title: Tax induces the recruitment of NF-kB to unintegrated HIV-1 DNA to rescue viral gene expression and replication

abstract:
We have previously reported that the normally essential step of integration of the HIV-1 proviral DNA intermediate into the host cell genome becomes dispensable in T cells that express the Human T cell leukemia virus 1 (HTLV-1) Tax protein. The rescue of integrase (IN) deficient HIV-1 replication by Tax results from the strong activation of transcription from the long terminal repeat (LTR) promoter on episomal HIV-1 DNA, an effect that is closely correlated with the recruitment of activating epigenetic marks, such as H3Ac, and depletion of repressive epigenetic marks, such as H3K9me3, from chromatinized unintegrated proviruses. In addition, activation of transcription from unintegrated HIV-1 DNA coincides with the recruitment of NF-kB to the two NF-kB binding sites found in the HIV-1 LTR enhancer. Here we report that the recruitment of NF-kB to unintegrated viral DNA precedes, and is a prerequisite for, Tax-induced changes in epigenetic marks, so that an IN-HIV-1 mutant lacking both LTR NF-kB sites is entirely non-responsive to Tax and fails to undergo the epigenetic changes listed above. We also report that heterologous promoters introduced into IN-HIV-1-based vectors are transcriptionally active even in the absence of Tax. Finally, we failed to reproduce a recent report arguing that heterologous promoters introduced into IN-vectors based on HIV-1 are more active if the HIV-1 promoter and enhancer, located in the LTR U3 region, are deleted, in a so-called self inactivating or SIN lentivector design.

ImportanceIntegrase-deficient expression vectors based on HIV-1 are becoming increasingly popular as tools for gene therapy in vivo due to their inability to cause insertional mutagenesis. However, many IN-lentiviral vectors are able to achieve only low levels of gene expression and methods to increase this low level have not been extensively explored. Here we analyze how the HTLV-1 Tax protein is able to rescue the replication of IN-HIV-1 in T cells and describe IN-lentiviral vectors that are able to express a heterologous gene effectively.


title: Biomolecular recognition of the glycan neoantigen CA19-9 by distinct antibodies

abstract: Glycans decorate cell surface, secreted glycoproteins and glycolipids. Altered glycans are often found in cancers. Despite their high diagnostic and therapeutic potentials, glycans are polar and flexible molecules that are quite challenging for the development and design of high-affinity binding antibodies. To understand the mechanisms by which glycan neoantigens are specifically recognized by antibodies, we analyze the biomolecular recognition of a single tumor-associated carbohydrate antigen CA19-9 by two distinct antibodies using X-ray crystallography. Despite the plasticity of glycans and the very different antigen-binding surfaces presented by the antibodies, both structures reveal an essentially identical extended CA19-9 conformer, suggesting that this conformers stability selects the antibodies. Starting from the bound structure of one of the antibodies, we use the AbLIFT computational method to design a variant with seven core mutations that exhibited tenfold improved affinity for CA19-9. The results reveal strategies used by antibodies to specifically recognize glycan antigens and show how automated antibody-optimization methods may be used to enhance the clinical potential of existing antibodies.

category: molecular biology


title: Regulation of ribosomal protein synthesis in mycobacteria: autogenous control of rpsO

abstract:
Autogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. Our main goal was to develop techniques for investigating the r-protein synthesis regulation in mycobacteria, Gram-positive organisms with a high GC-content, which has never been addressed. We started with the rpsO gene known to be autoregulated by its product, r-protein S15, in a broad range of bacterial species. To study the in vivo regulation of rpsO from Mycobacterium smegmatis (Msm), we first applied an approach based on chromosomally integrated Msm rpsO-lacZ reporters by using E. coli as a surrogate host. The {beta}-galactosidase assay has shown that mycobacterial rpsO expression is feedback regulated at the translation level in the presence of Msm S15 in trans, like in E. coli. Next, to overcome difficulties caused by the inefficiency of mycobacterial gene expression in E. coli, we created a fluorescent reporter system based on M. smegmatis. To this end, the integrative shuttle plasmid pMV306 was modified to provide insertion of the Msm or Mtb (M. tuberculosis) rpsO-egfp reporters into the Msm chromosome, and a novel E. coli-mycobacteria replicative shuttle vector, pAMYC, a derivative of pACYC184, was built. Analysis of the eGFP expression in the presence of the pAMYC derivative expressing Msm rpsO vs an empty vector confirms the autogenous regulation of the rpsO gene in mycobacteria. Additionally, we have revealed that the mycobacterial rpsO core promoters are rather weak and require upstream activating elements to enhance their strength.

IMPORTANCEBacterial ribosomes are targets for a majority of as-yet reported antibiotics, hence ribosome biogenesis and its regulation are central for development of new antimicrobials. One of the key mechanisms regulating ribosome biogenesis in bacteria is the autogenous control of r-protein synthesis, which has been so far explored for E. coli and Bacillus spp. but not yet for mycobacteria. Here, we describe experimental approaches for in vivo analysis of mechanisms regulating r-protein synthesis in mycobacteria, including M. tuberculosis, and show, for the first time, that the autogenous control at the translation level is really functioning in these microorganisms. The developed system paves the way for studying various regulatory circuits involving proteins or sRNAs as mRNA- targeting trans-regulators in mycobacteria as well as in other actinobacterial species.


title: Hierarchical reactivation of transcription during mitosis-to-G1 transition by Brn2 and Ascl1 in neural stem cells

abstract: During mitosis, chromatin condensation is accompanied by a global arrest of transcription. Recent studies suggest transcriptional reactivation upon mitotic exit occurs in temporally coordinated waves, but the underlying regulatory principles have yet to be elucidated. In particular, the contribution of sequence-specific transcription factors (TFs) remains poorly understood. Here we report that Brn2, an important regulator of neural stem cell identity, associates with condensed chromatin throughout cell division, as assessed by live-cell imaging of proliferating neural stem cells. By contrast, the neuronal fate determinant Ascl1 dissociates from mitotic chromosomes. ChIP-seq analysis reveals that Brn2 mitotic-chromosome binding does not result in sequence-specific interactions prior to mitotic exit, relying mostly on electrostatic forces. Nevertheless, surveying active transcription using single-molecule RNA-FISH against immature transcripts, indicates the differential presence of TF near chromatin when exiting mitosis is associated with early (anaphase) versus late (early G1) reactivation of key targets of Brn2 and Ascl1, respectively. Moreover, by using a mitotic-specific dominant negative approach, we show that competing with Brn2 binding during mitotic exit reduces the transcription of its target gene Nestin. Our study shows an important role for differential binding of TFs to mitotic chromosomes, governed by their electrostatic properties, in defining the temporal order of transcriptional reactivation during mitosis-to-G1 transition.

category: developmental biology


title: Simons Collaborative Marine Atlas Project (Simons CMAP): an open-source portal to share, visualize and analyze ocean data

abstract: Simons Collaborative Marine Atlas Project (Simons CMAP) is an open-source data portal that interconnects large, complex, and diverse public data sets currently dispersed in different formats across different Oceanography discipline-specific databases. Simons CMAP is designed to streamline the retrieval of custom subsets of data, the generation of data visualizations, and the analyses of diverse data, thus expanding the power of these potentially underutilized data sets for cross-disciplinary studies of ocean processes. We describe a unified architecture that allows numerical model outputs, satellite products, and field observations to be readily shared, mined, and integrated regardless of data set size or resolution. A current focus of Simons CMAP is integration of physical, chemical, and biological data sets essential for characterizing the biogeography of key marine microbes across ocean basins and seasonal cycles. Using a practical example, we demonstrate how our unifying data harmonization plans significantly simplifies and allows for systematic data integration across all Simons CMAP data sets.

category: ecology


title: Spatial model of water quality in Southeast Asian urban lake based on HBI using macrozoobenthos diversity as a proxy

abstract: Urban lake is one of ecosystem that has experienced anthropogenic pressures and this can affect its water quality. One of a robust approach to assess the water quality is by using Hilsenhoff Biotic Index (HBI). This tool is quite versatile since it can be applied by using any aquatic organism as proxy including macrozoobenthos. This invertebrate group also has an advantage since it is common and easy to collect. Here this study is first, aiming to provide HBI based water quality spatial model using macrozoobenthos as a proxy applied in urban lake in West Java in Southeast Asia and second to seek the best model that can represent the water quality variables in particular dissolved oxygen (DO), pH, and temperature. Based on the spatial model and HBI, either inlet or outlet parts of the lake, it has better water quality in comparison to central parts. Based on HBI values, water quality in inlet and outlet parts (HBI = 6.7) is categorized as fair and poor (HBI = 6.9) for the central parts of the lake. The increase in HBI and decrease in water quality are positively correlated with the increase in water temperature variable in comparison to water DO and pH variables. Akaike model selection confirms that the macrozoobenthos diversity can be used as a proxy for increase in water temperature ({Psi})HBI (~temp)(AIC = -10.264) followed by combination of water temperature increase and decrease in DO({Psi})HBI (~temp+DO)(AIC = -9.042398).


title: Polymorphism of Genetic Ambigrams

abstract: Double synonyms in the genetic code can be used as a tool to test competing hypotheses regarding ambigrammatic narnavirus genomes. Applying the analysis to recent observations of Culex narnavirus 1 and Zhejiang mosquito virus 3 ambigrammatic viruses indicates that the open reading frame on the complementary strand of the segment coding for RNA-dependent RNA polymerase does not code for a functional protein. Culex narnavirus 1 has been shown to possess a second segment, also ambigrammatic, termed  Robin. We find a comparable segment for Zhejiang mosquito virus 3, a moderately diverged relative of Culex narnavirus 1. Our analysis of Robin polymorphisms suggests that its reverse open reading frame also does not code for a functional protein. We make a hypothesis about its role.

category: genomics


title: Synthesizing VERDICT maps from standard diffusion mp-MRI data using GANs

abstract: VERDICT maps have shown promising results in clinical settings discriminating normal from malignant tissue and identifying specific Gleason grades non-invasively. However, the quantitative estimation of VERDICT maps requires a specific diffusion-weighed imaging (DWI) acquisition. In this study we investigate the feasibility of synthesizing VERDICT maps from standard DWI data from multi-parametric (mp)- MRI by employing conditional generative adversarial networks (GANs). We use data from 67 patients who underwent both standard DWI-MRI and VERDICT MRI and rely on correlation analysis and mean squared error to quantitatively evaluate the quality of the synthetic VERDICT maps. Quantitative results show that the mean values of tumour areas in the synthetic and the real VERDICT maps were strongly correlated while qualitative results indicate that our method can generate realistic VERDICT maps that could supplement mp-MRI assessment for better diagnosis.

category: bioengineering


title: Synthesizing VERDICT maps from standard DWI data using GANs


title: Robust clustering and interpretation ofscRNA-seq data using reference componentanalysis

abstract:
MotivationThe transcriptomic diversity of the hundreds of cell types in the human body can be analysed in unprecedented detail using single cell (SC) technologies. Though clustering of cellular transcriptomes is the default technique for defining cell types and subtypes, single cell clustering can be strongly influenced by technical variation. In fact, the prevalent unsupervised clustering algorithms can cluster cells by technical, rather than biological, variation.

ResultsCompared to de novo (unsupervised) clustering methods, we demonstrate using multiple benchmarks that supervised clustering, which uses reference transcriptomes as a guide, is robust to batch effects. To leverage the advantages of supervised clustering, we present RCA2, a new, scalable, and broadly applicable version of our RCA algorithm. RCA2 provides a user-friendly framework for supervised clustering and downstream analysis of large scRNA-seq data sets. RCA2 can be seamlessly incorporated into existing algorithmic pipelines. It incorporates various new reference panels for human and mouse, supports generation of custom panels and uses efficient graph-based clustering and sparse data structures to ensure scalability. We demonstrate the applicability of RCA2 on SC data from human bone marrow, healthy PBMCs and PBMCs from COVID-19 patients. Importantly, RCA2 facilitates cell-type-specific QC, which we show is essential for accurate clustering of SC data from heterogeneous tissues. In the era of cohort-scale SC analysis, supervised clustering methods such as RCA2 will facilitate unified analysis of diverse SC datasets.

AvailabilityRCA2 is implemented in R and is available at github.com/prabhakarlab/RCAv2

category: bioinformatics


title: Polyelectrolyte chain at thermal equilibrium: A comparison between the density functional theory framework (DFT) and the molecular dynamic simulations (MD)

abstract:
By means of the density functional theory framework (DFT) as well as the molecular dynamic simulations (MD), a polyelectrolyte chain (PE) in the good solvent conditions at thermal equilibrium is studied. The strength of the electrostatic interactions is varied by the Bjerrum length of the solvent. It turns out that average extension of a PE scales with the degree of polymerization, very much similar to a neutral polymer chain in good solvent. Remarkably, the difference between a PE and a neutral chain appears to be solely in the correlations among monomers which are stored in the Virial coefficients. Interestingly, upon increasing the Bjerrum length of solvent, the chain shrinks. This outcome is confirmed by the DFT framework as well as the MD simulations.

SIGNIFICANCEThe significance of this study is that it strongly criticizes the idea (already mentioned in T. Kreer, Soft Matter, 12, 3479 (2016)) that the PEs behave similar to a neutral ideal chain. This study could be useful in our understanding of biopolymers.

category: biophysics


title: Mapping densely distributed membrane receptors in blood platelets with expansion microscopy

abstract:
Interrogating small platelets and their densely packed, highly abundant receptor landscape is key to understand platelet clotting, a process that can save lives when stopping blood loss after an injury, but also kill when causing heart attack, stroke or pulmonary embolism. The underlying key receptor distributions and interactions, in particular the relevance of integrin clustering, are not fully understood is because of highly abundant and densely distributed GPIIb/IIIa receptors. This makes receptor distributions difficult to assess even by super-resolution fluorescence microscopy. Here, we combine dual-color expansion and confocal microscopy with colocalization analysis to assess platelet receptor organization without the need of a super-resolution microscope. We show that 4x expansion is highly straight-forward for super-resolution microscopy of platelets, while 10x expansion provides higher precision at the price of increased efforts in sample preparation and imaging. Quantifying various receptor colocalization scenarios we demonstrate that expansion microscopy can pinpoint receptor distributions and interactions in resting and activated platelets being superior to conventional methods that fail in such dense 3D scenarios with highly abundant receptors. We reveal the presence of GPIIb/IIIa clusters in resting platelets, which are not affected by platelet activation indicating that they contribute to the rapid platelet response during platelet clotting.

Graphical Abstract

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title: Mapping densely packed GPIIb/IIIa receptors in murine blood platelets with expansion microscopy


title: Structural basis for the Pr-Pfr long-range signaling mechanism of a full-length bacterial phytochrome at the atomic level

abstract:
Light sensing allows organisms to adapt to constantly changing environmental factors. Phytochromes constitute a widespread biological photoreceptor family that typically interconvert between two photostates called Pr (red light-absorbing) and Pfr (far-red light-absorbing). Despite the vast structural information reported on phytochromes, the lack of full-length structures at the (near-)atomic level in both pure Pr and Pfr states leaves gaps in the structural mechanisms involved in the signal transmission pathways during the photoconversion. Here we present three crystallographic structures from the plant pathogen Xanthomonas campestris virulence regulator bacteriophytochrome, including two full-length proteins, in the Pr and Pfr states. The structural findings, combined with mutational, biochemical and computational studies, allow us to describe the signaling mechanism of a full-length bacterial phytochrome at the atomic level, from the isomerization of the chromophore and the {beta}-sheet/-helix tongue transition to the remodeling of the quaternary assembly of the protein.

TEASERCrystal structures of the full-length bacteriophytochrome XccBphP in both Pr and Pfr states unveil photoswitching mechanism


title: Discovery of a molecular glue that enhances UPRmt to restore proteostasis via TRKA-GRB2-EVI1-CRLS1 axis

abstract: Lowering proteotoxicity is a potentially powerful approach for the treatment of neurological disorders, such as Parkinsons disease. The unfolded protein response (UPR) is a major mechanism that preserves the network maintaining cellular proteostasis. In the present study, we developed the screening strategy to discover compounds that significantly enhanced the activation of mitochondrial UPR (UPRmt) through increasing cardiolipin content. We identified that ginsenoside Rg3 (Rg3) increased cardiolipin depending on cardiolipin synthase 1 (CRLS1) in both worms and in human neural cells. Using LiP-SMap (limited proteolysis-mass spectrometry) strategy, we identified GRB2 (growth factor receptor bound protein 2) as a direct target of Rg3 in human neural cells. Rg3 enhances the binding between GRB2 and TRKA, that transduces signals via phosphrorylation of ERK. We provide bioinformatic and experimental evidence that EVI1, the critical oncogenic transcriptional regulator in leukemia, binds to CRLS1 promoter region and stimulated CRLS1 expression and subsequently increased cardiolipin content in the presence of Rg3. In a Parkinsons disease mouse model, Rg3 restores motor function by protecting nigral dopaminergic neurons dependent on Grb2. Our data recapitulate the TRKA-GRB2-EVI1-CRLS1 axis in maintaining proteostasis in Parkinsons disease via UPRmt.

category: pharmacology and toxicology


title: Dynamic Auditory Remapping Across the Sleep-Wake Cycle

abstract: In a single day we transition from vigilant wakefulness to unconscious sleep and dreaming, undergoing diverse behavioural, physiological and neural changes. While during the awake state, exogenous stimuli and endogenous changes lead to sensory reorganisation, this remapping has not been charted throughout the sleep-wake cycle. We recorded neural activity in response to a range of tones using electroencephalography during a full nights sleep, and examined whether auditory responses become more similar, dissimilar or remain unchanged between wakefulness, non-rapid (NREM) and rapid eye movement (REM) sleep. We found that neural similarities between pairs of auditory evoked potentials differed by conscious state in both early and late auditory processing stages. Furthermore, tone-pairs neural similarities were modulated by conscious state as a function of tone frequency, where some tone-pairs changed similarity between states and others continued unaffected. These findings demonstrate a state-, stimulus- and time-dependent functional reorganization of auditory processing across the sleep-wake cycle.

category: neuroscience


title: The spatial signal in area LIP is not an obligatory correlate of perceptual evidence during informed saccadic choices

abstract: The lateral intraparietal area (LIP) contains spatially selective neurons that are partly responsible for determining where to look next and are thought to serve a variety of sensory, motor planning, and cognitive control functions within this role1,2,3. Notably, according to numerous studies in monkeys4,5,6,7,8,9,10,11,12, area LIP implements a fundamental perceptual process, the gradual accumulation of sensory evidence in favor of one choice (e.g., look left) over another (look right), which manifests as a slowly developing spatial signal during a motion discrimination task. However, according to recent inactivation experiments13,14, this signal is unnecessary for accurate task performance. Here we reconcile these contradictory findings. We designed an urgent version of the motion discrimination task in which there is no systematic lag between the perceptual evaluation and the motor action reporting it, and such that the evolution of the subjects choice can be tracked millisecond by millisecond15,16,17,18. We found that while choice accuracy increased steeply with increasing sensory evidence, at the same time, the spatial selection signal in LIP became progressively weaker, as if it hindered performance. In contrast, in a similarly urgent task in which the discriminated stimuli and the choice targets were spatially coincident, this neural signal seemed to facilitate performance. The data suggest that the LIP activity traditionally interpreted as evidence accumulation may correspond to a slow, post-decision shift of spatial attention from one location (where the motion occurs) to another (where the eyes land).


title: Similar neural and perceptual masking effects of low-power optogenetic stimulation in primate V1

abstract: Can direct stimulation of primate V1 substitute for a visual stimulus and mimic its perceptual effect? To address this question, we developed an optical-genetic toolkit to "read" neural population responses using widefield calcium imaging, while simultaneously using optogenetics to "write" neural responses into V1 of behaving macaques. We focused on the phenomenon of visual masking, where detection of a dim target is significantly reduced by a co-localized medium-brightness pedestal. Using our toolkit, we tested whether V1 optogenetic stimulation can recapitulate the perceptual masking effect of a visual pedestal. We find that, similar to a visual pedestal, low-power optostimulation can significantly reduce visual detection sensitivity, that a sublinear interaction between visual and optogenetic evoked V1 responses could account for this perceptual effect, and that these neural and behavioral effects are spatially selective. Our toolkit and results open the door for further exploration of perceptual substitutions by direct stimulation of sensory cortex.


title: F-type Pyocins are Diverse Non-Contractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa

abstract:
Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or non-contractile phage tails known as R-type and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3-end of the F-type pyocin cluster are divergent in sequence, and likely determine bactericidal specificity. We use sequence similarities among these proteins to define 11 distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly re-assorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins emerged earlier or have been subject to distinct evolutionary pressures. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as anti-bacterial therapeutics.

IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes a broad spectrum of antibiotic resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great interest in the development of alternative therapeutics. One alternative is protein-based antimicrobials called bacteriocins, which are produced by one strain of bacteria to kill other strains. In this study, we investigate F-type pyocins, bacteriocins naturally produced by P. aeruginosa that resemble non-contractile phage tails. We show that they are potent killers of P. aeruginosa, and distinct pyocin groups display different killing specificities. We have identified the probable specificity determinants of F-type pyocins, which opens up the potential to engineer them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well characterized phage tails will greatly facilitate their development into effective antibacterials.

category: microbiology


title: Host preferences of coexisting Perkinsea parasitoids during coastal dinoflagellate blooms

abstract: Parasites in aquatic systems are highly diverse and ubiquitous. In marine environments, parasite-host interactions contribute substantially to shaping microbial communities, but their nature and complexity remain poorly understood. In this study, we examined the relationship between Perkinsea parasitoids and bloom-forming dinoflagellate species. Our aim was to determine whether parasite-host species interactions are specific and whether the diversity and distribution of parasitoids are shaped by their dinoflagellate hosts. Several locations along the Catalan coast (NW Mediterranean Sea) were sampled during the blooms of five dinoflagellate species and the diversity of Perkinsea was determined by combining cultivation-based methods with metabarcoding of the V4 region of 18S rDNA. Most known species of Parviluciferaceae, and others not yet described, were detected, some of them coexisting in the same coastal location, and with a wide distribution. The specific parasite-host interactions determined for each of the studied blooms demonstrated the host preferences exhibited by parasitoids in nature. The dominance of a species within the parasitoid community is driven by the presence and abundances of its preferred host(s).The absence of parasitoid species, often associated with a low abundance of their preferred hosts, suggested that high infection rates are reached only under conditions that favour parasitoid propagation, especially dinoflagellate blooms.


title: Chromosome segregation fidelity is controlled by small changes in kinetochore phospho-occupancy

abstract: Kinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e. all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded [~]50% phospho-occupancy, and the cumulative phospho-occupancy changed by only [~]20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained by ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. Thus, networks of kinases and phosphatases maintain low inherent phospho-occupancy to promote microtubule attachment to kinetochores while providing for high sensitivity of kinetochore-microtubule attachments to very small changes in phospho-occupancy to ensure high mitotic fidelity.

category: cell biology


title: Temporal differentiation of resource capture and biomass accumulation as a driver of yield increase in intercropping

abstract:
- Intercropping, i.e. the simultaneous cultivation of different crops on the same field, has demonstrated yield advantages compared to monoculture cropping. These yield advantages have often been attributed to complementary resource use, but few studies quantified the temporal complementarity of nutrient acquisition and biomass production. Our understanding of how nutrient uptake rates of nitrogen (N) and phosphorous (P) and biomass accumulation change throughout the growing season and between different neighbors is limited.
- We conducted weekly destructive harvests to measure temporal trajectories of N and P uptake and biomass production in three crop species (oat, lupin and camelina) growing either as isolated single plants, in monocultures or as intercrops. Additionally, we quantified organic acid exudation in the rhizosphere and biological N2-fixation of lupin throughout the growing season. Logistic models were fitted to characterize nutrient acquisition and biomass accumulation trajectories.
- Nutrient uptake and biomass accumulation trajectories were curtailed by competitive interactions, resulting in earlier peak rates and lower total accumulated nutrients and biomass compared to cultivation as isolated single plants. Different pathways led to overyielding in the two mixtures. The oat-camelina mixture was characterized by a shift from belowground temporal niche partitioning of resource uptake to aboveground competition for light during the growing season. The oat-lupin mixture showed strong competitive interactions, where lupin eventually overyielded due to reliance on atmospheric N and stronger competitiveness for soil P.
- Synthesis: This study demonstrates temporal shifts to earlier peak rates of plants growing with neighbors compared to those growing alone, suggesting that the observed temporal shifts in our experiment are driven by competitive interactions rather than active plant behavior to reduce competition. The two differing pathways to overyielding in the two mixtures highlight the importance of examining temporal dynamics in intercropping systems to understand the underlying mechanisms of overyielding.


title: A tissue-aware machine learning framework enhances the mechanistic understanding and genetic diagnosis of Mendelian and rare diseases

abstract: Genetic studies of Mendelian and rare diseases face the critical challenges of identifying pathogenic gene variants and their modes-of-action. Previous efforts rarely utilized the tissue-selective manifestation of these diseases for their elucidation. Here we introduce an interpretable machine learning (ML) platform that utilizes heterogeneous and large-scale tissue-aware datasets of human genes, and rigorously, concurrently and quantitatively assesses hundreds of candidate mechanisms per disease. The resulting tissue-aware ML platform is applicable in gene-specific, tissue-specific, or patient-specific modes. Application of the platform to selected Mendelian disease genes pinpointed mechanisms that lead to tissue-specific disease manifestation. When applied jointly to diseases that manifest in the same tissue, the models revealed common known and previously underappreciated factors that underlie tissue-selective disease manifestation. Lastly, we harnessed our ML platform toward genetic diagnosis of tissue-selective rare diseases. Patient-specific models of candidate disease-causing genes from 50 patients successfully prioritized the pathogenic gene in 86% of the cases, implying that the tissue-selectivity of rare diseases aids in filtering out unlikely candidate genes. Thus, interpretable tissue-aware ML models can boost mechanistic understanding and genetic diagnosis of tissue-selective heritable diseases. A webserver supporting gene prioritization is available at https://netbio.bgu.ac.il/trace/.

category: genetics


title: Defining the Yeast Resistome through in vitro Evolution and Whole Genome Sequencing

abstract: In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in the model microbe, Saccharomyces cerevisiae. Analysis of 355 curated, laboratory-evolved clones, resistant to 80 different compounds, demonstrates differences in the types of mutations that are identified in selected versus neutral evolution and reveals numerous new, compound-target interactions. Through enrichment analysis we further identify a set of 137 genes strongly associated with or conferring drug resistance as indicated by CRISPR-Cas9 engineering. The set of 25 most frequently mutated genes was enriched for transcription factors and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function, single nucleotide variants found in 170-amino-acid domains in two Zn2C6 transcription factors, YRR1 and YRM1 (p < 1x 10 -100). This remarkable enrichment for transcription factors as drug resistance genes may explain why it is challenging to develop effective antifungal killing agents and highlights their important role in evolution.


title: Evidence for a force favouring GC over AT at short intronic sites in Drosophila simulans and D. melanogaster

abstract: Population genetics studies often make use of a class of nucleotide site free from selective pressures in order to make inferences about population size changes or natural selection at other sites. If such neutral sites can be identified, they offer the opportunity to avoid any confounding effects of selection. Here we investigate evolution at putatively neutrally evolving short intronic sites in natural populations of Drosophila melanogaster and D. simulans, in order to understand the properties of spontaneous mutations and the extent of GC-biased gene conversion in these species. Use of data on the genetics of natural populations is advantageous because it integrates information from large numbers of individuals over long timescales. In agreement with direct evidence from observations of spontaneous mutations in Drosophila, we find a bias in the spectrum of mutations towards AT basepairs. In addition, we find that this bias is stronger in the D. melanogaster lineage than the D. simulans lineage. The evidence for GC-biased gene conversion in Drosophila has been equivocal. Here we provide evidence for a weak force favouring GC in both species, which is stronger in D. simulans. Some homologous short intronic sites have diverged in GC content between the two species, which may have been caused by lineage-specific changes in the extent to which different regions of the genome are subject to a GC (or AT)-favouring force.


title: High-throughput and dosage-controlled intracellular delivery of large cargos by an acoustic-electric micro-vortices platform

abstract: Intracellular delivery of cargos for cell engineering plays a pivotal role in transforming medicine and biomedical discoveries. Recent advances in microfluidics and nanotechnology have opened up new avenues for efficient, safe, and controllable intracellular delivery, as they improve precision down to the single-cell level. Based on this capability, several promising micro- and nanotechnology approaches outperform viral and conventional non-viral techniques in offering dosage-controlled delivery and/or intracellular delivery of large cargos. However, to achieve this level of precision and effectiveness, they are either low in throughput, limited to specific cell types (e.g., adherent vs. suspension cells), or complicated to operate with. To address these challenges, here we introduce a versatile and simple-to-use intracellular delivery microfluidic platform, termed Acoustic-Electric Shear Orbiting Poration (AESOP). Hundreds of acoustic microstreaming vortices form the production line of the AESOP platform, wherein hundreds of thousands of cells are trapped, permeabilized, and mixed with exogenous cargos. Using AESOP, we show intracellular delivery of a wide range of molecules (from <1 kDa to 2 MDa) with high efficiency, cell viability, and dosage-controlled capability into both suspension and adherent cells and demonstrate throughput at 1 million cells/min per single chip. In addition, we demonstrate AESOP for two gene editing applications that require delivery of large plasmids: i) eGFP plasmid (6.1 kbp) transfection, and ii) CRISPR-Cas9-mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and sgRNA. Compared to alternative platforms, AESOP not only offers dosage-controlled intracellular delivery of large plasmids (>6kbp) with viabilities over 80% and comparable delivery efficiencies, but also is an order of magnitude higher in throughput, compatible with both adherent and suspension cell lines, and simple to operate.


title: TuMV infection alters miR168/AGO1 and miR403/AGO2 systems regulation in Arabidopsis

abstract: In plants, Argonaute (AGO) proteins, essentially AGO1 and 2, restrict viral infections. These AGO proteins also participate in developmental processes and are endogenously tightly regulated by microRNAs. Under viral infections, the regulatory loop comprising miR168/AGO1 is well studied, but much less the miR403/AGO2 system. Here, we simultaneously studied the miR168/AGO1 and miR403/AGO2 regulatory systems under TuMV infection in Arabidopsis. TuMV induced downregulation of miRNAs precursor molecules, concomitantly with enhanced mature miRNAs accumulation, without evidence of transcriptional alteration. Under infection, AGO1 protein remained at basal levels whereas AGO2 overaccumulated. These results share both similarities and differences with previous reports studying abiotic and biotic impact on microRNA biogenesis and AGO-dependent antiviral defense, and expand our knowledge of the miR403/AGO2 regulatory system.

category: plant biology


title: The role of genus and life span in predicting seed and vegetative trait variation and correlation in Lathyrus, Phaseolus, and Vicia (Fabaceae)

abstract:
PREMISE OF THE STUDYAnnual and perennial life history transitions are abundant among angiosperms, and understanding the phenotypic variation underlying life span shifts is a key endeavor of plant evolutionary biology. Comparative analyses of trait variation and correlation networks among annual and perennial plants is increasingly important as new perennial crops are being developed in a predominately annual-based agricultural setting. However, it remains unclear how seed to vegetative growth trait relationships may correlate with life span.

METHODSWe measured 29 annual and perennial congeneric species of three herbaceous legume genera (Lathyrus, Phaseolus, and Vicia) for seed size and shape, germination proportion, and early vegetative height and leaf growth over three months in order to assess relative roles of genus and life span in predicting phenotypic variation and correlation.

KEY RESULTSGenus was the greatest predictor of seed size and shape variation, while life span consistently predicted static vegetative growth traits. Correlation networks revealed that annual species had significant associations between seed traits and vegetative traits, while perennials had no significant seed-vegetative associations. Each genus also differed in the extent of integration between seed and vegetative traits, as well as within-vegetative trait correlation patterns.

CONCLUSIONSGenus and life span were important for predicting aspects of early life stage phenotypic variation and trait relationships. Differences in phenotypic correlation may indicate selection on seed size traits will impact vegetative growth differently depending on life span, which has important implications for nascent perennial breeding programs.


title: Improved exercise anaerobic power and less central fatigue in CD73 knockout mice

abstract: Ecto-5-nucleotidase or CD73 is the main source of extracellular adenosine involved in the activation of adenosine A2A receptors, responsible for the ergogenic effects of caffeine. We now investigated the role of CD73 in exercise by comparing female wild-type (WT) and CD73 knockout (KO) mice in a treadmill graded test to evaluate running power, oxygen uptake [Formula], and respiratory exchange ratio (RER) - the gold standards characterizing physical performance. Spontaneous locomotion in the open field and submaximal running power and [Formula] in the treadmill were similar between CD73-KO and WT mice; [Formula] also demonstrated equivalent aerobic power, but CD73-KO mice displayed a 43.7{+/-}4.2% larger critical power (large effect size, P<0.05) and 3.8{+/-}0.4% increase of maximum RER (small effect size, P<0.05). Thus, KO of CD73 was ergogenic, i.e., it increased physical performance.


title: Deletion of CD73 increases exercise power in mice


title: Systemic administration of Ivabradine, an HCN channel inhibitor, blocks spontaneous absence seizures

abstract:
ObjectiveHyperpolarization-activated cyclic nucleotide-gated (HCN) channels are known to be involved in the generation of absence seizures (ASs), and there is evidence that cortical and thalamic HCN channel dysfunctions may have a pro-absence role. Many HCN channel blockers are available, but their role in ASs has been investigated only by localized brain injection or in in vitro model systems due to their limited brain availability. Here, we investigated the effect on ASs of orally administered ivabradine (an HCN channel blocker approved for the treatment of heart failure in humans) following injection of the P-glycoprotein inhibitor elacridar, that is known to increase penetration into the brain of drug substrates for this efflux transporter. The action of ivabradine was also tested following in vivo microinjection in the cortical initiation network (CIN) of the somatosensory cortex and in the thalamic ventrobasal nucleus (VB) as well as on cortical and thalamocortical neurons in brain slices.

MethodsWe used EEG recordings in freely moving Genetic Absence Epilepsy from Strasbourg Rats (GAERS) to assess the action of oral administration of ivabradine, with and without elacridar, on ASs. Ivabradine was also microinjected in the CIN and VB of GAERS in vivo and applied to Wistar CIN and GAERS VB slices while recording patch-clamped cortical layer 5/6 and thalamocortical neurons, respectively.

ResultsOral administration of ivabradine markedly and dose-dependently reduced ASs. Ivabradine injection in CIN abolished ASs and elicited small-amplitude 4-7 Hz waves (without spikes), whereas in the VB it was less potent. Moreover, ivabradine applied to GAERS VB and Wistar CIN slices selectively decreased HCN-channel-dependent properties of cortical layer 5/6 pyramidal and thalamocortical neurons, respectively.

SignificanceThese results provide the first demonstration of the anti-absence action of a systemically administered HCN channel blocker, indicating the potential of this class of drugs as a novel therapeutic avenue for ASs.


title: Phospholipid peroxidation fuels ExoU phospholipase-dependent cell necrosis and supports Pseudomonas aeruginosa-driven pathology

abstract: Regulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.

category: immunology


title: Delayed induction of type I and III interferons and nasal epithelial cell permissiveness to SARS-CoV-2

abstract: The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we applied single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrated widespread tropism for nasal epithelial cell types. The host response was dominated by type I and III IFNs and interferon-stimulated gene products. This response was notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response began to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFN{beta} or IFN{lambda}1 induced an efficient antiviral state that potently restricted SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data suggest that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.


title: Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2


title: SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys

abstract: Betacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.


title: Eliciting a potent antitumor immune response by expressing tumor antigens in a skin commensal

abstract: Immune modulation has become central to treating cancer. However, global immune stimulation is only effective in a subset of patients and can lead to serious complications, including colitis and type I diabetes. Newer modalities like engineered T cells and tumor vaccines are more specific, but they have shown limited efficacy in solid tumors and are difficult to scale. Bacterial strains from the human microbiome can induce antigen-specific T cells to help maintain barrier function. Here, we redirect CD8+ and CD4+ T cells elicited by the skin commensal Staphylococcus epidermidis to recognize tumor cells by expressing tumor-derived antigens in the bacterial cell. S. epidermidis expressing the model antigen ovalbumin (S. epidermidis-OVA) stimulates antigen-specific CD8+ and CD4+ T cells in vitro. The subcellular localization of the antigen skews the response: cell wall-attached OVA preferentially stimulates CD8+ T cells whereas secreted OVA predominantly induces CD4+ T cells. In a syngeneic tumor model (OVA-expressing B16 melanoma), mice colonized topically with S. epidermidis-OVA exhibit a marked reduction in subcutaneous tumor volume compared to mice colonized with S. epidermidis expressing mCherry; this effect is dependent on live bacteria and a combination of CD8+ and CD4+ T cells. S. epidermidis-OVA also reduces tumor burden when tumor cells are injected intravenously (a model of metastasis), demonstrating that the antitumor effect operates in tissues distant from the site of bacterial colonization. S. epidermidis strains expressing neoantigen peptides from the B16 tumor cell line exhibit potent antitumor efficacy without inducing an autoimmune response against melanocytes in healthy tissue. Antigen-expressing colonists are a simple but powerful strategy to elicit a targeted T cell response in the context of cancer and other diseases.


title: Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants

abstract: The protective efficacy of neutralizing antibodies (nAbs) elicited during natural infection with SARS-CoV-2 and by vaccination based on its spike protein has been compromised with emergence of the recent SARS-CoV-2 variants. Residues E484 and K417 in the receptor-binding site (RBS) are both mutated in lineages first described in South Africa (B.1.351) and Brazil (B.1.1.28.1). The nAbs isolated from SARS-CoV-2 patients are preferentially encoded by certain heavy-chain germline genes and the two most frequently elicited antibody families (IGHV3-53/3-66 and IGHV1-2) can each bind the RBS in two different binding modes. However, their binding and neutralization are abrogated by either the E484K or K417N mutation, whereas nAbs to the cross-reactive CR3022 and S309 sites are largely unaffected. This structural and functional analysis illustrates why mutations at E484 and K417 adversely affect major classes of nAbs to SARS-CoV-2 with consequences for next-generation COVID-19 vaccines.


title: HES1 protein oscillations are necessary for neural stem cells to exit from quiescence

abstract: Quiescence is a dynamic process of reversible cell-cycle arrest. High-level sustained expression of the HES1 transcriptional repressor, which oscillates with an ultradian periodicity in proliferative neural stem cells (NSCs), is thought to mediate quiescence. However, it is not known whether this is due to a change in levels or in dynamics. Here, we induce quiescence in NSCs with BMP4, which does not increase HES1 level, and we find that HES1 continues to oscillate. To assess the role of HES1 dynamics, we express sustained HES1 under a moderate-strength promoter, which overrides the endogenous oscillations while maintaining the total HES1 level within physiological range. We find that sustained HES1 does not affect proliferation or entry into quiescence, however, exit from quiescence is impeded. Thus, oscillatory expression of HES1 is specifically required for NSCs to exit quiescence, a finding of potential importance for controlling reactivation of stem cells in tissue regeneration and cancer.


title: A rigorous framework for detecting SARS-CoV-2 spike protein mutational ensemble from genomic and structural features

abstract: The recent release of SARS-CoV-2 genomic data from several countries has provided clues into the potential antigenic drift of the coronavirus population. In particular, the genomic instability observed in the spike protein necessitates immediate action and further exploration in the context of viralhost interactions. By temporally tracking 527,988 SARS-CoV-2 genomes, we identified invariant and hypervariable regions within the spike protein. We evaluated combination of mutations from SARS-CoV-2 lineages and found that maximum number of lineage-defining mutations were present in the N-terminal domain (NTD). Based on mutant 3D-structural models of known Variants of Concern (VOCs), we found that structural properties such as accessibility, secondary structural type, and intra-protein interactions at local mutation sites are greatly altered. Further, we observed significant differences between intra-protein networks of wild-type and Delta mutant, with the latter showing dense intra-protein contacts. Extensive molecular dynamics simulations of D614G mutant spike structure with hACE2 further revealed dynamic features with 47.7% of mutations mapping on flexible regions of spike protein. Thus, we propose that significant changes within spike protein structure have occurred that may impact SARS-CoV-2 pathogenesis, and repositioning of vaccine candidates is required to contain the spread of COVID-19 pathogen.


title: A comprehensive fitness landscape model reveals the evolutionary history and future evolvability of eukaryotic cis-regulatory DNA sequences

abstract: Mutations in non-coding cis-regulatory DNA sequences can alter gene expression, organismal phenotype, and fitness. Fitness landscapes, which map DNA sequence to organismal fitness, are a long-standing goal in biology, but have remained elusive because it is challenging to generalize accurately to the vast space of possible sequences using models built on measurements from a limited number of endogenous regulatory sequences. Here, we construct a sequence-to-expression model for such a landscape and use it to decipher principles of cis-regulatory evolution. Using tens of millions of randomly sampled promoter DNA sequences and their measured expression levels in the yeast Sacccharomyces cerevisiae, we construct a deep transformer neural network model that generalizes with exceptional accuracy, and enables sequence design for gene expression engineering. Using our model, we predict and experimentally validate expression divergence under random genetic drift and strong selection weak mutation regimes, show that conflicting expression objectives in different environments constrain expression adaptation, and find that stabilizing selection on gene expression leads to the moderation of regulatory complexity. We present an approach for detecting selective constraint on gene expression using our model and natural sequence variation, and validate it using observed cis-regulatory diversity across 1,011 yeast strains, cross-species RNA-seq from three different clades, and measured expression-to-fitness curves. Finally, we develop a characterization of regulatory evolvability, use it to visualize fitness landscapes in two dimensions, discover evolvability archetypes, quantify the mutational robustness of individual sequences and highlight the mutational robustness of extant natural regulatory sequence populations. Our work provides a general framework that addresses key questions in the evolution of cis-regulatory sequences.


title: A cell atlas of chromatin accessibility across 25 adult human tissues

abstract: Current catalogs of regulatory sequences in the human genome are still incomplete and lack cell type resolution. To profile the activity of human gene regulatory elements in diverse cell types and tissues in the human body, we applied single cell chromatin accessibility assays to 25 distinct human tissue types from multiple donors. The resulting chromatin maps comprising [~]500,000 nuclei revealed the status of open chromatin for over 750,000 candidate cis-regulatory elements (cCREs) in 54 distinct cell types. We further delineated cell type-specific and tissue-context dependent gene regulatory programs, and developmental stage specificity by comparing with a recent human fetal chromatin accessibility atlas. We finally used these chromatin maps to interpret the noncoding variants associated with complex human traits and diseases. This rich resource provides a foundation for the analysis of gene regulatory programs in human cell types across tissues and organ systems.


title: Structural basis for the specificity of PPM1H phosphatase for Rab GTPases

abstract: LRRK2 serine/threonine kinase is associated with inherited Parkinsons disease. LRRK2 phosphorylates a subset of Rab GTPases within their switch 2 motif to control their interactions with effectors. Recent work has shown that the metal-dependent protein phosphatase PPM1H counteracts LRRK2 by dephosphorylating Rabs. PPM1H is highly selective for LRRK2 phosphorylated Rabs, and closely related PPM1J exhibits no activity toward substrates such as Rab8a phosphorylated at Thr72 (pThr72). Here we have identified the structural determinant of PPM1H specificity for Rabs. The crystal structure of PPM1H reveals a structurally conserved phosphatase fold that strikingly has evolved a 110-residue flap domain adjacent to the active site. The flap domain distantly resembles tudor domains that interact with histones in the context of epigenetics. Cellular assays, crosslinking and 3-D modelling suggest that the flap domain encodes the docking motif for phosphorylated Rabs. Consistent with this hypothesis, a PPM1J chimera with the PPM1H flap domain dephosphorylates pThr72 of Rab8a both in vitro and in cellular assays. Therefore, PPM1H has acquired a Rab-specific interaction domain within a conserved phosphatase fold.

category: biochemistry


title: Multiple Sites on SARS-CoV-2 Spike Protein are Susceptible to Proteolysis by Cathepsins B, K, L, S, and V

abstract: SARS-CoV-2 is the coronavirus responsible for the COVID-19 pandemic. Proteases are central to the infection process of SARS-CoV-2. Cleavage of the spike protein on the viruss capsid causes the conformational change that leads to membrane fusion and viral entry into the target cell. Since inhibition of one protease, even the dominant protease like TMPRSS2, may not be sufficient to block SARS-CoV-2 entry into cells, other proteases that may play an activating role and hydrolyze the spike protein must be identified. We identified amino acid sequences in all regions of spike protein, including the S1/S2 region critical for activation and viral entry, that are susceptible to cleavage by furin and cathepsins B, K, L, S, and V using PACMANS, a computational platform that identifies and ranks preferred sites of proteolytic cleavage on substrates, and verified with molecular docking analysis and immunoblotting to determine if binding of these proteases can occur on the spike protein that were identified as possible cleavage sites. Together, this study highlights cathepsins B, K, L, S, and V for consideration in SARS-CoV-2 infection and presents methodologies by which other proteases can be screened to determine a role in viral entry. This highlights additional proteases to be considered in COVID-19 studies, particularly regarding exacerbated damage in inflammatory preconditions where these proteases are generally upregulated.


title: How the replication and transcription complex of SARS-CoV-2 functions in leader-to-body fusion

abstract:
BackgroundCoronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although unprecedented efforts are underway to develop therapeutic strategies against this disease, scientists have acquired only a little knowledge regarding the structures and functions of the CoV replication and transcription complex (RTC) and 16 non-structural proteins, named NSP1-16.

ResultsIn the present study, we proposed a two-route model to answer how the RTC functions in the jumping transcription of CoVs. The key step leading to this model was that the motif AAACH for METTL3 recognition flanking the transcription regulatory sequence (TRS) motif was discovered to determine the m6A methylation of SARS-CoV-2 RNAs, by reanalyzing public Nanopore RNA-seq data. As the most important finding, TRS hairpins were reported for the first time to interpret NSP15 cleavage, RNA methylation of CoVs and their association at the molecular level. In addition, we reported canonical TRS motifs of all CoVs to prove the importance of our findings.

ConclusionsThe main conclusions are: (1) TRS hairpins can be used to identify recombination regions in CoV genomes; (2) RNA methylation of CoVs participates in the determination of the RNA secondary structures by affecting the formation of base pairing; and (3) The eventual determination of the CoV RTC global structure needs to consider METTL3 in the experimental design. Our findings enrich fundamental knowledge in the field of gene expression and its regulation, providing a crucial basis for future studies.


title: How the replication and transcription complex functions in jumping transcription of SARS-CoV-2


title: Gram-negative outer membrane proteins with multiple β-barrel domains

abstract: Outer membrane beta barrels (OMBBs) are found in the outer membrane of Gram-negative bacteria and eukaryotic organelles. OMBBs fold as antiparallel {beta}-sheets that close onto themselves, forming pores that traverse the membrane. Currently known structures include only one barrel, of 8-36 strands, per chain. The lack of multi-OMBB chains is surprising, as most OMBBs form oligomers and some function only in this state. Using a combination of sensitive sequence-comparison methods and co-evolutionary analysis tools, we identify many proteins combining multiple beta barrels within a single chain; combinations that include 8-stranded barrels prevail. These multi-barrels seem to be the result of independent, lineage-specific fusion and amplification events. The absence of multi-barrels that are universally conserved in bacteria with an outer membrane, coupled with their frequent de novo genesis suggests that their functions are not essential, but rather beneficial in specific environments. Adjacent barrels of complementary function within the same chain may allow for new functions beyond those of the individual barrels.


title: Ezh2 as an epigenetic checkpoint regulator during monocyte differentiation: a potential target to improve cardiac repair after myocardial infarction

abstract: Epigenetic regulation of histone H3K27 methylation has recently emerged as a key step during M2-like macrophage polarization, essential for cardiac repair after Myocardial Infarction (MI). We demonstrate for the first-time that EZH2, responsible for H3K27 methylation, has an ectopic cytoplasmic localization during monocyte differentiation in M2 macrophages. Moreover, we show that pharmacological EZH2 inhibition, with GSK-343, enhances bivalent genes, expression to promote human monocyte repair functions. GSK-343 treatment accelerated cardiac inflammatory resolution preventing infarct expansion and subsequent cardiac dysfunction after MI in vivo. In conclusion, our study reveals that epigenetic modulation of cardiac-infiltrating immune cells may hold promise to limit adverse cardiac remodeling after MI.

category: physiology


title: Escherichia coli robustly expresses ATP synthase at growth rate maximizing concentrations

abstract: Improved protein expression is an important evolutionary adaptation of bacteria. A key question is whether evolution has led to optimal protein expression that maximizes immediate growth rate (short-term fitness) across conditions. Alternatively, fitter genetic variants could display suboptimal short-term fitness, because they cannot do better or because they strive for long-term fitness maximization by, for instance, anticipating future conditions. To answer this question, we focus on the ATP-producing enzyme F1F0 H+-ATPase, which is an abundant enzyme and ubiquitously expressed across conditions. We tested the optimality of H+-ATPase expression in Escherichia coli across 27 different nutrient conditions. In all tested conditions, wild-type E. coli expresses its H+- ATPase remarkably close to optimal concentrations that maximize immediate growth rate. This work indicates that bacteria can achieve robust optimal protein expression for immediate growth- rate.

category: systems biology


title: Affinity Requirements For Control Of Synaptic Targeting And Neuronal Cell Survival By Heterophilic IgSF Cell Adhesion Molecules

abstract: Neurons in the developing brain express many different cell adhesion molecules (CAMs) on their surfaces, and CAM interactions are essential for the determination of synaptic connectivity patterns. CAM binding affinities can vary by more than 200-fold, but the significance of affinity differences among CAMs is unknown. Here we provide a systematic characterization of the in vivo consequences of altering CAM affinity. Interactions between DIP- and its binding partners Dpr6 and Dpr10 control synaptic targeting and cell survival for Drosophila optic lobe neurons. We generated mutations that change DIP-::Dpr10 binding affinity and introduced these into the endogenous loci. We show that cell survival and synaptic targeting have different affinity requirements, and that there is a threshold affinity required for targeting. Reducing affinity causes graded loss-of-function phenotypes, while increasing affinity rescues cells that would normally die. Affinity reduction can be compensated for by increasing gene copy number.


title: Diversity and Function of Motile Ciliated Cell Types within Ependymal Lineages of the Zebrafish Brain

abstract: Motile cilia defects impair cerebrospinal fluid (CSF) flow, and can cause brain and spine disorders. To date, the development of ciliated cells, their impact on CSF flow and their function in brain and axial morphogenesis are not fully understood. Here, we have characterized motile ciliated cells within the zebrafish brain ventricles. We show that the ventricular surface undergoes significant restructuring through development, involving a transition from mono- to multiciliated cells (MCCs) driven by gmnc. MCCs are translationally polarized, co-exist with monociliated cells and generate directional flow patterns. Moreover, these ciliated cells have different developmental origins, and are genetically heterogenous with respect to expression of the Foxj1 family of ciliary master regulators. Finally, we show that cilia loss from specific brain regions or global perturbation of multiciliation does not affect overall brain or spine morphogenesis, but results in enlarged ventricles. Our findings establish that motile ciliated cells are generated by complementary and sequential transcriptional programs to support ventricular development.


title: Epitranscriptomic Dysregulation in Stress-induced Psychopathologies

abstract: To date, over 100 different chemical modifications to RNA have been identified. Collectively known as the epitranscriptome, these modifications function to regulate RNA stability and as such, represent another mechanistic layer of post-transcriptional gene regulation. N6-methyladenosine (m6A) is the most common RNA modification in the mammalian brain and has been implicated in a number of processes relevant to neurodevelopment, brain function and behaviour. Here, following brief descriptions on epitranscriptomic mechanisms, we will review the literature on the potential functions of the m6A-methylome in fine-tuning gene expression which include prescribing localisation of transcripts in distal compartments as well as interactions with microRNAs and long non-coding RNAs. We will then discuss findings from rodent and human studies for stress-induced disorders - major depression and post-traumatic stress disorder - which support a hypothesis for a dysregulation of the m6A-methylome and the m6A-machinery in the pathophysiology. To support this, we have included a bioinformatic analysis of publicly available single-cell RNA-sequencing and bulk transcriptomics datasets which suggests an altered m6A-methylome as a consequence of dysregulated cell- and regionally-specific expression of key enzymes involved in the  writing, reading and erasing of m6A. We hope this review will generate further interest in the field of epitranscriptomics, opening up new lines of research into its involvement in psychiatric disorders.


title: The MS remyelinating drug bexarotene (an RXR agonist) promotes induction of human Tregs and suppresses Th17 differentiation in vitro

abstract:
The retinoid X receptor (RXR) agonist bexarotene has recently been shown to promote remyelination in individuals with multiple sclerosis. Murine studies demonstrated that RXR agonists can have anti-inflammatory effects by enhancing the ability of all-trans-retinoic acid (tRA), the primary active metabolite of vitamin A, to promote T regulatory cell (Treg) induction and reduce Th17 differentiation in vitro, following stimulation of naive CD4 cells in the presence of TGF-{beta}.

Stimulating naive human CD4 T cells for 7 days, in the presence of either Treg or Th17 skewing cytokines {+/-} bexarotene (1 M), {+/-} other RXR agonists (9CisRA and NRX 194204), or {+/-} tRA (100 nM) shows that RXR agonists, including bexarotene, are capable of tipping the human Treg/Th17 axis in favour of Treg induction. Furthermore, this occurs independently of tRA and retinoic acid receptor (RAR) signalling. Tregs induced in the presence of bexarotene express many of the canonical markers of T cell regulation and are functionally suppressive in vitro.

These findings support a potential immunomodulatory role for bexarotene and highlight the possible therapeutic application of RXR agonists in autoimmune disease, with bexarotenes pro-remyelinating effects making multiple sclerosis a particularly attractive disease target.

Significance StatementThe pan-retinoid X receptor (RXR) agonist bexarotene has recently been shown to promote remyelination in patients with multiple sclerosis. Here we demonstrate that bexarotene, and other RXR agonists have immunomodulating effects, tipping the Th17/T regulatory cell (Treg) differentiation axis in favour of Treg development.

These findings lend support to the idea of developing RXR agonists as treatments of autoimmune diseases, in particular multiple sclerosis.


title: Mechanical stress determines morphogenesis and cell ordering in confined bacterial biofilms

abstract:
Biofilms are aggregates of bacterial cells surrounded by an extracellular matrix. Much progress has been made in studying biofilm growth on solid substrates; however, little is known about the biophysical mechanisms underlying biofilm development in three-dimensional confined environments, in which the biofilm-dwelling cells must push against and even damage the surrounding environment to proliferate. Here, combining single-cell imaging, mutagenesis, and rheological measurement, we reveal the key morphogenesis steps of Vibrio cholerae biofilms embedded in hydrogels as they grow by four orders of magnitude from their initial size. We show that the morphodynamics and cell ordering in embedded biofilms are fundamentally different from those of biofilms on flat surfaces. Treating embedded biofilms as inclusions growing in an elastic medium, we quantitatively show that the stiffness contrast between the biofilm and its environment determines biofilm morphology and internal architecture, selecting between spherical biofilms with no cell ordering and oblate ellipsoidal biofilms with high cell ordering. When embedded in stiff gels, cells self-organize into a bipolar structure that resembles the molecular ordering in nematic liquid crystal droplets. In vitro biomechanical analysis shows that cell ordering arises from stress transmission across the biofilm-environment interface, mediated by specific matrix components. Our imaging technique and theoretical approach are generalizable to other biofilm-forming species, and potentially to biofilms embedded in mucus or host tissues as during infection. Our results open an avenue to understand how confined cell communities grow by means of a compromise between their inherent developmental program and the mechanical constraints imposed by the environment.

Significance StatementBiofilms are microbial cities in which bacterial cells reside in a polymeric matrix. They are commonly found inside soft confining environments such as food matrices and host tissues, against which bacteria must push to proliferate. Here, by combining single-cell live imaging and mechanical characterization, we show that the confining environment determines the dynamics of biofilm shape and internal structure. This self-organized evolution of biofilm architecture is caused by force transmission between the environment and the biofilm, mediated by the extracellular matrix secreted by the cells. Our findings lead to new ways to understand how bacterial communities develop under mechanical constraints, and potentially to new strategies for preventing and controlling biofilm growth in three-dimensional environments.


title: Morphogenesis and cell ordering in confined bacterial biofilms


title: Stony Coral Tissue Loss Disease biomarker bacteria identified in corals and overlying waters using a rapid field-based sequencing approach

abstract: Stony Coral Tissue Loss Disease (SCTLD) is a devastating disease. Since 2014, it has spread along the entire Florida Reef Tract, presumably via a water-borne vector, and into the greater Caribbean. It was first detected in the United States Virgin Islands (USVI) in January 2019. To more quickly identify disease biomarker microbes, we developed a rapid pipeline for microbiome sequencing. Over a span of 10 days we collected, processed, and sequenced coral tissue and near-coral seawater microbiomes from diseased and apparently healthy Colpophyllia natans, Montastraea cavernosa, Meandrina meandrites and Orbicella franksi. Analysis of the resulting bacterial and archaeal 16S ribosomal RNA sequences revealed 25 biomarker amplicon sequence variants (ASVs) enriched in diseased tissue. These biomarker ASVs were additionally recovered in near-coral seawater (within 5 cm of coral surface), a potential recruitment zone for pathogens. Phylogenetic analysis of the biomarker ASVs belonging to Vibrio, Arcobacter, Rhizobiaceae, and Rhodobacteraceae revealed relatedness to other coral disease-associated bacteria and lineages novel to corals. Additionally, four ASVs (Algicola, Cohaesibacter, Thalassobius and Vibrio) were exact sequence matches to microbes previously associated with SCTLD. This work represents the first rapid coral disease sequencing effort and identifies biomarkers of SCTLD that could be targets for future SCTLD research.


title: A draft genome of Alliaria petiolata (garlic mustard) as a model system for invasion genetics

abstract: The emerging field of invasion genetics examines the genetic causes and consequences of biological invasions, but few study systems are available that integrate deep ecological knowledge with genomic tools. Here we report on the de novo assembly and annotation of a genome for the biennial herb Alliaria petiolata (M. Bieb.) Cavara & Grande (Brassicaceae), which is widespread in Eurasia and invasive across much of temperate North America. Our goal was to sequence and annotate a genome to complement resources available from hundreds of published ecological studies, a global field survey, and hundreds of genetic lines maintained in Germany and Canada. We sequenced a genotype (EFCC-3-20) collected from the native range near Venice, Italy and sequenced paired-end and mate pair libraries at ~70 x coverage. A de novo assembly resulted in a highly continuous draft genome (N50 = 121 Mb; L50 = 2) with 99.7 % of the 1.1 Gb genome mapping to scaffolds of at least 50 Kb in length. A total of 64,770 predicted genes in the annotated genome include 99 % of plant BUSCO genes and 98 % of transcriptome reads. Consistent with previous reports of (auto)hexaploidy in western Europe, we found that almost one third of BUSCO genes (390/1440) mapped to two or more scaffolds despite < 2 % genome-wide average heterozygosity. The continuity and gene space quality of our draft assembly will enable molecular and functional genomic studies of A. petiolata to address questions relevant to invasion genetics and conservation strategies.


title: Traditional use of Cissampelos pareira L. for hormone disorder and fever provides molecular links of ESR1 modulation to viral inhibition

abstract: Bioactive fractions or compounds obtained from medicinal plants have been used for the treatment of multiple diseases. This effect could be due to common pathways underlying these conditions that are targeted by such medicines. In this study, we explored the molecular basis of action of one such herbal formulation Cissampelos pareira, used for the treatment of female hormone disorders and fever. Genome-wide expression studies on MCF7 cell lines treated with Cipa extract were carried out using Affymetrix arrays. Transcriptome analysis revealed a downregulation of signatures of estrogen response governed by estrogen receptor  (ER). Molecular docking analysis identified 38 constituent molecules in Cipa that potentially bind ({Delta}G< -7.5) with ER at the same site as estrogen. Cipa transcriptome signatures show high positive connectivity (https://clue.io/) scores with protein translation inhibitors such as emetine (score: 99.61) and knockdown signatures of genes linked to the antiviral response such as ribosomal protein RPL7 (score: 99.92), which is also an ER coactivator. Cipa exhibits antiviral activity in dengue infected MCF7 cells that is decreased upon ESR1 (estrogen receptor 1) gene knockdown. This approach reveals a novel pathway involving ESR1-RPL7 axis that could be a potential target in dengue viral infection.


title: Transcriptome analysis and connectivity mapping of Cissampelos pareira L. provides molecular links of ESR1 modulation to viral inhibition


title: Phagocytosed polyhedrin-cytokine co-crystal nanoparticles provide sustained secretion of bioactive cytokines from macrophages

abstract: Many cells possess the ability to engulf and incorporate particles by phagocytosis. This active process is characteristic of microorganisms as well as higher order species. In mammals, monocytes, macrophages and microglia are among so-called professional phagocytes. In addition, cells such as fibroblast and chondrocytes are classified as non-professional phagocytes. Professional phagocytes play important roles in both the innate and adaptive immune response, wound healing and tissue homeostasis. Consequently, these cells are increasingly studied as targets and vectors of therapeutic intervention to treat a range of diseases. Professional phagocytes are notoriously difficult to transfect limiting their study and manipulation. Consequently, efforts have shifted towards the development of nanoparticles to deliver a cargo to phagocytic cells via phagocytosis. However, this approach carries significant technical challenges, particularly for protein cargos. We have focused on the development of nanoscale co-crystalline protein depots, known as PODS(R), that contain protein cargos, including cytokines. Here, we show that PODS are readily phagocytosed by non-professional as well as professional phagocytic cells and have attributes, such as highly sustained release of cargo, that suggest potential utility for the study and exploitation of phagocytic cells for drug delivery. Monocytes and macrophages that ingest PODS retain normal characteristics including a robust chemotactic response. Moreover, the PODS-cytokine cargo is secreted by the loaded cell at a level sufficient to modulate the behavior of surrounding non-phagocytic cells. The results presented here demonstrate the potential of PODS nanoparticles as a novel molecular tool for the study and manipulation of phagocytic cells and for the development of Trojan horse immunotherapy strategies to treat cancer and other diseases.


title: Psilocybin induces rapid and persistent growth of dendritic spines in frontal cortex in vivo

abstract: Psilocybin is a serotonergic psychedelic with untapped therapeutic potential. There are hints that the use of psychedelics can produce neural adaptations, although the extent and time scale of the impact in a mammalian brain are unknown. In this study, we used chronic two-photon microscopy to image longitudinally the apical dendritic spines of layer 5 pyramidal neurons in the mouse medial frontal cortex. We found that a single dose of psilocybin led to [~]10% increases in spine size and density, driven by an elevated spine formation rate. The structural remodeling occurred quickly within 24 hours and was persistent 1 month later. Psilocybin also ameliorated stress-related behavioral deficit and elevated excitatory neurotransmission. Overall, the results demonstrate that psilocybin-evoked synaptic rewiring in the cortex is fast and enduring, potentially providing a structural trace for long-term integration of experiences and lasting beneficial actions.


title: Broadly reactive human monoclonal antibodies targeting the pneumococcal histidine triad protein protect against fatal pneumococcal infection

abstract: Streptococcus pneumoniae remains a leading cause of bacterial pneumonia despite the widespread use of vaccines. While vaccines are effective at reducing the incidence of most vaccine-included serotypes, a rise in infection due to non-vaccine serotypes, and moderate efficacy against some vaccine included serotypes have contributed to high disease incidence. Additionally, numerous isolates of S. pneumoniae are antibiotic or multi-drug resistant. Several conserved pneumococcal proteins prevalent in the majority of serotypes have been examined as vaccines in preclinical and clinical trials. An additional, yet unexplored tool for disease prevention and treatment is the use of human monoclonal antibodies (mAbs) targeting conserved pneumococcal proteins. Here, we isolate the first human mAbs (PhtD3, PhtD6, PhtD7, PhtD8, PspA16) against the pneumococcal histidine triad protein (PhtD), and the pneumococcal surface protein A (PspA), two conserved and protective antigens. mAbs to PhtD target diverse epitopes on PhtD, and mAb PspA16 targets the N-terminal segment of PspA. The PhtD-specific mAbs bind to multiple serotypes, while mAb PspA16 serotype breadth is limited. mAbs PhtD3 and PhtD8 prolong the survival of mice infected with pneumococcal serotype 3. Furthermore, mAb PhtD3 prolongs the survival of mice in intranasal and intravenous infection models with pneumococcal serotype 4, and in mice infected with pneumococcal serotype 3 when administered 24 hours after pneumococcal infection. All PhtD and PspA mAbs demonstrate opsonophagocytic activity, suggesting a potential mechanism of protection. Our results provide new human mAbs for pneumococcal disease prevention and treatment, and identify epitopes on PhtD and PspA recognized by human B cells.


title: Potent neutralization antibodies induced by a recombinant trimeric Spike protein vaccine candidate containing PIKA adjuvant for COVID-19

abstract: Neutralizing antibodies are critical to prevent corona virus infection. The structures of immunogens to elicit most potent neutralization antibodies are still under investigation. Here we tested the immunogenicity of the trimeric, full length Spike protein with 2 proline mutations to preserve its prefusion conformation. Recombinant trimeric Spike protein expressed by CHO cells was used with polyI:C (PIKA) adjuvant to immunize mice by 0-7-14 day schedule. The results showed that Spike-specific antibody was induced at day 21 with titer of more than 50,000 in average as measured by direct binding to Spike protein. The titer of neutralization reached more than 1000 in average when tested by a pseudo-virus system, using monoclonal antibodies (40592-MM57 and 40591-MM43) with neutralizing IC50 at 1 g/ml as standards. Protein/peptide array showed that the antibodies induced by trimeric S protein vaccine bind similarly to natural infection with the receptor binding domain (RBD) as major immunodominant region. No linear epitopes were found in RBD, although several linear epitopes were found in C-terminal domain right after RBD, and heptad repeat regions. Our study supports the efficacy of recombinant trimeric Spike protein vaccine candidate for COVID-19, with excellent safety and readiness for storage and distribution in developing countries.


title: The Amyloid Precursor Protein regulates human cortical neurogenesis

abstract: The approximately 16 billion neurons of the human neocortex are derived from a relatively limited number of developmental neural stem cells (NSCs). During embryogenesis, human cortical NSCs initially generate neurons at a particularly slow rate while preserving their progenitor state for a relatively long time. How this balance between the progenitor state and neurogenic state is regulated, and whether it contributes to species-specific brain patterning, is poorly understood. Here we show that the characteristic potential of human NSCs to remain in a progenitor state as they generate neurons for a prolonged amount of time requires the Amyloid Precursor Protein (APP). In contrast, APP is dispensable in mouse NSCs, which undergo neurogenesis at a much faster rate. Mechanistically, loss of APP cell-autonomously accelerates neurogenesis through activation of the AP1 transcription factor and repression of WNT signaling. We propose that the fine balance between self-renewal and differentiation is homeostatically regulated by APP, which may contribute to human-specific temporal patterns of neurogenesis.


title: The temporal balance between self-renewal and differentiation of human neural stem cells requires the Amyloid Precursor Protein


title: Predators affect a plant virus through direct and trait-mediated indirect effects on vectors

abstract: Arthropods that vector plant pathogens often interact with predators within food webs. Predators affect vectors by eating them (consumptive effects) and by inducing antipredator behaviors (non-consumptive effects), and these interactions may affect transmission of vector-borne pathogens. However, it has proven difficult to experimentally tease apart the effects of predators on vector fitness and behavior as they are often correlated. We addressed this problem by assessing how both aphids and an aphid-borne pathogen were affected by variable predation risk. Specifically, we experimentally manipulated ladybeetle predators mouthparts to isolate consumptive, and non-consumptive, effects of predators on aphid fitness, movement, and virus transmission. We show that although lethal predators decreased aphid vector abundance, they increased pathogen transmission by increasing aphid movement among hosts. Moreover, aphids responded to risk of predation by moving to younger plant tissue that was more susceptible to the pathogen. Aphids also responded to predator risk through compensatory reproduction, which offset direct consumptive effects. Our results support predictions of disease models showing alterations of vector movement due to predators can have greater effects on transmission of pathogens than vector consumption. Broadly, our study shows isolating direct and indirect predation effects can reveal novel pathways by which predators affect vector-borne pathogens.


title: Tetrazoles as PPARγ ligands: Structural and Computational Investigation.

abstract: Diabetes is an important chronic disease affecting about 10% of the adult population in the US and over 420 million people worldwide, resulting in 1.6 million deaths every year, according to the World Health Organization. The most common type of the disease, type 2 diabetes, can be pharmacologically managed using oral hypoglycemic agents or thiazolidinediones (TZDs), such as pioglitazone, which act by activating the Peroxisome Proliferated-Activated Receptor {gamma}. Despite their beneficial effects in diabetes treatment, TZDs like rosiglitazone and troglitazone were withdrawn due to safety reasons, creating a void in the pharmacological options for the treatment of this important disease. Here, we explored a structure-based approach in the screening for new chemical probes for a deeper investigation of the effects of PPAR{gamma} activation. A class of tetrazole compounds was identified and the compounds named T1, T2 and T3 were purchased and evaluated for their ability to interact with the PPAR{gamma} ligand binding domain (LBD). The compounds were binders with micromolar range affinity, as determined by their IC50 values. A Monte Carlo simulation of the compound T2 revealed that the tetrazole ring makes favorable interaction with the polar arm of the receptor binding pocket. Finally, the crystal structure of the PPAR{gamma}-LBD-T2 complex was solved at 2.3 [A], confirming the binding mode for this compound. The structure also revealed that, when the helix H12 is mispositioned, an alternative binding conformation is observed for the ligand suggesting an H12-dependent binding conformation for the tetrazole compound.


title: Tetrazoles as PPARγ ligands: A Structural and Computational Investigation.


title: GATA3 is essential for separating patterning domains during facial morphogenesis

abstract:
Neural crest cells (NCCs) within the mandibular and maxillary portions of the first pharyngeal arch are initially competent to respond to signals from either region. However, mechanisms that are only partially understood establish developmental tissue boundaries to ensure spatially correct patterning. In the Hinge and Caps model of facial development, signals from both ventral prominences, referred to as the caps, pattern the adjacent tissues while the intervening region, known as the hinge, maintains separation of the mandibular and maxillary domains. One cap signal is GATA3, a member of the GATA family of zinc-finger transcription factors with a distinct expression pattern in the ventral-most part of the mandibular and maxillary portions of the first arch. Here we show that disruption of Gata3 in mouse embryos leads to hemifacial microsomia, facial bone hypoplasia and syngnathia (bony fusion of the upper and lower jaws). These changes are preceded by gene expression changes in post-migratory NCCs around the maxillomandibular junction (the hinge). GATA3 is thus a crucial component in establishing the network of factors that functionally separate the upper and lower jaws during development.

Summary StatementLoss of Gata3 leads to BMP-mediated disruption of Fgf8 expression at the maxillomandibular junction during development, resulting in later fusion of the upper and lower jaws.


title: Measuring the unknown: an estimator and simulation study for assessing case reporting during epidemics

abstract:
The fraction of cases reported, known as  reporting, is a key performance indicator in an outbreak response, and an essential factor to consider when modelling epidemics and assessing their impact on populations. Unfortunately, its estimation is inherently difficult, as it relates to the part of an epidemic which is, by definition, not observed.

We introduce a simple statistical method for estimating reporting, initially developed for the response to Ebola in Eastern Democratic Republic of the Congo (DRC), 2018-2020. This approach uses transmission chain data typically gathered through case investigation and contact tracing, and uses the proportion of investigated cases with a known, reported infector as a proxy for reporting. Using simulated epidemics, we study how this method performs for different outbreak sizes and reporting levels. Results suggest that our method has low bias, reasonable precision, and despite sub-optimal coverage, usually provides estimates within close range (5-10%) of the true value.

Being fast and simple, this method could be useful for estimating reporting in real-time in settings where person-to-person transmission is the main driver of the epidemic, and where case investigation is routinely performed as part of surveillance and contact tracing activities.

Author summaryWhen responding to epidemics of infectious diseases, it is essential to estimate how many cases are not being reported. Unfortunately reporting, the proportion of cases actually observed, is difficult to estimate during an outbreak, as it typically requires large surveys to be conducted on the affected populations. Here, we introduce a method for estimating reporting from case investigation data, using the proportion of cases with a known, reported infector. We used simulations to test the performance of our approach by mimicking features of a recent Ebola epidemic in the Democratic Republic of the Congo. We found that despite some uncertainty in smaller outbreaks, our approach can be used to obtain informative ballpark estimates of reporting under most settings. This method is simple and computationally inexpensive, and can be used to inform the response to any epidemic in which transmission events can be uncovered by case investigation.


title: Gene expression profiling of skeletal muscles

abstract: Soleus and tibialis anterior are two well-characterized skeletal muscles commonly utilized in skeletal muscle-related studies. Next-generation sequencing provides an opportunity for an in-depth biocomputational analysis to identify the gene expression patterns between soleus and tibialis anterior and analyze those genes functions based on past literature. This study acquired the gene expression profiles from soleus and tibialis anterior murine skeletal muscle biopsies via RNA-sequencing. Read counts were processed through edgeRs differential gene expression analysis. Differentially expressed genes were filtered down using a false discovery rate less than 0.05c, a fold-change value larger than twenty, and an association with overrepresented pathways based on the Reactome pathway over-representation analysis tool. Most of the differentially expressed genes associated with soleus encoded for components of lipid metabolism and unique contractile elements. Differentially expressed genes associated with tibialis anterior encoded mostly for glucose and glycogen metabolic pathways regulatory enzymes and calcium-sensitive contractile components. These gene expression distinctions partly explain the genetic basis for muscle specialization and may help to explain skeletal muscle susceptibility to disease and drugs and refine tissue engineering approaches.