title: Expanded transcriptomic analysis of human hepatic stellate cells links novel coding and noncoding products to human liver fibrosis

abstract: End stage liver disease and liver failure occur primarily as a consequence of progressive fibrosis resulting from chronic liver injury. Hepatic stellate cells (HSCs) are the primary cell type responsible for production of the extracellular matrix (ECM) that forms the fibrotic scar. While the essential role of HSCs is understood, there remain no treatments that target HSCs to inhibit the development or progression of fibrosis. We have performed analysis of the transcriptome of human HSCs to define the long noncoding (lnc) RNAs expressed in this cell type, including many not previously annotated. Through analysis of full-length RNA transcripts, we identified additional lncRNAs that were not assembled by short reads. We also discovered new isoforms of proteincoding genes that encode amino acid sequences that are not present in annotated isoforms. Analysis of non-polyadenylated RNAs did not identify additional genes encoding long noncoding RNA transcripts, but did reveal the presence of hundreds of circular (circ) RNAs, including those with potential for translation. Incorporating these transcripts and genes into analysis of a published dataset of human liver fibrosis revealed the induction of lncRNAs, novel protein isoforms, and circRNAs associated with development of disease. These results identify RNAs and amino acid sequences expressed in HSCs and associated with human liver disease that may serve as therapeutic targets to inhibit fibrosis or biomarkers to benchmark progression of disease.


title: Core and Flanking bHLH-PAS:DNA interactions mediate specificity and drive obesity

abstract:
The basic-Helix-Loop-Helix Per-Arnt-Sim (PAS) homology domain (bHLH-PAS) transcription factor (TF) family comprises critical biological sensors of physiological (hypoxia, tryptophan metabolites, neuronal activity, and appetite) and environmental (diet derived metabolites and environmental pollutants) stimuli to regulate genes involved in signal adaptation and homeostasis1. bHLH TFs bind DNA as homo or heterodimers via E-box (CANNTG) response elements, however the DNA binding specificity of the PAS domain-containing bHLH subfamily remains unresolved1. We systematically analysed cognate DNA binding hierarchies of prototypical bHLH-PAS family members (ARNT, ARNT2, HIF1, HIF2, AhR, NPAS4, SIM1) and demonstrate distinct core (NNCGTG) specificities for different heterodimer classes. The results also show that bHLH-PAS TFs bind over a large footprint 12-15bp and recognise preferential DNA sequences flanking the core. For example, specificity beyond otherwise identical core binding by SIM1 and the HIFs is mediated through N-terminal HIF-DNA interactions. We also reveal an intimate relationship between DNA shape and both core and flanking TF binding allowing motif sequence flexibility and underpinning TF binding specificity. Furthermore, DNA-shape affinity relationships revealed that novel downstream PAS-A-loop DNA interactions are associated with AT-rich sequences that lead to high-affinity binding, and that loss of this function underpins a monogenic cause of human hyperphagic obesity in a recapitulated SIM1.R171H knock-in mouse model.

Importantly, models of protein-DNA binding accurately predict in vivo occupancy, while response element methylation blocks DNA binding and predicts cell type specific chromatin occupancy. These data provide a definitive and accurate map of bHLH-PAS TF specificity and target selectivity through novel flanking protein-DNA interactions that are crucial for in vivo biological function.

category: molecular biology


title: Determining the targeting specificity of the selective peroxisomal targeting factor Pex9

abstract: Targeting proteins to their correct cellular location is a fundamental process that allows them to carry out their cellular functions. Peroxisomes utilize two paralog targeting factors, Pex5 and Pex9, for proteins with a Peroxisomal Targeting Signal 1 (PTS1). However, in spite of their similarity, Pex9 targets only a subset of Pex5 cargo proteins. Here, we studied the properties that facilitate the targeting specificity of Pex9, both by unbiased screens and by site-directed mutagenesis of the PTS1 motifs of either binders or non-binders. We find that the binding specificity of Pex9 is largely determined by the hydrophobic nature of the amino acid preceding the PTS1 tripeptide of its cargos. This is in line with structural modeling of the PTS1-binding cavity of the two factors, showing that while Pex5 has large negative electrostatic patches at the area surrounding the PTS1 binding cavity, Pex9 is mostly hydrophobic. Our work outlines the mechanism by which targeting specificity is achieved, enabling dynamic rewiring of the peroxisomal proteome in changing metabolic needs.

category: cell biology


title: Breasi-CRISPR: an efficient genome editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex

abstract: In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. This can be challenging when the researched protein lacks reliable antibodies. Here, we combine Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. This method is called Breasi-CRISPR (Brain Easi-CRISPR), and enables knock-in of both short and long epitope tag sequences in genes of interest with high efficiency. Using Breasi-CRISPR, we were able to visualize epitope tagged proteins known to have either high or low expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1, and RPL22. Detection was possible by immunohistochemistry as soon as one day after electroporation at embryonic day 13 (E13). Two and five days after electroporation, we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices two days after electroporation. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores in an efficient manner, allowing the visualization of endogenous proteins via live-imaging in organotypic brain slices two days after electroporation. Finally, we used Breasi-CRISPR to perform co-IP mass-spectrometry analyses of tagged autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data show Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

category: developmental biology


title: An ancient endogenous DNA virus in the human genome

abstract:
The genomes of eukaryotes preserve a striking diversity of ancient viruses in the form of endogenous viral elements (EVEs). Study of this genomic fossil record provides insights into the diversity, origin and evolution of viruses across geological timescales. In particular, Mavericks have emerged as one of the oldest groups of viruses infecting vertebrates ([≥]419 My). They have been found in the genomes of fish, amphibians and non-avian reptiles but had been overlooked in mammals. Thus, their evolutionary history and the causes of their demise in mammals remain puzzling questions. Here, we conduct a detailed evolutionary study of two Maverick-like integrations found on human chromosomes 7 and 8. We performed a comparative analysis of the integrations and determined their orthology across placental mammals (Eutheria) via the syntenic arrangement of neighbouring genes. The integrations were absent at the orthologous sites in the genomes of marsupials and monotremes. These observations allowed us to reconstruct a time-calibrated phylogeny and infer the age of their most recent common ancestor at 268.61 (199.70-344.54) My. In addition, we estimate the age of the individual integrations at ~105 My which represent the oldest non-retroviral EVEs found in the human genome. Our findings suggest that active Mavericks existed in the ancestors of modern mammals ~172 My ago (Jurassic Period) and potentially to the end of the Early Cretaceous. We hypothesise Mavericks could have gone extinct in mammals from the evolution of an antiviral defence system or from reduced opportunities for transmission in terrestrial hosts.

ImportanceThe genomes of vertebrates preserve an enormous diversity of endogenous viral elements (remnants of ancient viruses that accumulate in host genomes over evolutionary time). Although retroviruses account for the vast majority of these elements, diverse DNA viruses have also been found and novel lineages are being described. Here we analyse two elements found in the human genome belonging to an ancient group of DNA viruses called Mavericks. We study their evolutionary history, finding that the elements are shared between humans and many different species of placental mammals. These observations suggest the elements inserted at least ~105 Mya in the most recent common ancestor of placentals. We further estimate the age of the viral ancestor around 268 My. Our results provide evidence for some of the oldest viral integrations in the human genome and insights into the ancient interactions of viruses with the ancestors of modern-day mammals.

category: evolutionary biology


title: Genomic and phenotypic analyses suggest moderate fitness differences among Zika virus lineages

abstract: RNA viruses have short generation times and high mutation rates, allowing them to undergo rapid molecular evolution during epidemics. However, the extent of RNA virus phenotypic evolution within epidemics and the resulting effects on fitness and virulence remain mostly unknown. Here, we screened the 2015-2016 Zika epidemic in the Americas for lineage-specific fitness differences. We engineered a library of recombinant viruses representing twelve major Zika virus lineages and used them to measure replicative fitness within disease-relevant human primary cells and live mosquitoes. We found that two of these lineages conferred significant in vitro replicative fitness changes among human primary cells, but we did not find fitness changes in Aedes aegypti mosquitoes. Additionally, we found evidence for elevated levels of positive selection among five amino acid sites that define major Zika virus lineages. While our work suggests that Zika virus may have acquired several phenotypic changes during a short time scale, these changes were relatively moderate and do not appear to have enhanced virulence or transmission during the epidemic.


title: Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq

abstract: Pooled CRISPR screens coupled with single-cell RNA-sequencing have enabled systematic interrogation of gene function and regulatory networks. Here, we introduce Cas13 RNA Perturb-seq (CaRPool-seq) which leverages the RNA-targeting CRISPR/Cas13d system and enables efficient combinatorial perturbations alongside multimodal single-cell profiling. CaRPool-seq encodes multiple perturbations on a cleavable array which is associated with a detectable barcode sequence, allowing for the simultaneous targeting of multiple genes. We compared CaRPool-seq to existing Cas9-based methods, highlighting its unique strength to efficiently profile combinatorially perturbed cells. Finally, we apply CaRPool-seq to perform multiplexed combinatorial perturbations of myeloid differentiation regulators in an acute myeloid leukemia (AML) model system and identify extensive interactions between different chromatin regulators that can enhance or suppress AML differentiation phenotypes.

category: genomics


title: Exploring different virulent proteins of human respiratory syncytial virus for designing a novel epitope-based polyvalent vaccine: Immunoinformatics and molecular dynamics approaches

abstract: Human Respiratory Syncytial Virus (RSV) is one of the most prominent causes of lower respiratory tract infections (LRTI), contributory to infecting people from all age groups - a majority of which comprises infants and children. The implicated severe RSV infections lead to numerous deaths of multitudes of the overall population, predominantly the children, every year. Consequently, despite several distinctive efforts to develop a vaccine against the RSV as a potential countermeasure, there is no approved or licensed vaccine available yet, to control the RSV infection effectively. Therefore, through the utilization of immunoinformatics tools, a computational approach was taken in this study, to design and construct a multi-epitope polyvalent vaccine against the RSV-A and RSV-B strains of the virus. Potential predictions of the T-cell and B-cell epitopes were followed by extensive tests of antigenicity, allergenicity, toxicity, conservancy, homology to human proteome, transmembrane topology, and cytokine-inducing ability. The most promising epitopes (i.e. 13 CTL epitopes, 9 HTL epitopes, and 10 LBL epitopes) exhibiting full conservancy were then selected for designing the peptide fusion with appropriate linkers, having hBD-3 as the adjuvant. The peptide vaccine was modeled, refined, and validated to further improve the structural attributes. Following this, molecular docking analysis with specific TLRs was carried out which revealed excellent interactions and global binding energies. Additionally, molecular dynamics (MD) simulation was conducted which ensured the stability of the interactions between vaccine and TLR. Furthermore, mechanistic approaches to imitate and predict the potential immune response generated by the administration of vaccines were determined through immune simulations. Owing to an overall evaluation, in silico cloning was carried out in efforts to generate recombinant pETite plasmid vectors for subsequent mass production of the vaccine peptide, incorporated within E.coli. However, more in vitro and in vivo experiments can further validate its efficacy against RSV infections.

category: immunology


title: Bile Salts and N-Acetyl-D-Glucosamine induce transcription of the vieSAB Operon in Vibrio cholerae El Tor

abstract: The vieSAB operon regulates motility, biofilm formation and cholera toxin production in the classical biotype of V. cholerae. Classical biotype mutants lacking the cyclic-di-GMP phosphodiesterase and response regulator vieA or its cognate sensor kinase vieS fail to effectively colonize the infant mouse small intestine, are less motile than the wild-type strain, and are hyper-biofilm formers. However, deletion of vieA or vieS in the currently-circulating El Tor biotype does not have a demonstrable effect on these phenotypes. To begin to understand the role of vieSAB in the El Tor biotype, we studied its regulation and the effect of vieA expression on bacterial physiology, separating the functional roles of VieAs enzymatic and DNA-binding activities. We identify bile salts and N-acetylglucosamine, which can be found at high concentration in the small intestine, as stimulating expression of vieSAB during in vitro growth. We show that, in contrast to many response regulators, VieA does not regulate its own expression. Together, these data indicate that V. cholerae upregulates vieSAB in response to small-intestinal signals, allowing VieA to alter the physiology of the cell through its transcriptional regulation and cyclic-di-GMP signaling activities in order to adapt to the human host.

category: microbiology


title: Insect asynchronous flight requires neural circuit de-synchronization by electrical synapses

abstract:
Despite profound mechanistic insight into motor pattern generation, for asynchronous insect flight - the most prevalent form of flight employed by >600.000 species - architecture and function of the underlying central pattern generating (CPG) neural network remain elusive. Combining electro- and optophysiology, Drosophila genetics, and mathematical modelling, we uncover a miniaturized circuit solution of motoneurons interconnected by electrical synapses that, contrary to doctrine, serve to de-synchronize network activity. This minimal gap-junctional motoneuron network suffices to translate unpatterned premotor input into stereotyped firing sequences which are conserved across species and generate stable wingbeat power. Mechanistically, network de-synchronization requires weak electrical coupling in conjunction with specific postsynaptic excitability dynamics, revealing an unexpected, generic feature in the control of neural circuit dynamics by electrical synapses.

One Sentence SummaryElectrical synapses de-synchronize neural network firing to enable stable wingbeat power during insect flight.

category: neuroscience


title: Dendritic compartmentalization of input-specific integration and plasticity rules across cortical development

abstract: Lifelong learning is the ability to continually acquire new information and build upon existing knowledge. Biological and artificial neurons gain and refine internal representations by modifying synaptic weights, but it is unclear how previous knowledge remains stable in spite of ongoing plasticity. Here we show that cortical pyramidal neurons solve this plasticity-stability dilemma by differentially regulating synaptic plasticity within dendritic compartments. We found that oblique dendrites of layer 5 pyramidal neurons in adult mouse primary visual cortex (V1) selectively receive monosynaptic input from dorsal lateral geniculate nucleus (dLGN), integrate linearly, and--surprisingly--lack synaptic potentiation. In contrast, basal dendrites, which do not receive dLGN input, exhibit conventional NMDA receptor (NMDAR)-mediated supralinear integration and synaptic potentiation. Both dendritic compartments initially express NMDAR-mediated supralinear integration and synaptic potentiation during development, but oblique dendritic properties diverge after a critical period of early visual experience. This developmental transition was accompanied by a decrease in relative NMDAR receptor activity and expression at spiny synapses on oblique dendrites. Our results demonstrate a new biological mechanism for how single neurons can safeguard inputs from ongoing experience-dependent plasticity by altering synaptic properties of dendritic domains.


title: Functional analysis of a novel de novo variant in PPP5C associated with microcephaly, seizures, and developmental delay

abstract: We describe a proband evaluated through the Undiagnosed Diseases Network (UDN) who presented with microcephaly, developmental delay, and refractory epilepsy with a de novo p.Ala47Thr missense variant in the protein phosphatase gene, PPP5C. This gene has not previously been associated with a Mendelian disease, and based on the population database, gnomAD, the gene has a low tolerance for loss-of-function variants (pLI=1, o/e=0.07). We functionally evaluated the PPP5C variant in C. elegans by knocking the variant into the orthologous gene, pph-5, at the corresponding residue, Ala48Thr. We employed assays in three different biological processes where pph-5 was known to function through opposing the activity of genes, mec-15 and sep-1. We demonstrated that, in contrast to control animals, the pph-5 Ala48Thr variant suppresses the neurite growth phenotype and the GABA signaling defects of mec-15 mutants, and the embryonic lethality of sep-1 mutants. The Ala48Thr variant did not display dominance and behaved similarly to the reference pph-5 null, indicating that the variant is likely a strong hypomorph or complete loss-of-function. We conclude that pph-5 Ala48Thr is damaging in C. elegans. By extension in the proband, PPP5C p.Ala47Thr is likely damaging, the de novo dominant presentation is consistent with haplo-insufficiency, and the PPP5C variant is likely responsible for one or more of the probands phenotypes.

category: genetics


title: PAX6 LIMITS THE COMPETENCE OF DEVELOPING CEREBRAL CORTICAL CELLS

abstract: The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6s main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side-effects of morphogens engaged contemporaneously in other essential functions.


title: Presenilin and APP regulate synaptic kainate receptors

abstract: Kainate receptors (KARs) form a family of ionotropic glutamate receptors which regulate the activity of neuronal networks by both pre- and post-synaptic mechanisms. Their implication in pathologies is well documented for epilepsy. The higher prevalence of epileptic symptoms in Alzheimer disease (AD) patients questions the role of KARs in AD. Here we investigated whether the synaptic expression and function of KARs was impaired in mouse models of AD. We addressed this question by immunostaining and electrophysiology at synapses between mossy fibers and CA3 pyramidal cells, in which KARs are abundant and play a prominent physiological role. We observed a decrease of the immunostaining for GluK2 in the stratum lucidum in CA3, and of the amplitude of synaptic currents mediated by GluK2-containing KARs in an amyloid mouse model (APP/PS1) of AD. Interestingly, a similar phenotype was observed in CA3 pyramidal cells with a genetic deletion of either presenilin or APP/APLP2 as well as in organotypic cultures treated with {gamma}-secretase inhibitors. Finally, the GluK2 protein interacts with full-length and C-terminal fragments of APP. Overall, our data suggest that APP stabilizes KARs at synapses, possibly through a trans-synaptic mechanism, and this interaction is under the control the {gamma}-secretase proteolytic activity of presenilin.


title: 6S RNA-dependent antibiotic susceptibility

abstract: Bacterial small RNAs (sRNAs) contribute to a variety of regulatory mechanisms that modulate wide ranging pathways, including metabolism, virulence, and antibiotic resistance. We investigated the involvement of sRNAs in rifampicin resistance in the opportunistic pathogen Staphylococcus aureus. Using a competition assay with an sRNA mutant library, we identified 6S RNA as being required for protection against low concentrations of rifampicin, an RNA polymerase (RNAP) inhibitor. This effect applied to rifabutin and fidaxomicin, two other RNAP-targeting antibiotics. 6S RNA is highly conserved in bacteria and its absence in two other major pathogens, Salmonella enterica and Clostridioides difficile, also impaired susceptibility to RNAP inhibitors. In S. aureus, 6S RNA is produced from an autonomous gene and accumulates in stationary phase. In contrast to what was reported in Escherichia coli, S. aureus 6S RNA does not appear to play a critical role in the transition from exponential to stationary phase, but affects {sigma}B-regulated expression in prolonged stationary phase. Nevertheless, its protective effect against rifampicin is independent of alternative sigma factor {sigma}B activity. Our results suggest that 6S RNA helps maintain RNAP-{sigma}A integrity in S. aureus, which could in turn help bacteria withstand low concentrations of RNAP inhibitors.


title: InvL, an invasin-like adhesin, is a type II secretion system substrate required for Acinetobacter baumannii uropathogenesis.

abstract:
Acinetobacter baumannii is an opportunistic pathogen of growing concern, as isolates are commonly multidrug resistant. While A. baumannii is most frequently associated with pulmonary infections, a significant proportion of clinical isolates come from urinary sources, highlighting its uropathogenic potential. The type II secretion system (T2SS) of commonly used model Acinetobacter strains is important for virulence in various animal models, but the potential role of the T2SS in urinary tract infection (UTI) remains unknown. Herein, we used a catheter-associated UTI (CAUTI) model to demonstrate that a modern urinary isolate, UPAB1, requires the T2SS for full virulence. A proteomic screen to identify putative UPAB1 T2SS effectors revealed an uncharacterized lipoprotein with structural similarity to the intimin-invasin family, which serve as type V secretion system (T5SS) adhesins required for the pathogenesis of several bacteria. This protein, designated InvL, lacked the {beta}-barrel domain associated with T5SSs, but was confirmed to require the T2SS for both surface localization and secretion. This makes InvL the first identified T2SS effector belonging to the intimin-invasin family. InvL was confirmed to be an adhesin, as the protein bound to extracellular matrix components and mediated adhesion to urinary tract cell lines in vitro. Additionally, the invL mutant was attenuated in the CAUTI model, indicating a role in Acinetobacter uropathogenesis. Finally, bioinformatic analyses revealed that InvL is present in nearly all clinical isolates belonging to international clone 2, a lineage of significant clinical importance. In all, we conclude that the T2SS substrate InvL is an adhesin required for A. baumannii uropathogenesis.

IMPORTANCEWhile pathogenic Acinetobacter can cause various infections, we recently found that 20% of clinical isolates come from urinary sources. Despite the clinical relevance of Acinetobacter as a uropathogen, few virulence factors involved in urinary tract colonization have been defined. Herein, we identify a novel type II secretion system effector, InvL, which is required for full uropathogenesis by a modern urinary isolate. Though InvL has predicted structural similarity to the intimin-invasin family of autotransporter adhesins, InvL is predicted to be anchored to the membrane as a lipoprotein. Similar to other invasin homologs however, we demonstrate that InvL is a bona fide adhesin capable of binding extracellular matrix components and mediating adhesion to urinary tract cell lines. In all, this work establishes InvL as an adhesin important for Acinetobacters urinary tract virulence, and represents the first report of a type II secretion system effector belonging to the intimin-invasin family.


title: Wall teichoic acids facilitate the release of toxins from the surface of Staphylococcus aureus.

abstract:
A major feature of the pathogenicity of Staphylococcus aureus is its ability to secrete cytolytic toxins. This process involves the translocation of the toxins from the cytoplasm, through the bacterial membrane and the cell wall to the external environment. The process of their movement through the membrane is relatively well defined, involving both general and toxin-specific secretory systems. Movement of the toxins through the cell wall was considered to involve the passive diffusion of the proteins through the porous cell wall structures, however, recent work suggests that this is more complex, and here we demonstrate a role for the wall teichoic acids (WTA) in this process. Utilising a genome-wide association approach we identified a polymorphism in the locus encoding the WTA biosynthetic machinery as associated with the cytolytic activity of the bacteria. We verified this association using an isogenic mutant set and found that WTA is required for the release of several cytolytic toxins from the bacterial cells. We show this effect is mediated by a change in the electrostatic charge across the cell envelope that results from the loss of WTA. As a major target for the development of novel therapeutics, it is important that we fully understand the entire process of cytolytic toxin production and release. These findings open up a new aspect to this process that requires in-depth investigation, while also demonstrating that clinical isolates can utilise WTA production to vary their cytotoxicity, thereby altering their pathogenic capabilities.

ImportanceThe production and release of cytolytic toxins is a critical aspect to the pathogenicity of many bacterial pathogens. In this study we demonstrate a role for wall teichoic acids, molecules that are anchored to the peptidoglycan of the bacterial cell wall, in the release of toxins from S. aueus cells into the extracellular environment. Our findings suggest this effect is mediated by a gradient of electrostatic charge the presence of the negatively charged WTA molecules create across the cell envelope. This work brings an entirely new aspect to our understanding of the cytotoxicity of S. aureus and demonstrates a further means by which this major human pathogen can adapt its pathogenic capabilities.


title: Trpc6 gain-of-function disease mutation enhances phosphatidylserine exposure in murine platelets

abstract: Platelets enhance coagulation by exposing phosphatidylserine (PS) on their cell surface in response to strong agonist activation. Transient receptor potential channels, including TRPC6, have been implicated in the calcium influx central to this process. Here, we characterize the effect of a Trpc6 gain-of-function (GOF) disease-associated, and a dominant negative (DN), mutation on murine platelet activation. Platelets from mice harboring Trpc6E896K/E896K (GOF) and Trpc6DN/DN mutations were subject to in vitro analysis. Trpc6E896K/E896K and Trpc6DN/DN mutant platelets show enhanced and absent calcium influx, respectively, upon addition of the TRPC3/6 agonist GSK1702934A (GSK). GSK was sufficient to induce integrin IIb{beta}3 activation, P-selection and PS exposure, talin cleavage, and MLC2 phosphorylation in Trpc6E896K/E896K, but not in wild-type, platelets. Thrombin-induced calcium influx and PS exposure were enhanced, and clot retraction delayed, by GOF TRPC6, while no differences were noted between wild-type and Trpc6DN/DN platelets. In contrast, Erk activation upon GSK treatment was absent in Trpc6DN/DN, and enhanced in Trpc6E896K/E896K, platelets, compared to wild-type. The positive allosteric modulator, TRPC6-PAM-C20, and fluoxetine maintained their ability to enhance and inhibit, respectively, GSK-mediated calcium influx in Trpc6E896K/E896K platelets. The data demonstrate that gain-of-function mutant TRPC6 channel can enhance platelet activation, including PS exposure, while confirming that TRPC6 is not necessary for this process. Furthermore, they suggest that Trpc6 GOF disease mutants do not simply increase wild-type TRPC6 responses, but can affect pathways not usually modulated by TRPC6 channel activity, displaying a true gain-of-function phenotype.


title: Behavioural analysis of loss of function zebrafish supports baz1b as master regulator of domestication

abstract: Domestication is associated with both morphological and behavioural phenotypic changes that differentiate domesticated species from their wild counterparts. Some of the traits are those purposely targeted by the selection process, whilst others co-occur as a result of selection. The combination of traits is referred to as the  domestication syndrome and their shared characteristics has given rise to the neural crest domestication syndrome (NCDS) hypothesis. According to this hypothesis, the phenotypic changes are a consequence of a selection towards animals with mild underdevelopment of the neural crest. A similar mechanism is suggested to affect some species of "self-domesticated" animals, including humans. Recent research supports a role for BAZ1B, one of the haplo-insufficient genes in Williams syndrome (WS), in the evolution of craniofacial features of modern humans. Interestingly, WS recapitulates some of the traits observed in the domestication syndrome, including hypersociability and reduced facial bones. However, the evidence linking BAZ1B to behavioural phenotypes associated with domestication is presumptive. Here, we use zebrafish as a model to test the hypothesis that baz1b loss-of-function leads to both morphological and behavioural phenotypes associated with the domestication syndrome by influencing the development of the neural crest. Our research provides further evidence supporting the NCDS hypothesis and bazlbs role in this process.

category: animal behavior and cognition


title: Structure-based electron-confurcation mechanism of the Ldh-EtfAB complex

abstract: Lactate oxidation with NAD+ as electron acceptor is a highly endergonic reaction and some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) using a lactate dehydrogenase (Ldh) in concert with the electron transferring proteins EtfA and EtfB. The electron cryo-microscopically (cryo-EM) characterized (Ldh-EtfAB)2 complex of Acetobacterium woodii at 2.43 [A] resolution consists of a mobile EtfAB shuttle located between the rigid central Ldh and the peripheral EtfAB base units. The FADs of Ldh and the EtfAB shuttle contact each other thereby forming the D (dehydrogenase conducting) state. The intermediary Asp37 and Asp139 may harmonize the redox potentials between the FADs and the pyruvate/lactate pair crucial for FBEC. A plausible novel B (bifurcation conducting) state with the EtfAB base and shuttle FADs in a productive electron transfer distance was derived by integrating Alphafold2 calculations. Kinetic analysis of enzyme variants shed light on the connection between NAD binding/release and D-to-B state transition. The FBEC inactivity when truncating the ferredoxin domain of EtfA substantiates its role as redox relay. Lactate oxidation in Ldh is based on the catalytic base His423 and a metal center. On this basis, a comprehensive catalytic mechanism of the FBEC process was outlined.

category: biochemistry


title: Sentinel Cards Provide Practical SARS-CoV-2 Monitoring in School Settings

abstract:
Accurate, high-resolution environmental monitoring of SARS-CoV-2 traces indoors through sentinel cards is a promising approach to help students safely return to in-person learning. Because SARS-CoV-2 RNA can persist for up to a week on several indoor surface types, there is a need for increased temporal resolution to determine whether consecutive surface positives arise from new infection events or continue to report past events. Cleaning sentinel cards after sampling would provide the needed resolution, but might interfere with assay performance. We tested the effect of three cleaning solutions (BZK wipes, wet wipes, RNase Away) at three different viral loads: "high" (4 x 104 GE/mL), "medium" (1 x 104 GE/mL), and "low" (2.5 x 103 GE/mL). RNAse Away, chosen as a positive control, was the most effective cleaning solution on all three viral loads. Wet wipes were found to be more effective than BZK wipes in the medium viral load condition. The low viral load condition was easily reset with all three cleaning solutions. These findings will enable temporal SARS-CoV-2 monitoring in indoor environments where transmission risk of the virus is high and the need to avoid individual-level sampling for privacy or compliance reasons exists.

ImportanceBecause SARS-CoV-2, the virus that causes COVID-19, persists on surfaces, testing swabs taken from surfaces is useful as a monitoring tool. This approach is especially valuable in school settings, where there are cost and privacy concerns that are eliminated by taking a single sample from a classroom. However, the virus persists for days to weeks on surface samples, so it is impossible to tell whether positive detection events on consecutive days are persistent signal or new infectious cases, and therefore whether the positive individuals have been successfully removed from the classroom. We compare several methods for cleaning "sentinel cards" to show that this approach can be used to identify new SARS-CoV-2 signals day to day. The results are important for determining how to monitor classrooms and other indoor environments for SARS-CoV-2 virus.

category: bioinformatics


title: SRSF1 as a novel interacting partner for IFITM1/3 unravels the emergent role of IFITM1/3 mediating protein expression

abstract:
IFITM proteins play a role in cancer cell progression through undefined mechanisms. Here, we propose an emergent role of IFITM1/3 regulating protein synthesis. SBP-tagged IFITM1 protein was used to identify an association of IFITM1 protein with the cytosolic isoform of SRSF1 that transports mRNA to the ribosome. This cytosolic association was confirmed in situ using proximity ligation assays for SRSF1 and IFITM1/3, suggesting a role associated with translation. Accordingly, IFITM1/3 were shown to interact with HLA-B mRNA in response to IFN{gamma} stimulation using RNA-protein proximity ligation assays. In addition, shotgun RNA sequencing in IFITM1/IFITM3 null cells and wt-SiHa cells indicated that reduced HLA-B gene expression does not account for lowered HLA-B protein synthesis in response to IFN{gamma}. Furthermore, ribosome profiling using sucrose gradient sedimentation identified a reduction in 80S ribosomal fraction an IFITM1/IFITM3 null cells compared to their wild-type counterpart, partially reverted by IFITM1/3 complementation. Our data all together link the binding of IFITM1/3 proteins to HLA-B mRNA and SRSF1 as a mechanism to catalyze the synthesis of target proteins, suggesting an RNA chaperonin role for IFITM1/3 proteins.

SignificanceIFITMs are widely studied for their role in inhibiting viruses, and multiple studies have associated IFITMs with cancer progression. However, mechanistic insights are not well understood. Our study proposes that IFITMs have a role regulating protein synthesis, a pivotal function highly relevant for viral infection and cancer progression. Our results suggest that IFITM1/3 is present in the ribosomal fraction and regulates particular protein expression; among them, we identified HLA-B. Changes in HLA-B expression could impact the presentation and recognition of oncogenic antigens on the cell surface by cytotoxic T cells and, ultimately, limit tumor cell eradication. In addition, the role of IFITMs mediating protein translation is relevant, as has the potential of regulating the expression of viral and oncogenic proteins.

category: cancer biology


title: Knock-out of CmNAC-NOR affects melon climacteric fruit ripening

abstract: Fruit ripening is an important process that affects fruit quality. In melon, ETHQV6.3, a QTL involved in climacteric ripening regulation, was previously found to be encoded by CmNAC-NOR, a homologue of the tomato NOR gene. To further investigate CmNAC-NOR function we have obtained two CRISPR/Cas9 mediated mutants (nor-3 and nor-1) in the climacteric Vedrantais background. nor-3, containing a 3-bp deletion altering the NAC domain A, resulted in ~8 days delay of ripening without affecting fruit quality. In contrast, nor-1 contained a 1-bp deletion resulting in a fully disrupted NAC domain, which completely blocked climacteric ripening. nor-1 fruits did not produce ethylene, abscission layer was not formed and there was no external color change. Additionally, volatile components were dramatically altered, seeds were not well developed and flesh firmness was also altered. nor-1 allele in heterozygosis showed ~20 days delay of fruit ripening. Our results provide new information regarding the function of CmNAC-NOR in melon fruit ripening, suggesting that it as a potential target to modulate shelf life in climacteric melon commercial varieties.

category: plant biology


title: High temperature limits leaf size via direct control of cell cycle by coordinated functions of PIF4 and TCP4

abstract:
High ambient temperature suppresses Arabidopsis rosette leaf area, in contrast to elongation of stem and petiole. While the mechanism underlying temperature-induced elongation response is extensively studied, the genetic basis of temperature-regulation of leaf size is largely unknown. Here we show that warm temperature inhibits cell proliferation in the Arabidopsis leaves resulting in fewer cells compared to the control condition. Cellular phenotyping, genetic, and biochemical analyses established the key roles of PIF4 and TCP4 transcription factors in the suppression of Arabidopsis leaf area under high temperature by a reduction in cell number. We show that temperature-mediated suppression of cell proliferation requires PHYTOCHROME INTERACTING FACTOR 4 (PIF4). PIF4 interacts with TEOSINTE BRANCHED1/CYCLOIDEA/PCF4 (TCP4) and regulates the expression of cell cycle inhibitor, KIP-RELATED PROTEIN 1 (KRP1), to control the leaf size under high temperature. Warm temperature induces binding of both PIF4 and TCP4 to the KRP1 promoter. PIF4 binding to KRP1 under high temperature is TCP4 dependent as TCP4 regulates PIF4 levels under high temperature. We propose a model where a warm temperature-mediated accumulation of PIF4 in the leaf cells promotes its binding to the KRP1 promoter in a TCP4-dependent way to regulate cell production and leaf size. Our finding of high temperature-mediated transcriptional upregulation of KRP1 integrates a developmental signal with an environmental signal that converges on a basal cell regulatory process.

One sentence summaryHigh ambient temperature suppresses Arabidopsis leaf area by reducing cell proliferation via PIF4- and TCP4-dependent transcription control of KRP1.


title: Synthetic genetic circuits enable reprogramming of plant roots

abstract: The shape of a plants root system influences its ability to reach essential nutrients in the soil and to acquire water during drought. Progress in engineering plant roots to optimize water and nutrient acquisition has been limited by our capacity to design and build genetic programs that alter root growth in a predictable manner. Here, we construct synthetic genetic circuits to control gene expression with high spatial precision across root tissues. These circuits produce specific patterns of gene expression by combining the activity of multiple native promoters using logical operations. We then use the circuits to predictably alter root structure. This work demonstrates the ability of synthetic genetic circuits to control gene expression across tissues and offers an exciting means to reprogram plant growth.

category: synthetic biology


title: Regulation of adult neurogenesis and neuronal differentiation by Neural Cell Adhesion Molecule 2 (NCAM2)

abstract: Adult neurogenesis persists in mammals in the neurogenic zones where newborn neurons are incorporated into existing neuronal circuits. Relevant molecular elements of the neurogenic niches include the family of Cell Adhesion Molecules (CAM), which participate in signal transduction and regulate radial glial progenitors (RGPs) survival, division and differentiation. The Neural Cell Adhesion Molecule 2 (NCAM2) is expressed in brain development and in adult stages, and controls dendrite arborisation and synaptic formation and maintenance during development. Nevertheless, the role of NCAM2 in neurogenesis and lineage progression is not well understood. Here we analyse the functions of NCAM2 in the regulation of RGPs in adult neurogenesis in the dentate gyrus and during corticogenesis, by using different lentiviral-mediated genetic approaches to modulate its expression, both in vivo and in vitro. First, we characterized the expression of NCAM2 among the main actors of the neurogenic process revealing different levels of NCAM2 amid the progression of RGPs and the formation of juvenile neurons. Further, we show that overexpression of NCAM2 arrest infected cells in a RGP-like state, with characteristic morphological, immunocytochemical and electron microscopy features. In contrast, NCAM2 overexpression in embryonic cortical progenitors does not seems to alter cell fate, but causes transient migration deficits. These results reveal a differential role of NCAM2 in the regulation of adult and embryonic RGPs, and specifically, a significant implication of NCAM2 in the regulation and progression of RGPs during adult neurogenesis in the hippocampus.


title: CACNB4 overexpression decreases dendritic spine density in sex-specific manner

abstract: The canonical voltage-gated calcium channel (VGCC) subunit complex is comprised of the 1 subunit, the ion permeable channel, plus three auxiliary subunits: {beta}, 2{delta} and {gamma}. {beta} is the most extensively studied auxiliary subunit and is necessary for proper forward trafficking of the 1 subunit to the plasma membrane. 1 subunits mediate voltagedependent movement of calcium ions into the cytoplasm of neurons, including at dendritic sites, where increased intracellular calcium initiates signaling cascades that shape structural and functional plasticity of dendritic spines. Genetic studies strongly implicate calcium signaling dysfunction in the etiology of neurodevelopmental disorders including schizophrenia. Dendritic spine density (DSD) is significantly decreased in schizophrenia in primary auditory cortex where DSD is driven by loss of small spines, and small spine loss is associated with increased peptide levels of ALFDFLK found in the VGCC {beta} subunit {beta}4. Overexpessing CACNB4 to increase {beta}4 levels selectively reduced small spine density in cortical neuron cultures. The studies described herein set out to validate this in vitro observation in an intact mammalian system within a neurodevelopmental context. We overexpressed CACNB4 in neurodevelopment and assessed DSD and morphology in cerebral cortex of male and female mice at an adult timpoint. We then characterized {beta} protein levels and {beta}4 protein-protein interactions in male and female mouse cortex. Overexpression selectively reduced small dendritic spine density but this effect was present only in female mice and did not appear to result from estrous stage. Instead, the sex-dependent effect on DSD corresponded to sex differences in the {beta}4 interactome of male versus female mice: the VGCC {beta} subunit {beta}1b was significantly enriched in the {beta}4 interactome of brain tissue of male mice, and thus may have served to mitigate VGCC overexpression-mediated spine loss in male mice.


title: Replicating RNA platform enables rapid response to the SARS-CoV-2 Omicron variant and elicits enhanced protection in naïve hamsters compared to ancestral vaccine

abstract:
In late 2021, the SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VoC) was reported with many mutations in the viral spike protein that were predicted to enhance transmissibility and allow viral escape of neutralizing antibodies. Within weeks of the first report of B.1.1.529, this VoC has rapidly spread throughout the world, replacing previously circulating strains of SARS-CoV-2 and leading to a resurgence in COVID-19 cases even in populations with high levels of vaccine- and infection-induced immunity. Initial studies have shown that B.1.1.529 is less sensitive to protective antibody conferred by previous infections and vaccines developed against earlier lineages of SARS-CoV-2. The ability of B.1.1.529 to spread even among vaccinated populations has led to a global public health demand for updated vaccines that can confer protection against B.1.1.529. We report here the rapid development of a replicating RNA vaccine expressing the B.1.1.529 spike and show that this B.1.1.529-targeted vaccine is immunogenic in mice and hamsters. Interestingly, we found that mice previously immunized with A.1-specific vaccines failed to elevate neutralizing antibody titers against B.1.1.529 following B.1.1.529-targeted boosting, suggesting pre-existing immunity may impact the efficacy of B.1.1.529-targeted boosters. Furthermore, we found that our B.1.1.529-targeted vaccine provides superior protection compared to the ancestral A.1-targeted vaccine in hamsters challenged with the B.1.1.529 VoC after a single dose of each vaccine.

One Sentence SummaryRapidly developed RNA vaccine protects against SARS-CoV-2 Omicron variant


title: Nutrient gradients mediate creation of highly resistant layers in structured microbial populations during antibiotic exposures

abstract: Antibiotic treatments often fail to eliminate bacterial populations due to heterogeneity in how individual cells respond to the drug. In structured bacterial populations such as biofilms, bacterial metabolism and environmental transport processes lead to an emergent phenotypic structure and self-generated nutrient gradients towards the interior of the colony, which can affect cell growth, gene expression and susceptibility to the drug. Even in single cells, survival depends on a dynamic interplay between the drugs action and the expression of resistance genes. How expression of resistance is coordinated across populations in the presence of such spatiotemporal environmental coupling remains elusive. Using a custom microfluidic device, we observe the response of spatially extended microcolonies of tetracycline-resistant E. coli to precisely defined dynamic drug regimens. We find an intricate interplay between drug-induced changes in cell growth and growth-dependent expression of resistance genes, resulting in the redistribution of nutrients and the reorganization of growth patterns. This dynamic environmental feedback affects the regulation of drug resistance differently across the colony, generating dynamic phenotypic structures that maintain colony growth during exposure to high drug concentrations and increase population-level resistance to subsequent exposures. A mathematical model linking metabolism and the regulation of gene expression is able to capture the main features of spatiotemporal colony dynamics. Uncovering the fundamental principles that govern collective mechanisms of antibiotic resistance in spatially extended populations will allow the design of optimal drug regimens to counteract them.


title: Minilungs from hESCs to study the interaction of Streptococcus pneumoniae with the respiratory tract

abstract:
The new generation of organoids derived from human pluripotent stem cells holds a promising strategy for modeling host-bacteria interaction studies. Organoids recapitulate the composition, diversity of cell types and, to some extent, the functional features of the native organ. We have generated lung bud organoids derived from human embryonic stem cells to study the interaction of Streptococcus pneumoniae (pneumococcus) with the alveolar epithelium. Invasive pneumococcal disease is an important health problem that may occur as a result of the spread of pneumococcus from the lower respiratory tract to sterile sites. We show here an efficient experimental approach to model the main events of the pneumococcal infection that occur in the human lung exploring bacterial adherence to the epithelium, internalization, and triggering of an innate response that includes the interaction with the surfactant and the expression of representative cytokines and chemokines. Thus, this model, based on human minilungs, can be used to study pneumococcal virulence factors, the pathogenesis of different serotypes and it will allow therapeutic interventions in a reliable human context.

IMPORTANCEStreptococcus pneumoniae is responsible for high morbidity and mortalities rates worldwide affecting mainly children and adults older than 65 years. Pneumococcus is also the most common etiologic agent of bacterial pneumonia, non-epidemic meningitis, and a frequent cause of bacterial sepsis. Although the advent of pneumococcal vaccines has decreased the burden of the diseases caused by pneumococcus, the emerging of antibiotic-resistant strains and non-vaccine types by serotype replacement, is worrisome. To study the biology of pneumococcus and to establish a reliable human model for pneumococcal pathogenesis, we have generated human minilungs from embryonic stem cells. The results show that these organoids can be used to model some events occurring during the interaction of pneumococcus with the lung such as adherence, internalization, and the initial alveolar innate response. This model also represents a great alternative to study virulence factors involved in pneumonia, drug screening, and other therapeutic interventions.


title: Recruitment of transcriptional effectors by Cas9 creates cis regulatory elements and demonstrates distance-dependent transcriptional regulation

abstract: It is essential to regulate the expression of genes, such as those encoding the proteins of the cardiac sarcomere. This regulation is often mediated by cis regulatory elements termed enhancers and repressors that recruit transcription factors to gene-distal sites. However, the relationship between transcription factors recruitment to gene-distant sites and the regulation of gene expression is not fully understood. Specifically, it is unclear if such recruitment to any genomic site is sufficient to form an enhancer or repressor at the site, and what is the relationship between the cis regulatory elements position and its ability to control the transcription of distant genes. Using dead Cas9 to recruit either viral or endogenous transcription factor activation domains, we demonstrate that targeting  naive genomic sites lacking open chromatin or active enhancer marks is sufficient to alter the chromatin signature of the target site, the distant gene promoter, and significantly induce the distant gene expression, even across chromatin insulating loci. The magnitude of induction is affected by the distance between the activation site and the cognate gene in a non-linear manner. Dead Cas9 mediated recruitment of repression domains behave similarly to activation in that targeting of non-regulatory regions could repress gene expression with a nonlinear distance dependence and across chromatin insulating loci. These findings expand the models of enhancer generation and function by showing that an arbitrary genomic site can become a regulatory element and interact epigenetically and transcriptionally with a distant promoter. They also provide new fundamental insights into the rules governing gene expression.


title: Requirement of cholesterol for calcium-dependent vesicle fusion by stabilizing synaptotagmin-1-induced membrane bending

abstract: Cholesterol is essential for neuronal activity and function. Cholesterol depletion in the plasma membrane impairs synaptic transmission. However, the molecular mechanisms by which cholesterol deficiency leads to defects in vesicle fusion remain poorly understood. Here we show that cholesterol is required for Ca2+-dependent fusion using the in-vitro reconstitution of vesicle fusion, atomic force microscopy (AFM), and amperometry to monitor exocytosis in chromaffin cells. Purified native vesicles were crucial for the complete reconstitution of physiological Ca2+-dependent fusion, whereas vesicle-mimicking liposomes failed to reproduce the cholesterol effect. Intriguingly, cholesterol had no effect on membrane insertion of synaptotagmin-1, a Ca2+ sensor for ultrafast fusion. Cholesterol stabilizes local membrane bending induced by synaptotagmin-1 insertion, thereby lowering the energy barrier for Ca2+-dependent fusion to occur. Our data provide evidence that cholesterol depletion abolishes Ca2+-dependent vesicle fusion by disrupting synaptotagmin-1-induced membrane bending, and suggests that cholesterol is a master regulator for Ca2+-dependent fusion.


title: Fish oil Nano-emulsion Kills Macrophage: Ferroptosis and Autophagy Triggered by Catalase-catalysed Superoxide Eruption

abstract: Fish oil is increasingly utilized in the form of nano-emulsion as nutrient and function fortifier. The nano-emulsions high polyunsaturated fatty acids content and electron donors at oil/water interface could be a potential site of redox reaction, if a previously unrecognised trigger was provided. Here we report that a vigorous superoxide production occurred in fish oil nano-emulsion in the presence of mammalian catalase in both acellular and cellular systems. Furthermore, the resulting superoxide increased cytosolic ROS and membrane lipid peroxidation of murine macrophage, and eventually caused fatal oxidative damage, which involves autophagy and ferroptosis but not apoptosis. The cell death was significantly inhibited by a catalase-specific inhibitor. The ferroptosis was independent of protease Caspase-3 activation or glutathione peroxidase suppression. Our findings discover a hidden risk factor of the widely acclaimed fish oil emulsion and suggest a novel mechanism of cellular damage by dietary lipids on mucosal layer of the alimentary tract.


title: Distinct silencer states determine epigenetic states of heterochromatin

abstract: A remarkable property of heterochromatin is that a given heterochromatic locus can exhibit different transcriptional states in genetically identical cells. Studies demonstrate that epigenetic inheritance of the silenced state requires silencers and therefore cannot be driven by the inheritance of modified histones alone. To address these observations, we surveyed the chromatin architectures of strong and weak silencers in Saccharomyces. We found that strong silencers recruited Sir proteins and silenced the locus in all cells. Strikingly, weakening these silencers reduced Sir protein recruitment and stably silenced the locus in some cells; however, this silenced state could probabilistically convert to an expressed state that lacked Sir protein recruitment. Additionally, changes in the constellation of silencer-bound proteins or the concentration of a structural Sir protein modulated the probability that a locus existed in the silenced or expressed state. These findings argued that distinct states of silencers generate epigenetic states and regulate their dynamics.


title: Modeling of Alpha-1 Antitrypsin Deficiency with Syngeneic Human iPSC-Hepatocytes Reveals Metabolic Dysregulation and Cellular Heterogeneity in PiMZ and PiZZ Hepatocytes

abstract: Individuals homozygous for the pathogenic "Z" mutation in alpha-1 antitrypsin deficiency (AATD) are known to be at increased risk for chronic liver disease. That some degree of risk is similarly conferred by the heterozygous state, estimated to affect 2% of the US population, has also become clear. A lack of model systems that recapitulate heterozygosity in human hepatocytes has limited the ability to study the impact of expressing a single ZAAT allele on hepatocyte biology. Here, through the application of CRISPR-Cas9 editing, we describe the derivation of syngeneic induced pluripotent stem cells (iPSCs) engineered to determine the effects of ZAAT heterozygosity in iPSC-derived hepatocytes (iHeps) relative to homozygous mutant (ZZ) or corrected (MM) cells. We find that heterozygous MZ iHeps exhibit an intermediate disease phenotype and share with ZZ iHeps alterations in AAT protein processing and downstream perturbations in hepatic metabolic function including ER and mitochondrial morphology, reduced mitochondrial respiration, and branch-specific activation of the unfolded protein response in subpopulations of cells. Our cellular model of MZ heterozygosity thus provides evidence that expression of a single Z allele is sufficient to disrupt hepatocyte homeostatic function and suggest a mechanism underlying the increased risk of liver disease observed among MZ individuals.


title: Molecular mechanics underlying flat-to-round membrane budding in live secretory cells

abstract: Membrane budding entails forces to transform flat membrane into vesicles essential for cell survival. Accumulated studies have identified coat-proteins (e.g., clathrin) as potential budding factors. However, forces mediating many non-coated membrane buddings remain unclear. By visualizing proteins in mediating endocytic budding in live neuroendocrine cells, performing in vitro protein reconstitution and physical modelling, we discovered how non-coated-membrane budding is mediated: actin filaments and dynamin generate a pulling force transforming flat membrane into {Lambda}-shape; subsequently, dynamin helices surround and constrict {Lambda}-profiles base, transforming {Lambda}- to {Omega}-profile, and then constrict {Omega}-profiles pore, converting {Omega}-profiles to vesicles. These mechanisms control budding speed, vesicle size and number, generating diverse endocytic modes differing in these parameters. Their impact is widespread beyond secretory cells, as the unexpectedly powerful functions of dynamin and actin, previously thought to mediate fission and overcome tension, respectively, may contribute to many dynamin/actin-dependent non- coated-membrane buddings, coated-membrane buddings, and other membrane remodelling processes.


title: A quiescent resident progenitor pool is the central organizer of tendon healing

abstract: A tendons ordered extracellular matrix (ECM) is integral for transmitting force and highly prone to injury. How tendon cells, or tenocytes, embedded within this dense ECM mobilize and contribute to healing is unknown. Here, we identify a specialized Axin2+ population in mouse and human tendons that remains latent in homeostasis yet initiates the healing response and serves as a major source of tendon progenitors. Axin2+ tenocytes readily expand in vitro and express stem cell markers. In vivo, Axin2+-descendants are major functional contributors to repair: Axin2+ tenocytes proliferate, acquire injury responsive states, and re-adopt a tenocyte fate post-injury. Specific loss of Wnt secretion in Axin2+ cells alters their progenitor identity, disrupts their activation upon injury, and severely compromises any healing response. Our work highlights an unusual paradigm, wherein quiescent Axin2+ tenocytes self-regulate their identity and mobilization upon injury and provide the key initiating signal to organize a tendon-wide healing response.


title: Phase separating RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions

abstract:
Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation, especially at low endogenous concentrations found in cells, are thought to follow the tenets of classical nucleation theory describing a sharp transition between a dense phase and a dilute phase characterized by dispersed monomers. Here, we used in vitro biophysical studies to study subsaturated solutions of phase separating RNA binding proteins with intrinsically disordered prion like domains (PLDs) and RNA binding domains (RBDs). Surprisingly, we find that subsaturated solutions are characterized by heterogeneous distributions of clusters comprising tens to hundreds of molecules. These clusters also include low abundance mesoscale species that are several hundreds of nanometers in diameter. Our results show that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. Interestingly, however, cluster formation and phase separation can be decoupled from one another using solutes that impact the solubilities of phase separating proteins. They can also be decoupled by specific types of mutations. Overall, our findings implicate the presence of distinct, sequence-specific energy scales that contribute to the overall phase behaviors of RNA binding proteins. We discuss our findings in the context of theories of associative polymers.

Significance StatementMembraneless biomolecular condensates are molecular communities with distinct compositional preferences and functions. Considerable attention has focused on phase separation as the process that gives rise to condensates. Here, we show that subsaturated solutions of RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions. The formation of clusters in subsaturated solutions and condensates in supersaturated solution are coupled through sequence-specific interactions. Given the low endogenous concentrations of phase separating proteins, our findings suggest that clusters in subsaturated conditions might be of functional relevance in cells.

category: biophysics


title: Binding of His-tagged fluorophores to lipid bilayers and giant vesicles

abstract: His-tagged molecules can be attached to lipid bilayers via certain anchor lipids, a method that has been widely used for the biofunctionalization of membranes and vesicles. To measure the coverage by the membrane-bound molecules, it is useful to study molecules that are fluorescent as well. Here, we use two such molecules, green fluorescence protein (GFP) and green-fluorescent fluorescin isothiocyanate (FITC), both of which are tagged with a chain of six histidines that bind to achor lipids within the bilayers. This His-tag is much smaller in size than the GFP molecule but somewhat larger than the FITC dye. The lipid bilayers form giant unilamellar vesicles (GUVs), the behavior of which can be directly observed in the optical microscope. Several protocols for the preparation of GUVs have been developed. We apply and compare three well-established protocols based on polyvinyl alcohol (PVA) hydrogel swelling, electroformation on platinum wires, and electroformation on indium tin oxide (ITO) glass. For the same nanomolar concentration in the exterior solution, the coverage by His-tagged FITC is much lower than the one by His-tagged GFP. However, for both GFP and FITC, we find that the binding of the His-tagged molecules to the anchor lipids depends strongly on the preparation method. The highest binding affinitiy is obtained for electroformation on platinum wires. PVA gel swelling gives rise to a somewhat smaller binding affinity whereas electroformation on ITO glass leads to essentially no binding. Furthermore, the binding affinitiy is also observed to depend on the pH of the aqueous solution, with a relatively weak and strong pH-dependence for His-tagged GFP and His-tagged FITC, respectively.


title: Cooperativity and folding kinetics in a multi-domain protein with interwoven chain topology

abstract: Although a large percentage of eukaryotic proteomes consist of proteins with multiple domains, not much is known about their assembly mechanism, especially those with complicated native state architectures. Some have complex topology in which the structural elements along the sequence are interwoven in such a manner that the domains cannot be separated by cutting at any location along the sequence. We refer to such proteins as Multiply connected Multidomain Proteins (MMPs). The phoshotransferase enzyme Adenylate Kinase (ADK) with three domains (NMP, LID, and CORE), the subject of this study, is an example of MMP. We devised a coarse-grained model to simulate ADK folding initiated by changing either the temperature or guanidinium chloride (GdmCl) concentration. The simulations reproduce the experimentally measured melting temperatures that are associated with two equilibrium transitions, FRET efficiency as a function of GdmCl concentration, and the global folding times nearly quantitatively. Although the NMP domain orders independently, cooperative interactions between the LID and the CORE domains are required for complete assembly of the enzyme. The kinetic simulations show that on the collapse time scale, but less than the global folding time, multiple interconnected metastable states are populated, attesting to the folding heterogeneity. The network connectivity between distinct states shows that the CORE domain folds only after the NMP and LID domains are formed, reflecting the interwoven nature of the chain topology. We propose that the rules for MMP folding must also hold for the folding of RNA enzymes.


title: Augmented glucose dependency of autoreactive B cells provides a treatment target forlupus

abstract: Heightened glycolysis is inherent to immune/inflammatory disorders, but little is known of its role in the pathogenesis of systemic lupus erythematosus (lupus). Here, we profile key autoimmune populations in acute and chronic lupus-prone models and their response to glycolytic inhibition. We demonstrate that glycolysis is specifically required for autoreactive germinal center B cells (GCB), but not for T follicular helper cells (Tfh) to survive. This augmented reliance on glucose oxidation to maintain ATP production in pathogenic GCB renders them highly susceptible to oxidative stress-induced apoptosis triggered by glycolysis blockade via 2-deoxyglucose (2DG). We show that 2DG can preferentially reduce GCB in lupus-prone mice, while sparing other autoreactive populations, including Tfh, but still significantly improving lifespan and kidney function. Furthermore, the subset of GCB expressing B-cell maturation antigen (BCMA) exhibits an exaggerated dependence on glycolysis to sustain their growth. Depletion of these cells with a proliferation-inducing ligand-based CAR T-cells leads to greatly prolonged lifespan of mice with severe autoimmune activation. These results reveal that glycolysis dependent GCB, especially those expressing BCMA, are key lupus mediators and highlight that they can be selectively targeted to improve disease outcomes for lupus patients.


title: Dynamically regulated Focal adhesions coordinate endothelial cell remodelling in developing vasculature

abstract: The assembly of a mature vascular network involves the coordinated control of cell shape changes to regulate morphogenesis of a complex vascular network. Cellular changes include a process of endothelial cell (EC) elongation which is essential for establishing appropriately sized lumens during vessel maturation1-3. However how EC elongation is dynamically regulated in vivo is not fully understood since live monitoring of this event can be challenging in animal models. Here, we utilise the live imaging capacity of the zebrafish to explore how integrin adhesion complexes, known as Focal Adhesions (FAs), control EC dynamics in live flow pressured vasculature. To do this, we generated a zebrafish mutant, deficient for the integrin adaptor protein Talin1. Notably, unlike the severe cardiovascular defects that arise in Talin1 knockout mice4, vasculogenesis still occurs normally talin1 mutants and cardiac output remains sufficient up to two days post fertilisation (dpf). This allowed us to uncouple primary roles for FAs in ECs during subsequent morphogenesis events, including angiogenesis and vessel remodelling, without interference of secondary effects that might occur due to systemic vessel failure or loss of blood flow. We further established a FA marker line, expressing endothelial Vinculin-eGFP, and demonstrated that FAs are lost in our talin1 mutants. This Vinculin transgene represents the first in vivo model to monitor endothelial FA dynamics. Loss of FAs in talin1 mutants, leads to compromised F-actin rearrangements, which perturb EC elongation and cell-cell junction linearisation during vessel remodelling. Chemical induction of actin polymerisation can restore these cellular phenotypes, suggesting a recovery of actin rearrangements that are sufficient to allow cell and junction shape changes. Together, we have identified that FAs are essential for active guidance of EC elongation and junction linearisation in flow pressured vessels. These observations can explain the severely compromised vessel beds, haemorrhage and vascular leakage that has been observed in mouse models that lack integrin signalling4-8.


title: Functional crosstalk between the carrier translocase machinery and YME1 complex maintains mitochondrial proteostasis and integrity

abstract: The TIM22 pathway cargos are essential for sustaining mitochondrial proteostasis as an excess of these proteins leads to proteostatic stress and cell death. Yme1 is an inner membrane metalloprotease that regulates proteostasis with its chaperone-like and proteolytic activities. Although the mitochondrial translocase and protease machinery are critical for organelle health, the functional link between these complexes remains unexplored. The present study unravels a novel genetic connection between the TIM22 complex and YME1 machinery in maintaining mitochondrial proteostasis and quality control. Our genetic analyses indicate that impairment in the TIM22 complex rescues the respiratory growth defects of cells without Yme1. We further demonstrate that Yme1 is essential for the stability of the TIM22 complex and regulating the proteostasis of the TIM22 pathway substrates. Moreover, impairment in the TIM22 complex suppressed the mitochondrial structural and functional defects of Yme1 devoid cells. Notably, the functional dependence between the TIM22 and YME1 complexes remains functionally conserved from yeast to humans. Our findings suggest that excessive levels of the TIM22 pathway substrates could be one of the reasons for the respiratory growth defects of cells lacking Yme1 and compromising the TIM22 complex compensate for the imbalance in mitochondrial proteostasis caused by loss of Yme1.


title: Nucleation of the destruction complex on the centrosome accelerates degradation of β-catenin and regulates Wnt signal transmission

abstract: Wnt signal transduction is mediated by a protein assembly called the Destruction Complex (DC) made from scaffold proteins and kinases that are essential for transducing extracellular Wnt ligand concentrations to changes in nuclear {beta}-catenin, the pathways transcriptional effector. Recently, DC scaffold proteins have been shown to undergo liquid-liquid phase separation in vivo and in vitro providing evidence for a mesoscale organization of the DC. However, the mesoscale organization of DC at endogenous expression levels and how that organization could play a role in {beta}-catenin processing is unknown. Here we find that the native mesoscale structure is a dynamic biomolecular condensate nucleated by the centrosome. Through a combination of advanced microscopy, CRISPR-engineered custom fluorescent tags, finite element simulations, and optogenetic tools, that allow for independent manipulation of the biophysical parameters that drive condensate formation, we find that a function of DC nucleation by the centrosome is to drive efficient processing of {beta}-catenin by co-localizing DC components to a single reaction hub. We demonstrate that simply increasing the concentration of a single DC kinase onto the centrosome controls {beta}-catenin processing. This simple change in localization completely alters the fate of the Wnt-driven human embryonic stem cell differentiation to mesoderm. Our findings demonstrate the role of nucleators in dynamically controlling the activities of biomolecular condensates and suggest a tight integration between cell cycle progression and Wnt signal transduction.


title: Complementing the EGFR dynamic interactome using live-cell proximity labeling

abstract: The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family (RTK) of transmembrane receptors, known to regulate many key cellular processes, including growth, proliferation, and differentiation. Its expression, activation, trafficking, and degradation have been extensively studied, as dysregulation of EGFR activation has been linked to a vast number of cancers. Activation of EGFR by different ligands results in distinct cellular responses, and the relative distribution of EGFR in different endosome pools in a process called endosomal sorting, leading to lysosomal degradation, or cell surface recycling, respectively, is considered a fundamental process in EGFR stimulation outcome. The EGFR interactome is therefore an essential element in the study of RTK functional selectivity. Here, we aimed to complement the existing EGFR interactome with spatio-temporal information on EGFR, its interactors, and phosphorylation state. We identified and quantified EGFR stable and transient interactions at different time points after stimulation using an EGFR-APEX2 fusion construct expressed in HEK293T cells and were able to use bystander proteins to map EGFR subcellular location at each time point. Utilizing the fast and concise biotinylation of proximity proteins by APEX2, we were able to detect slight differences in early signaling kinetics between TGF- and EGF, thereby increasing our knowledge on RTK signaling and differential trafficking.


title: IZUMO1 is a sperm fusogen

abstract:
Mammalian sperm-egg adhesion depends on the trans-interaction between the sperm-specific type I glycoprotein IZUMO1 and its oocyte-specific GPI-anchored receptor JUNO. However, the mechanisms and proteins (fusogens) which mediate the following step of gamete fusion remain unknown. Using live imaging and content mixing assays in a heterologous system and structure-guided mutagenesis, we unveil an unexpected function for IZUMO1 in cell-to-cell fusion. We show that IZUMO1 alone is sufficient to induce fusion, and that this ability is retained in a mutant unable to bind JUNO. On the other hand, a triple mutation in exposed aromatic residues prevents this fusogenic activity without impairing JUNO interaction. Our findings suggest a second, crucial function for IZUMO1 as a unilateral mouse gamete fusogen.

HighlightsO_LIIZUMO1 expression in somatic cells in culture induces cell-to-cell fusion
C_LIO_LIThe fusogenic activity of IZUMO1 is unilateral
C_LIO_LICell fusion is independent of the binding of IZUMO1 to JUNO
C_LIO_LIIZUMO1-mediated cell merger depends on its transmembrane domain, and three solvent-exposed aromatic residues
C_LI

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/478669v2_ufig1.gif" ALT="Figure 1">
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title: Clathrin mediates both internalization and vesicular release of triggered T cell receptor at the immunological synapse through distinct adaptors

abstract: Ligation of the T cell receptor (TCR) to peptide-MHC complexes initiates signaling leading to T cell activation. Regulation of T cell responses also requires mechanisms to stop this signaling and to downregulate surface expression of the receptor. T cells achieve this both by TCR internalization and by releasing TCR loaded vesicles directly from the plasma membrane. How these distinct fates are coordinated is unknown. Here we show that clathrin is recruited to TCR microclusters by HRS and STAM2 and that this process is essential for TCR release. Subsequently, EPN1 recruits clathrin to the remaining antigen conjugated TCRs to enable a late wave of TCR endocytosis. With these results we demonstrate two clathrin-dependent mechanisms and show how the clathrin machinery participates in membrane evagination and invagination depending upon the adaptor recruiting it. These sequential mechanisms mediate bi-directional membrane exchange at the immunological synapse, providing a scaffold for critical communication.


title: Control of Craniofacial Development by the Collagen Receptor, Discoidin Domain Receptor 2

abstract: Development of the craniofacial skeleton requires interactions between progenitor cells and the collagen-rich extracellular matrix (ECM). The mediators of these interactions are not well-defined. Mutations in discoidin domain receptor 2 (DDR2), a non-integrin collagen receptor, are associated with craniofacial abnormalities, such as midface hypoplasia and open fontanels. However, the exact role of DDR2 in craniofacial morphogenesis is not known. As will be shown, Ddr2-deficient mice exhibit defects in craniofacial bones including impaired calvarial growth and frontal suture formation, cranial base hypoplasia due to aberrant chondrogenesis and delayed ossification at growth plate synchondroses. As established by localization and lineage tracing studies, Ddr2 is expressed in progenitor cell-enriched craniofacial regions including sutures and synchondrosis resting zone cartilage, overlapping with Gli1+ cells, and contributing to chondrogenic and osteogenic lineages during skull growth. Tissue-specific knockouts further established the requirement for Ddr2 in Gli1+ skeletal progenitors and chondrocytes and suggest important functions in chondrocyte proliferation and orientation as well as ECM organization. These studies establish a cellular basis for regulation of craniofacial morphogenesis by this understudied collagen receptor.


title: Impact of cell size on morphogen gradient precision

abstract: Patterning during embryonic development is remarkably precise and reproducible. How high patterning precision is achieved is still largely elusive. Here, we use a novel simulation framework to determine the impact of cell size on the variability of morphogen gradients and the positional information they convey. We find that both variability in the cell diameter and spatial averaging of the local concentration affect the sensed gradient variability only little. In contrast, the mean cell diameter has a profound impact on gradient variability and on the resulting positional accuracy. We reveal the corresponding scaling relationships between positional error, cell diameter and gradient length. Consistent with this observation, apical cell areas are small in tissues that are patterned by morphogen gradients. We conclude that tissues achieve higher patterning precision with small cross-sectional cell areas.


title: AlphaFold accurately predicts distinct conformations based on oligomeric state of a de novo designed protein

abstract: Using the molecular modeling program Rosetta, we designed a de novo protein, called SEWN0.1, that binds the heterotrimeric G protein Gq. The design is helical, well-folded, and primarily monomeric in solution at a concentration of 10 uM. However, when we solved the crystal structure of SEWN0.1, we observed a dimer in a conformation incompatible with binding Gq. Unintentionally, we had designed a protein that adopts alternate conformations depending on its oligomeric state. Recently, there has been tremendous progress in the field of protein structure prediction as new methods in artificial intelligence have been used to predict structures with high accuracy. We were curious if the structure prediction method AlphaFold could predict the structure of SEWN0.1 and if the prediction depended on oligomeric state. When AlphaFold was used to predict the structure of monomeric SEWN0.1, it produced a model that resembles the Rosetta design model and is compatible with binding Gq, but when used to predict the structure of a dimer, it predicted a conformation that closely resembles the SEWN0.1 crystal structure. AlphaFolds ability to predict multiple conformations for a single protein sequence should be useful for engineering protein switches.


title: Oncogenic c-Myc activity increases tolerance to proteotoxic and genotoxic stress through regulation of HSF1

abstract: Oncogenes, such as c-Myc, enhance growth and proliferative signaling to promote continuous cell cycle divisions, the hallmark of cancer. The inadvertent consequence of this is an increase in cellular stresses. However, whether and how oncogenes can directly contribute to cellular stress tolerance, and how much cancer cells rely on these mechanisms for survival, remains poorly understood. Here we show that c-Mycdependent proteotoxic stress contributes to the generation of genotoxic stress. We reveal an important role for the transcription factor Heat Shock Factor 1 (HSF1) in the tolerance to both these c-Myc-induced stresses. c-Myc upregulates HSF1 directly, by activating its expression, and indirectly, via c-Mycdependent proteotoxic stress activation. In addition to relieving c-Myc-induced proteotoxic and genotoxic stress, HSF1 also enables DNA damage response signalling through{gamma} H2AX. Consequently, acute depletion of HSF1 significantly increases c-Myc-driven genome instability and decreases cell viability. Our results establish that c-Myc-dependent regulation of HSF1 ensures that proteotoxic and genotoxic stress, resulting from c-Myc-induced enhanced growth and proliferation, are compatible with cell survival.


title: A Spiking Neural Network Model of Rodent Head Direction calibrated with Landmark Free Learning.

abstract:
Maintaining a stable estimate of head direction requires both self motion (ideothetic) information and environmental (allothetic) anchoring. In unfamiliar or dark environments ideothetic drive can maintain a rough estimate of heading but is subject to inaccuracy, visual information is required to stabilise the head direction estimate. When learning to associate visual scenes with head angle, animals do not have access to the  ground truth of their head direction, and must use egocentrically derived imprecise head direction estimates.

We use both discriminative and generative methods of visual processing to learn these associations without extracting explicit landmarks from a natural visual scene, finding all are sufficiently capable at providing corrective signal. Further, we present a spiking continuous attractor model of head direction (SNN), which when driven by ideothetic input is subject to drift. We show that head direction predictions made by the chosen model-free visual learning algorithms can correct for drift, even when trained on a small training set of estimated head angles self-generated by the SNN. We validate this model against experimental work by reproducing cue rotation experiments which demonstrate visual control of the head direction signal.


title: Pyruvate supports RET-dependent mitochondrial ROS production necessary to control Mycobacterium avium infection in human primary macrophages

abstract: Macrophages deploy a variety of antimicrobial programs to contain mycobacterial infection. Upon activation, they undergo extensive metabolic reprogramming to meet an increase in energy demand, but also to support immune effector functions such as secretion of cytokines and antimicrobial activities. Here, we report that mitochondrial import of pyruvate is linked to production of mitochondrial ROS and control of Mycobacterium avium infection in human primary macrophages. Using chemical inhibition, targeted mass spectrometry and single cell image analysis, we show that macrophages infected with M. avium switched to aerobic glycolysis without any major imbalances in the tricarboxylic acid cycle or changes in the energy charge. Instead, we found that pyruvate import contributed to hyperpolarization of mitochondria in infected cells and increased production of mitochondrial reactive oxygen species by the complex I via reverse electron transport, which reduced the macrophage burden of M. avium. While mycobacterial infections are extremely difficult to treat and notoriously resistant to antibiotics, this work stresses out that compounds specifically inducing mitochondrial reactive oxygen species could present themself as valuable adjunct treatments.


title: Maternal transfer and long-term population effects of PCBs in Baltic grey seals using a new toxicokinetic-toxicodynamic population model

abstract: Empirical evidence has shown that historical exposure of polychlorinated biphenyls (PCBs) to Baltic grey seals not only severely affected individual fitness, but also population growth rates and most likely caused the retarded recovery rate of the depleted population for decades. We construct a new model which we term a toxicokinetic-toxicodynamic (TKTD) population model to quantify these effects. The toxicokinetic sub model describes in detail the bioaccumulation, elimination, and vertical transfer from mother to offspring of PCBs, and was linked to a toxicodynamic model for estimation of PCB-related damage, hazard, and stress impacts on fertility and survival rates. Both sub-models were then linked to a Leslie matrix population model to calculate changes in population growth rate and age structure given different rates of PCB exposure. Toxicodynamic model parameters related to reproductive organ lesions were calibrated using published historical data on observed pregnancy rates in Baltic grey seal females. Our model showed that increased PCB exposure caused reduced fertility, decreased vertical transfer, and increased biomagnification. Compared to empirical data, the TKTD population model described well the age-specific bioaccumulation pattern of PCBs in Baltic grey seals, and thus, the toxicokinetic parameters, deduced from literature, are believed to be reliable. The model also captured well the general effects of PCBs on historical population growth rates. The developed model can be used to perform population viability analyses of Baltic grey seals with multiple stressors, also including by-catches and different hunting regimes. The model can also be extended to other marine mammals and other contaminants than PCB by adjustments of model parameter values and thus provides a test bed in silico for new substances.

category: ecology


title: Positive and relaxed selective pressures have both strongly influenced the evolution of cryonotothenioid fishes during their radiation in the freezing Southern Ocean

abstract: Evolution in the chronic cold of the Southern Ocean has had a profound influence on the physiology of cryonotothenioid fishes. However, the suite of genetic changes underlying the physiological gains and losses in these fishes is still poorly surveyed. Using molecular evolution techniques, this study aimed to identify which functional classes of genes changed during the cryonotothenioid radiation in a polar ocean. The influences of both positive and relaxed selective pressures were isolated following two major physiological transitions: the onset of freezing temperatures, and the loss of hemoproteins. Looking at the influence of cold temperatures, positive selective pressure was most prominently found to act on biosynthetic processes (the machinery of transcription and translation) as well as on protein polymerization, cell adhesion, and cell cycle progression, suggesting these are key challenges to life in freezing waters. Relaxation of selective pressure produced a more complex pattern of change, affecting several homeostatic processes, suggesting their attenuation in cold-stable and oxygen-rich waters, but also seemingly acting as a route to key genetic change behind the morphological and ecological diversification seen in the clade. Finally, while chronic cold-water temperatures appear to have instigated clear genetic change, the loss of hemoproteins led to little observable change relative to their red-blooded relatives. Combined, the influence of positive and relaxed selection show that long-term exposure to cold has led to profound changes in cryonotothenioid genomes, which may make it challenging for them to respond to unpredictable heat waves and to adapt to a rapidly changing climate.


title: Widespread contribution of transposable elements to the rewiring of mammalian 3D genomes and gene regulation

abstract:
Transposable elements (TEs) are major contributors of genetic material in mammalian genomes. These often include binding sites for architectural proteins, including the multifarious master protein, CTCF. These TE-derived architectural protein binding sites shape the 3D genome by creating loops, domains, and compartments borders as well as RNA-DNA chromatin interactions, all of which play a role in the compact packaging of DNA in the nucleus and have the potential to facilitate regulatory function.

In this study, we explore the widespread contribution of TEs to mammalian 3D genomes by quantifying the extent to which they give rise to loops and domain border differences across various cell types and species using a variety of 3D genome mapping technologies. We show that specific (sub-)families of TEs have significantly contributed to lineage-specific 3D chromatin structures in specific mammals. In many cases, these loops have the potential to facilitate interaction between distant cis-regulatory elements and target genes, and domains have the potential to segregate chromatin state to impact gene expression in a lineage-specific and cell-type-specific manner. Backing our extensive conformation study cataloguing and computational analyses, we perform experimental validation using CRISPR-Cas9 to delete one such candidate TE and show disruption of species-specific 3D chromatin structure.

Taken together, we comprehensively quantify and selectively validate our finding that TEs contribute significantly to 3D genome organization and continuously shape it to affect gene regulation during the course of mammalian evolution over deep time.


title: Interphase chromosomes of the Aedes aegypti mosquito are liquid crystalline and can sense mechanical cues

abstract: We use data-driven physical simulations to study the three-dimensional architecture of the Aedes aegypti genome. Hi-C maps exhibit both a broad diagonal and compart-mentalization with telomeres and centromeres clustering together. Physical modeling reveals that these observations correspond to an ensemble of 3D chromosomal structures that are folded over and partially condensed. Clustering of the centromeres and telomeres near the nuclear lamina appears to be a necessary condition for the formation of the observed structures. Further analysis of the mechanical properties of the genome reveals that the chromosomes of Aedes aegypti, by virtue of their atypical structural organization, are highly sensitive to the deformation of the nuclei. This last finding provides a possible physical mechanism linking mechanical cues to gene regulation.


title: Plakoglobin regulates adipocyte differentiation independently of the Wnt/β-catenin signalling pathway

abstract: The scaffold protein 14-3-3{zeta} is an established regulator of adipogenesis and postnatal adiposity. We and others have demonstrated that the 14-3-3{zeta} interactome to be diverse and dynamic, and it can be examined to identify novel regulators of physiological processes, including adipogenesis. In the present study, we sought to determine if factors that influence adipogenesis could be identified in the 14-3-3{zeta} interactome found in white adipose tissue of lean or obese TAP-tagged-14-3-3{zeta} overexpressing mice. Using mass spectrometry, changes in the abundance of novel, as well as established, adipogenic factors within the 14-3-3{zeta} interactome could detected. One novel candidate is plakoglobin, the homolog of the known adipogenic inhibitor {beta}-catenin, and herein, we report that plakoglobin is involved in adipocyte differentiation. Plakoglobin is expressed in murine 3T3-L1 cells and is primarily localized to the nucleus, where its abundance decreases during adipogenesis. Ectopic overexpression and siRNA-mediated depletion of plakoglobin had dual effects on inhibiting adipogenesis and reducing PPAR{gamma}2 expression. Plakoglobin depletion in human adipose-derived stem cells also impaired adipogenesis and reduced lipid accumulation post-differentiation. Transcriptional assays indicated that plakoglobin does not participate in Wnt/{beta}-catenin signaling, as its depletion did not affect Wnt3a-mediated SUPERTOPFlash activity. Taken together, our results establish plakoglobin as a novel regulator of adipogenesis in vitro and highlights the ability of using the 14-3-3{zeta} interactome to discover undiscovered pro-obesogenic factors.


title: Decoding rRNA Sequences for Improved Metagenomics in Sylvatic Mosquito Species

abstract:
BackgroundRNA-seq metagenomics on mosquitoes for surveillance and microbiome or pathogen discovery allows us to understand the disease ecology of arboviruses. A major hurdle in these studies is the depletion of overabundant ribosomal RNA (rRNA), commonly achieved using oligo-based protocols. The lack of publicly available complete reference rRNA sequences for many mosquito vector species narrows the range of such studies, causing a knowledge bias in mosquito vector biology. Here we describe a strategy to assemble full-length 28S and 18S rRNA sequences of 29 sylvatic and peri-urban mosquito species sampled from Cambodia, the Central African Republic, Madagascar, and French Guiana.

ResultsOur score-based strategy successfully parses rRNA reads into insect and non-insect sources, leading to the assembly of complete rRNA sequences for all specimens in the study. We then evaluated the functionality of rRNA sequences as barcodes for taxonomy and phylogeny relative to the mitochondrial cytochrome c oxidase I (COI) gene marker system. rRNA- and COI-based phylogenetic inferences share little congruity. However, the former allowed for molecular species identification when COI sequences were ambiguous or unavailable and revealed better supported intergeneric evolutionary histories concordant with contemporary mosquito systematics.

ConclusionsThe presented assembly strategy and the expansion of the rRNA reference library in public databases by 234 novel complete 28S and 18S rRNA sequences provide a new tool in the form of an RNA marker system to improve mosquito RNA-seq metagenomics. More holistic insights on mosquito vector ecology will benefit the design of public health measures against arboviral diseases.