title: Profiling of the human intestinal microbiome and bile acids under physiologic conditions using an ingestible sampling device

abstract: The spatiotemporal structure of the human microbiome and metabolome reflects and determines regional intestinal physiology and may have implications for disease. Yet, we know little about the distribution of microbes and their products in the gut because of reliance on stool samples and limited access only to some regions of the gut using endoscopy in fasting or sedated individuals. To address these deficiencies, we developed and evaluated a safe, ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion. The collection of 240 intestinal samples from 15 healthy individuals using the device revealed significant differences between microbes and metabolites present in the intestines versus stool. Certain microbial taxa were differentially enriched, and bile acid profiles varied along the intestines and were highly distinct from those of stool. Correlations between gradients in bile acid concentrations and microbial abundance predicted species that altered the bile acid pool through deconjugation. Overall, we identified heterogeneous intestinal profiles of bacterial taxa and metabolites indicating that non-invasive multi-regional sampling of the intestinal tract under physiological conditions can help elucidate the roles of the gut microbiome and metabolome in human physiology and disease.


title: SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation

abstract: Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a novel mechanism by which the SARS-CoV-2 virus co-opts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-{beta}. We reveal that the SARS-CoV-2 encoded Non-Structural Protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-{beta} production, leading to reduced viral infection. Our findings reveal a new target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.

category: microbiology


title: Pore-C Simultaneously Captures Genome-wide Multi-way Chromatin Interaction and Associated DNA Methylation Status in Arabidopsis

abstract: In the past decade, genome-wide characterization of the three-dimensional chromatin structure in plants using high-throughput methods has greatly advanced our knowledge in plant genome architecture (Liu and Weigel, 2015; Ouyang et al., 2020). However, due to the limitation of Illumina short-read sequencing, the genome-wide contact map obtained by Hi-C/ChIA-PET is pairwise, and the multi-way interaction can only be inferred from the two-way data. To directly capture multi-way interaction in Arabidopsis, we applied a long-read-based method called Pore-C that directly sequences the DNA fragments joined by proximity-based ligation (Ulahannan et al., 2019).

category: molecular biology


title: Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming

abstract: The expression of the pluripotency factors OCT4, SOX2, KLF4 and MYC (OSKM) can convert somatic differentiated cells into pluripotent stem cells in a process known as reprogramming. Notably, cycles of brief OSKM expression do not change cell identity but can reverse markers of aging in cells and extend longevity in progeroid mice. However, little is known about the mechanisms involved. Here, we have studied changes in the DNA methylome, transcriptome and metabolome in naturally aged mice subject to a single period of transient OSKM expression. We found that this is sufficient to reverse DNA methylation changes that occur upon aging in the pancreas, liver, spleen and blood. Similarly, we observed reversion of transcriptional changes, especially regarding biological processes known to change during aging. Finally, some serum metabolites altered with aging were also restored to young levels upon transient reprogramming. These observations indicate that a single period of OSKM expression can drive epigenetic, transcriptomic and metabolomic changes towards a younger configuration in multiple tissues and in the serum.

category: cell biology


title: CONTEXT-DEPENDENT ENHANCER FUNCTION REVEALED BYTARGETED INTER-TAD RELOCATION

abstract: The expression of genes with a key function during development is frequently controlled by large regulatory landscapes containing multiple enhancer elements. These landscapes often match Topologically Associating Domains (TADs) and sometimes integrate range of similar enhancers, thus leading to TADs having a global regulatory specificity. To assess the relative functional importance of enhancer sequences versus the regulatory domain they are included in, we set out to transfer one particular enhancer sequence from its native domain into a TAD with a closely related, yet different functional specificity. We used Hoxd genes and their biphasic regulation during limb development as a paradigm, since they are first activated in proximal limb cells by enhancers located in one TAD, which is then silenced at the time when the neighboring TAD starts to activate its enhancers in distal limb cells. We introduced a strong distal limb enhancer into the  proximal limb TAD and found that its new context strongly suppresses its distal specificity, even though it continues to be bound by HOX13 transcription factors, which normally are responsible for this activity. Using local genetic alterations and chromatin conformation measurements, we see that the enhancer is capable of interacting with target genes, with a pattern comparable to its adoptive neighborhood of enhancers. Its activity in distal limb cells can be rescued only when a large portion of the surrounding environment is removed. These results indicate that, at least in some cases, the functioning of enhancer elements is subordinated to the local chromatin context, which can exert a dominant control over its activity.

category: developmental biology


title: Understanding the development of oral epithelial organs through single cell transcriptomic analysis

abstract: During vertebrate craniofacial development, the oral epithelium begins as a simple and morphologically homogeneous tissue. It then gives rise to locally complex structures, including the developing teeth, salivary glands, and taste buds. While there is significant knowledge about the molecular mechanisms regulating the morphogenesis of these organs at later stages, how the epithelium is initially patterned and specified to generate diverse cell types and organs remains largely unknown. To elucidate the genetic programs that direct the formation of distinct oral epithelial populations, we mapped the transcriptional landscape of embryonic day (E) 12 mouse mandibular epithelia at single cell resolution. Our analysis identified key transcription factors and gene regulatory networks that define different epithelial cell types as well as regions patterned along the oral-aboral axis. By examining the spatiotemporal expression of region-specific markers in embryonic mandibles, our results pointed to a model where the dental field is progressively confined to its position by the formation of the aboral epithelium anteriorly and the non-dental oral epithelium posteriorly. Using our data, we also identified Ntrk2 as a promoter of cell proliferation in the forming incisor, contributing to its invagination. Together, our results provide a detailed transcriptional atlas of the developing mandibular epithelium and unveil new genetic markers and regulators that are present during the specification of various oral epithelial structures.


title: Identification and characterization of a dlx2b cis-regulatory element both sufficient and necessary for correct transcription during zebrafish tooth development

abstract:
Much remains to be learned about how cis-regulatory elements such as enhancers function, especially during vertebrate organ development. To increase knowledge in this area, we have examined the cis-regulation of the transcription factor dlx2b during zebrafish larval tooth formation. We have created a GFP knock-in line that replicates dlx2b expression during tooth development and have also isolated a minimal enhancer of dlx2b (MTE1) sufficient for activating most of the tooth germ expression pattern. We have found that four evolutionarily conserved predicted transcription factor binding sites are required for the function of this minimal enhancer in both contexts. When the conserved sequences are mutated in a transgene it eliminates the activity of the enhancer and when they are deleted at the dlx2b locus it causes a dramatic alteration in the expression pattern. We hypothesize that disabling this enhancer at the dlx2b locus may be enabling other nearby cis-regulatory elements to take control of the promoter. These experiments reveal details of how cis-regulatory elements are working to control gene expression during organogenesis and highlight how much remains to be learned by empirical studies of gene regulation.

Graphical Abstract

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title: High Hedgehog signaling is transduced by a multikinase-dependent switch controling the apico-basal distribution of the GPCR Smoothened.

abstract: The oncogenic GPCR Smoothened is a key transducer of the Hedgehog morphogen, which plays essential roles in the patterning of epithelial structures. Here, we examine how Hedgehog controls Smoothened subcellular localization and activity in a polarized epithelium using the Drosophila wing imaginal disc as a model. We provide evidence that Hedgehog promotes the stabilization of Smoothened by switching its fate after endocytosis toward recycling. This effect involves the sequential and additive action of Protein Kinase A, Casein Kinase I and the Fused kinase. Moreover, in the presence of very high levels of Hedgehog, a second effect of Fused leads to the local enrichment of Smoothened in the most basal domain of the cell membrane. Together, these results link the morphogenetic effects of Hedgehog to the apico-basal distribution of Smoothened and provide a novel mechanism for regulation of a GPCR by plasma membrane subcompartimentalisation.


title: An ancient role for the Hippo pathway in axis formation and morphogenesis

abstract: How did cells of early metazoan organisms first organize themselves to form a body axis? The canonical Wnt pathway has been shown to be sufficient for induction of axis in Cnidaria, a sister group to Bilateria, and is important in bilaterian axis formation. Here, we provide experimental evidence that in cnidarian Hydra the Hippo pathway regulates the formation of a new axis during budding upstream of the Wnt pathway. The target of Hippo pathway, the transcriptional co-activator YAP, inhibits the initiation of budding in Hydra, and is regulated by Hydra LATS. In addition, we show functions of Hippo pathway in regulation of actin organization and cell proliferation in Hydra. We hypothesize that Hippo pathway served as a link between continuous cell division, cell density and axis formation early in metazoan evolution.


title: A key role for p60-Katanin in axon navigation is conditioned by the tubulin polyglutamylase TTLL6

abstract: Selective interplay between microtubules and their regulatory proteins emerged as major regulator of neuronal circuit development. However, how microtubule posttranslational modifications influence this interplay to govern axon guidance and targeting remains elusive. Using loss-of-function and rescue experiments during zebrafish development, we show that the microtubule-severing enzyme p60-Katanin controls motor axon pathfinding and larval mobility in a dose-dependent manner, and plays non-overlapping roles with the related protein Spastin in these processes. Notably, we uncover the tubulin glutamylase TTLL6 as a selective and key regulator of p60-Katanin activity during axon navigation. In contrast, our study demonstrates that, although critical for motor circuit wiring, the highly similar glutamylase TTLL11 fails to influence p60-Katanin and to rescue the axon targeting errors associated with TTLL6 knockdown, which discloses the functional specificity of these long-chain glutamylases at a physiological level. Altogether, our work provides in vivo proofs of concept that the tubulin code acts in a highly selective manner to tune the activity of key microtubule-severers in neuronal circuit wiring of a vertebrate model.


title: Cell cycle dynamics controls fluidity of the developing mouse neuroepithelium

abstract: As organs are remodelled by morphogenetic changes and pattern formation during development, their material properties may change. To address whether and how this occurs in the mouse neural tube, we combined highly resolved mosaic analysis, biophysical modelling and perturbation experiments. We found that at early developmental stages the neuroepithelium surprisingly maintains both high junctional tension and high fluidity. This is achieved via a previously unrecognized mechanism in which interkinetic nuclear movements generate cell area dynamics that drive extensive cell rearrangements. Over time, the proliferation rate declines, effectively solidifying the tissue. Thus, unlike well-studied jamming transitions, the solidification we uncovered resembles a glass transition that depends on the dynamics of stresses generated by proliferation and differentiation. This new link between epithelial fluidity, interkinetic movements and cell cycle dynamics has implications for the precision of pattern formation and could be relevant to multiple developing tissues.


title: Lysosomes are Required for Early Dorsal Signaling in the Xenopus Embryo

abstract:
Lysosomes are the digestive center of the cell and play important roles in human disease, including cancer. Previous work has suggested that late endosomes, also known as multivesicular bodies (MVBs), and lysosomes are essential for canonical Wnt pathway signaling. Sequestration of Glycogen Synthase 3 (GSK3) and of {beta}{square}catenin destruction complex components in MVBs is required for sustained canonical Wnt signaling. Little is known about the role of lysosomes during early development. In the Xenopus egg, a Wnt-like cytoplasmic determinant signal initiates formation of the body axis following a cortical rotation triggered by sperm entry. Here we report that cathepsin D was activated in lysosomes specifically on the dorsal marginal zone of the embryo at 64-cell stage, long before zygotic transcription starts. Expansion of the multivesicular body (MVB) compartment with low-dose Hydroxychloroquine (HCQ) greatly potentiated the dorsalizing effects of the Wnt agonist Lithium chloride (LiCl) in embryos, and this effect required macropinocytosis. Formation of the dorsal axis required lysosomes, as indicated by brief treatments with the vacuolar ATPase (V-ATPase) Bafilomycin A1 inhibitor at the 32-cell stage. Inhibiting the MVB-forming machinery with a dominant-negative point mutation in Vacuolar Protein Sorting 4 (Vps4-EQ) also interfered with the endogenous dorsal axis. The Wnt-like activity of the dorsal cytoplasmic determinant Huluwa (Hwa), and that of microinjected xWnt8 mRNA, also required lysosome acidification and the MVB-forming machinery. We conclude that lysosome function is essential for early dorsal axis development in Xenopus. The results highlight the intertwining between membrane trafficking, lysosomes, and vertebrate axis formation.

SignificanceThe dorsal axis of the vertebrate Xenopus embryo is established by an early Wnt signal generated by a rotation of the cortex of the egg towards the opposite side of the sperm entry point. In this study, we report that lysosomal Cathepsin D becomes activated on the dorsal marginal zone of the embryo already at the 64-cell stage, and that this asymmetry is enhanced by increasing Wnt signaling levels. We present experiments showing that lysosome activity, macropinocytosis, and multivesicular body formation are required for the dorsal signal provided maternally in the egg, and for twinning by microinjected huluwa and Wnt8 mRNA. The results indicate that the cell biology of lysosomes plays a fundamental role in vertebrate development.


title: Research on the temporal evolution track and influence of green development in the last decade

abstract: Green development is important to realize the coordinated development of local ecological environment, economy and society, but also an important way to promote the harmony between human and land. This study utilizes projection pursuit model (PPM) measure, as well as the system gray prediction model GM (1, N), and other measurement methods, build an evaluation system from three dimensions of green fortune, green growth and green benefits to analyze the temporal evolution, development trend and its influencing factors of Baoshans ecological engineering of project acres, the results show: (1)The overall level of the green development system in Baoshan, Yunnan Province, is relatively good and shows an increasing trend year by year, with an average growth rate of 18.3%, and there is a synergistic coupling effect among the three subsystems. (2) Regional green development has achieved remarkable progress, but problems and pressure among the three subsystems are still grim. The sub-system of green growth and green benefits declined from 2010 to 2016, with an annual decline rate of 12%, and then picked up slightly in the next three years, reflecting the pressure of resources and ecological environment in this region and the difficulty of the sub-system of green wealth growth. (3) The prediction analysis shows that the green development index in Baoshan, Yunnan Province will keep a steady rise in the next two years, and the predicted value will be 4.51 in 2020, with an average growth rate of 15.1%, and the regional green development system is in good condition. (4) Green development is mainly affected by environmental pollution, industrial structure, urbanization, population, market and other factors from the three subsystems of local ecology, ecology, economy and social benefits. This paper puts forward suggestions in terms of popularizing the concept of green development, improving the level of ecological economic development and promoting the regional environment bearing capacity, green development model of ecological engineering area is also built. It aims to have some practical reference value for prompting regional ecological civilization construction and green development. And provide regional support for the post-2020 Global Biodiversity Framework and the Achievement of the United Nations Sustainable Development Goals.

category: ecology


title: Genomic databanks and targeted assays help characterise mosquito incursions within Australia

abstract: Biosecurity strategies aiming to restrict the spread of invasive pests can benefit from knowing where new incursions have come from. This knowledge can be acquired using genomic databanks, where genetic variation in incursion samples is compared against variation in reference samples. Here, we use genomic databanks to investigate domestic incursions of two mosquito species in Australia. We used a deep learning method to trace a 2021 invasion of Aedes aegypti in Tennant Creek, Northern Territory, to Townsville, Queensland, and to trace Ae. albopictus incursions between several invaded and uninvaded islands in the Torres Strait. We observed high precision of tracing despite 30-70 generations separating incursions and source samples. Targeted assays also improved precision, in this case by comparing Wolbachia infection data to mitochondrial DNA variation in Ae. aegypti. For Ae. albopictus, incursions detected in 2021 were traced to different islands than incursions detected in 2019. Patterns of relatedness and inbreeding indicated that Tennant Creek was likely invaded by Ae. aegypti from one family, whereas Torres Strait incursions involved distinct kinship groups. Our results highlight the value of repeated tracing with genomic databanks that remain informative over years, and demonstrate how additional targeted assays (e.g. Wolbachia) can improve inferences.

category: genomics


title: Genomic databanks and targeted assays help characterise domestic mosquito incursions


title: Nucleosome topology and DNA sequence modulate the engagement of pioneer factors SOX2 and OCT4

abstract: Nucleosomes in eukaryotic genomes present a barrier to the competent binding of many regulatory proteins. Pioneer transcription factors (pTFs) can bind their target sites on nucleosomal DNA and collaborate with other factors to locally open chromatin and promote transcription. While the interaction of pluripotency pioneer factors and functional partners Sox2 and Oct4 with nucleosomes has been widely studied, molecular details about their engagement in different nucleosome contexts remain elusive. Here, using high-resolution nuclear magnetic resonance (NMR) spectroscopy and biochemical studies, we reveal site-specific structural and dynamic information about pTF interaction with nucleosomes. We find that the affinity of Sox2 and Oct4 to the nucleosome and their synergistic binding correlates with solvent-exposed sites but is highly position and DNA sequence dependent and linked to distinct pTF conformation and dynamics. Sox2 alone forms a super-stable complex near superhelical location 5 (SHL5) with similar affinity and conformation to that of naked DNA but shows elevated dynamics at suboptimal positions. Oct4 strongly favors positions near SHL5.5 and SHL6.5 and both of its DNA binding modules, POUS or POUHD, are required for stable complex formation. A ternary complex forms efficiently on canonical Sox2-Oct4 composite motifs (no spacing) near nucleosome ends but is sparse at spaced motifs and absent at internal sites. Moreover, the ability of Sox2 to fold and bend DNA plays a key role in the formation of a stable nucleosome complex and cooperative Oct4 binding. Collectively, our findings describe diverse binding modes of Sox2 and Oct4 on nucleosomes that could guide their site selection and potential interaction with other chromatin factors in vivo.

category: biochemistry


title: Tethering designer short double-stranded RNA to mRNA for co-delivery of molecularly-targeted adjuvants and antigens towards cancer vaccination

abstract: Immunostimulatory adjuvants play pivotal roles in mRNA cancer vaccines. Herein, mRNA architectonics-based approach was employed to enhance immunostimulatory properties of mRNA, by tethering immunostimulatory short 24-base-pair double-stranded RNA (dsRNA) to mRNA via hybridization, for specific stimulation of retinoic acid-inducible gene-I (RIG-I), an innate immune receptor triggering effective cellular immunity. This approach ensures the co-delivery of mRNA and adjuvants to obtain antigen expression and immune activation in the same antigen presenting cells for effective vaccination, and allows the tuning of immunostimulatory effects by controlling dsRNA number to avoid safety concerns associated with excessive immunostimulation. Thorough in vitro screening provided optimal mRNA structure that induces the secretion of a broad spectrum of proinflammatory molecules and effectively activated mouse and human dendritic cells, with minimal effect on antigen translation efficiency of mRNA. The optimal mRNA formulation improved the potency of mRNA vaccine to induce cytotoxic immune responses after encapsulation into mRNA lipoplex, and exhibited high therapeutic efficiency in lung metastatic model of melanoma using mRNA encoding tumor associated antigen. Furthermore, this system is versatile to potentiate vaccination effect with the use of mRNA polyplex micelle.

category: bioengineering


title: Phase separation driven by interchangeable properties in the intrinsically disordered regions of protein paralogs

abstract: Paralogs, arising from gene duplications, increase the functional diversity of proteins. Protein functions in paralog families have been extensively studied, but little is known about the roles of intrinsically disordered regions (IDRs), even though more than half of eukaryotic proteins have them. Using the RNA-binding protein Musashi family as an example, we applied multiple structural techniques and phylogenetic analysis to show how members in a paralog family have evolved their IDRs to different physicochemical properties but converge to the same function. In this example, the lower prion-like tendency of Musashi-1s IDRs, rather than Musashi-2s, is compensated by its higher -helical propensity to assist their assembly. Without a folded structure to restraint, IDRs sequences mutate faster along with evolution. No matter how fast they change, our work suggests that IDRs evolve different traits to a converged function, such as liquid-liquid phase separation.

category: biophysics


title: Improving Small Molecule pKa Prediction Using Transfer Learning with Graph Neural Networks

abstract: Enumerating protonation states and calculating micro-state pKa values of small molecules is an important yet challenging task for lead optimization and molecular modeling. Commercial and non-commercial solutions have notable limitations such as restrictive and expensive licenses, high CPU/GPU hour requirements, or the need for expert knowledge to set up and use. We present a graph neural network model that is trained on 714,906 calculated mico-state pKa predictions from molecules obtained from the ChEMBL database. The model is fine-tuned on a set of 5,994 experimental pKa values significantly improving its performance on two challenging test sets. Combining the graph neural network model with Dimorphite-DL, an open-source program for enumerating ionization states, we have developed the open-source Python package pkasolver, which is able to generate and enumerate protonation states and calculate micro-state pKa values with high accuracy.


title: Dynamics and structural changes of calmodulin upon interaction with its potent antagonist calmidazolium

abstract:
Calmodulin (CaM) is a eukaryotic multifunctional, calcium-modulated protein that regulates the activity of numerous effector proteins involved in a variety of physiological processes. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Here, we report the structural characterization of CaM:CDZ complexes using combined SAXS, X-ray crystallography, HDX-MS and NMR approaches. Our results provide molecular insights into the CDZ-induced dynamics and structural changes of CaM leading to its inhibition. CDZ-binding induces an open-to-closed conformational change of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from protein targets, despite their distant chemical nature. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. These results open the way to rationally design new CaM-selective drugs.

Figure and text for the Table of Contents (ToC)

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/474921v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@b0c76org.highwire.dtl.DTLVardef@15f462forg.highwire.dtl.DTLVardef@1f8e57forg.highwire.dtl.DTLVardef@1a33575_HPS_FORMAT_FIGEXP  M_FIG C_FIG Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results open the way to rationally design new and more selective inhibitors of calmodulin.


title: A molecular dynamics study of antimicrobial peptide translocation across the outer membrane of Gram-negative bacteria

abstract: With rising bacterial resistance, antimicrobial peptides (AMPs) have been widely investigated as potential antibacterial molecules to replace conventional antibiotics. Our understanding of the molecular mechanism for membrane disruption are largely based on AMP interactions with the inner phospholipid bilayers of both Gram-negative and Grampositive bacteria. Mechanisms for AMP translocation across the outer membrane of Gram-negative bacteria composed of lipopolysaccharides and the asymmetric lipid bilayer are incompletely understood. In the current study, we have employed atomistic molecular dynamics and umbrella sampling simulations with an aggregate duration of ~ 8 microseconds to understand the free energy landscape of CM15 peptide within the OM of Gram-negative bacteria, E. coli. The peptide has a favourable binding free energy (-130 kJ mol-1) in the O-antigen region with a large barrier (150 kJ mol-1) at the interface between the anionic coresaccharides and upper bilayer leaflet made up of lipid A molecules. We have analyzed the peptide and membrane properties at each of the 100 ns duration umbrella sampling windows to study variations in the membrane and the peptide structure during the translocation through the OM. Interestingly the peptide is seen to elongate, adopting a membrane perpendicular orientation in the phospholipid region resulting in the formation of a transient water channel during its translocation through the bilayer. The presence of the peptide at the lipid A and core-saccharide interface results in a 11% increase in the membrane area with the peptide adopting a predominantly membrane parallel orientation in this cation rich region. Additionally, the lateral displacement of the peptide is significantly reduced in this region, and increases toward the inner phospholipid leaflet and the outer O-antigen regions of the membrane. The peptide is found to be sufficiently hydrated across both the hydrophilic as well as hydrophobic regions of the membrane and remains unstructured without any gain in helical content. Our study unravels the complex free energy landscape for the translocation of the AMP CM15 across the outer membrane of Gram-negative bacteria and we discuss the implications of our findings with the broader question of how AMPs overcome this barrier during antimicrobial activity.


title: Membrane-mediated interactions between hinge-like particles

abstract: Adsorption of nanoparticles on a membrane can give rise to interactions between particles, mediated by membrane deformations, that play an important role in self-assembly and membrane remodeling. Previous theoretical and experimental research has focused on nanoparticles with fixed shapes, such as spherical, rod-like, and curved nanoparticles. Recently, hinge-like DNA origami nanostructures have been designed with tunable mechanical properties. Inspired by this, we investigate the equilibrium properties of hinge-like particles adsorbed on an elastic membrane using Monte Carlo and umbrella sampling simulations. The configurations of an isolated particle are influenced by competition between bending energies of the membrane and the particle, which can be controlled by changing adsorption strength and hinge stiffness. When two adsorbed particles interact, they effectively repel one another when the strength of adhesion to the membrane is weak. However, a strong adhesive interaction induces an effective attraction between the particles, which drives their aggregation. The configurations of the aggregate can be tuned by adjusting the hinge stiffness: Tip-to-tip aggregation occurs for flexible hinges, whereas tip-to-middle aggregation also occurs for stiffer hinges. Our results highlight the potential for using the mechanical features of deformable nanoparticles to influence their self-assembly when the particles and membrane mutually influence one another.


title: ORIGIN OF THREE RELATED MOVEMENT GENE MODULES IN PLANT VIRUSES: EVOLUTIONARY RADIATIONS OF BENYVIRUS-LIKE RNA REPLICONS

abstract: Previous studies have shown that the RNA genomes of some plant viruses encode two related genetic modules required for the virus movement over host body, containing 2 or 3 genes and named as triple gene block (TGB) and binary movement block (BMB). In this paper, we revealed a novel related movement gene module, called Tetra-Cistron Movement Block (TCMB). It was found to be encoded by virus-like transcriptome assemblies of moss Dicranum scoparium and Antarctic flowering plant Colobanthus quitensis. These TCMBs are encoded by second RNA components of the putative two-component viruses related to plant benyviruses. Like RNA-2 of benyviruses, TCMB RNA-2 contains the 5-terminal coat protein gene. General organization of TCMB is very similar to TGB: it includes RNA helicase gene which is followed by two small overlapping cistrons encoding hydrophobic proteins with a distant sequence similarity to TGB2 and TGB3 genes. However, TCMB includes also forth 5-terminal gene coding for protein with an obvious similarity to double-stranded RNA-binding proteins belonging to the DSRM AtDRB-like superfamily. Finally, we suggest the proposed involvement of replicative beny-like helicases in evolution of the BMB and TCMB movement genetic modules.


title: A single, improbable B cell receptor mutation confers potent neutralization against cytomegalovirus

abstract:
Cytomegalovirus (CMV) is a leading cause of infant hearing loss and neurodevelopmental delay, but vaccine candidates have faced challenges eliciting neutralizing antibodies. One of the most well-studied targets for CMV vaccines is the viral fusogen glycoprotein B (gB), which is required for viral entry into host cells. Within gB, antigenic domain 2 site 1 (AD-2S1) is a target of potently neutralizing antibodies, but gB-based candidate vaccines have yet to elicit robust responses against this region. We mapped the genealogy of B cells encoding potently neutralizing anti-gB AD-2S1 antibodies from their inferred unmutated common ancestor (UCA) and characterized the binding and function of early lineage ancestors. Remarkably, we found that the single amino acid heavy chain mutation A33N, an improbable mutation rarely generated by somatic hypermutation machinery, conferred broad CMV neutralization to the UCA antibody. Structural studies revealed that this mutation mediated key contacts with the gB AD-2S1 epitope. Collectively, these results provide insight into potently neutralizing gB-directed antibody evolution and a foundation for designing next-generation CMV vaccines.

One-sentence summaryThis manuscript identifies an early B cell lineage mutation that confers neutralizing function to antibodies targeting CMV fusogen gB.


title: Myogenic regulatory factors MyoD and Myf5 are required for dorsal aorta formation and angiogenic sprouting

abstract:
The vertebrate embryonic midline vasculature forms in close proximity to the developing skeletal muscle, which originates in the somites. Angioblasts migrate from bilateral positions along the ventral edge of the somites until they meet at the midline, where they sort and differentiate into the dorsal aorta and the cardinal vein. This migration occurs at that the same time that myoblasts in the somites are beginning to differentiate into skeletal muscle, a process which requires the activity of the basic helix loop helix (bHLH) transcription factors Myod and Myf5. Here we examined vasculature formation in myod and myf5 mutant zebrafish. In the absence of skeletal myogenesis, angioblasts migrate normally to the midline but form only the cardinal vein and not the dorsal aorta. The phenotype is due to the failure to activate vascular endothelial growth factor ligand vegfaa expression in the somites, which in turn is required in the adjacent angioblasts for dorsal aorta specification. Myod and Myf5 cooperate with Hedgehog signaling to activate and later maintain vegfaa expression in the medial somites, which is required for angiogenic sprouting from the dorsal aorta. Our work reveals that the early embryonic skeletal musculature in teleosts evolved to organize the midline vasculature during development.

Summary statementThe myogenic transcription factors MyoD and Myf5 have a novel function in inducing the artery through regulation of Vegf.


title: Mutation of Vsx genes in zebrafish highlights the robustness of the retinal specification network.

abstract:
Genetic studies in human and mice have established a dual role for Vsx genes in retina development: an early function in progenitors specification, and a later requirement for bipolar-cells fate determination. Despite their conserved expression patterns, it is currently unclear to which extent Vsx functions are also conserved across vertebrates, as mutant models are available only in mammals. To gain insight into vsx function in teleosts, we have generated vsx1 and vsx2 CRISPR-Cas9 double knockouts (vsxKO) in zebrafish. Our electrophysiological and histological analyses indicate severe visual impairment and bipolar cells depletion in vsxKO larvae, with retinal precursors being rerouted towards photoreceptor or Muller glia fates. Surprisingly, neural retina is properly specified and maintained in mutant embryos, which do not display microphthalmia. We show that although important cis-regulatory remodelling occurs in vsxKO retinas during early specification, this has little impact at a transcriptomic level. Our observations point to genetic redundancy as an important mechanism sustaining the integrity of the retinal specification network, and to Vsx genes regulatory weight varying substantially among vertebrate species.

Brief Summary Statement for use in emailed and online tables of content alertsThe mutation of vsx genes in zebrafish confirms a conserved role in bipolar cells specification across vertebrates, but do not interfere with the specification of the neural retina domain. Our data reveal the unexpected robustness of the genetic network sustaining the identity of the neural retina.


title: Nulliparity affects the expression of a limited number of genes and pathways in Day 8 equine embryos

abstract: Nulliparous mares produce lighter and smaller foals compared to mares having previously foaled, with effects observed at least until 4 months of age. The need for a first gestation priming for the uterus to reach its full capacity has been proposed to explain this observation. Embryo developmental defects could be hypothesized but effects of maternal parity on the embryo have only been described once, in old mares, thus combining effects of parity and old age. The aim of this study was to determine effects of mare parity on embryo gene expression. Day-8 post ovulation blastocysts were collected from young (5/6 years old) nulliparous (YN, N=6) or multiparous (YM, N=4) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure (TE_part) or inner-cell-mass-enriched (ICMandTE) trophoblast were obtained by embryo bisection for RNA sequencing (paired end, non-oriented, Illumina, NextSeq500). Deconvolution was performed on the ICMandTE dataset. Differential expression, with embryo sex and diameter as cofactors and gene set enrichment analysis (GO BP, KEGG, REACTOME databases) were performed using a false discovery rate <0.05 cutoff. Only a few genes were altered (ICM: n=18; TE: n=6) but several gene sets were perturbed (ICM: n=62; TE: n=50) by maternal parity. In YM, only pathways related to transcription, RNA processing and vesicle transport functions were enriched in the ICM whereas only pathways related to RNA localization were enriched in TE. In YN, while only gene sets related to ribosomes and extracellular matrix were enriched in the ICM, functions related to energy and lipid metabolism, lipid transport and interleukin-1 signaling were enriched in the TE. In conclusion, several genes and pathways are affected in embryos collected from nulliparous mares, with different effects on TE and ICM. Embryo development is altered in nulliparous mares, which could partially explain the term phenotype. Whether differences in gene expression result/induce poor embryo-maternal communication remains to be determined.


title: m6A reader Pho92 is recruited co-transcriptionally and couples translation efficacy to mRNA decay to promote meiotic fitness in yeast

abstract: N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.


title: Spatially coherent diffusion of human RNA Pol II depends on transcriptional state rather than chromatin motion

abstract: Gene transcription by RNA polymerase II (RNAP II) is a tightly regulated process in the genomic, temporal, and spatial context. Transcriptionally active genes often spatially cluster at RNA Pol II foci, called transcription factories, causing long-range interactions between distal sites of the genome. Recently, we have shown that chromatin exhibits spatially long-range coherently moving regions over the entire nucleus, and transcription enhances this long-range correlated DNA movement. Yet, it remains unclear how the mobility of RNA Pol II molecules is affected by transcription regulation and whether this response depends on the coordinated chromatin movement. We applied our Dense Flow reConstruction and Correlation method to analyze nucleus-wide coherent movements of RNA Pol II in living human cancer cells. We quantify the spatial correlation length of RNA Pol II in the context of DNA motion. We observe a spatially coherent movement of RNA Pol II molecules over ~1 m, considerably less than for DNA, suggesting that spatially coherent RNA Pol II motion does not solely result from the DNA motion. In contrast to DNA, inducing transcription in quiescent cells decreased the coherent motion of RNA Pol II, while the inhibition of transcription elongation by using DRB slightly increased coherent RNA Pol II motion. The spatially coherent movement of RNA Pol II domains is affected by the transcriptional state and largely independent of the underlying chromatin domains. Our study reveals the nucleus-wide interplay between chromatin and RNA Pol II in the dynamic regulation of chromatin organization.


title: Caulobacter requires anionic sphingolipids and deactivation of fur to lose lipid A

abstract: Lipid A, the membrane-anchored portion of lipopolysaccharide, is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here, we identify regulatory and structural factors that together permit Caulobacter crescentus to eliminate lipid A from its OM. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog which we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to eliminate lipid A. Fitness of lipid A-deficient Caulobacter requires a previously uncharacterized anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.


title: A mixed generative model of auditory word repetition

abstract: In this paper, we introduce a word repetition generative model (WORM), which--when combined with an appropriate belief updating scheme--is capable of inferring the word that should be spoken when presented with an auditory cue. Our generative model takes a deep temporal form, combining both discrete and continuous states. This allows a (synthetic) WORM agent to perform categorical inference on continuous acoustic signals, and--based on the same model--to repeat heard words at the appropriate time. From the perspective of word production, the model simulates how high-level beliefs about discrete lexical, prosodic and context attributes give rise to continuous acoustic signals at the sensory level. From the perspective of word recognition, it simulates how continuous acoustic signals are recognised as words and, how (and when) they should be repeated. We establish the face validity of our generative model by simulating a word repetition paradigm in which a synthetic agent or a human subject hears a target word and subsequently reproduces that word. The repeated word should be the target word but differs acoustically. The results of these simulations reveal how the generative model correctly infers what must be repeated, to the extent it can successfully interact with a human subject. This provides a formal process theory of auditory perception and production that can be deployed in health and disease. We conclude with a discussion of how the generative model could be scaled-up to include a larger phonetic and phonotactic repertoire, complex higher-level attributes (e.g., semantic, concepts, etc.), and produce more elaborate exchanges.

category: neuroscience


title: Neurodevelopmental oscillatory basis of speech processing in noise

abstract:
Humans extraordinary ability to understand speech in noise relies on multiple processes that develop with age. Using magnetoencephalography (MEG), we characterize the underlying neuromaturational basis by quantifying how cortical oscillations in 144 participants (aged 5 to 27 years) track phrasal and syllabic structures in connected speech mixed with different types of noise. While the extraction of prosodic cues from clear speech was stable during development, its maintenance in a multi-talker background matured rapidly up to age 9 and was associated with speech comprehension. Furthermore, while the extraction of subtler information provided by syllables matured at age 9, its maintenance in noisy backgrounds progressively matured until adulthood. Altogether, these results highlight distinct behaviorally relevant maturational trajectories for the neuronal signatures of speech perception. In accordance with grain-size proposals, neuromaturational milestones are reached increasingly late for linguistic units of decreasing size, with further delays incurred by noise.

TeaserThe neural signature of speech processing in silence and noise features multiple behaviorally relevant developmental milestones


title: A genetically-defined population in the lateral and ventrolateral periaqueductal gray selectively promotes flight to safety

abstract: When encountering external threats, survival depends on the engagement of appropriate defensive reactions to minimize harm. There are major clinical implications for identifying the neural circuitry and activation patterns that produce such defensive reactions, as maladaptive overactivation of these circuits underlies pathological human anxiety and fear responses. A compelling body of work has linked activation of large glutamatergic neuronal populations in the midbrain periaqueductal gray (PAG) to defensive reactions such as freezing, flight and threat-induced analgesia. These pioneering data have firmly established that the overarching functional organization axis of the PAG is along anatomically-defined columnar boundaries. Accordingly, broad activation of the dorsolateral column induces flight, while activation of the lateral or ventrolateral (l and vl) columns induces freezing. However, the PAG contains a diverse arrangement of cell types that vary in neurochemical profile and location. How these cell types contribute to defensive responses remains largely unknown, indicating that targeting sparse, genetically-defined populations can lead to a deeper understanding of how the PAG generates a wide array of behaviors. Though several prior works showed that broad excitation of the lPAG or vlPAG causes freezing, we found that activation of lateral and ventrolateral PAG (l/vlPAG) cholecystokinin-expressing (cck) cells selectively causes flight to safer regions within an environment. Furthermore, inhibition of l/vlPAG-cck cells reduces avoidance of a predatory threat without altering other defensive behaviors like freezing. Lastly, l/vlPAG-cck activity increases away from threat and during movements towards safer locations. In contrast, activating l/vlPAG cells pan-neuronally promoted freezing and these cells were activated near threat. These data underscore the importance of investigating genetically-identified PAG cells. Using this approach, we found a sparse population of cck-expressing l/vlPAG cells that have distinct and opposing function and neural activation motifs compared to the broader local ensemble defined solely by columnar anatomical boundaries. Thus, in addition to the anatomical columnar architecture of the APG, the molecular identity of PAG cells may confer an additional axis of functional organization, revealing unexplored functional heterogeneity.


title: Heterogeneity in Neuronal Dynamics is Learned by Gradient Descent for Temporal Processing Tasks

abstract: Individual neurons in the brain have complex intrinsic dynamics that are highly diverse. We hypothesize that the complex dynamics produced by networks of complex and heterogeneous neurons may contribute to the brains ability to process and respond to temporally complex data. To study the role of complex and heterogeneous neuronal dynamics in network computation, we develop a rate-based neuronal model, the generalized-leaky-integrate-and-firing-rate (GLIFR) model, which is a rate-equivalent of the generalized-leaky-integrate-and-fire model. The GLIFR model has multiple dynamical mechanisms which add to the complexity of its activity while maintaining differentiability. We focus on the role of after-spike currents, currents induced or modulated by neuronal spikes, in producing rich temporal dynamics. We use machine learning techniques to learn both synaptic weights and parameters underlying intrinsic dynamics to solve temporal tasks. The GLIFR model allows us to use standard gradient descent techniques rather than surrogate gradient descent, which has been utilized in spiking neural networks. After establishing the ability to optimize parameters using gradient descent in single neurons, we ask how networks of GLIFR neurons learn and perform on temporally challenging tasks, such as sinusoidal pattern generation and sequential MNIST. We find that these networks learn a diversity of parameters, which gives rise to diversity in neuronal dynamics. We also observe that training networks on the sequential MNIST task leads to formation of cell classes based on the clustering of neuronal parameters. GLIFR networks have mixed performance when compared to vanilla recurrent neural networks but appear to be more robust to random silencing. When we explore these performance gains further, we find that both the ability to learn heterogeneity and the presence of after-spike currents contribute. Our work both demonstrates the computational robustness of neuronal complexity and diversity in networks and demonstrates a feasible method of training such models using exact gradients.


title: Depletion of Scleraxis-lineage cells accelerates tendon ECM aging and promotes retention of a specialized remodeling tenocyte population that drives enhanced tendon healing

abstract: Tendons connect muscle to bone, enabling skeletal movement and joint stability. During natural aging, tendons exhibit impaired homeostasis, increased risk of injury and impaired healing capacity. Understanding the mechanisms responsible for maintenance of tendon homeostasis and how these mechanisms are diminished with aging is crucial for developing therapeutics that can inhibit age-related impairments in tendon function and healing capacity. Here we show that depletion of Scleraxis-lineage (ScxLin) cells, the predominant tenocyte population in adult tendons, recapitulates age-related impairments in cell density, ECM structure and organization. Proteomic analysis demonstrated comparable ECM compositional shifts with ScxLin depletion in young adult tendon, and wild type (WT) aged tendons and identify the specific ECM components that are lost during disrupted tendon homeostasis. Consistent with this, single cell RNA sequencing demonstrates loss of tenocyte subpopulations associated with "ECM synthesis" in both aging and ScxLin depletion, identifying the requirement for these cells in maintaining tendon homeostasis. However, aging and ScxLin depletion results in differential retention of other tenocyte populations, with retention of a specialized remodeling tenocyte population concomitant with mechanically superior healing in ScxLin depleted tendons relative to WT. In contrast, aged tendon retains a pro-inflammatory tenocyte population, which may drive the impaired healing outcomes observed during aging. Collectively, this study defines ScxLin cell depletion as a novel model of accelerated tendon ECM aging and identifies tenocyte subpopulations that are associated with disrupted tendon homeostasis and differential healing outcomes.


title: Active Predictive Coding Networks: A Neural Solution to the Problem of Learning Reference Frames and Part-Whole Hierarchies

abstract: We introduce Active Predictive Coding Networks (APCNs), a new class of neural networks that solve a major problem posed by Hinton and others in the fields of artificial intelligence and brain modeling: how can neural networks learn intrinsic reference frames for objects and parse visual scenes into part-whole hierarchies by dynamically allocating nodes in a parse tree? APCNs address this problem by using a novel combination of ideas: (1) hypernetworks are used for dynamically generating recurrent neural networks that predict parts and their locations within intrinsic reference frames conditioned on higher object-level embedding vectors, and (2) reinforcement learning is used in conjunction with backpropagation for end-to-end learning of model parameters. The APCN architecture lends itself naturally to multi-level hierarchical learning and is closely related to predictive coding models of cortical function. Using the MNIST, Fashion-MNIST and Omniglot datasets, we demonstrate that APCNs can (a) learn to parse images into part-whole hierarchies, (b) learn compositional representations, and (c) transfer their knowledge to unseen classes of objects. With their ability to dynamically generate parse trees with part locations for objects, APCNs offer a new framework for explainable AI that leverages advances in deep learning while retaining interpretability and compositionality.


title: Astrocyte glutamate transport is modulated by motor learning and regulates neuronal correlations and movement encoding by motor cortex neurons

abstract: While motor cortex is crucial for learning precise and reliable movements, whether and how astrocytes contribute to its plasticity and function during motor learning is unknown. Here we report that primary motor cortex (M1) astrocytes in mice show in vivo plasticity during learning of a lever push task, as revealed by transcriptomic and functional modifications. In particular, we observe changes in expression of glutamate transporter genes and increased coincidence of intracellular calcium events. Astrocyte-specific manipulations of M1 are sufficient to alter motor learning and execution, and neuronal population coding, in the same task. Mice expressing decreased levels of the astrocyte glutamate transporter GLT1 show impaired and variable movement trajectories. Mice with increased astrocyte Gq signaling show decreased performance rates, delayed response times and impaired trajectories, along with abnormally high levels of GLT1. In both groups of mice, M1 neurons have altered inter-neuronal correlations and impaired population representations of task parameters, including response time and movement trajectories. Thus, astrocytes have a specific role in coordinating M1 neuronal activity during motor learning, and control learned movement execution and dexterity through mechanisms that importantly include fine regulation of glutamate transport.


title: Basal amygdala inputs to the medial prefrontal cortex mediate fear memory strengthening

abstract: Survival critically depends on the memorization of stimuli recurrently predicting pleasant or aversive experiences. Memory strengthening by additional learning is a critical process allowing the long-term stabilization of learned aversive experience. However, the underlying neuronal circuits and mechanisms are still largely unknown. Using a combination of single unit recordings and optogenetic manipulations, we observed that strengthened fear responses following successive fear learning episodes led to a remapping in the population of basal amygdala (BA) neurons encoding the conditioned stimulus. Moreover, strengthened fear responses were preferentially carried by BA neurons projecting to the medial prefrontal cortex (mPFC). Selective optogenetic inhibition of BA terminals in the mPFC during a second learning episode prevented fear memory strengthening. Together, our findings suggest that fear memory strengthening with additional training critically depends on the selective remapping of a subpopulation of BA neurons projecting to the mPFC.


title: GM1a functions as a coreceptor/ attachment factor for Dengue virus during infection in mammalian systems

abstract:
Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, its true receptor(s) for internalization into live cells has not yet been identified, and no successful antivirals or treatments have been isolated to this date. This study aims to improve our understanding of virus entry routs by exploring the sialic acid-based cell surface molecule GM1a and its role in DENV infection. The interaction of the virus with GM1a was studied using fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), imaging FCS (ImFCS) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS), and the effect on infectivity and movement of the virus during infection was explored using plaque assays and fluorescence-based imaging and single particle tracking (SPT). GM1a was deemed to interact with DENV at domain I (DI) and domain II (DII) of the E protein of the protein coat at quaternary contacts of a fully assembled virus, leading to a ten-fold increase and seven-fold increase in infectivity for DENV1 and DENV2 in mammalian cell systems respectively. The interaction of virus with GM1a triggers a speeding up of virus movement on live cell surfaces, possibly resulting from a reduction in rigidity of cellular rafts during infection, and functions as a coreceptor/ attachment factor for DENV during infection in mammalian systems.

Author SummaryDengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, no successful antivirals or treatments have been isolated to this date. This may be due to the incomplete understanding of the virus infection mechanism, including a lack of an identified  true receptor and entry related attachment factors or co-receptors responsible for internalization of the virus. This work focuses on the early infection stage of DENV1 and DENV2 strains, to identify how the virus moves on cell surfaces in its search for its receptors, and identifies the critical role of the sialic acid ganglioside GM1a during internalization of the virus.


title: Distinctive mechanisms of epilepsy-causing mutants discovered by measuring S4 movement in KCNQ2 channels

abstract: Neuronal KCNQ channels mediate the muscarine-regulated M-current, a key regulator of membrane excitability in the central and peripheral nervous systems. Mutations in KCNQ2 channels cause severe neurodevelopmental disorders, including epileptic encephalopathies. However, the impact that mutations have on channel function remain poorly defined, largely because of our limited understanding of the voltage sensing mechanisms that trigger channel gating. Here, we present measurements of voltage sensor movements in wt-KCNQ2 and channels bearing epilepsy-causing mutations using mutagenesis, cysteine accessibility, and voltage clamp fluorometry (VCF). Cysteine modification reveals that a stretch of 8-9 amino acids in the S4 become exposed upon opening of KCNQ2 channels. VCF shows that the voltage dependence and kinetics of S4 movement and channel opening/closing closely correlate, suggesting an activation scheme in which channel opening does not require multiple voltage-sensor movements. VCF and kinetic modeling reveal different mechanisms by which epilepsy-causing mutations affect KCNQ2 channel voltage-dependent gating. This study provides insight into KCNQ2 channel function, which will aid in uncovering the mechanisms underlying channelopathies.


title: An atlas of amyloid aggregation: the impact of substitutions, insertions, deletions and truncations on amyloid beta fibril nucleation

abstract: Multiplexed assays of variant effects (MAVEs) guide clinical variant interpretation and reveal disease mechanisms. To date, MAVEs have focussed on a single mutation type - amino acid (AA) substitutions - despite the diversity of coding variants that cause disease. Here we use Deep Indel Mutagenesis (DIM) to generate the first comprehensive atlas of diverse variant effects for a disease protein, the amyloid beta (A{beta}) peptide that aggregates in Alzheimers disease (AD) and is mutated in familial AD (fAD). The atlas identifies known fAD mutations and reveals many variants beyond substitutions accelerate A{beta} aggregation and are likely to be pathogenic. Truncations, substitutions, insertions, single- and internal multi-AA deletions differ in their propensity to enhance or impair aggregation, but likely pathogenic variants from all classes are highly enriched in the polar N-terminus of A{beta}. This first comparative atlas highlights the importance of including diverse mutation types in MAVEs and provides important mechanistic insights into amyloid nucleation.


title: BrainStat: A toolbox for brain-wide statistics and neuroscientific contextualization

abstract: AO_SCPLOWBSTRACTC_SCPLOWAnalysis and interpretation of neuroimaging datasets has become a multidisciplinary endeavor, relying not only on statistical methods, but increasingly on associations with respect to other brain-derived features such as gene expression, histological data, and functional as well as cognitive architectures. Here, we introduce BrainStat - a toolbox for (i) univariate and multivariate linear models in volumetric and surface-based brain imaging datasets, and (ii) multidomain feature association of results with respect to spatial maps of post-mortem gene expression and histology, task-based fMRI meta-analysis, as well as resting-state fMRI motifs across several common surface templates. The combination of statistics and feature associations into a turnkey toolbox streamlines analytical processes and accelerates cross-modal research. The toolbox is implemented in both Python and MATLAB, two widely used programming languages in the neuroimaging and neuroinformatics communities. BrainStat is openly available and complemented by an expandable documentation.


title: Excessive self-grooming of Shank3 mutant mice is associated with gene dysregulation and imbalance between the striosome and matrix compartments in the striatum

abstract: Autism spectrum disorders (ASD) are characterised by atypical social communication and stereotyped behaviours. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are detected in 1-2% of patients with ASD and intellectual disability (ID), but the mechanisms underpinning the symptoms remain largely unknown. Here, we characterized the behaviour of Shank3 mutant mice deleted for exon 11 (Shank3{Delta}11/{Delta}11) from three to twelve months of age. We observed decreased locomotor activity, increased stereotyped self-grooming and atypical socio-sexual interaction compared to wild-type littermates. We then used RNAseq on four brain regions of the same animals to identify differentially expressed genes (DEG). DEGs were identified mainly in the striatum and were associated with synaptic transmission (e.g. Grm2, Dlgap1), G-protein-signalling pathways (e.g. Gnal, Prkcg1, and Camk2g), as well as excitation/inhibition balance (e.g. Gad2). Downregulated and upregulated genes were respectively enriched in the gene clusters of medium-sized spiny neurons expressing the dopamine 1 (D1-MSN) and the dopamine 2 receptor (D2-MSN). Moreover, expression of DEGs reported as striosome markers within the striatum (Cnr1, Gnal1, Gad2, and Drd4) were positively correlated with excessive self-grooming. Finally, we showed that the striosome compartment of Shank3{Delta}11/{Delta}11 mice was enlarged and displayed higher expression of GAD65 compared to wild-type mice. Altogether, these results shed light on a possible role of the striosomes/matrix imbalance in excessive self-grooming in Shank3{Delta}11/{Delta}11 mice. Such striatal alterations could be present in a subgroup of patients with ASD and ID and could open the way to new therapeutic approaches.


title: An extended reservoir of class-D beta-lactamases in non-clinical bacterial strains

abstract:
Bacterial genes coding for antibiotic resistance represent a major issue in the fight against bacterial pathogens. Among those, genes encoding beta-lactamases target penicillin and related compounds such as carbapenems, which are critical for human health. Beta-lactamases are classified into classes A, B, C and D, based on their amino acid sequence. Class D enzymes are also known as OXA beta-lactamases, due to the ability of the first enzymes described in this class to hydrolyze oxacillin. While hundreds of class D beta-lactamases with different activity profiles have been isolated from clinical strains, their nomenclature remains very uninformative. In this work, we have carried out a comprehensive survey of a reference database of 80,490 genomes and identified 24,916 OXA-domain containing proteins. These were deduplicated and their representative sequences clustered into 45 non-singleton groups derived from a phylogenetic tree of 1413 OXA-domain sequences, including five clusters that include the C-terminal domain of the BlaR membrane receptors. Interestingly, 801 known class D beta-lactamases fell into only 18 clusters. To probe the unknown diversity of the class, we selected ten protein sequences in ten uncharacterized clusters and studied the activity profile of the corresponding enzymes. A beta-lactamase activity could be detected for seven of them. Three enzymes were active against oxacillin and two against imipenem. These results indicate that, as already reported, environmental bacteria constitute a large reservoir of resistance genes that can be transferred to clinical strains, whether through plasmid exchange or hitchhiking with the help of transposase genes.

ImportanceThe transmission of genes coding for resistance factors from environmental to nosocomial strains is a major component in the development of bacterial resistance towards antibiotics. Our survey of class D beta-lactamase genes in genomic databases highlighted the high sequence diversity of the enzymes that are able to recognize and/or hydrolyze beta-lactam antibiotics. Among those, we could also identify new beta-lactamases that are able to hydrolyze carbapenems, one of the last resort antibiotic families used in human chemotherapy. Therefore, it can be expected that the use of this antibiotic family will fuel the emergence of new beta-lactamases into clinically relevant strains.


title: Bone marrow age dictates clonality of smooth muscle-derived cells in the atherosclerotic plaque

abstract:
Aging is the predominant risk factor for atherosclerosis, the leading cause of death. Rare smooth muscle cell (SMC) progenitors clonally expand giving rise to up to [~]70% of atherosclerotic plaque cells; however, the effect of age on SMC clonality is not known. Our results indicate that aged bone marrow (BM)-derived cells non-cell autonomously induce SMC polyclonality and worsen atherosclerosis. Indeed, in myeloid cells from aged mice and humans, TET2 levels are reduced which epigenetically silences integrin {beta}3 resulting in increased tumor necrosis factor [TNF]- signaling. In turn, TNF signals through TNF receptor 1 on SMCs to promote proliferation and induces recruitment and expansion of multiple SMC progenitors into the atherosclerotic plaque. Notably, integrin {beta}3 overexpression in aged BM preserves dominance of the lineage of a single SMC progenitor and attenuates plaque burden. Our results demonstrate a molecular mechanism of aged macrophage-induced SMC polyclonality and atherogenesis and suggest novel therapeutic strategies.

Graphical abstractAge of BM-derived monocytes/macrophages determines clonality of SMC lineage in the atherosclerotic plaque. Atherogenesis is depicted in a young (a) or aged (b) host. Aged monocytes/macrophages have decreased levels of the epigenetic regulator TET2, leading to reduction of the 5-hydroxymethylcytosine (5hmC) mark on the Itgb3 promoter. The resulting low integrin {beta}3 levels in aged monocytes/macrophages induces high TNF- levels, facilitating recruitment and expansion of multiple SMC progenitors (polyclonality) in the atherosclerotic plaque and worse disease burden. In contrast, the young control is characterized by mono/oligoclonal SMC expansion in a smaller plaque.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/476756v2_ufig1.gif" ALT="Figure 1">
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title: Age of the bone marrow dictates clonality of smooth muscle-derived cells in the atherosclerotic plaque


title: The Intrinsically Disordered Region of Coronins Fine-tunes Oligomerization and Actin Polymerization

abstract: Coronins are highly conserved actin-binding proteins (ABPs) in the eukaryotic kingdom for polymerizing actin cytoskeleton. The biochemical activity of coronins is primarily mediated by the structural N-terminal {beta}-propeller and the C-terminal helical coiled-coil (CC) domains, but less is known about the function of a middle nonconserved region, the "unique region (UR)". The coronin UR is an intrinsically disordered region (IDR). Herein, we demonstrate that the low complexity of the UR is a conserved signature of the coronin protein family, and the UR/IDR exhibits a striking evolutionary correlated pattern associated with sequence length. By analyzing the role of the IDR in coronins via coarse-grained simulations, we reveal that evolutionary selection of IDR length is coupled with the oligomerization of IDR-containing proteins (IDPs) to provide optimal functional output. By integrating biochemical and cell biology experiments and protein engineering, we found that the IDR regulates Crn1 biochemical activity, both in vivo and in vitro, by fine-tuning CC domain oligomerization and maintaining Crn1 in a tetrameric state. The IDR-guided optimization of Crn1 oligomerization is critical for Arp2/3-mediated actin polymerization.


title: The synaptic vesicle protein Mover/TPRG1L is associated with lipid droplets in astrocytes

abstract:
Crucial brain functions such as neurotransmission, myelination, and signaling pose a high demand for lipids. Lipid dysregulation is associated with neuroinflammation and neurodegeneration. Astrocytes protect neurons from lipid induced damage by accumulating and metabolizing toxic lipids in organelles called lipid droplets (LDs). LDs have long been considered as lipid storage compartments in adipocytes, but less is known about their biogenesis and composition in the brain. In particular, proteins covering the LD surface are not yet fully identified.

Here, we report that the synaptic vesicle protein Mover/TPRG1L, which regulates the probability of neurotransmitter release in neurons, is a component of the LD coat in astrocytes. Using conventional and super resolution microscopy, we demonstrate that Mover surrounds naive and oleic acid induced astrocytic LDs. We confirm the identity of astrocytic LDs using the neutral lipid stains Bodipy and LipidTox, as well as immunofluorescence for perilipin-2, a known component of the LD coat. In astrocytes, recombinant Mover was sufficient to induce an accumulation of LDs. Furthermore, we identified point mutations that abolish targeting to LDs and show similarities in the required binding sequences for association to the presynapse and LDs.

Our results show that Mover is not only a presynaptic protein but also a candidate for LD regulation. This highlights the dual role of Mover in synaptic transmission and regulation of astrocytic LDs, which may be particularly important in the context of lipid-related neurological disorders.

Main points- Mover is a lipid droplet protein
- Overexpression of Mover drives lipid droplet accumulation
- The F206 is important for LD targeting


title: Effects of transcranial magnetic stimulation on the human brain recorded with intracranial electrocorticography: First-in-human study

abstract: Transcranial magnetic stimulation (TMS) is increasingly used as a noninvasive technique for neuromodulation in research and clinical applications, yet its mechanisms are not well understood. Here, we present the first in-human study evaluating the effects of TMS using intracranial electrocorticography (iEEG) in neurosurgical patients. We first evaluated safety in a gel-based phantom. We then performed TMS-iEEG in 20 neurosurgical participants with no adverse events. Next, we evaluated brain-wide intracranial responses to single pulses of TMS to the dorsolateral prefrontal cortex (dlPFC) (N=10, 1414 electrodes). We demonstrate that TMS preferentially induces neuronal responses locally within the dlPFC at sites with higher electric field strength. Evoked responses were also noted downstream in the anterior cingulate and anterior insular cortex, regions functionally connected to the dlPFC. These findings support the safety and promise of TMS-iEEG in humans to examine local and network-level effects of TMS with higher spatiotemporal resolution than currently available methods.


title: Sialic acid and fucose residues on the SARS-CoV-2 receptor binding domain modulate IgG reactivity

abstract: The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of recombinant RBD produced in different mammalian cells. We found a higher degree of complex type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in Human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it might be possible to enhance the immunogenicity of recombinant viral proteins.


title: JWA deficiency accelerates aging through disrupting intestinal epithelial homeostasis via Notch1/PPARγ/Stat5 axis

abstract: Aging usually suppresses the renewal and regeneration of intestinal epithelium. The imbalance of intestinal epithelial homeostasis may also be a promoter for aging. JWA responds to oxidative stress and repairs damaged DNA; it participates in multiple cellular processes like cell proliferation and differentiation. Here we identified JWA as a new aging-associated gene, whose deletion-accelerated aging in mice was related to intestinal epithelium atrophy. We further knocked out intestinal epithelial JWA and found it disrupted intestinal epithelial homeostasis, thus promoting aging in mice. Mechanistically, we discovered that JWA deficiency promoted Notch1 ubiquitination degradation via ERK/Fbxw7 cascade and interfered with the PPAR{gamma}/Stat5 signal axis. This reduced the intestinal stem cell function and altered the intestinal epithelial cell lineage distribution, finally suppressing the renewal and regeneration of intestinal epithelium. Our results demonstrated that JWA is a new aging-associated gene essential for the renewal and regeneration of intestinal epithelium. We also provide a new idea that maintaining intestinal epithelial homeostasis may be a potential anti-aging strategy in humans or mammals.


title: Alternative splicing changes are associated with pre-birth adaptation during lung development

abstract:
Gas exchanges are ensured by lung alveoli, which are mainly composed by epithelial alveolar type 1 (AT1), alveolar type 2 (AT2) and capillary endothelial cells (ECs). Alveologenesis starts during late embryonic development and continues after birth and relies on extensive biochemical crosstalk between these cell types. How this crosstalk is modulated to anticipate and accommodate the radical changes occurring at birth is still unclear.

We investigated the alternative splicing (AS) changes occurring during lung development at the embryonic to postnatal transition by performing RNAseq of mouse lungs at distinct developmental stages. We found that most of the AS changes occur at the embryonic to postnatal transition. In addition, we identified hnRNP A1, Cpeb4 and Elavl2/HuB as putative splicing regulators of this transition. We show that the AS of a major pro- angiogenic chemokine, vascular endothelial growth factor A (VEGFA), is differentially regulated at this transition. Remarkably, we found that there is a switch from the predominance of Vegfa 164 to Vegfa 188 just before birth specifically in AT1 cells, whilst in other cell populations Vegfa does not undergo AS changes. Moreover, we identified a novel Vegfa isoform generated by the retention of intron 5, Vegfa i5.

Our results reveal a cell type-specific regulation of Vegfa AS that may constitute a pre- birth adaptation mechanism of the epithelial-endothelial crosstalk, which may be fundamental for the adaptation to breathing and may have implications for pathological conditions.


title: Complex genetic architecture underlying the plasticity of maize agronomic traits

abstract: Phenotypic plasticity is the property of a given genotype to produce multiple phenotypes in response to changing environmental conditions. Understanding the genetic basis of phenotypic plasticity and establishing a predictive model is highly relevant for future agriculture under changing climate. Here, we report findings on the genetic basis of phenotypic plasticity for 23 complex traits using a maize diverse population, planted at five sites with distinct environmental conditions and genotyped with ~ 6.60 million SNPs. We found that altitude-related environmental factors were main drivers for across site variation in flowering time traits but not plant architecture and yield traits. For 23 traits, we detected 109 QTLs, of which 29 was for mean, 66 was for plasticity, and 14 for both parameters, besides, 80% of the QTLs were interreacted with the environment. The effects of several QTLs changed in magnitude or sign, driving variation in phenotype plasticity, and we further experimentally validated one plastic gene ZmTPS14.1 whose effect was likely mediated by the compensation effect of ZmSPL6 which was from the downstream pathway probably. By integrating genetic diversity, environmental variation, and their interaction in a joint model, we could provide site-specific predictions with increased accuracy by as much as 15.5%, 3.8%, and 4.4% for DTT, PH, and EW, respectively. Overall, we revealed a complex genetic architecture involving multiallelic, pleiotropy, and genotype by environment interaction underlying maize complex trait mean and plasticity variation. Our study thus provided novel insights into the dynamic genetic architectures of agronomic traits in response to changing environments, paving a practical route to precision agriculture.

category: genetics


title: Ancient herpes simplex 1 genomes reveal recent viral structure in Eurasia

abstract: Human herpes simplex virus 1 (HSV-1), a life-long infection spread by oral contact, today infects a majority of adults globally1, yet no ancient HSV-1 genomes have yet been published. Phylogeographic clustering of sampled diversity into European, pan-Eurasian, and African groups2, 3 has suggested that the virus co-diverged with anatomically modern humans migrating out of Africa4, although a much younger origin has also been proposed5. The lack of ancient HSV-1 genomes, high rates of recombination, and high mobility of humans in the modern era have impeded the understanding of HSV-1s evolutionary history. Here we present three full ancient European HSV-1 genomes and one partial genome, dating to between the 3rd and 17th century CE, sequenced to up to 9.5x with paired human genomes up to 10.16x. These HSV-1 strains fall within modern Eurasian diversity. We estimate a mean mutation rate of 7.6 x 10-7 - 1.13 x 10-6 for non-African diversity leading to an estimated age of sampled modern Eurasian diversity to 4.68 (3.87 - 5.65) kya. Extrapolation of these rates indicate the age of sampled HSV-1 to 5.29 (4.60-6.12 kya, suggesting lineage replacement coinciding with late Neolithisation and implicating Bronze Age migrations6 in the distribution of HSV-1 through Eurasia.


title: Optimus: a general purpose adaptive optimisation engine in R

abstract:
MotivationMany calculations in computational biology necessitate a use of a probabilistic optimisation protocol to determine a set of parameters that capture the system at a desired state in the configurational space. Here, we developed a flexible optimisation engine in R that can be plugged to any, simple or complex, modelling initiative through a few lucid interfacing functions, to perform a seamless optimisation with rigorous parameter sampling.

ResultsOptimus features an acceptance ratio simulated annealing, acceptance ratio replica exchange, and adaptive thermoregulation, thus driving a Monte Carlo optimisation process in a flexible manner, through constrained acceptance frequency but unconstrained adaptive pseudo temperature regiments. We show the applicability of our R optimiser to a wide variety of problems spanning data analyses and computational biology tasks.

Availability and ImplementationOptimus is written and implemented in R, and is freely available from the http://github.com/SahakyanLab/Optimus repository.

Contactaleksandr.sahakyan@imm.ox.ac.uk

Supplementary InformationSupplementary information with more details, tutorials, and developer instructions is available.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/476810v1_ufig1.gif" ALT="Figure 1">
View larger version (10K):
org.highwire.dtl.DTLVardef@749b4aorg.highwire.dtl.DTLVardef@17f439eorg.highwire.dtl.DTLVardef@303ad6org.highwire.dtl.DTLVardef@66dda7_HPS_FORMAT_FIGEXP  M_FIG The Optimus software logo depicting two gears ("Op") that drag the system "s", trapped in a "u" minimum, through a rough solution landscape into a more favourable solution with a deeper pseudo-energy minimum ("p").

C_FIG

category: bioinformatics


title: Pareto-optimal trade-off for phenotypic switching of populations in a stochastic environment

abstract:
Finding optimal survival strategies of living systems embedded in fluctuating environments generally involves a balance between phenotypic diversification and sensing. If we neglect sensing mechanisms, it is known that slow, resp. fast, environmental transitions favor a regime of heterogeneous, resp. homogeneous, phenotypic response.

We focus here on the simplest non-trivial case, i.e. two randomly switching phenotypes subjected to two stochastically switching environments. The optimal asymptotic (long term) growth rate of this model was studied elsewhere; we further expand these results by discussing finite time growth rate fluctuations. An exact asymptotic expression for the variance, alongside with approximations valid in different regimes, are tested numerically in details. Our simulations of the dynamics suggest a close connection between this variance and the extinction probability, understood as risk for the population. Motivated by an earlier trade-off analysis between average capital growth rate and risk in Kellys gambling model, we study the trade-off between the average growth rate and the variance in the present model. Despite considerable differences between the two models, we find similar optimal trade-off curves (Pareto fronts), suggesting that our conclusions are robust, and broadly applicable in various fields ranging from biology/ecology to economics.


title: High-resolution map of plastid encoded polymerase binding patterns demonstrates a major role of transcription in chloroplast gene expression

abstract: Plastids are endosymbiotic organelles containing their own genomes, which are transcribed by two types of RNA polymerases. One of those enzymes is a bacterial-type, multi-subunit polymerase encoded by the plastid genome. The plastid encoded RNA polymerase (PEP) is required for efficient expression of genes encoding proteins involved in photosynthesis. Despite the importance of PEP, its DNA binding locations have not been studied on the genome-wide scale at high resolution. We established a highly specific approach to detect the genome-wide pattern of PEP binding to chloroplasts DNA using ptChIP-seq. We found that in mature Arabidopsis thaliana chloroplasts, PEP has a complex DNA binding pattern with preferential association at genes encoding rRNA, tRNA, and a subset of photosynthetic proteins. Sigma factors SIG2 and SIG6 strongly impact PEP binding to a subset of tRNA genes and have more moderate effects on PEP binding throughout the rest of the genome. PEP binding is commonly enriched on gene promoters, around transcription start sites. Finally, the levels of PEP binding to DNA are correlated with the levels of RNA accumulation, which allowed estimating the quantitative contribution of transcription to RNA accumulation.

category: plant biology


title: Cold Tolerance is Governed by Diverse Genetic Mechanisms Including Those Regulated by NB-LRR-type Receptor Proteins in Arabidopsis.

abstract:
Cold stress adversely affects the growth and development of plants and limits the geographical distribution of crop plants. Accumulation of spontaneous mutations shapes the adaptation of plant species to diverse climatic conditions. Genome-wide association study of the phenotypic variation gathered by a newly designed phenomic platform with that of the single nucleotide polymorphic (SNP) loci across the genomes of 417 Arabidopsis natural variants collected from various geographical regions revealed 33 candidate genes for cold tolerance. Investigation of at least two independent mutants for 29 of these genes identified 16 cold tolerance genes controlling diverse genetic mechanisms. This study identified five genes encoding novel leucine-rich repeat domain-containing proteins, including three nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins. Among the 16 identified cold tolerance genes, ADS2 and ACD6 are the only two cold tolerance genes identified earlier. The comparatively little overlap between the genes identified in this genome-wide association study of natural variants with those discovered previously through forward and reverse genetic approaches suggests that cold tolerance is a complex physiological process governed by a large number of genetic mechanisms.

Short SummaryCold stress adversely affects the growth and development of plants and limits the geographical distribution of crop plants. Genome-wide association study of the phenotypic variation of Arabidopsis natural variants with that of the single nucleotide polymorphic loci followed by T-DNA insertion mutant analyses of 29 candidate genes led to assigning cold tolerance function for the first time to 14 genes including three nucleotide-binding sites leucine repeat region (NB-LRR) protein genes. The comparatively little overlap between the genes identified in this study with those discovered previously suggests that cold tolerance is governed by a complex network of multiple genetic mechanisms.


title: Outbreak.info: A standardized, searchable platform to discover and explore COVID-19 resources and data

abstract: To combat the ongoing COVID-19 pandemic, scientists have been conducting research at breakneck speeds, producing over 52,000 peer reviewed articles within the first 12 months. In contrast, a little over 1,000 peer reviewed articles were published within the first 12 months of the SARS-CoV-1 pandemic starting in 2002. In addition to publications, there has also been an upsurge in clinical trials to develop vaccines and treatments, scientific protocols to study SARS-CoV-2, methodology for epidemiological modeling, and datasets spanning molecular studies to social science research. One of the largest challenges has been keeping track of the vast amounts of newly generated disparate data and research that exist in independent repositories. To address this issue, we developed outbreak.info, which provides a standardized, searchable interface of heterogeneous resources on COVID-19 and SARS-CoV-2. Unifying metadata from twelve repositories, we have assembled a collection of over 260,000 publications, clinical trials, datasets, protocols, and other resources as of March 2022. We used a rigorous schema to enforce a consistent format across different data sources and resource types, and linked related resources where possible. This enables users to quickly retrieve information across data repositories, regardless of resource type or repository location. Outbreak.info also combines the combined research library with spatiotemporal genomics data on SARS-CoV-2 variants. The web interface provides interactive visualizations and reports to explore the unified data and generate hypotheses. In addition to providing a web interface, we also publish the data we have assembled and standardized in a high performance public API and an R package. Finally, we discuss the challenges inherent in combining metadata from scattered and heterogeneous resources and provide recommendations to streamline this process to aid scientific research.


title: Outbreak.info Research Library: A standardized, searchable platform to discover and explore COVID-19 resources and data


title: Outbreak.info Research Library: A standardized, searchable platform to discover and explore COVID-19 resources


title: Promoting Fc-Fc interactions between anti-capsular antibodies provides strong complement-dependent immune protection against Streptococcus pneumoniae

abstract: Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and an important cause of childhood mortality. Despite the introduction of successful vaccines, the global spread of both non-vaccine serotypes and antibiotic-resistant strains reinforce the development of alternative therapies against this pathogen. One possible route is the development of monoclonal antibodies (mAbs) that induce killing of bacteria via the immune system. Here we investigate whether mAbs can be used to induce killing of pneumococcal serotypes for which the current vaccines show unsuccessful protection. Our study demonstrates that when human mAbs against pneumococcal capsule polysaccharides (CPS) have a poor capacity to induce complement activation, a critical process for immune protection against pneumococci, their activity can be strongly improved by hexamerization-enhancing mutations. Our data indicate that anti-capsular antibodies may have a low capacity to form higher-order oligomers (IgG hexamers) that are needed to recruit complement component C1. Indeed, specific point mutations in the IgG-Fc domain that strengthen hexamerization strongly enhance C1 recruitment and downstream complement activation on encapsulated pneumococci. Specifically, hexamerization-enhancing mutations E430G or E345K in CPS6-IgG strongly potentiate complement activation on S. pneumoniae strains that express capsular serotype 6 (CPS6), and the highly invasive serotype 19A strain. Furthermore, these mutations improve complement activation via mAbs recognizing CPS3 and CPS8 strains. Importantly, hexamer-enhancing mutations enable mAbs to induce strong phagocytosis and intracellular killing by human neutrophils. Finally, passive immunization with CPS6-IgG1-E345K protected mice from developing severe pneumonia. Altogether, this work provides insights that are crucial for optimizing antibody therapies against encapsulated S. pneumoniae.


title: Claspin is required for growth recovery from serum starvation through regulating the PI3K-PDK1-mTOR pathway

abstract: Growth recovery from serum starvation requires the activation of PI3 kinase (PI3K)-PDK1-Akt-mTOR pathways. Claspin plays multiple important roles in regulation of DNA replication as a mediator for the cellular response to replication stress, an integral replication fork factor that facilitates replication fork progression and a factor that promotes initiation by recruiting Cdc7 kinase. Here, we report a novel role of Claspin in growth recovery from serum starvation. In the absence of Claspin, cells do not proceed into S phase and eventually die. Claspin interacts with PI3K and mTOR, and is required for activation of PI3K-PDK1-mTOR and for that of mTOR downstream factors, p70S6K and 4E-BP1, but not for p38 MAPK cascade during the recovery from serum starvation. PDK1 interacts with Claspin, notably with CKBD, in a manner dependent on phosphorylation of the latter protein, and is required for interaction of mTOR with Claspin. p53 and ROS (Reactive Oxygen Species) inhibitors increased survival of Claspin-deficient cells released from serum starvation. Thus, Claspin plays a novel role as a mediator/protein platform for nutrition-induced proliferation/survival signaling by activating the mTOR pathway.


title: Knockout of Bbs10 results in lack of cone electrical function and progressive retinal degeneration of rods and cones

abstract: Bardet Biedl Syndrome (BBS) is an autosomal recessive disorder caused by mutations in at least 22 different genes. A constant feature is early onset retinal degeneration leading to blindness, with variable central obesity, polydactyly, renal failure, and developmental anomalies. BBS type 10 (BBS10) is a common form caused by mutations in the BBS10 gene encoding a chaperonin-like protein. There are currently no treatments for the progressive vision loss. To aid in treatment development, a BBS10 mouse model was developed by knocking out the Bbs10 gene. Using optical coherence tomography (OCT), electroretinography (ERG), and a visually guided swim assay (VGSA), we demonstrate that Bbs10-/- mice have progressive retinal degeneration. Cone electrical function was absent although cones were anatomically present on histology and retained partial function based on VGSA. The retinal outer nuclear layer (photoreceptor nuclei) progressively thinned as demonstrated on OCT and histology, and rod electrical activity decreased over time on ERG. These phenotypes are more rapidly progressive than retinal degeneration in the Bbs1M390R/M390R knock-in mouse. They are consistent with a cone-rod dystrophy distinct from typical rod-cone degeneration in retinitis pigmentosa and recapitulate aspects of retinal degeneration observed in humans with BBS10. This study has implications for BBS10 gene therapy.