glycoproteins, gB, gD, gH, and gL. The current model posits that the glycoproteins do not interact prior to receptor engagement and that binding of gD to its receptor causes a "cascade" of sequential pairwise interactions, first activating the gH/gL complex and subsequently activating gB, the viral fusogen. But how these glycoproteins interact remains unresolved. Here, using a quantitative split-luciferase approach, we show that pairwise HSV-1 glycoprotein complexes form prior to fusion, remain stable throughout fusion, and do not depend on the presence of the cellular receptor. Based on our findings, we propose a revised "conformational cascade" model of HSV-1 entry. We hypothesize that all 4 glycoproteins assemble into a complex prior to fusion, with gH/gL positioned between gD and gB. Once gD binds to a cognate receptor, the proximity of the glycoproteins within this complex allows for efficient transmission of the activating signal from the receptor-activated gD to gH/gL to gB through sequential conformational changes, ultimately triggering the fusogenic refolding of gB. Our results also highlight previously unappreciated contributions of the transmembrane and cytoplasmic domains to glycoprotein interactions and fusion. Similar principles could be at play in other multicomponent viral entry systems, and the split luciferase approach used here is a powerful tool for investigating protein-protein interactions in these and a variety of other systems.
IMPORTANCEHerpes simplex virus 1 (HSV-1) infects the majority of humans for life and can cause diseases ranging from painful sores to deadly brain inflammation. No vaccines or curative treatments currently exist. HSV-1 infection of target cells requires coordinated efforts of four viral glycoproteins. But how these glycoproteins interact remains unclear. Using a quantitative protein interaction assay, we found that HSV-1 glycoproteins form stable, receptor-independent complexes. We propose that the 4 proteins form a complex, which could facilitate transmission of the entry-triggering signal from the receptor-binding component to the membrane fusogen component through sequential conformational changes. Similar principles could be applicable across other multicomponent protein systems. A revised model of HSV-1 entry could facilitate the development of therapeutics targeting this process. | microbiology |
Conservation of energetic pathways for electroautotrophy in the uncultivated candidate order Tenderiales Electromicrobiology can be used to understand extracellular electron uptake in previously undescribed chemolithotrophs. Enrichment and characterization of the uncultivated electroautotroph "Candidatus Tenderia electrophaga" using electromicrobiology led to the designation of the order Tenderiales. Representative Tenderiales metagenome assembled genomes (MAGs) have been identified in a number of environmental surveys, yet a comprehensive characterization of conserved genes for extracellular electron uptake has thus far not been conducted. Using comparative genomics we identified conserved orthologous genes within the Tenderiales and nearest neighbor orders important for extracellular electron uptake based on a previously proposed pathway from "Ca. Tenderia electrophaga". The Tenderiales contained a conserved cluster we designated uetABCDEFGHIJ, which encodes proteins containing features that would enable transport of extracellular electrons to cytoplasmic membrane bound energy transducing complexes such as two conserved cytochrome cbb3 oxidases. For example, UetJ is predicted to be an extracellular undecaheme c-type cytochrome that forms a heme wire. We also identified clusters of genes predicted to facilitate assembly and maturation of electron transport proteins, as well as cellular attachment to surfaces. Autotrophy among the Tenderiales is supported by the presence of carbon fixation and stress response pathways that could allow cellular growth by extracellular electron uptake. Key differences between the Tenderiales and other known neutrophilic iron oxidizers were revealed, including very few Cyc2 genes in the Tenderiales. Our results reveal a possible conserved pathway for extracellular electron uptake and suggests the Tenderiales have an distribution unlimited ecological role coupling metal or mineral redox chemistry and the carbon cycle in marine and brackish sediments.
ImportanceElectromicrobiology enables enrichment and identification of chemolithotrophic bacteria capable of extracellular electron uptake to drive energy metabolism and CO2 fixation. The recently described order Tenderiales contains the uncultivated electroautotroph "Candidatus Tenderia electrophaga". The "Ca. Tenderia electrophaga" genome contains genes proposed to make up a previously undescribed extracellular electron uptake pathway. Here we use comparative genomics to show that this pathway is well conserved among Tenderiales spp. recovered by metagenome assembled genomes. This conservation extends to near neighbors of the Tenderiales, but not to other well-studied chemolithotrophs including iron and sulfur oxidizers. Our findings suggest that extracellular electron uptake may be pervasive among the Tenderiales and the geographic location from which metagenome assembled genomes were recovered offers clues to their natural ecological niche. | microbiology |
The DNA replication protein Orc1 from the yeast Torulaspora delbrueckii is required for heterochromatin formation but not as a silencer-binding protein To understand the process by which new protein functions emerge, we examined how the yeast heterochromatin protein Sir3 arose through gene duplication from the conserved DNA replication protein Orc1. Orc1 is a subunit of the origin recognition complex (ORC), which marks origins of DNA replication. In Saccharomyces cerevisiae, Orc1 also promotes heterochromatin assembly by recruiting the structural proteins Sir1-4 to silencer DNA. In contrast, the paralog of Orc1, Sir3, is a nucleosome-binding protein that spreads across heterochromatic loci in conjunction with other Sir proteins. We previously found that a non-duplicated Orc1 from the yeast Kluyveromyces lactis behaved like ScSir3 but did not have a silencer-binding function like ScOrc1. Moreover, K. lactis lacks Sir1, the protein that interacts directly with ScOrc1. Here, we searched for the presumed intermediate state in which non-duplicated Orc1 possesses both the silencer-binding and spreading functions. In the non-duplicated species Torulaspora delbrueckii, which has an ortholog of Sir1 (TdKos3), we found that TdOrc1 spreads across heterochromatic loci independently of ORC, as ScSir3 and KlOrc1 do. This spreading is dependent on the nucleosome binding BAH domain of Orc1 and on Sir2 and Kos3. However, TdOrc1 does not have a silencer-binding function: T. delbrueckii silencers do not require ORC binding sites to function, and Orc1 and Kos3 do not appear to interact. Instead, Orc1 and Kos3 both spread across heterochromatic loci with other Sir proteins. Thus, Orc1 and Sir1/Kos3 originally had different roles in heterochromatin formation than they do now in S. cerevisiae. | genetics |
Comparative and longitudinal analysis of axial and retinal biometry in prospective models of hyperopia Structured AbstractO_ST_ABSPurposeC_ST_ABSTo quantify changes in axial and retinal biometry in aging hyperopic mouse models.
MethodsFundus photographs and ocular biometric measurements from Mfrprd6, Prss56glcr4, Adipor1tm1Dgen, C1qtnf5tm1.1(KOMP)Vlcg and Prss56em2(IMPC)J homozygotes and C57BL/6J control mice were ascertained longitudinally up to one year of age. Parameters including axial length (AL), central corneal thickness (CCT), anterior chamber depth (ACD), lens thickness (LT), outer nuclear layer thickness (ONLT), retinal thickness (RT), vitreous chamber depth (VCD) and posterior length (PL) were measured using Spectral Domain-Optical Coherence Tomography imaging. Mixed-model analysis of variance and factorial analysis of covariance, using body size as a covariate, followed by post-hoc analysis was performed to identify significant strain differences.
ResultsStrain specific changes in axial and retinal biometry along with significant effects of age, sex and body size on AL were noted. Mfrprd6, Prss56glcr4, Adipor1tm1Dgen and Prss56em2(IMPC)J homozygotes had significantly shorter AL than controls. While a comparable decrease in PL was observed in Mfrprd6, Prss56glcr4, and Adipor1tm1Dgen homozygotes, the decrease was attributed to changes in different posterior components from each mutant. Mfrprd6 and Adipor1tm1Dgen homozygotes developed regularly sized fundus spots across the ocular globe, which differed from the large bright spots seen in aged Prss56glcr4 and Prss56em2(IMPC)J homozygotes. While ONLT of C1qtnf5tm1.1(KOMP)Vlcg mice was less than controls, AL and fundus images appeared normal.
ConclusionsThis study highlights differences in contributions of ocular components to AL among hyperopic mouse models with decreased AL. Understanding the mechanisms through which these proteins function, will help to elucidate their role in controlling ocular growth. | genetics |
Comparative single-cell transcriptomes of dose and time dependent epithelial-mesenchymal spectrums Epithelial-mesenchymal transition (EMT) is a key cellular process involved in development and disease progression. Single-cell transcriptomes can characterize intermediate EMT states observed in tumors and fibrotic tissues, but previous in vitro models focused on time-dependent responses after stimulation with single dose of EMT signals. It was therefore unclear whether single-cell transcriptomes support stable intermediate EMT phenotypes crucial for disease progression. We performed single-cell RNA-sequencing with human mammary epithelial cells treated with various concentrations of TGF-{beta}. We found that the dose-dependent EMT harbors multiple intermediate states at the single-cell level after two weeks of treatment, suggesting a stable continuum. After correcting batch effects from experiments, we performed comparative analyses of the dose- and time-dependent EMT. We found that the dose-dependent EMT shows a stronger anti-correlation between epithelial and mesenchymal transcriptional programs and a better resolution of transition stages compared to the time-dependent process. These properties enable higher sensitivity to detect genes whose expressions are associated with core EMT regulatory networks. Nonetheless, we found cell clusters unique to the time-dependent EMT, which correspond to en route cell populations that do not appear at steady states. Furthermore, combining dose- and time-dependent cell clusters gave rise to more accurate prognosis for cancer patients compared to individual EMT spectrum. Our new data and analyses reveal a stable EMT continuum at the single-cell resolution and the transcriptomic level. The dose-dependent experimental model can complement the widely used time-course experiments to reflect physiologically or pathologically relevant EMT phenotypes in a comprehensive manner. | genomics |
Discovery of diverse human BH3-only and non-native peptide binders of pro-apoptotic BAK indicate that activators and inhibitors use asimilar binding mode and are not distinguished by binding affinity or kinetics Apoptosis is a programmed form of cell death important for the development and maintenance of tissue homeostasis. The BCL-2 protein family controls key steps in apoptosis, dysregulation of which can lead to a wide range of human diseases. BCL-2 proteins comprise three groups: anti-apoptotic proteins, pro-apoptotic proteins, and BH3-only proteins. BAK is one of two pro-apoptotic proteins, and previous work has shown that binding of certain BH3-only proteins such as truncated BID (tBID), BIM, or PUMA to BAK leads to mitochondrial outer membrane permeabilization, the release of cytochrome c, and ultimately cell death. This process, referred to as activation, involves the BH3-stimulated conversion of BAK from monomer to dimer and then to oligomers that promote membrane disruption. Crystal structures of putative intermediates in this pathway, crosslinking data, and in vitro functional tests have provided insights into the activation event, yet the sequence-function relationships that make some but not all BH3-only proteins function as activators remain largely unexamined. In this work, we used computational protein design, yeast surface-display screening of candidate BH3-like peptides, and structure-based energy scoring to identify ten new binders of BAK that span a large sequence space. Among the new binders are two peptides from human proteins BNIP5 and PXT1 that promote BAK activation in liposome assays and induce cytochrome-c release from mitochondria, expanding current views of how BAK-mediated cell death may be triggered in cells. High-resolution crystal structures and binding experiments revealed a high degree of similarity in binding geometry, affinity, and association kinetics between peptide activators and inhibitors, including peptides described previously and those identified in this work. We propose a model for BAK activation that is based on differential engagement of BAK monomers vs. the BAK activation transition state that integrates our observations with previous reports of BAK binders, activators, and inhibitors. | biochemistry |
Comparative genomic and crystal structure analyses identify a collagen glucosyltransferase from Acanthamoeba Polyphaga Mimivirus Humans and Acanthamoeba Polyphaga Mimivirus share numerous homologous genes, including collagens and collagen-modifying enzymes. To explore the homology, we performed a genome-wide comparison between human and mimivirus using DELTA-BLAST (Domain Enhanced Lookup Time Accelerated BLAST) and identified 52 new mimiviral proteins that are homologous with human proteins. To gain functional insights into mimiviral proteins, their human protein homologs were organized into Gene Ontology (GO) and REACTOME pathways to build a functional network. Collagen and collagen-modifying enzymes form the largest subnetwork with most nodes. Further analysis of this subnetwork identified a putative collagen glycosyltransferase R699. Protein expression test suggested that R699 is highly expressed in E coli, unlike the human collagen-modifying enzymes. Enzymatic activity assays showed that R699 catalyzes the conversion of unique galactosylhydroxylysine within the GXXXUG motif (U=galactosylhydroxylysine) to glucosylgalactosylhydroxylysine on collagen using uridine diphosphate glucose (UDP-Glc) as a sugar donor, suggesting R699 is a mimiviral collagen galactosylhydroxylysyl glucosyltransferase (GGT) with defined substrate specificity. Structural study of R699 produced the first crystal structure of a collagen GGT with a visible UDP-Glc. Sugar moiety of the UDP-Glc resides in a previously unrecognized pocket. Mn2+ coordination and nucleoside-diphosphate binding site are conserved among GGT family members and critical for R699s collagen GGT activity. To facilitate further analysis of human and mimiviral homologous proteins, we presented an interactive and searchable genome-wide comparison website for quickly browsing human and Acanthamoeba Polyphaga Mimivirus homologs, which is available at RRID Resource ID: SCR_022140 or https://guolab.shinyapps.io/app-mimivirus-publication/. | biochemistry |
The metastatic capacity of high-grade serous ovarian cancer cells changes along disease progression: inhibition by mifepristone Background: Reductionist two-dimensional (2D) in vitro assays have long been the standard for studying the metastatic abilities of cancer cells. However, tri-dimensional (3D) organotypic models provide a more complex environment, closer to that seen in patients, and thereby provide a more accurate representation of their true capabilities. Our laboratory has previously shown that the antiprogestin and antiglucocorticoid mifepristone can reduce the growth, adhesion, migration, and invasion of various aggressive cancer cells assessed using 2D assays. In this study, we characterize the metastatic capabilities of high-grade serous ovarian cancer cells generated along disease progression, in both 2D and 3D assays, and the ability of cytostatic doses of mifepristone to inhibit them. Methods: High-grade serous ovarian cancer cells collected from two separate patients at different stages of their disease were used throughout the study. The 2D wound healing and Boyden chamber assays were used to study migration, while a layer of extracellular matrix was added to the Boyden chamber to study invasion. A 3D organotypic model, composed of fibroblasts embedded in collagen I and topped with a monolayer of mesothelial cells was used to further study cancer cell adhesion and mesothelial displacement. All assays were studied in cells representing different stages of disease progression in the absence or presence of cytostatic doses of mifepristone. Results: 2D in vitro assays demonstrated that the migration and invasive rates of the cells isolated from both patients decreased along disease progression. Conversely, in both patients, cells representing late-stage disease demonstrated a higher adhesion capacity to the 3D organotypic model than those representing an early-stage disease. This adhesive behaviour is associated with the in vivo tumor capacity of the cells. Regardless of these differences in adhesive, migratory, and invasive behaviour among the experimental protocols used, cytostatic doses of mifepristone were able to inhibit the adhesion, migration, and invasion rates of all cells studied, regardless of their basal capabilities over reductionist or organotypic metastatic in vitro model systems. Finally, we demonstrate that when cells acquire the capacity to grow spontaneously as spheroids, they do attach to a 3D organotypic model system when pre-incubated with conditioned media. Of relevance, mifepristone was able to cause dissociation of these multicellular structures. Conclusion: Differences in cellular behaviours were observed between reductionist 2D and 3D assays when studying the metastatic capabilities of high-grade serous ovarian cancer cells representing disease progression. Mifepristone inhibited these metastatic capabilities in all assays studied. | cancer biology |
NetAct: a computational platform to construct core transcription factor regulatory networks using gene activity A major question in systems biology is how to identify the core gene regulatory circuit that governs the decision-making of a biological process. Here, we develop a computational platform for constructing core transcription-factor regulatory networks, named NetAct, using both transcriptomics data and literature-based transcription factor-target databases. NetAct robustly infers the activities of regulators using target expression, constructs networks based on transcriptional activity, and integrates mathematical modeling for validation. We show that NetAct outperforms existing algorithms in inferring transcriptional activity from our benchmark tests. We illustrate the application of NetAct to model networks driving TGF-beta induced Epithelial-Mesenchymal transition and macrophage polarization. | systems biology |
The proteomic and transcriptomic landscapes altered by Rgg2/3 activity in Streptococcus pyogenes Streptococcus pyogenes, otherwise known as Group A Streptococcus (GAS), is an important and highly adaptable human pathogen with the ability to cause both superficial and severe diseases. Understanding how S. pyogenes senses and responds to its environment will likely aid in determining how it causes a breadth of diseases. One regulatory network involved in GASs ability to sense and respond to the changing environment is the Rgg2/3 quorum sensing (QS) system, which responds to metal and carbohydrate availability and regulates changes to the bacterial surface. To better understand the impact of Rgg2/3 QS on S. pyogenes physiology, we performed RNA-seq and TMT-LC-MS/MS analysis on cells in which this system was induced or disrupted. Primary findings confirmed that pheromone stimulation in wildtype cultures is limited to the induction of operons whose promoters contain previously determined Rgg2/3 binding sequences. However, supplementing exogenous pheromone to a deletion mutant of rgg3, a strain that endogenously produces elevated amounts of pheromone, led to extended alterations of the transcriptome and proteome, ostensibly by stress-induced pathways. Under such exaggerated pheromone conditions (Drgg3+SHP), a connection was identified between Rgg2/3 and the stringent response. Mutation of relA, the bifunctional guanosine tetra- and penta-phosphate nucleoside synthetase/hydrolase, and alarmone synthase genes sasA and sasB, impacted culture doubling times and disabled induction of Rgg2/3 in response to mannose, while manipulation of Rgg2/3 signaling modestly altered nucleotide levels. Our findings indicate that excessive pheromone production or exposure places stress on GAS resulting in an indirect altered proteome and transcriptome beyond primary pheromone signaling. | microbiology |
Temporally integrated transcriptome analysis reveals ASFV pathology and host response dynamics African swine fever virus (ASFV) causes a lethal swine hemorrhagic disease and is currently responsible for widespread damage to the pig industry. The molecular mechanisms of ASFV pathogenicity and its interaction with host responses remain poorly understood. In this study, we profiled the temporal viral and host transcriptomes in porcine alveolar macrophages (PAMs) infected at 6, 12, 24 and 48 hours with highly virulent (SY18) and low virulent (HuB20) ASFV strains. We first identified profound differences in the virus expression programs between SY18 and HuB20, while the transcriptome dynamics in host cells were dominated by infection time. Through integrated computational analysis and experimental validation, we identified differentially expressed genes and related biological processes, and elaborated differential usage of the NF-kappaB related pathways by the two virus strains. In addition, we observed that compared to the highly virulent SY18 strain, HuB20 infection quickly activates expression of receptors, sensors, regulators, as well as downstream effectors, including cGAS, STAT1/2, IRF9, MX1/2, suggesting rapid induction of a strong immune response. Lastly, we constructed a host-virus coexpression network, which shed light on pathogenic functions of several ASFV genes. Taken together, these results will provide a basis for further mechanistic studies on the functions of both viral and cellular genes that are involved in different responses.
Author SummarySince it was first described in Kenya in 1921, ASF has spread across sub-Saharan Africa, the Caribbean, the Western Europe, the Trans-Caucasus region, and the Russian Federation. Recent out-breaks have also been reported in Asia, which has devastated the pig industry, resulting in an approximately 40% reduction in pork worldwide. In the absence of effective vaccine or treatment, the mortality for infections with highly virulent strains approaches 100%, while low virulent strains causing less mortality spreads fast recently. Nevertheless, the mechanisms of ASFV pathogenicity, especially the differences between highly and low virulent strains remain poorly understood. Here, we used RNA-seq to analyze the viral and host transcriptome changes in PAMs infected with a virulent strain (SY18) or an attenuated strain (HuB20) at different stages. We found that the presence of ASFV significantly affected the cellular transcriptome profile. In addition, we did temporal and described the dynamic expression programs induced in the host cells by ASFV infection of different virulence strains. In particular, we identified differential gene expression patterns in host innate immune responses and expressed cytokines and chemokines between ASFV strains of different virulence. Our study provides new insights into ASFV pathogenicity research and novel drug or vaccine targets. | microbiology |
Highly specialized carbohydrate metabolism capability in Bifidobacterium strain associated with intestinal barrier maturation in early preterm infants "Leaky gut", or high intestinal barrier permeability, is common in preterm newborns. The role of microbiota in this process remains largely uncharacterized. We employed both short- and long-read sequencing of the 16S rRNA gene and metagenomes to characterize the intestinal microbiome of a longitudinal cohort of 113 preterm infants born between 240/7-326/7 weeks of gestation. Enabled by enhanced taxonomic resolution, we found significantly increased abundance of Bifidobacterium breve and a diet rich in mothers breastmilk to be associated with intestinal barrier maturation during the first week of life. We combined these factors using genome-resolved metagenomics and identified a highly specialized genetic capability of the Bifidobacterium strains to assimilate human milk oligosaccharides and host-derived glycoproteins. Our study proposed mechanistic roles of breastmilk feeding and intestinal microbial colonization in postnatal intestinal barrier maturation; these observations are critical towards advancing therapeutics to prevent and treat hyperpermeable gut-associated conditions, including necrotizing enterocolitis. | microbiology |
A comprehensive list of the replication promoters of Bunyavirales reveals a unique promoter structure in Nairoviridae differing from other virus families Bunyaviruses belong to the order Bunyavirales, the largest group of RNA viruses. They infect a wide variety of host species around the world, including plants, animals and humans, and pose a major threat to public health. Major families in the order Bunyavirales have tri-segmented negative-sense RNA genomes, the 5 and 3 ends of which form complementary strands that serve as a replication promoter. Elucidation of the mechanisms by which viral RNA-dependent RNA polymerase recognizes the promoter to initiates RNA synthesis is important for understanding viral replication and pathogenesis, and for developing antivirals. A list of replication promoter configuration patterns may provide details on the differences in the replication mechanisms among bunyaviruses. Here, by using public sequence data of all known bunyavirus species, we constructed a comprehensive list of the replication promoters comprising 40 nucleotides in both the 5 and 3 ends of the genome that form a specific complementary strand. We showed that among tri-segmented bunyaviruses, viruses belonging to the family Nairoviridae, including the highly pathogenic Crimean-Congo hemorrhagic fever virus, have evolved a GC-rich promoter structure that differs from that of other bunyaviruses. The unique promoter structure might be related to the large genome size of the family Nairoviridae among bunyaviruses. It is possible that the large genome architecture confers a pathogenic advantage. The promoter list provided in this report is expected to be useful for predicting virus family-specific replication mechanisms of segmented negative-sense RNA viruses. | microbiology |
Quorum sensing regulation of a major facilitator superfamily transporter affects multiple streptococcal virulence factors Cell-cell signaling mediated by Rgg-family transcription factors and their cognate pheromones is conserved in Firmicutes, including all streptococci. In Streptococcus pyogenes, or Group A strep, one of these systems, the Rgg2/3 quorum sensing (QS) system, has been shown to regulate phenotypes including cellular aggregation and biofilm formation, lysozyme resistance, and macrophage immunosuppression. Here, we show that the abundance of several secreted virulence factors (streptolysin O, SpyCEP, and M protein) decreases upon induction of QS. The main mechanism underlying the changes in protein levels appears to be transcriptional, occurs downstream of the QS circuit and is dysregulated by the deletion of an Rgg2/3 QS-regulated major facilitator superfamily (MFS) transporter. Additionally, we identify this MFS transporter as the factor responsible for a previously observed increase in aminoglycoside sensitivity in QS-induced cells.
ImportanceThe production of virulence factors is a tightly regulated process in bacterial pathogens. Efforts to elucidate the mechanisms by which genes are regulated may advance the understanding of factors influencing pathogen behavior or cellular physiology. This work finds that expression of a major facilitator superfamily (MFS) transporter, which is governed by a quorum sensing (QS) system, impacts the expression of multiple secreted virulence factors and accounts for a documented QS-dependent antibiotic susceptibility. Although the mechanism underlying this effect is not clear, MFS orthologs with high sequence similarity from S. pneumoniae and S. porcinus were unable to substitute indicating substrate specificity of the GAS MFS gene. These findings demonstrate novel associations between the expression of a transmembrane transporter and virulence factor expression and aminoglycoside transport. | microbiology |
Stem Cell Secretome Promotes Scarless Corneal Wound Healing and Rescues Corneal Sensory Nerves Corneal scarring is a leading cause of blindness in the world. In this study, we explored the therapeutic potential of corneal stromal stem cell (CSSC)-derived secretome in a mechanical debridement mouse model of corneal scarring. CSSC secretome was able to promote scarless corneal wound healing. The mechanisms include 1) dampening inflammation with reduced CD45+, CD11b+/GR1+, and CD11b+/F4/80+ inflammatory cells in the wounded corneas; 2) reducing fibrotic extracellular matrix deposition such as collagen IV, collagen 3A1, SPARC, and alpha-SMA; 3) rescuing sensory nerves. The proteomic analysis shows upregulated proteins related to wound healing and cell adhesion which boost scarless wound healing. It also shows upregulated neuroprotective proteins in CSSC secretome related to axon guidance, neurogenesis, neuron projection development, and neuron differentiation. Four unique complement inhibitory proteins CD59, vitronectin, SERPING1, and C1QBP found in CSSC secretome contribute to reducing a complement cascade mediating cell death and membrane attacking complex autoantibodies after corneal injury. This study provides novel insights into mechanisms of stem cell secretome induced scarless corneal wound healing and neuroprotection and identifies regenerative proteins in the CSSC secretome. | cell biology |
The rescue of epigenomic abnormalities in ICF1 patient iPSCs following DNMT3B correction is incomplete at a residual fraction of H3K4me3-enriched regions Background: Bi-allelic hypomorphic mutations in DNMT3B disrupt DNA methyltransferase activity and lead to Immunodeficiency, Centromeric instability, Facial anomalies syndrome, type 1 (ICF1). While several ICF1 phenotypes have been linked to abnormally hypomethylated repetitive regions, the unique genomic regions responsible for the remaining disease phenotypes remain largely uncharacterized. Here we explored two ICF1 patient-induced pluripotent stem cells (iPSCs) and their CRISPR/Cas9 corrected clones to determine whether gene correction can overcome DNA methylation defects and related/associated changes in the epigenome of non-repetitive regions. Results: Hypomethylated regions throughout the genome are highly comparable between ICF1 iPSCs carrying different DNMT3B variants, and significantly overlap with those in ICF1-peripheral blood and lymphoblastoid cell lines. These regions include large CpG island domains, as well as promoters and enhancers of several lineage-specific genes, in particular immune-related, suggesting that they are pre-marked during early development. The gene corrected ICF1 iPSCs reveal that the majority of phenotype-related hypomethylated regions re-acquire normal DNA methylation levels following editing. However, at the most severely hypomethylated regions in ICF1 iPSCs, which also display the highest increased H3K4me3 levels and enrichment of CTCF-binding motifs, the epigenetic memory persisted, and hypomethylation was uncorrected. Conclusions: Restoring the catalytic activity of DNMT3B rescues the majority of the aberrant ICF1 epigenome. However, a small fraction of the genome is resilient to this reversal, highlighting the challenge of reverting disease states that are due to genome-wide epigenetic perturbations. Uncovering the basis for the persistent epigenetic memory will promote the development of strategies to overcome this obstacle. | genomics |
Chemical structures of cyclic ADP ribose (cADPR) isomers and the molecular basis of their production and signaling Cyclic ADP ribose (cADPR) isomers are important signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via NAD hydrolysis, yet their chemical structures are unknown. We show that v-cADPR (2cADPR) and v2-cADPR (3-cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of v-cADPR (2-cADPR)-producing TIR domains reveal that conformational changes are required for the formation of the active assembly that resembles those of Toll-like receptor adaptor TIR domains, and mutagenesis data demonstrate that a conserved tryptophan is essential for cyclization. We show that v2-cADPR (3-cADPR) is a potent activator of ThsA effector proteins from Thoeris anti-phage defence systems and is responsible for suppression of plant immunity by the effector HopAM1. Collectively, our results define new enzymatic activities of TIR domains, reveal the molecular basis of cADPR isomer production, and establish v2-cADPR (3-cADPR) as an antiviral signaling molecule and an effector-mediated signaling molecule for plant immunity suppression. | biochemistry |
Microfluidic chemostatic bioreactor for high-throughput screening and sustainable co-harvesting of biomass and biodiesel in microalgae As a renewable and sustainable source for energy, environment, and biomedical applications, microalgae and microalgal biodiesel have attracted great attention. However, their applications are confined due to the cost-efficiency of microalgal mass production. One-step strategy and continuous culturing systems could be solutions. However, current studies for optimization throughout microalgae-based biofuel production pipelines are generally derived from the batch culture process. Better tools are needed to study algal growth kinetics in continuous systems. A microfluidics chemostatic bioreactor array was presented, providing low-adhesion cultivation for algae in the gas, nutrition, and temperature (GNT) well-controlled environment with high throughput. The chip wasused to mimic the continuous culture environment of bioreactors. It allowed simultaneously studying of 8x8 different chemostatic conditions on algal growth and oil production in parallel on a 7x7 cm2 footprint. On-chip experiments of batch and continuous cultures of Chlorella. sp. were performed to study growth and lipid accumulation under different nitrogen concentrations. The results demonstrated that microalgal cultures can be regulated to grow and accumulate lipids concurrently, thus enhancing lipid productivity in one step. The developed on-chip culturing condition screening, which was more suitable for continuous bioreactor, was achieved at a half shorter time, 64-times higher throughput, and less reagent consumption. It could be used to establish chemostat cultures in continuous bioreactors which can dramatically accelerate the development of renewable and sustainable algal for CO2 fixation and biosynthesis and related systems for advanced sustainable energy, food, pharmacy, and agriculture with enormous social and ecological benefits.
TEASERSustainable microfluidic bioreactor for 64 times higher-throughput screening CO2 fixation and biomass and biodiesel production in microalgae. | bioengineering |
A Standardized Ontology for Naming tRNA-derived RNAs Based on Molecular Origin Small RNAs processed from transfer RNA are recognized to have regulatory functions distinct from protein synthesis. This rapidly advancing field has led to a constellation of transcript nomenclatures. Building upon the accepted tRNA naming system and linking tRNA-derived small RNAs to their molecular sources, we propose an improved nomenclature. Facilitated by the tDRnamer system, it will promote inter-study comparisons leveraging a biologically-rooted nomenclature for this emerging class of intriguing regulators. | bioinformatics |
Structural heterogeneity of amyloid aggregates identified by spatially resolved nanoscale infrared spectroscopy Amyloid plaques, composed of aggregates of the amyloid beta (A{beta}) protein, are one of the central manifestations of Alzheimers disease pathology. Aggregation of A{beta} from amorphous oligomeric species to mature fibrils has been extensively studied. However, significantly less in known about early-stage aggregates compared to fibrils. In particular, structural heterogeneities in prefibrillar species, and how that affects the structure of later stage aggregates are not yet well understood. Conventional spectroscopies cannot attribute structural facets to specific aggregates due to lack of spatial resolution, and hence aggregates at any stage of aggregation must be viewed as having the same average structure. The integration of infrared spectroscopy with Atomic Force Microscopy (AFM-IR) allows for identifying the signatures of individual nanoscale aggregates by spatially resolving spectra. In this report, we use AFM-IR to demonstrate that amyloid oligomers exhibit significant structural variations as evidenced in their infrared spectra, ranging from ordered beta structure to disordered conformations with predominant random coil and beta turns. This heterogeneity is transmitted to and retained in protofibrils and fibrils. We show for the first time that amyloid fibrils do not always conform to their putative ordered structure and structurally different domains can exist in the same fibril. We further show the implications of these results in amyloid plaques in Alzheimers tissue using infrared imaging, where these structural heterogeneities manifest themselves as lack of expected beta sheet structure. | biophysics |
The trade-off function of photorespiration in a changing environment The photorespiratory pathway in plants comprises metabolic reactions distributed across several cellular compartments. It emerges from the dual catalytic function of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) which either carboxylates or oxygenates ribulose-1,5-bisphosphate (RuBP). Carboxylation reactions produce 3-phospho-glycerate (3PGA) molecules which are substrate for central carbohydrate metabolism while oxygenation forms 2-phosphoglycolate (2PG) molecules which are substrate for the multicompartmental recovery process of photorespiration. Further, 2PG is a strong inhibitor of several enzymes involved in the Calvin-Benson-Bassham cycle which challenges the experimental and theoretical study of carbon assimilation, photorespiration and metabolic regulation in vivo. Here, an approach of structural kinetic modeling (SKM) is presented to investigate the extend of stabilization of CBC and carbohydrate metabolism by photorespiration. Further, our approach highlights the importance of feedback regulation by 2-PG for alleviation of environmental perturbation. Our findings indicate that oxygenation of RuBP by Rubisco significantly stabilizes CBC activity and, thus, carbohydrate metabolism. Based on our findings, we suggest a trade-off function of photorespiration which reduces carbon assimilation rates but simultaneously stabilizes metabolism by increasing plasticity of metabolic regulation within the chloroplast. Furthermore, our analysis suggests a stabilizing effect of increasing the partition of newly assimilated carbon going towards sucrose biosynthesis. With this, our analysis sheds light on the role of a multicompartmental metabolic pathway in stabilizing plant metabolism within a changing environment. | plant biology |
A task-general connectivity model reveals variation in convergence of cortical inputs to functional regions of the cerebellum While resting-state fMRI studies have provided a broad picture of the connectivity between human neocortex and cerebellum, the degree of convergence of cortical inputs onto cerebellar circuits remains unknown: Does each cerebellar region receive input from a single cortical area or convergent inputs from multiple cortical areas? Here we use task-based fMRI data to build a range of cortico-cerebellar connectivity models, each allowing for a different degree of convergence. We compared these models by their ability to predict cerebellar activity patterns for novel task sets. Models that allow some degree of convergence provided the best predictions, arguing for convergence of multiple cortical inputs onto single cerebellar voxels. Importantly, the degree of convergence varied across the cerebellum with the highest convergence observed in areas linked to language, working memory, and social cognition. These findings suggest important differences in the way that functional subdivisions of the cerebellum support motor and cognitive function. | neuroscience |
Splashed E-box and AP-1 motifs cooperatively drive regeneration-response and shape regeneration abilities Injury triggers genetic program to induce gene expression for regeneration. Several studies have recently reported the identification of regeneration-response enhancers (RREs) in zebrafish; however, it remains unclear whether a common mechanism operates in RREs. Here, we show that E-box and activator protein 1 (AP-1) motifs cooperatively function as RREs. We identified three RREs from the fn1b promoter by a search of conserved sequences and an in vivo transgenic assay for regeneration-response in zebrafish. Two of them derived from transposons displayed RRE activity only when combined with the -0.7 kb fn1b promoter, while another non-transposable element functioned as a standalone enhancer. A search for transcription factor-binding motifs and validation by transgenic assay revealed that both of E-box and AP-1 motifs are necessary and sufficient for RREs. Such RREs responded to variety of tissue injuries including zebrafish heart and Xenopus limb bud regenerations. Our findings highlight that regeneration is regulated by merging two activating signals evoked by tissue injuries. It is speculated that a large pool of potential enhancers in the genome shaped regenerative capacities during evolution.
SUMMARY STATEMENTThe study revealed that regeneration-response enhancer is composed of two transcription factor-binding motifs. The fidelity of regeneration-dependent gene expression is ensured by merging two activating signals evoked by injuries. | developmental biology |
Climate and ant richness explain the global distribution of ant-plant mutualisms Biotic interactions are known to play an important role in shaping species geographic distributions and diversity patterns. However, the role of mutualistic interactions in shaping global diversity patterns remains poorly quantified, particularly with respect to interactions with invertebrates. Moreover, it is unclear how the nature of different mutualisms interacts with abiotic drivers and affects diversity patterns of mutualistic organisms. Here, we present a global-scale biogeographic analysis of three different ant-plant mutualisms, differentiating between plants bearing domatia, extrafloral nectaries (EFNs), and elaiosomes, based on comprehensive geographic distributions of ~15,000 flowering plants and ~13,000 ant species. Domatia and extrafloral nectaries involve indirect plant defenses provided by ants, while elaiosomes attract ants to disperse seeds. Our results show distinct biogeographic patterns of different ant-plant mutualisms, with domatium- and EFN-bearing plant richness decreasing sharply from the equator towards the poles, while elaiosome-bearing plants prevail at mid-latitudes. Contemporary climate, especially mean annual temperature and precipitation, emerge as the most important predictor of ant-associated plant diversity. In hot and moist regions, typically the tropics, domatium- and EFN-bearing plant richness increases with related ant guild richness, while in warm regions plants with elaiosomes are strongly linked to interacting ants. Our results suggest that ant richness in combination with climate drives the spatial variation of plants bearing domatia, extrafloral nectaries, and elaiosomes, highlighting the importance of mutualistic interactions for understanding plant biogeography and its response to global change. | ecology |
Analysis of CheW-like domains provides insights into organization of prokaryotic chemotaxis systems The ability to control locomotion in a dynamic environment provides a competitive advantage for microorganisms, thus driving the evolution of sophisticated regulatory systems. Nineteen known categories of chemotaxis systems control motility mediated by flagella and Type IV pili, plus other cellular functions. A key feature that distinguishes chemotaxis systems from generic two-component regulatory systems is separation of receptor and kinase functions into distinct proteins, linked by CheW scaffold proteins. This arrangement allows for formation of varied arrays with remarkable signaling properties. We recently analyzed sequences of CheW-like domains found in CheA kinases and CheW and CheV scaffold proteins. Sixteen Architectures of CheA, CheW, and CheV proteins contain ~94% of all CheW-like domains, forming six Classes with likely functional specializations. We surveyed chemotaxis system categories and proteins containing CheW-like domains in ~1900 prokaryotic species, the most comprehensive analysis to date. The larger sample size revealed previously unknown insights. Co-occurrence analyses suggested that chemotaxis systems occur in non-random combinations within species, increasing our understanding of evolution of chemotaxis. Furthermore, many Types of CheW-like domains occurred predominantly with specific categories of chemotaxis systems, suggesting specialized functional interactions. For example, Class 2 (Type CheW.IC) domains exhibit properties spanning the primary Classes of CheW-like domains in CheA and CheW proteins. CheW.IC frequently co-occurred with methyl-accepting coiled coil (MAC) proteins, which contain both receptor and kinase functions. Although MAC proteins should not need CheW scaffolds to connect receptor and kinase functions, co-occurrence suggested that MAC systems may nevertheless benefit from array formation facilitated by CheW.IC domains. | bioinformatics |
PHi-C2: interpreting Hi-C data as the dynamic 3D genome state Hi-C is a widely used assay for studying three-dimensional (3D) genome organization across the whole genome. Here, we present PHi-C2, a Python package supported by mathematical and biophysical polymer modeling, that converts an input Hi-C matrix data into the polymer models dynamics, structural conformations, and rheological features. The updated optimization algorithm to regenerate a highly similar Hi-C matrix provides a fast and accurate optimal solution compared to the previous version by eliminating a computational bottleneck in the iterative optimization process. Besides, we newly set up the availability on Google Colab workflow to run, easily change parameters and check the results in the notebook. Overall, PHi-C2 can be a valuable tool to mine the dynamic 3D genome state embedded in Hi-C data.
Availability and ImplementationPHi-C2 as the phic Python package is freely available under the GPL license and can be installed from the Python package index. The source code is available from GitHub at https://github.com/soyashinkai/PHi-C2. Without preparing a Python environment, PHi-C2 can run on Google Colab (https://bit.ly/3rlptGI).
[email protected] or [email protected] | bioinformatics |
Inhibition of Aromatase by Hops, Licorice Species, and their bioactive compounds in Postmenopausal Breast Tissue Breast cancer risk continues to rise post menopause. Endocrine therapies are employed to prevent postmenopausal breast cancer in high-risk women. However, their adverse effects have reduced acceptability and overall success in cancer preventionatural products such as hops (Humulus lupulus) and pharmacopoeial licorice (Glycyrrhiza) species, which are used for managing menopausal symptoms, have demonstrated estrogenic and chemopreventive properties. Their beneficial effects on aromatase activity and expression as important factors in postmenopausal breast carcinogenesis are understudied. The presented data show that Gycyrrhiza inflata (GI) has the highest aromatase inhibition potency among these plants. Moreover, phytoestrogens such as 8-prenylnaringenin from hops as well as liquiritigenin and 8-prenylapigenin from licorice are shown to be potent bioactives, in line with computational docking studies. 8-Prenylnaringenin, GI extract, liquiritigenin, and licochalcone A all suppress aromatase expression in postmenopausal womens breast tissue. Collectively, these data suggest that these natural products may have breast cancer prevention potential for high-risk postmenopausal women. | pharmacology and toxicology |
Composition structures and biologically meaningful logics: plausibility and relevance in bipartite models of gene regulation Boolean network models have widely been used to study the dynamics of gene regulatory networks. However, such models are coarse-grained to an extent that they abstract away molecular specificities of gene regulation. In contrast, bipartite Boolean network models of gene regulation explicitly distinguish genes from transcription factors (TFs). In such models, multiple TFs may simultaneously contribute to the regulation of a gene by forming heteromeric complexes. The formation of heteromeric complexes gives rise to composition structures in the corresponding bipartite network. Remarkably, composition structures can severely restrict the number of Boolean functions (BFs) that can be assigned to a gene. The introduction of bipartite Boolean network models is relatively recent, and so far an empirical investigation of their biological plausibility is lacking. Here, we estimate the prevalence of composition structures arising through heteromeric complexes in Homo sapiens. Moreover, we present an additional mechanism by which composition structures arise as a result of multiple TFs binding to the cis-regulatory regions of a gene and we provide empirical support for this mechanism. Next, we compare the restriction in BFs imposed by composition structures and by biologically meaningful properties. We find that two types of minimally complex BFs, namely nested canalyzing functions (NCFs) and read-once functions (RoFs), are more restrictive than composition structures. Finally, using a compiled dataset of 2687 BFs from published models, we find that composition structures are highly enriched in real biological networks, but that this enrichment is most likely driven by NCFs and RoFs. | systems biology |
Pattern dynamics and stochasticity of the brain rhythms Our current understanding of brain rhythms is based on quantifying their instantaneous or time-averaged characteristics. What remains unexplored, is the actual structure of the waves--their shapes and patterns over finite timescales. To address this, we used two independent approaches to link wave forms to their physiological functions: the first is based on quantifying their consistency with the underlying mean behavior, and the second assesses "orderliness" of the waves features. The corresponding measures capture the waves characteristic and abnormal behaviors, such as atypical periodicity or excessive clustering, and demonstrate coupling between the patterns dynamics and the animals location, speed and acceleration. Specifically, we studied patterns of{theta} and{gamma} waves, and Sharp Wave Ripples, and observed speed-modulated changes of the waves cadence, an antiphase relationship between orderliness and acceleration, as well as spatial selectiveness of patterns. Further-more, we found an interdependence between orderliness and regularity: larger deviations from steady oscillatory behavior tend to accompany disarrayed temporal cluttering of peaks and troughs. Taken together, our results offer a complementary--mesoscale--perspective on brain wave structure, dynamics, and functionality. | neuroscience |
Reinstatement of Pavlovian responses to alcohol cues by stress RationaleStress may contribute to relapse to alcohol use in part by enhancing reactivity to cues previously paired with alcohol. Yet, standard models of stress-induced reinstatement generally use contingent presentations of alcohol-paired cues to reinforce instrumental behaviors, making it difficult to isolate the ability of cues to invigorate alcohol-seeking.
ObjectiveHere we sought to test the impact of stress on behavioral responses to alcohol-paired cues, using a model of stress-induced reinstatement of Pavlovian conditioned approach, inspired by Nadia Chaudhris work on context-induced reinstatement.
MethodsLong Evans rats were trained to associate one auditory cue with delivery of alcohol or sucrose and an alternative auditory cue with no reward. Following extinction training, rats were exposed to a stressor prior to being re-exposed to the cues under extinction conditions. We assessed the effects of yohimbine, intermittent footshock and olfactory cues paired with social defeat on responses to alcohol-paired cues, and the effects of yohimbine on responses to sucrose-paired cues.
ResultsThe pharmacological stressor, yohimbine, enhanced Pavlovian responses to both alcohol and sucrose cues, but intermittent footshock and social defeat cues did not.
ConclusionsWhile yohimbine elicited reinstatement of Pavlovian conditioned responses, these effects may be unrelated to activation of stress systems. | neuroscience |
habCluster: Identifying Geographical Boundary among Intraspecific Units Using Community Detection Algorithm in R Conservation management for a species generally rests on intraspecific units, while identification of their geographic boundaries is necessary for the implementation. Intraspecific units can be discriminated using population genetic methods, yet an analytical approach is still lacking for detecting their geographic boundaries. Here, based on landscape connectivity, we present a geographical boundary delineation method, habCluster, using community detection algorithm. Community detection is an algorithm in graph theory used to identify clusters of highly connected nodes within a network. We assume that the habitat raster cells with better connections tend to form a continuous habitat patch than the others, thus making the range of an intraspecific unit. The method is tested on grey wolf (Canis lupus) habitat in Europe and on giant panda (Ailuropoda melanoleuca) habitat in China. The habitat suitability for grey wolves and giant pandas were evaluated using species distribution modeling. Each cell in the habitat suitability index (HSI) raster is treated as a node and directly connected with its eight neighbor cells. The edge weight between nodes is the distance between the center of them weighted by the average of their HSI values. We implement habCluster using R programming language with inline C++ code to speed up the computing. The geographical clusters detected were compared with the HSI maps for both species and with the nature reserves for giant panda. We found the boundaries of the clusters delineated using habCluster could serve as a good indicator of habitat patches, and they match generally well with nature reserves in the giant panda case. habCluster can provide spatial analysis basis for conservation management plans such as monitoring, translocation and reintroduction, as well as for population structure research. | ecology |
Life history traits and a method for continuous mass rearing of the planthopper Pentastiridius leporinus, a vector of the causal agent of syndrome "basses richesses" in sugar beet BACKGROUNDThe planthopper Pentastiridius leporinus (Hemiptera: Cixiidae) is the main vector of the {gamma}-3 proteobacterium Candidatus Arsenophonus phytopathogenicus which causes the syndrome "basses richesses" (SBR) in sugar beet. SBR is a new and fast spreading disease in Central Europe that leads to high yield losses. To date the development of management strategies is hampered by insufficient knowledge about general life history traits of the planthopper and, most importantly, the year round availability of insects reared under controlled conditions. Rearing of P. leporinus has been considered challenging and to date no protocol exists.
RESULTSHere we describe a method for mass rearing P. leporinus on sugar beet from egg to adult, which has produced five generations and >20,000 individuals between June 2020 and March 2022. An alternative host such as wheat is not necessary for completing the life cycle. No-choice experiments showed that P. leporinus lays 139.1 {+/-} 132.9 eggs on sugar beet, whereas no oviposition was observed on its nymphal host wheat. Head capsule width was identified as a trait that unequivocally distinguished the five nymphal instars. Developmental time from first instar to adult was 193.6 {+/-} 35.8 days for males and 193.5 {+/-} 59.2 days for females. Infection rates of adults were tested with nested polymerase chain reaction (PCR). The results demonstrated that 70-80% of reared planthoppers across all generations carried the SBR proteobacterium.
CONCLUSIONThe mass rearing protocol and life history data will help overcome an important bottleneck in SBR research and enhance efforts in developing integrated pest management tools. | ecology |
DeepLUAD: An efficient approach for lung adenocarcinoma pattern classification Histopathological analysis of whole-slide images is the gold standard technique for diagnosis of lung cancer and classifying it into types and subtypes by specialized pathologists. This labor-based approach is time and effort consuming, which led to development of automatic approaches to assist in reducing the time and effort. Deep learning is a supervised classification approach that is well adapted for automatic classification of histopathological images. We aimed to develop a deep learning-based approach for lung adenocarcinoma pattern classification and generalize the proposed approach to the classification of the major non-small cell lung cancer types. Three publicly available datasets were used in this study. A deep learning approach for histopathological image analysis using convolutional neural networks was developed and incorporated into automatic pipelines to accurately classify the predominant patterns on the whole-slide images level and non-small cell lung cancer types on patch-level. The models were evaluated using the confusion matrix to perform an error analysis and the classification report to compute F1-score, recall and precision. As results, the three models have shown an excellent performance with best combination of hyper-parameters for training models. First and second models predicted adenocarcinoma predominant patterns on two different datasets with an accuracy, respectively, of 96.15% and 89.51%. The third model has exceeded an accuracy of 99.72% in classifying major non-small cell lung cancer types. The proposed deep learning-based lung cancer classification approach can be used to assist pathologists in identifying of lung adenocarcinomas patterns. | bioinformatics |
Generalizable strategy to analyze domains in the context of parent protein architecture: A CheW case study Domains are the three-dimensional building blocks of proteins. An individual domain can be found in a variety of protein architectures that perform unique functions and are subject to different evolutionary selective pressures. We describe an approach to evaluate the variability in amino acid sequences of a single domain across architectural contexts. The ability to distinguish the different evolutionary paths of one protein domain can help determine whether existing knowledge about a specific domain will apply to an uncharacterized protein. Such discrimination can lead to insights and hypotheses about function, as well as guide experimental priorities. We developed and tested our approach on CheW-like domains (PF01584), which mediate protein/protein interactions and are difficult to compare experimentally. CheW-like domains occur in CheW scaffolding proteins, CheA kinases, and CheV proteins that regulate bacterial chemotaxis. We chose 16 protein Architectures that included 94% of all CheW-like domains found in nature. Because some Architectures had more than one CheW-like domain, CheW-like domains were divided into 21 distinct Contexts. The CheW-like domain sequences were closely related within most Contexts; however, one Context was subdivided into three Types. The resulting 23 sequence Types coalesced into five or six Classes of CheW-like domains, which we described in detail. In addition, we created SimpLogo, an innovative method for visualizing amino acid composition across large sets of multiple sequence alignments of arbitrary length. SimpLogo offers substantial advantages over standard sequence logos for comparison and analysis of related protein sequences. The R package for SimpLogo is freely available. | bioinformatics |
The funny current If is essential for the fight-or-flight response in cardiac pacemaker cells The sympathetic nervous system fight-or-flight response is characterized by a rapid increase in heart rate, which is mediated by an increase in the spontaneous action potential (AP) firing rate of pacemaker cells in the sinoatrial node. Sympathetic neurons stimulate sinoatrial myocytes (SAMs) by activating {beta} adrenergic receptors ({beta}ARs) and increasing cAMP. The funny current (If) is among the cAMP-sensitive currents in SAMs. If is critical for pacemaker activity, however, its role in the fight-or-flight response remains controversial. In this study, we used AP waveform analysis, machine learning, and dynamic clamp experiments in acutely-isolated SAMs from mice to quantitatively define the AP waveform changes and role of If in the fight-or-flight response. We found that while {beta}AR stimulation significantly altered nearly all AP waveform parameters, the increase in AP firing rate was only correlated with changes in a subset of parameters (diastolic duration, late AP duration, and diastolic depolarization rate). Dynamic clamp injection of the {beta}AR-sensitive component of If showed that it accounts for approximately 41% of the fight-or-flight increase in AP firing rate and 60% of the decrease in the interval between APs. Thus, If is an essential contributor to the fight-or-flight increase in heart rate. | physiology |
Learning consistent subcellular landmarks to quantify changes in multiplexed protein maps Highly multiplexed quantitative subcellular imaging holds enormous promise for understanding how spatial context shapes the activity of our genome and its products at multiple scales. Yet unbiased analysis of subcellular organisation across experimental conditions remains challenging, because differences in molecular profiles between conditions confound differences in molecular profiles across space. Here, we introduce a deep-learning framework called CAMPA (Conditional Autoencoder for Multiplexed Pixel Analysis), which uses a variational autoencoder conditioned on cellular states and perturbations to learn consistent molecular signatures. Clustering the learned representations into subcellular landmarks allows quantitative comparisons of landmark sizes, shapes, molecular compositions and relative spatial organisation between conditions. By performing high-resolution multiplexed immunofluorescence on human cells, we use CAMPA to reveal how subnuclear organisation changes upon different perturbations of RNA production or processing, and how different membraneless organelles scale with cell size. Furthermore, by integrating information across the cellular and subcellular scales, we uncover new links between the molecular composition of membraneless organelles and bulk RNA synthesis rates of single cells. We anticipate that CAMPA will greatly accelerate the systematic mapping of multiscale atlases of biological organisation to identify the rules by which context shapes physiology and disease. | systems biology |
Single-cell RNA sequencing reveals molecular features of postnatal maturation in the murine retinal pigment epithelium Transcriptomic analysis of the mammalian retinal pigment epithelium (RPE) aims to identify cellular networks that influence ocular development, maintenance, function, and disease. However, available evidence points to RPE cell heterogeneity in the native tissue, which adds complexity to transcriptomic analysis. Here, to assess cell heterogeneity, we performed single-cell RNA sequencing of RPE cells from two young adult male C57BL/6J mice. Following quality control to ensure robust transcript identification limited to cell singlets, we detected 13,858 transcripts among 2,667 and 2,846 RPE cells, respectively. Dimensional reduction by principal component analysis and uniform manifold approximation and projection revealed six distinct cell popu-lations. All clusters expressed transcripts typical of RPE cells; the smallest (C1, containing 1-2% of total cells) exhibited hallmarks of stem and/or progenitor cells. Placing C1-6 along a pseudotime axis suggested a relative decrease in melanogenesis and stem/progenitor gene expression, and a corresponding increase in visual cycle gene expression upon RPE maturation. K-means testing of all detected transcripts identified additional expression patterns that may advance understanding of RPE stem/pro-genitor cell maintenance and the evolution of cellular metabolic networks during development. This work provides new insights into the transcriptome of the mouse RPE and a baseline for identifying experimentally induced transcriptional changes in future studies of this tissue. | systems biology |
Allogeneic testes transplanted into castrated adult medaka (Oryzias latipes) are engrafted and can function over a long period for production of donor-derived offspring by natural mating Germ cell transplantation protocols generally require induced immunosuppression in the recipient to avoid rejection of the transplanted cells; cytotoxic drugs may also be used to deprive the recipient germ cells and create a germ cell niche that allows donor-derived germ cells to thrive. In the present study, we demonstrate that in medaka, allogeneic adult testicular tissue will engraft in adult recipients immediately after the rough castration of the recipients and without use of immunosuppressive drugs. The allografted testes are retained in the recipient body for at least three months and are able to produce viable gametes that yield offspring after natural mating. Some recipients showed a high frequency (over 60%) of offspring derived from spermatozoa produced by the transplanted germ cell tissues. Histological analyses showed that allografted testicular tissues included both germ cells and somatic cells that had established within an immunocompetent recipient testis. The relative simplicity of this new approach will benefit investigations of the basic processes of reproductive immunology and will improve the technique of gonadal tissue transplantation. | zoology |
Validation of tWO novel primers for THE promising amplification of the mitogenomic Cytochrome c Oxidase subunit I (COI) barcoding region in Artemia aff. sinica (Branchiopoda, Anostraca) Due to the lack of a taxonomic key for the identification of Artemia species, molecular markers have been increasingly used for phylogenetic studies. The mtCOI marker is a regularly considered marker for the molecular systematics of Artemia populations. The proposed universal and specific primers have mostly failed to amplify the Artemia aff. sinica mtCOI marker, and on the whole, the successfully amplified products of the PCR were inefficient, primarily through the representation of poly-peak or incomplete sequences. We presumed that if a forward primer could be developed regarding the joint regions of the last part of the previous gene (tRNATyr) and the beginning of the target gene mtCOI, the sequence could be relevant to the target-sequence of mtCOI. Thus, here, we describe a new set of primers, which could be used to amplify the mtCOI barcoding region of Artemia aff. sinica Cai, 1989, with a high performance of sequencing. The new primer set worked well also for other Artemia bisexual species, as well as for parthenogenetic populations. It is recommended that joint regions between the previous/next gene(s) and the target marker, could be aimed at when designing specific primers for other markers and taxa. | zoology |
Cyst size variability in invasive American Artemia franciscana Kellogg, 1906 (Branchiopoda: Anostraca) in Asia: a commercial approach Artemia is the most common live food which is used in aquaculture worldwide. This study reports on biometrical variation of introduced American Artemia franciscana cyst from 24 non-native localities and two native habitats in Asia and USA, respectively. Results showed the largest diameter of untreated cyst, diameter of decapsulated cyst and thicker chorion ordinarily belong to invasive populations. Because of the small cysts, which have an effect on increasing quantity per unit weight and could be the cause of increased hatching efficiency, commercial productions of A. franciscana cyst from native sources should potentially be considered higher quality than productions from non-indigenous environments. Principal Component Analysis revealed that all cyst batches from San Francisco Bay were classified in one group and the most invasive populations could arrange in other separated group. Although, diameter of decapsulated cyst and chorion thickness showed a negative and significant correlation among invasive populations, there was no significant relationship within native populations. These observations contrast with biometrical patterns of parthenogenetic populations. | zoology |
Oxytocin attenuates microglial activation and restores social and non-social memory in the APP/PS1 mouse model of Alzheimer's disease Alzheimers disease (AD) is the main cause of dementia in the elderly and is characterized by memory loss, social withdrawal and neurodegeneration, eventually leading to death. Brain inflammation has emerged as a key pathogenic mechanism in AD. We hypothesized that oxytocin, a pro-social hypothalamic neuropeptide with anti-inflammatory properties, could have therapeutic actions in AD. We investigated oxytocin production in mouse models of AD, and evaluated the therapeutic potential of intranasal oxytocin. We observed lower levels of hypothalamic oxytocin in wild-type mice following brain infusion of amyloid-{beta} oligomers (A{beta}Os), as well as in APP/PS1 AD model mice. Treatment of APP/PS1 mice with intranasal oxytocin reduced microglial activation and favored deposition of A{beta} in dense core plaques, a potentially neuroprotective mechanism. Oxytocin further alleviated social and non-social memory impairments in APP/PS1 mice. Our findings point to oxytocin as a potential therapeutic target to reduce brain inflammation and correct memory deficits in AD. | neuroscience |
ATXN2 is a target of N-terminal proteolysis Spinocerebellar ataxia 2 (SCA2) is a neurodegenerative disorder caused by the expansion of the poly-glutamine (polyQ) tract of Ataxin-2 (ATXN2). Other polyQ-containing proteins such as ATXN7 and huntingtin are associated with the development of neurodegenerative diseases when their N-terminal polyQ domains are expanded. Furthermore, they undergo proteolytic processing events that produce N-terminal fragments that include the polyQ stretch, which are implicated in pathogenesis. Interestingly, N-terminal ATXN2 fragments were reported in a brain extract from a SCA2 patient, but it is currently unknown whether an expanded polyQ domain contributes to ATXN2 proteolytic susceptibility. Here, we used transient expression in HEK293 cells to determine whether ATXN2 is a target for specific N-terminal proteolysis. We found that ATXN2 proteins with either normal or expanded polyQ stretches undergo proteolytic cleavage releasing an N-terminal polyQ-containing fragment. We identified a short amino acid sequence downstream of the polyQ domain that is necessary for N-terminal cleavage of full-length ATXN2 and sufficient to induce proteolysis of a heterologous protein. However, this sequence is not required for cleavage of a short ATXN2 isoform produced from an alternative start codon located just upstream of the CAG repeats encoding the polyQ domain. Our study extends our understanding of ATXN2 posttranslational regulation by revealing that this protein can be the target of specific proteolytic cleavage events releasing polyQ-containing products that are modulated by the N-terminal domain of ATXN2. N-terminal ATXN2 proteolysis of expanded polyQ domains might contribute to SCA2 pathology, as observed in other neurodegenerative disorders caused by polyQ domain expansion. | neuroscience |
Alone in a crowd: Effect of a nonfunctional lateral line on expression of the social hormone parathyroid hormone 2. Parathyroid hormone 2 (Pth2) is a vertebrate-specific neuropeptide whose thalamic expression is upregulated by social contact with conspecifics. However, social interactions fail to stimulate pth2 expression in isolated zebrafish whose lateral line hair cells have been chemically ablated. These results suggest that modulation of pth2 by social context is acutely dependent on mechanosensory information from the lateral line. However, it is unclear how a congenital loss of lateral line function influences the ability of zebrafish to interpret their social environment. In this study, we measure pth2 levels in zebrafish mutants lacking hair cell function in either the lateral line only, or in both the inner ear and lateral line. Socially-raised lateral line mutants express lower levels of pth2 relative to wild type siblings, but there is no further reduction when all sensory hair cells are nonfunctional. However, social isolation of hair cell mutants causes a further reduction in pth2 expression, pointing to additional unidentified sensory cues that influence pth2 production. Lastly, we report that social context modulates fluorescent transgenes driven by the pth2 promoter. Altogether, these data suggest that lateral line mutants experience a chronic sense of isolation, even when raised in a social environment.
SUMMARY STATEMENTExpression of the pro-social neuropeptide pth2 is downregulated in larval zebrafish with a congenital loss of lateral line function. Thus, even in social environments, fish with compromised lateral lines may experience chronic loneliness. | neuroscience |
Phase separation of competing memories along the human hippocampal theta rhythm. Competition between overlapping memories is considered one of the major causes of forgetting and it is still unknown how the human brain resolves such mnemonic conflict. In the present MEG study, we empirically tested a computational model that leverages an oscillating inhibition algorithm to minimise overlap between memories. We used a proactive interference task, where a reminder word could be associated with either a single image (non-competitive condition) or two competing images, and participants were asked to always recall the most recently learned word-image association. Time-resolved pattern classifiers were trained to detect the reactivated content of target and competitor memories from MEG sensor patterns, and the timing of these neural reactivations was analysed relative to the phase of the dominant hippocampal 3Hz theta oscillation. In line with our preregistered hypotheses, target and competitor reactivations locked to different phases of the hippocampal theta rhythm after several repeated recalls. Participants who behaviourally experienced lower levels of interference also showed larger phase separation between the two overlapping memories. The findings provide evidence that the temporal segregation of memories, orchestrated by slow oscillations, plays a functional role in resolving mnemonic competition by separating and prioritising relevant memories under conditions of high interference. | neuroscience |
Preconfigured dynamics in the hippocampus are guided by embryonic birthdate and rate of neurogenesis The incorporation of novel information into the hippocampal network is likely be constrained by its innate architecture and internally generated activity patterns. However, the origin, organization, and consequences of such patterns remain poorly understood. Here, we show that hippocampal network dynamics are affected by sequential neurogenesis. We birthdated CA1 pyramidal neurons with in-utero electroporation over 4 embryonic days encompassing the peak of hippocampal neurogenesis, and compared their functional features in freely moving, adult mice. Neurons of the same birthdate displayed distinct connectivity, coactivity across brain states, and assembly dynamics. Same birthdate hippocampal neurons were topographically organized, in that anatomically clustered (<500{micro}m) neurons exhibited overlapping spatial representations. Overall, the wiring and functional features of CA1 pyramidal neurons reflected a combination of birthdate and the rate of neurogenesis. These observations demonstrate that sequential neurogenesis in embryonic development shapes the preconfigured forms of adult network dynamics. | neuroscience |
Exploring the utility of recombinantly expressed snake venom serine protease toxins as immunogens for generating experimental snakebite antivenoms Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies. | immunology |
Pnpt1 mediates NLRP3 inflammasome activation by MAVS and metabolic reprogramming in macrophages Polyribonucleotide nucleotidyltransferase 1 (Pnpt1) plays critical roles in mitochondrial homeostasis by controlling mitochondrial RNA (mt-RNA) processing, trafficking and degradation. Pnpt1 deficiency results in mitochondrial dysfunction that triggers a Type I interferon response, suggesting a role in inflammation. However, the role of Pnpt1 in inflammasome activation remains largely unknown. In this study, we generated myeloid-specific Pnpt1-knockout mice, and demonstrated that Pnpt1 depletion enhanced interleukin-1 beta (IL-1{beta}) and interleukin-18 (IL-18) secretion in mouse sepsis models. Using cultured peritoneal and bone marrow-derived macrophages we demonstrated that Pnpt1 regulated NLRP3 inflammasome dependent IL-1{beta} release in response to lipopolysaccharides (LPS), followed by nigericin, ATP or poly (I:C) treatment. Pnpt1 deficiency in macrophages increased glycolysis after LPS, and mt-reactive oxygen species (mt-ROS) after NLRP3 inflammasome activation. Pnpt1 activation of the inflammasome was dependent on both increased glycolysis and expression of the mitochondrial antiviral-signaling protein (MAVS), but not NF-{kappa}B signaling. Collectively, these data strengthen the concept that Pnpt1 is an important mediator of inflammation as shown by activation of the NLRP3 inflammasome in mouse sepsis and cultured macrophages. | immunology |
Defining the HIV Capsid Binding Site of Nucleoporin 153 The interaction between the HIV-1 capsid (CA) and human nucleoporin 153 (NUP153) is vital for delivering the HIV-1 preintegration complex into the nucleus via the nuclear pore complex. The interaction with CA requires a phenylalanine/glycine-containing motif in the C-terminus of NUP153. This study used molecular modeling and biochemical assays to determine the amino acids of NUP153 that are essential for its interactions with CA. Molecular dynamics, FoldX, and PyRosetta simulations delineated the minimal CA binding motif of NUP153 based on the known structure of NUP153 bound to the HIV-1 CA hexamer. Computational predictions were experimentally validated by testing the interaction of NUP153 with CA using an in vitro binding assay and a cell-based TRIM-NUP153C restriction assay. This multidisciplinary approach identified eight amino acids from P1411 to G1418 that stably engage with CA, with significant correlations between molecular models and empirical experiments. Specifically, P1411, V1414, F1415, T1416, F1417, and G1418 were confirmed as critical amino acids required to interact NUP153 with CA. | molecular biology |
DNA metabarcoding of gut contents reveals key habitat and seasonal drivers of trophic networks involving generalist predators in agricultural landscapes BACKGROUND: Understanding the networks of trophic interactions into which generalist predators are embedded is key to assessing their ecological role of in trophic networks and the biological control services they provide. The advent of affordable DNA metabarcoding approaches greatly facilitates quantitative understanding of trophic networks and their response to environmental drivers. Here, we examine how key environmental gradients interact to shape predation by Lycosidae in highly dynamic vegetable growing systems in China. RESULTS: For the sampled Lycosidae, crop identity, pesticide use, and seasons shape the abundance of preydetected in spider guts. For the taxonomic richness of prey, local- and landscape-scale factors gradients were more influential. Multivariate ordinations confirm that these crop-abundant spiders dynamically adjust their diet to reflect environmental constraints and seasonal availability to prey. CONCLUSION: The plasticity in the diet composition is likely to account for the persistence of spiders in relatively ephemeral brassica crops. Our findings provide further insights into the optimization of habitat management for predator-based biological control practices. | ecology |
Food resources drive rodent population demography mediated by seasonality and inter-specific competition O_LIAs fast reproducing species, rodents present proximate numerical responses to resource availability that have been assessed by experimental manipulation of food, with contrasting results. Other intrinsic and extrinsic factors, such as climate severity, species life cycles, and sympatry of potential competitors in the community, may interplay to modulate such responses, but their effects have rarely been evaluated ensemble.
C_LIO_LIWe applied a niche-based approach to experimentally determine the effect of bottom-up (food availability) and top-down (climate severity) extrinsic factors, as well as intrinsic seasonal cycles, on rodent demography, also in presence of sympatry between species in the community.
C_LIO_LITo this end, we live-trapped rodents at two latitudinal extremes of the boreal-temperate gradient (Italian Alps and Norway) deploying control/treatment designs of food manipulation. We applied a multistate open robust design model to estimate population patterns and survival rate.
C_LIO_LIYellow-necked and wood mouse (Apodemus spp.) were sympatric with bank vole in Italy, while the latter was the only species trapped in Norway. At northern latitudes, where harsher climatic conditions occurred, vole survival was principally regulated by intrinsic seasonal cycles, with a positive effect of food also on population abundance. At southern latitudes, mice and voles exhibited asynchronous population patterns across seasons, with survival depending from seasonal cycles. When concentrated ad libitum food was experimentally provided, though, population size and survival of voles strongly decreased, while mice abundance benefited from food supplementation.
C_LIO_LIOur results evidence that rodent demography is regulated by a combination of top-down (climate severity) and bottom-up (food availability) extrinsic factors, together with intrinsic seasonal ones. Moreover, we showed that the seasonal niche partitioning of mice and voles could be disrupted by availability of abundant resources that favour the demography of the more opportunistic Apodemus spp. at the expense of Myodes glareolus, suggesting competitive mechanisms. We conclude putting our results in the context of climate change, where shifts in vegetation productivity may affect the diversity of the rodent community via demographic effects.
C_LI | ecology |
Potential future climate change effects on global reptile distribution and diversity AimUntil recently, complete information on global reptile distributions has not been widely available. Here, we provide the first comprehensive climate impact assessment for reptile distributions at a global scale.
LocationGlobal, excluding Antarctica
Time period1995, 2050, 2080
Major taxa studiedReptiles
MethodsWe performed species distribution models for 6296 reptile species and assessed potential global as well as realm-specific changes in species richness, the change in global species richness across climatic space and species-specific changes in distribution and range extent and overlap, under future climate change. To assess the future climatic impact of 3768 non-modeled species, we compared the future change in climatic conditions between both modeled and non-modeled species.
ResultsReptile richness was projected to decline significantly over time, globally but also for most zoogeographic realms, with the strongest decrease in Brazil, Australia and South Africa. Species richness was highest in warm, but moist regions, which were projected to shift further to climate extremes in the future. Extents of occurrence were projected to decline considerably in the future, with a low overlap between projected current and future ranges. Shifts in range centroids differed among realms and taxa, with a dominating global poleward shift. Non-modeled species were significantly stronger affected by climatic changes than modeled species.
Main conclusionsReptile richness was projected to decrease significantly across most parts of the world with ongoing future climate change. This effect is visible across lizards, snakes and turtles alike and has considerable impact on species extent of occurrence (EOO) and range distribution. Together with other anthropogenic impacts, such as habitat loss and harvesting, this is cause for concern. Given the historical lack of information on global reptile distributions, this calls for an re-assessment of global conservation efforts towards reptile species, with specific focus on anticipated future climatic changes. | ecology |
Significant phylogenetic signal is not enough to trust phylogenetic predictions In a recent study, Cantwell-Jones et al. (2022) proposed a list of 1044 species as promising key sources of B vitamins based primarily on phylogenetic predictions. To identify candidate plants, they fitted lambda models of evolution to edible species with known values in each of six B vitamins (232 to 280 species) and used the estimated parameters to predict B-vitamin profiles of edible plants lacking nutritional data (6460 to 6508 species). The latter species were defined as potential sources of a B vitamin if the predicted vitamin content was [≥]15% towards recommended dietary allowances for active females between 31-50 years per 100 g of fresh edible plant material consumed. Unfortunately, the reliability of the predictions that informed the list of candidate species is questionable due to insufficient phylogenetic signal in the data (Pagels {lambda} between 0.171 and 0.665) and a high incidence of species with missing values (over 95% of all the species analyzed in the study). We found that of the 1044 species proposed as promising B-vitamin sources, 626 to 993 species showed accuracies that were indistinguishable from those obtained under a white noise model of evolution (i.e. random predictions conducted in absence of any phylogenetic structure) in at least one of the vitamins, which proves the weakness of the inference drawn from imputed information in the original study. We hope this commentary serves as a cautionary note for future phylogenetic imputation exercises to carefully assess whether the data meet the requirements for the predictions to be valuable, or at least more accurate than expected by chance. | evolutionary biology |
Allele-specific knockouts reveal a role for apontic-like in the evolutionary loss of larval pigmentation in Bombyx mori The domesticated silkworm, Bombyx mori, and its wild progenitor, B. mandarina, are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in pigmentation in both larval and adult B. mori. Here we evaluate the efficiency of CRISPR/Cas9-targeted knockouts of pigment-related genes as a tool to understand their potential contributions to domestication-associated pigmentation loss in B. mori. To demonstrate the efficacy of targeted knockouts in B. mandarina, we generated a homozygous CRISPR/Cas9-targeted knockout of yellow-y. In yellow-y knockout mutants, black body color became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in pigment formation. Further, we performed allele-specific CRISPR/Cas9-targeted knockouts of the pigment-related transcription factor, apontic-like (apt-like) in B. mori x B. mandarina F1 hybrid individuals. Knockout of the B. mandarina allele of apt-like in F1 embryos results in depigmented patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F1 larval pigmentation. These results demonstrate a contribution of apt-like to the evolution of reduced pigmentation in B. mori. Together, our results demonstrate the feasibility of CRISPR/Cas9-targeted knockouts as a tool for understanding the genetic basis of traits associated with B. mori domestication.
Brief abstractBombyx mori and its wild progenitor are an important model for the study of phenotypic evolution associated with domestication. As proof-of-principle, we used CRISPR/Cas9 to generate targeted knockouts of two pigmentation-related genes. By generating a homozygous knockout of yellow-y in B. mandarina, we confirmed this gene"s role in pigment formation. Further, by generating allele-specific knockouts of apontic-like (apt-like) in B. mori x B. mandarina F1 hybrids, we establish that evolution of apt-like contributed to reduced pigmentation during B. mori domestication.
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MegaBayesianAlphabet: Mega-scale Bayesian Regression methods for genome-wide prediction and association studies with thousands of traits Large-scale phenotype data are expected to increase the accuracy of genome-wide prediction and the power of genome-wide association analyses. However, genomic analyses of high-dimensional, highly correlated data are challenging. We developed MegaBayesianAlphabet to simultaneously analyze genetic variants underlying thousands of traits using the flexible priors of the Bayesian Alphabet family. As a demonstration, we implemented the BayesC prior in the R package MegaLMM and applied it to both simulated and real data sets. Our analyses show that the resulting model MegaBayesC can effectively use high-dimensional phenotypic data to improve the accuracy of genetic value prediction, the reliability of marker discovery, and the accuracy of marker effect size estimation in genome-wide analyses. | genetics |
Limited overlap of eQTLs and GWAS hits due to systematic differences in discovery Most signals in genome-wide association studies (GWAS) of complex traits point to noncoding genetic variants with putative gene regulatory effects. However, currently identified expression quantitative trait loci (eQTLs) explain only a small fraction of GWAS signals. By analyzing GWAS hits for complex traits in the UK Biobank, and cis-eQTLs from the GTEx consortium, we show that these assays systematically discover different types of genes and variants: eQTLs cluster strongly near transcription start sites, while GWAS hits do not. Genes near GWAS hits are enriched in numerous functional annotations, are under strong selective constraint and have a complex regulatory landscape across different tissue/cell types, while genes near eQTLs are depleted of most functional annotations, show relaxed constraint, and have simpler regulatory landscapes. We describe a model to understand these observations, including how natural selection on complex traits hinders discovery of functionally-relevant eQTLs. Our results imply that GWAS and eQTL studies are systematically biased toward different types of variants, and support the use of complementary functional approaches alongside the next generation of eQTL studies. | genomics |
A detailed landscape of genomic alterations in malignant peripheral nerve sheath tumor cell lines challenges the current MPNST diagnosis Background: Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that arise from the peripheral nervous system. Half of the tumors develop in the context of the genetic disease Neurofibromatosis type 1 (NF1) and the rest are sporadic sarcomas. MPNSTs have a dismal prognosis due to their aggressiveness and tendency to metastasize, and new treatment options are needed. The diagnosis of MPNSTs can be challenging, especially outside of the NF1 context since specific histological criteria have not been completely established. Genomic analysis may both facilitate differential diagnoses and suggest precision medicine strategies. Methods: We generated a complete genomic resource of a set of widely used human NF1-related and sporadic MPNST cell lines by applying ploidy analysis, whole genome and whole exome sequencing and SNP-array analysis, complemented by methylome-based classification and immunofluorescence of cell identity markers (SOX9, SOX10, S100B). Results: NF1 MPNST cell lines faithfully recapitulated the genomic copy number profile of primary MPNSTs. Structural variants were key players in the complete inactivation of most recurrently altered tumor suppressor genes (TSGs) (NF1, CDKN2A, SUZ12/EED), while small variants played a minor role in the NF1 context, both concerning TSG inactivation and due to the absence of gain-of-function mutations. In clear contrast, the sporadic cell lines (STS-26T, HS-Sch-2, HS-PSS) did not recapitulate the copy number profile of primary MPNSTs. They carried different TSG inactivation and exhibited gain-of-function mutations by predicted kinase activation or generation of fusion genes. Mutational frequencies and signatures emerged as promising informative tools for aiding in MPNST differential diagnosis. Due to the multiple genomic differences exhibited, we complemented their characterization using a methylome-based classifier. All NF1-related cell lines were assigned within the MPNST group, while sporadic cell lines clustered either with melanomas or with an uncertain MPNST-like sarcoma group. The staining of cell identity markers reinforced the idea of a potential misdiagnose of the MPNSTs used to derive the sporadic cell lines analyzed. Conclusions: Deep genomic analysis, together with methylome-based sarcoma classification and cell identity marker analysis, challenged the MPNST identity of sporadic cell lines. Results presented here open an opportunity to revise MPNST differential diagnosis and classification. | genomics |
The first glimpse of Homo sapiens hereditary fusion genes Family-inherited fusion genes have been known to be associated with human disease for decades. However, only a small number of them have been discovered so far. In this report, monozygotic (MZ) twins are used as a genetic model to investigate hereditary fusion genes (HFG). We have analyzed RNA-Seq from 37 MZ twins and discovered 1,180 HFGs, the maximum of which is 608 per haploid genome. Eight HFGs associated with MZ twin inheritance range from 52.7% to 67.6%, some of which are previously-studied cancer fusion genes and indicate hereditary cancer genes. These data suggest that HFGs are major genetic factors for human diseases and complex traits. This study gives us the first glimpse of human HFGs and lays theoretical and technological foundations for future genetic and medical studies. | genomics |
Amplification is the Primary Mode of Gene-by-Sex Interaction in Complex Human Traits Sexual dimorphism is observed in many complex traits and diseases and is suspected to be in part due to widespread gene-by-sex interactions (GxSex). To date, empirical evidence for GxSex in GWAS data has been elusive. We hypothesized that GxSex may be pervasive but largely missed by current approaches if it acts primarily through sex differences in the magnitude of many genetic effects ("amplification"), regulated by a shared cue such as a sex hormone, rather than differences in the identity of causal variants or the direction of their effect. To test this hypothesis, we inferred the genetic covariance structure between males and females across 27 physiological traits in the UK Biobank. We found amplification to be a pervasive mode of GxSex across traits. As one example, we estimate that 38% of variants have a greater effect on urate levels in females than males. For some traits, notably those related to body mass, testosterone levels are associated with the magnitude of genetic effects in both males and females, but the association is opposite in sign between the sexes. Finally, we developed a novel test of sexually-antagonistic viability selection linking GxSex signals to allele frequency divergence between adult males and females. Using independent allele frequency data, we find marginally-significant evidence for contemporary sexually-antagonistic selection on genetic variation associated with testosterone. In summary, our results suggest that the systematic amplification of genetic effects is a common mode of GxSex that may contribute to sexual dimorphism and fuel its evolution. | genomics |
Computational prediction and characterization of cell-type-specific and shared binding sites Cell-type-specific gene expression is maintained in large part by transcription factors (TFs) selectively binding to distinct sets of sites in different cell types. Recent research works have provided evidence that such cell-type-specific binding is determined by TFs intrinsic sequence preferences, cooperative interactions with cofactors, cell-type-specific chromatin landscapes, and 3D chromatin interactions. However, computational prediction and characterization of cell-type-specific and shared binding sites is rarely studied. In this paper, we propose two computational approaches for predicting and characterizing cell-type-specific and shared binding sites by integrating multiple types of features, in which one is based on XGBoost and another is based on convolutional neural network (CNN). To validate the performance of our proposed approaches, ChIP-seq datasets of 10 binding factors were collected from the GM12878 (lymphoblastoid) and K562 (erythroleukemic) human hematopoietic cell lines, each of which was further categorized into cell-type-specific (GM12878-specific and K562-specific) and shared binding sites. Then, multiple types of features for these binding sites were integrated to train the XGBoost-based and CNN-based models. Experimental results show that our proposed approaches significantly outperform other competing methods on three classification tasks. To explore the contribution of different features, we performed ablation experiments and feature importance analysis. Consistent with previous studies, we find that chromatin features are major contributors in which chromatin accessibility is the best predictor. Moreover, we identified independent feature contribution for cell-type-specific and shared sites through SHAP values, observing that chromatin features play a main role in the cell-type-specific sites while motif features play a main role in the shared sites. Beyond these observations, we explored the ability of the CNN-based model to predict cell-type-specific and shared binding sites by excluding or including DNase signals, showing that chromatin accessibility significantly improves the prediction performance. Besides, we investigated the generalization ability of our proposed approaches to different binding factors in the same cellular environment or to the same binding factors in the different cellular environments. | bioinformatics |
Hemodynamic transient and functional connectivity follow structural connectivity and cell type over the brain hierarch The neural circuit of the brain is organized as a hierarchy of functional units with wide-ranging connections that support the information flow and functional connectivity. Studies using magnetic resonance imaging (MRI) indicate a moderate coupling between structural and functional connectivity at the system level. However, how do connections of different directions (feedforward and feedback) and regions with different excitatory and inhibitory (E/I) neurons shape the hemodynamic activity and functional connectivity over the hierarchy are unknown. Here, we used functional MRI to detect optogenetic-evoked and resting-state activities over a somatosensory pathway in the mouse brain and compared with axonal projection and E/I distribution. With a highly sensitive ultrafast imaging, we identified extensive activation in regions up to the third order of axonal projections following optogenetic excitation of the ventral posteriomedial nucleus of the thalamus. The evoked response and functional connectivity correlated with feedforward projections but less with the feedback and weakened with the hierarchy. The hemodynamic signal exhibited regional and hierarchical differences, with slower and more variable responses in high-order areas and bipolar responses predominantly in the contralateral cortex. Importantly, the positive and negative parts of hemodynamics correlated with E/I neuronal densities, respectively. Furthermore, resting-state functional connectivity more associated with E/I distribution whereas stimulus-evoked effective connectivity followed structural wiring. These findings indicate that structure-function relationship is projection-, cell-type- and hierarchy-dependent. Hemodynamic transients could reflect E/I activity and the increased complexity of hierarchical processing.
Significance statementThe neural circuit of the brain is organized as a hierarchy of functional units with complicated feedforward and feedback connections to selectively enhance (excitation) or suppress (inhibit) information from massive sensory inputs. How brain activity is shaped by the structural wiring and excitatory and inhibitory neurons are still unclear. We characterize how brain-wide hemodynamic responses reflect these structural constituents over the hierarchy of a somatosensory pathway. We find that functional activation and connectivity correlate with feedforward connection strengths and neuronal distributions. This association subsides with hierarchy due to slower and more variable hemodynamic responses, reflecting increased complexity of processing and neuronal compositions in high-order areas. Our findings indicate that hemodynamics follow the hierarchy of structural wiring and neuronal distribution. | neuroscience |
Multi-tasking Deep Network for Tinnitus Classification and Severity Prediction from Multimodal Structural Images Subjective tinnitus is an auditory phantom perceptual disorder without an objective biomarker. Fast and efficient diagnostic tools will advance clinical practice by detecting or confirming the condition, tracking change in severity, and monitoring treatment response. Motivated by evidence of subtle anatomical or functional morphological information in magnetic resonance images (MRI) of the brain, we examined data-driven machine learning methods for joint tinnitus classification (tinnitus or no tinnitus) and tinnitus severity prediction. We propose a deep multi-task multi-modal framework for joint functionalities using structural MRI (sMRI) data. To leverage cross-information multimodal neuroimaging data, we integrated two modalities of 3-dimensional sMRI - T1 weighted (T1w) and T2 weighted (T2w) images. To explore the key components in the MR images that drove task performance, we segmented both T1w and T2w images into three different components - cerebrospinal fluid (CSF), grey matter (GM) and white matter (WM), and examined performance of each segmented image. Results demonstrate that our multimodal framework capitalizes on the information across both modalities (T1w and T2w) for the joint task of tinnitus classification and severity prediction. Our model outperforms existing learning-based and conventional methods in terms of accuracy, sensitivity, specificity, and negative predictive value. | neuroscience |
Nitric oxide-induced tyrosine nitration of TrkB impairs BDNF signaling and restrains neuronal plasticity Nitric oxide has been long recognized as an important modulator of neural plasticity, but characterization of the molecular mechanisms involved - specially the guanylyl cyclase-independent ones - has been challenging. There is evidence that NO could modify BDNF-TRKB signaling, a key mediator of neuronal plasticity. However, the mechanism underlying the interplay of NO and TRKB remains unclear. Here we show that nitric oxide induces nitration of the tyrosine 816 in the TRKB receptor in vivo and in vitro, and that post-translational modification inhibits TRKB phosphorylation and binding of phospholipase C{gamma}1 (PLC{gamma}1) to this same tyrosine residue. Additionally, nitration triggers clathrin-dependent endocytosis of TRKB through the adaptor protein AP2M and ubiquitination, thereby increasing translocation of TRKB away from the neuronal surface and directing it towards lysosomal degradation. Accordingly, inhibition of nitric oxide increases TRKB phosphorylation and TRKB-dependent neurite branching in neuronal cultures. In vivo, chronic inhibition of neuronal nitric oxide synthase (nNOS) dramatically reduced TRKB nitration and facilitated TRKB signaling in the primary visual cortex, and promoted a shift in ocular dominance upon monocular deprivation in the visual cortex - an indicator of increased plasticity. Altogether, our data describe and characterize a new molecular brake on plasticity, namely nitration of TRKB receptors.
Significance statementWe described the nitration of TRKB receptors at the tyrosine residue 816 as a new post-translational modification (PTM) that restrains the signaling of the neurotrophic factor BDNF in neurons. This new PTM leads to endocytosis and degradation of the TRKB receptors. Intriguingly, this mechanism is tonically active under physiological conditions in vivo, and it is important for restricting ocular dominance plasticity in the visual cortex. This mechanism directly links two major systems involved in brain plasticity, BDNF/TRKB and nitric oxide. Our data provides a model for how NO production from nNOS can compromise TRKB function, and for the effects of nNOS inhibitors promoting plasticity. | neuroscience |
Reconstruction of the cell pseudo-space from single-cell RNA sequencing data with scSpace Tissues are highly complicated with spatial heterogeneity in gene expression. However, the cutting-edge single-cell RNA-seq technology eliminates the spatial information of individual cells, which contributes to the characterization of cell identities. Herein, we propose single-cell spatial position associated co-embeddings (scSpace), an integrative algorithm to distinguish spatially variable cell subclusters by reconstructing cells onto a pseudo-space with spatial transcriptome references (Visium, STARmap, Slide-seq, etc.). We demonstrated that scSpace can define biologically meaningful cell subpopulations neglected by single-cell RNA-seq or spatially resolved transcriptomics. The use of scSpace to uncover the spatial association within single-cell data, reproduced, the hierarchical distribution of cells in the brain cortex and liver lobules, and the regional variation of cells in heart ventricles and the intestinal villus. scSpace identified cell subclusters in intratelencephalic neurons, which were confirmed by their biomarkers. The application of scSpace in melanoma and Covid-19 exhibited a broad prospect in the discovery of spatial therapeutic markers. | bioinformatics |
Molecular dynamics of spike variants in the locked conformation: RBD interfaces, fatty acid binding and furin cleavage sites. Since December 2019 the SARS-CoV-2 virus has infected billions of people around the world and caused millions of deaths. The ability for this RNA virus to mutate has produced variants that have been responsible for waves of infections across the globe. The spike protein on the surface of the SARS-CoV-2 virion is responsible for cell entry in the infection process. Here we have studied the spike proteins from the Original, Alpha (B.1.1.7), Delta (B1.617.2), Delta-plus (B1.617.2-AY1), Omicron BA.1 and Omicron BA.2 variants. Using models built from cryo-EM structures with linoleate bound (6BZ5.pdb) and the N-terminal domain from 7JJI.pdb, each is built from the first residue, with missing loops modelled and 45 disulphides per trimer. Each spike variant was modified from the same Original model framework to maximise comparability. Three replicate, 200 ns atomistic molecular dynamics simulations were performed for each case. (These data also provide the basis for further, non-equilibrium molecular dynamics simulations, published elsewhere.) The analysis of our equilibrium molecular dynamics reveals that sequence variation at the closed receptor binding domain interface particularly for Omicron BA.2 has implications for the avidity of the locked conformation, with potential effects on Omicron BA.1 and Delta-plus. Linoleate binding has a mildly stabilizing effect on furin cleavage site motions in the Original and Alpha variants, but has no effect in Delta, Delta-plus and slightly increases motions at this site for Omicron BA.1, but not BA.2, under these simulation conditions. | biochemistry |
Fixation Can Change the Appearance of Phase Separation in Living Cells Fixing cells with paraformaldehyde (PFA) is an essential step in numerous biological techniques as it is thought to preserve a snapshot of biomolecular transactions in living cells. Fixed cell imaging techniques such as immunofluorescence have been widely used to detect liquid-liquid phase separation (LLPS) in vivo. Here, we compared images, before and after fixation, of cells expressing intrinsically disordered proteins that are able to undergo LLPS. Surprisingly, we found that PFA fixation can both enhance and diminish putative LLPS behaviors. For specific proteins, fixation can even cause their droplet-like puncta to artificially appear in cells that do not have any detectable puncta in the live condition. Fixing cells in the presence of glycine, a molecule that modulates fixation rates, can reverse the fixation effect from enhancing to diminishing LLPS appearance. We further established a kinetic model of fixation in the context of dynamic protein-protein interactions. Simulations based on the model suggest that protein localization in fixed cells depends on an intricate balance of protein-protein interaction dynamics, the overall rate of fixation, and notably, the difference between fixation rates of different proteins. Our work reveals that PFA fixation changes the appearance of LLPS from living cells, presents a caveat in studying LLPS using fixation-based methods, and suggests a mechanism underlying the fixation artifact. | cell biology |
On the limits of fitting complex models of population history to genetic data Our understanding of human population history in deep time has been assisted by fitting 'admixture graphs' to data: models that specify the ordering of population splits and mixtures which is the only information needed to capture the patterns of allele frequency correlation among populations. Not needing to specify population size changes, split times, or whether admixture events were sudden or drawn out simplifies the space of models that need to be searched. However, the space of possible admixture graphs relating populations is vast and cannot be sampled fully, and thus most published studies have identified fitting admixture graphs through a manual process driven by prior hypotheses, leaving the vast majority of alternative models unexplored. Here, we develop a method for systematically searching the space of all admixture graphs that can incorporate non-genetic information in the form of topology constraints. We implement this findGraphs tool within a software package, ADMIXTOOLS 2, which is a reimplementation of the ADMIXTOOLS software with new features and large performance gains. We apply this methodology to identify alternative models to admixture graphs that played key roles in eight published studies and find that graphs modeling more than six populations and two or three admixture events are often not unique, with many alternative models fitting nominally or significantly better than the published one. Our results suggest that strong claims about population history from admixture graphs should only be made when all well-fitting and temporally plausible models share common topological features. Our re-evaluation of published data also provides insight into the population histories of humans, dogs, and horses, identifying features that are stable across the models we explored, as well as scenarios of populations relationships that differ in important ways from models that have been highlighted in the literature, that fit the allele frequency correlation data, and that are not obviously wrong. | evolutionary biology |
Mosaic patterns of selection in genomic regions associated with diverse human traits Natural selection shapes the genetic architecture of many human traits. However, the prevalence of different modes of selection on genomic regions associated with variation in traits remains poorly understood. To address this, we developed an efficient computational framework to calculate enrichment of different evolutionary measures among regions associated with complex traits. We applied the framework to summary statistics from >900 genome-wide association studies (GWASs) and 11 evolutionary measures of sequence constraint, population differentiation, and allele age while accounting for linkage disequilibrium, allele frequency, and other potential confounders. We demonstrate that this framework yields consistent results across GWASs with variable sample sizes, numbers of trait-associated SNPs, and analytical approaches. The resulting evolutionary atlas maps diverse signatures of selection on genomic regions associated with complex human traits on an unprecedented scale. We detected positive enrichment for sequence conservation among trait-associated regions for the majority of traits (>77% of 290 high power GWASs), which was most dominant in reproductive traits. Many traits also exhibited substantial enrichment for population differentiation and recent positive selection, especially among hair, skin, and pigmentation traits. In contrast, we detected widespread negative enrichment for balancing selection (51% GWASs) and no evidence of enrichment for selection signals in regions associated with late-onset Alzheimer's disease. These results support a pervasive role for negative selection on regions of the human genome that contribute to variation in complex traits, but also demonstrate where diverse modes of selection have shaped trait-associated loci. This atlas of signatures of different modes of natural selection across the diversity of available GWASs will enable exploration of the relationship between the genetic architecture and selection in the human genome. | evolutionary biology |
Cell cycle independent role of cyclin D3 in host restriction of SARS-CoV-2 infection The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents a great threat to human health. The interplay between the virus and host plays a crucial role in successful virus replication and transmission. Understanding host-virus interactions is essential for development of new COVID-19 treatment strategies. Here we show that SARS-CoV-2 infection triggers redistribution of cyclin D1 and cyclin D3 from the nucleus to the cytoplasm, followed by its proteasomal degradation. No changes to other cyclins or cyclin dependent kinases were observed. Further, cyclin D depletion was independent from SARS-CoV-2 mediated cell cycle arrest in early S phase or S/G2/M phase. Cyclin D3 knockdown by small interfering RNA specifically enhanced progeny virus titres in supernatants. Finally, cyclin D3 co-immunoprecipitated with SARS-CoV-2 Envelope and Membrane proteins. We propose that cyclin D3 inhibits virion assembly and is depleted during SARS-CoV-2 infection to restore efficient assembly and release of newly produced virions. | microbiology |
FIB-SEM analysis on three-dimensional structures of growing organelles in wild Chlorella pyrenoidosa cells To clarify dynamic changes of organelle microstructures in Chlorella pyrenoidosa cells during photosynthetic growth with CO2 fixation, three-dimensional organelle microstructures in three growth periods of meristem, elongation and maturity were quantitatively determined and comprehensively reconstructed with focused ion beam scanning electron microscopy (FIB-SEM). The single round-pancake mitochondria in each cell split into a dumbbell and then into a circular ring, while barycenter distance of mitochondria to chloroplast and nucleus was reduced to 45.5% and 88.3% to strengthen energy transfer, respectively. The single pyrenoid consisting of a large part and another small part in each chloroplast gradually developed to a mature state in which the two parts were nearly equal in size. The nucleolus progressively became larger with euchromatin replication. The number of starch grains gradually increased, but average grain volume remained nearly unchanged. | cell biology |
The bacterial lysate Lantigen B reduces the expression of ACE2 on primary oropharyngeal cells Background: Vercelli and coworkers recently observed that a well-established bacterial lysate (OM-85, Vifor Pharma; CH) was able to downregulate the expression of Angiotensin-Converting Enzyme 2 (ACE2) on epithelial cells. This downregulation was also associated with a reduced infectivity of cells, resulting in a reduced viral titre. We evaluated whether another bacterial lysate (Lantigen B, Bruschettini Ltd; Italy) may have similar activities. However, while OM-85 is given per os and has a systemic effect after absorption at the gut level, Lantigen B is given locoregionally. Thus, the concentration that the bacterial lysate can reach at the mucosal level seems to be promising. Methods: Oropharyngeal cells were collected from healthy donors. After 24 hours of treatment in vitro with doses of Lantigen B comparable to those that are reached in vivo, the expression of ACE2 was evaluated by direct fluorescence and flow cytometry. Results: A reduction in the number of ACE2-positive cells was observed in 80% of treated samples. Only a few donors had poor expression of ACE2, and in these donors, the downregulation was less evident or absent. Conclusions: These results suggest that Lantigen B, at pharmacological doses, could be an interesting drug to reduce ACE2 expression on oropharyngeal cells, thus contributing to the prophylaxis of COVID-19 in humans. | cell biology |
Capacitation promotes a shift in the energy metabolism in murine sperm In mammals, sperm acquire fertilization ability after a series of physiological and biochemical changes, collectively known as capacitation, that occur inside the female reproductive tract. In addition to other requirements, sperm bioenergetic metabolism has been identified as a fundamental component in the acquisition of the capacitated status. Mammalian sperm produce ATP by means of two main metabolic processes, oxidative phosphorylation (OXPHOS) and aerobic glycolysis, that are localized in two different flagellar compartments, midpiece and principal piece, respectively. In mouse sperm, the occurrence of many events associated with capacitation depends on the activity of these two energy-producing pathways, leading to the hypothesis that some of these events may impose changes in sperm energetic demands. In the present study, we used extracellular flux analysis to evaluate the changes in the glycolytic and respiratory parameters of murine sperm that occur as a consequence of capacitation. Furthermore, we examined whether these variations affect sperm ATP sustainability. Our results show that capacitation promotes a shift in the usage ratio of the two main metabolic pathways, from oxidative to glycolytic. However, this metabolic rewiring does not seem to affect the rate at which the sperm consume ATP. We conclude that the probable function of the metabolic switch is to increase the ATP supply in the distal flagellar regions, thus sustaining the energetic demands that arise from capacitation. | cell biology |
A Drosophila larvae-inspired vacuum-actuated soft robot Peristalsis is one of the most common locomotion patterns in limbless animals. This motion is generated by propagating muscular contraction and relaxation along the body axis. While the kinematics of peristalsis has been examined intensively, the kinetics and mechanical control of peristalsis remain unclear, partially due to the lack of suitable physical models to analyse the force and temporal control in soft-bodied animals' locomotion. Here, based on a soft-bodied animal, Drosophila larvae, we proposed a vacuum-actuated soft robot replicating their crawling behaviour. The soft structure, made with hyperelastic silicon rubber, was designed to mimic the larval hydrostatic structure. To estimate the adequate range of pressures and time scales for control of the soft robots, a numerical simulation by the finite element method was conducted. Pulse-Width-Modulation (PWM) was used to generate time-series signals to control the vacuum pressure in each segment. Based on this control system, the soft robots could exhibit the peristaltic pattern resembling fly larval crawling. The soft robots reproduced two previous experimental results on fly larvae: slower crawling speed in backward crawling than in forward crawling, and the involvement of segmental contraction duration and intersegmental delay in crawling speed. Furthermore, the soft robot provided a novel prediction that the larger the contraction force, the faster the crawling speed. These observations indicate that the use of soft robots could serve to examine the kinetics and mechanical regulation of crawling behaviour in soft-bodied animals. | biophysics |
Simultaneous and sequential multi-species coronavirus vaccination Although successful COVID-19 vaccines have been developed, multiple pathogenic coronavirus species exist, urging for development of multi-species coronavirus vaccines. Here we developed prototype LNP-mRNA vaccine candidates against SARS-CoV-2 (Delta variant), SARS-CoV and MERS-CoV, and test how multiplexing of these LNP-mRNAs can induce effective immune responses in animal models. A triplex scheme of LNP-mRNA vaccination induced antigen-specific antibody responses against SARS-CoV-2, SARS-CoV and MERS-CoV, with a relatively weaker MERS-CoV response in this setting. Single cell RNA-seq profiled the global systemic immune repertoires and the respective transcriptome signatures of multiplexed vaccinated animals, which revealed a systemic increase in activated B cells, as well as differential gene expression signatures across major adaptive immune cells. Sequential vaccination showed potent antibody responses against all three species, significantly stronger than simultaneous vaccination in mixture. These data demonstrated the feasibility, antibody responses and single cell immune profiles of multi-species coronavirus vaccination. The direct comparison between simultaneous and sequential vaccination offers insights on optimization of vaccination schedules to provide broad and potent antibody immunity against three major pathogenic coronavirus species. | immunology |
Effects of larval and adult crowding on fitness components in Drosophila populations adapted to larval crowding experienced under different combinations of food amount and egg number Since the realization in the 1970s that simple discrete-time population growth models can show complex unstable dynamics of population size, many explanations were proposed for the evolution of enhanced population stability. The most plausible of these was density-dependent selection, suggested to favour greater stability due to r-K trade-offs. However, the first experiment aimed at testing this prediction revealed that Drosophila melanogaster populations adapted to larval crowding did not evolve greater constancy stability than their ancestral controls. A subsequent study showed that D. ananassae populations adapted to larval crowding had evolved greater constancy and persistence than ancestral controls. These D. ananassae populations had experienced chronic larval crowding in conditions of very low amounts of food, whereas the earlier studied D. melanogaster populations had experienced chronic larval crowding at fairly high food amounts. Further theoretical work also suggested that populations adapting to crowding could evolve greater stability even in the absence of r-K trade-offs. Most recently, studies in our laboratory showed that two sets of crowding adapted D. melanogaster populations, derived from a common ancestral lineage, which differed in the food amounts at which they experienced larval crowding, evolved different patterns of constancy and persistence stability. These two sets of populations also differed in the traits, e.g. larval feeding rate, that evolved as they became more competitive. Here, we examine the response of key fitness components to larval and adult densities in these two sets of populations, to see whether differences in their stability attributes can be explained by variation in how their life-histories respond to crowding at different life stages. Of all traits examined, only pre-adult survivorship responded differently to larval density across the two sets of populations. The populations that adapted to larval crowding at low food amounts showed reduced sensitivity of pre-adult survivorship to larval density, compared to those that adapted to larval crowding at high food amounts. We discuss our results in the context of different ways in which density-dependent selection may facilitate the evolution of greater constancy or persistence, depending on the ecological details of how crowding was experienced. | evolutionary biology |
High-throughput discovery of TRAF6-interacting peptides identifies determinants of positive and negative design and shows known and candidate human interaction partner motifs are not optimized for affinity TRAF6 is an adapter protein and E3 ubiquitin ligase that is involved in signaling downstream of cell receptors important for development and immune system activation and maintenance. TRAF6 participates in hundreds of protein-protein interactions, some of which are mediated by a C-terminal MATH domain that recruits TRAF6 to cell-surface receptors and associated proteins. The TRAF6 MATH domain binds to short peptide segments containing the motif PxExx[FYWHDE], where x is any amino acid. Blocking TRAF6 interactions is associated with favorable effects in a variety of disease models. To better define TRAF6 binding preferences, we generated a bacterial cell-surface peptide display library to sample the TRAF6 motif sequence space. We performed sorting experiments to identify 236 of the best TRAF6-interacting peptides and a set of 1,200 peptides that match the sequence PxE but do not bind TRAF6. Selected binders tested by single-clone bacterial display titrations and by bio-layer interferometry bound TRAF6 tighter than previously measured native peptides. We built all-atom structural models of the TRAF6 MATH domain in complex with high-affinity binders and motif-matching nonbinders identified by screening to elucidate the structural basis for TRAF6 interaction preferences. We identified motif features that favor binding to TRAF6 and also negative design elements, distributed across the motif, that can disfavor or preclude binding. Searching the human proteome for matches to the library screening-defined binding motif revealed that most known, biologically relevant TRAF6 binders occupy a different sequence space from the most enriched hits that we discovered in combinatorial library screening. Our experimentally determined binding preferences and structural models can support the design of peptide-based interaction inhibitors with higher affinities than endogenous TRAF6 ligands. | biochemistry |
AA-amyloidosis in cats (Felis catus) housed in shelters Systemic AA-amyloidosis is a protein-misfolding disease that is characterized by fibril deposition of serum amyloid-A protein (SAA) in several organs in humans and many animal species. Fibril deposits originate from abnormally high serum levels of SAA during chronic inflammation. In domestic short-hair cats, AA-amyloidosis has only been anecdotally reported and is considered a rare disease. Here we report that an astonishing 57-73% of early deceased short-hair cats kept in three independent shelters suffer from amyloid deposition in the liver, spleen, or kidney. Histopathology and mass spectrometry of post-mortem extracted deposits identified SAA as the major protein source. The duration of stay in the shelters was positively associated with a histological score of AA-amyloidosis (B=0.026, CI95%=0.007-0.046; p=0.010). Presence of SAA fragments in bile secretions raises the possibility of fecal-oral transmission of the disease. | pathology |
Cardiac patch treatment alleviates ischemic cardiomyopathy correlated with reverting Piezo1/2 expression by unloading left ventricular myocardium Pathologically elevated mechanical load promotes the adverse remodeling of left ventricle (LV) post myocardial infarction, which results in the progression from ischemic cardiomyopathy to heart failure. Cardiac patches could attenuate adverse LV remodeling by providing mechanical support to infarcted myocardium and border zone tissue. However, the mechanism of the translation from mechanical effects to favorable therapeutic outcome is still not clear. By transcriptome analysis, we found that the myocardial transcription levels of mechanosensitive ion channel proteins Piezo1 and Piezo2 significantly increased in patients with ischemic cardiomyopathy. In vitro tensile tests with local tissue information revealed a significant decrease in local strain and mechanical load in rat infarct. Cardiac function and geometry were preserved compared to non-treated control. Further, in LV myocardium of the patch-treated group, MI induced expression levels of Piezo1/2 were significantly reverted to the similar levels of the sham group, indicating that cardiac patch beneficial effects were correlated with suppressing mechanosensitive genes, particularly Piezo1/2. These findings demonstrated the potential of cardiac patches in treating ICM patients with remodeling risks, and could provide guidance for improvement in next generation of patch devices. | pathology |
Adipokine C1q/Tumor necrosis factor-related protein 3 (CTRP3) Attenuates Intestinal Inflammation via Histone Deacetylase Sirtuin 1 (SIRT1)/NF-κB Signaling BACKGROUND & AIMS: The adipokine C1q/tumor necrosis factor-related protein 3 (CTRP3) has anti-inflammatory effects in several non-intestinal disorders. Although CTRP3 is reduced in the serum of patients with inflammatory bowel disease (IBD), its function in IBD has not been established. We aimed to elucidate the function of CTRP3 and related molecular mechanisms in intestinal inflammation using a colitis model of genetically-modified CTRP3 mice and intestinal epithelial tissue from patients with Crohns disease (CD), one of the two main forms of IBD. METHODS: CTRP3 knockout (KO) and overexpressing transgenic (Tg) mice along with their corresponding wild-type (WT) littermates were subjected to drinking water containing dextran sulfate sodium (DSS) for 6-10 days to induce acute colitis. Mouse colitis symptoms and histological data were analyzed. CTRP3-mediated signaling was examined in the intestinal tissue of mice and patients with CD. RESULTS: CTRP3 mRNA and protein were detected in murine and human intestinal epithelial cells, as well as in murine intestinal smooth muscle cells and mesenteric fat. In DSS-induced acute colitis models, CTRP3 KO mice developed more severe colitis than their WT littermates, while CTRP3 overexpressing Tg mice developed less severe colitis than their WT littermates. In both water- and DSS-treated CTRP3 KO mice, reduced CTRP3 levels correlated with decreased levels of Sirtuin 1 (SIRT1), a histone deacetylase, increased levels of phosphorylated nuclear factor kappa B (NF-kB) subunit p65, resulting in increased expression of pro-inflammatory cytokines tumor necrosis factor-a (TNF-a) and interleukin 6 (IL-6). The results from CTRP3 Tg mice mirrored those from CTRP3 KO mice in most respects. This CTRP3/SIRT1/NF-kB relationship was also observed in the intestinal epithelial tissue of patients with active and inactive CD. CONCLUSIONS: CTRP3 expression levels correlate negatively with intestinal inflammation in mouse colitis models and CD patients. CTRP3 attenuates intestinal inflammation via SIRT1/NF-kB signaling to suppress pro-inflammatory cytokines in mouse colitis models and patients with IBD. The manipulation of CTRP3 signaling, including through the use of SIRT1 agonists, may offer translational potential in the treatment of IBD. | pathology |
A data-driven approach to quantifying meal characteristics influencing energy intake We used a data-driven approach to determine the influence of energy density, hyper-palatability, protein content, and eating rate on ad libitum non-beverage energy intake during 2733 meals consumed by 35 inpatient adults who participated in two 28-day feeding studies. All four meal characteristics significantly contributed to energy intake, but their relative importance varied by the prevailing dietary pattern according to macronutrient composition and degree of processing. | physiology |
Responses of Agricultural plants to Lithium pollution: Trends, Meta-Analysis, and Perspectives Lithium (Li) is gaining attention due to rapid rise of modern industries but their ultimate fingerprints on plants are not well established. Herein, we executed a meta-analysis of the existing recent literature investigating the impact of Li sources and levels on plant species under different growth conditions to understand the existing state of knowledge. Toxic effects of Li exposure in plants varies as a function of medium and interestingly, more negative responses are reported in hydroponic media as compared to soil and foliar application. Additionally, toxic effects of Li vary with Li source materials and LiCl more negatively affected plant development parameters such as plant germination (n=48) and root biomass (n=57) and recorded highly uptake in plants (n=78), while LiNO3 has more negative effects on shoot biomass. The Li at <50 mg L-1 concentrations significantly influenced the plant physiological indicators including plant germination and root biomass, while 50-500 mg L-1 Li concentration influence the biochemical parameters. The uptake potential of Li is dose dependent and their translocation/bioaccumulation remains unknown. Future work should include complete lifespan studies of the crop to elucidate the bioaccumulation of Li in edible tissues and to investigate possible trophic transfer of Li. | plant biology |
Dynamic emergence of relational structure network in human brains Reasoning the hidden relational structure from sequences of events is a crucial ability humans possess, which help them to predict the future and make inferences. Besides simple statistical properties, humans also excel in learning more complex relational networks. Several brain regions are engaged in the process, yet the time-resolved neural implementation of relational structure learning and its behavioral relevance remains unknown. Here human subjects performed a probabilistic sequential prediction task on image sequences generated from a transition graph-like network, with their brain activities recorded using electroencephalography (EEG). We demonstrate the emergence of two key aspects of relational knowledge - lower-order transition probability and higher-order community structure, which arise around 840 msec after image onset and well predict behavioral performance. Furthermore, computational modeling suggests that the formed higher-order community structure, i.e., compressed clusters in the network, could be well characterized by a successor representation operation. Overall, human brains are constantly computing the temporal statistical relationship among discrete inputs, based on which new abstract knowledge could be inferred. | neuroscience |
Predicting Working Memory performance based on specific individual EEG spatiotemporal features Working Memory (WM) is a limited capacity system for storing and processing information, which varies from subject to subject. Several works show the ability to predict the performance of WM with machine learning (ML) methods, and although good prediction results are obtained in these works, ignoring the intersubject variability and the temporal and spatial characterization in a WM task to improve the prediction in each subject. In this paper, we take advantage of the spectral properties of WM to characterize the individual differences in visual WM capacity and predict the subject's performance. Feature selection was implemented through the selection of electrodes making use of methods to treat unbalanced classes. The results show a correlation between the accuracy achieved with an Regularized Linear Discriminant Analysis (RLDA) classifier using the power spectrum of the EEG signal and the accuracy achieved by each subject in the behavioral experiment response of a WM task with retro-cue. The proposed methodology allows identifying spatial and temporal characteristics in the WM performance in each subject. Our methodology shows that it is possible to predict the WM performance in each subject. Finally, our results showed that by knowing the spatiotemporal characteristics that predict WM performance, it is possible to customize a WM task and optimize the use of electrodes for agile processing adapted to a specific subject. Thus, we pave the way for implementing neurofeedback through a Brain-Computer Interface. | neuroscience |
A sequential two-step priming scheme reproduces diversity in synaptic strength and short-term plasticity Glutamatergic synapses display variable strength and diverse short-term plasticity (STP), even for a given type of connection. Using non-negative tensor factorization (NTF) and conventional state modelling, we demonstrate that a kinetic scheme consisting of two sequential and reversible steps of release-machinery assembly and a final step of synaptic vesicle (SV) fusion reproduces STP and its diversity among synapses. Analyzing transmission at calyx of Held synapses reveals that differences in synaptic strength and STP are not primarily caused by variable fusion probability (pfusion) but determined by the fraction of docked synaptic vesicles equipped with a mature release machinery. Our simulations show, that traditional quantal analysis methods do not necessarily report pfusion of SVs with a mature release machinery but reflect both pfusion and the distribution between mature and immature priming states at rest. Thus, the approach holds promise for a better mechanistic dissection of the roles of presynaptic proteins in the sequence of SV docking, two-step priming and fusion and suggests a mechanism for activity-induced redistribution of synaptic efficacy. | neuroscience |
Opposing associations of Internet Use Disorder symptom domains with structural and functional organization of the striatum: a dimensional neuroimaging approach Background: Accumulating evidence suggests brain structural and functional alterations in Internet Use Disorder (IUD). However, conclusions are strongly limited due to the retrospective case-control design of the studies, small samples, and the focus on general rather than symptom-specific approaches. Methods: We here employed a dimensional multi-methodical MRI-neuroimaging design in a final sample of n = 203 subjects to examine associations between levels of IUD and its symptom-dimensions (loss of control/time management, craving/social problems) with brain structure, resting state and task-based (pain empathy, affective go/no-go) brain function. Results: Although the present sample covered the entire range of IUD, including normal, problematic as well as pathological levels, general IUD symptom load was not associated with brain structural or functional alterations. However, the symptom-dimensions exhibited opposing associations with the intrinsic and structural organization of the brain, such that loss of control/time management exhibited negative associations with intrinsic striatal networks and hippocampal volume, while craving/social problems exhibited a positive association with intrinsic striatal networks and caudate volume. Conclusions: Our findings provided the first evidence for IUD symptom-domain specific associations with progressive alterations in the intrinsic structural and functional organization of the brain, particularly of striatal systems involved in reward, habitual and cognitive control processes. | neuroscience |