E156G and Arg158, Phe-157/del mutation in NTD of spike protein in B.1.617.2 lineage of SARS-CoV-2 leads to immune evasion through antibody escape Emerging variants of SARS-CoV-2 with better immune escape mechanisms and higher transmissibility remains a persistent threat across the globe. B.1.617.2 (Delta) variant was first emerged from Maharashtra, India in December, 2020. This variant is classified to be a major cause and concern of the second wave of COVID-19 in India. In the present study, we explored the genomic and structural basis of this variant through computational analysis, protein modelling and molecular dynamics (MD) simulations approach. B.1.617.2 variant carried E156G and Arg158, Phe-157/del mutations in NTD of spike protein. These mutations in N-terminal domain (NTD) of spike protein of B.1.617.2 variant revealed more rigidity and reduced flexibility compared to spike protein of Wuhan isolate. Further, docking and MD simulation study with 4A8 monoclonal antibody which was reported to bind NTD of spike protein suggested reduced binding of B.1.617.2 spike protein compared to that of spike protein of Wuhan isolate. The results of the present study demonstrate the possible case of immune escape and thereby fitness advantage of the new variant and further warrants demonstration through experimental evidence. Our study identified the probable mechanism through which B.1.617.2 variant is more pathogenically evolved with higher transmissibility as compared to the wild-type.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/447321v3_ufig1.gif" ALT="Figure 1">
View larger version (23K):
org.highwire.dtl.DTLVardef@b92cborg.highwire.dtl.DTLVardef@1d261f7org.highwire.dtl.DTLVardef@11da73eorg.highwire.dtl.DTLVardef@1cef6ca_HPS_FORMAT_FIGEXP  M_FIG C_FIG
Curation, inference, and assessment of a globally reconstructed gene regulatory network for Streptomyces coelicolor Streptomyces coelicolor A3(2) is a model microorganism for the study of Streptomycetes, antibiotic production, and secondary metabolism in general. Even though S. coelicolor has an outstanding variety of regulators among bacteria, little effort to globally study its transcription has been made. We manually curated 29 years of literature and databases to assemble a meta-curated experimentally-validated gene regulatory network (GRN) with 5386 genes and 9707 regulatory interactions ([~]41% of the total expected interactions). This provides the most extensive and up-to-date reconstruction available for the regulatory circuitry of this organism. Only [~]6% (534/9707) are supported by experiments confirming the binding of the transcription factor to the upstream region of the target gene, the so-called "strong" evidence. While for the remaining interactions there is no confirmation of direct binding. To tackle network incompleteness, we performed network inference using several methods (including two proposed here) for motif identification in DNA sequences and GRN inference from transcriptomics. Further, we contrasted the structural properties and functional architecture of the networks to assess the reliability of the predictions, finding the inference from DNA sequence data to be the most trustworthy approach. Finally, we show two applications of the inferred and the curated networks. The inference allowed us to propose novel transcription factors for the key Streptomyces antibiotic regulatory proteins (SARPs). The curated network allowed us to study the conservation of the system-level components between S. coelicolor and Corynebacterium glutamicum. There we identified the basal machinery as the common signature between the two organisms. The curated networks were deposited in Abasy Atlas (https://abasy.ccg.unam.mx/) while the inferences are available as Supplementary Material.
Different contribution of the monkey prefrontal and premotor dorsal cortex in decision making during a transitive inference task Several studies have reported similar neural modulations between brain areas of the frontal cortex, such as the dorsolateral prefrontal (DLPFC) and the premotor dorsal (PMd) cortex, in tasks requiring encoding of the abstract rules for selecting the proper action. Here we compared the neuronal modulation of the DLPFC and PMd of monkeys trained to choose the higher rank from a pair of abstract images (target item), selected from an arbitrarily rank-ordered set (A>B>C>D>E>F) in the context of a transitive inference task. Once acquired by trial-and-error, the ordinal relationship between pairs of adjacent images (i.e., A>B; B>C; C>D; D>E; E>F), monkeys were tested in indicating the ordinal relation between items of the list not paired during learning. During these decisions, we observed that the choice accuracy increased and the reaction time decreased as the rank difference between the compared items enhanced. This result is in line with the hypothesis that after learning, the monkeys built an abstract mental representation of the ranked items, where rank comparisons correspond to the items position comparison on this representation. In both brain areas, we observed higher neuronal activity when the target item appeared in a specific location on the screen with respect to the opposite position and that this difference was particularly enhanced at lower degrees of difficulty. By comparing the time evolution of the activity of the two areas, we observed that the neural encoding of target item spatial position occurred earlier in the DLPFC than in the PMd. Analysis of science journalism reveals gender and regional disparities in coverage Science journalism is a critical way in which the public can remain informed and benefit from new scientific findings. Such journalism also shapes the publics view of the current state of scientific findings and legitimizes experts. Those covering science can only cite and quote a limited number of sources. Sources may be identified by the journalists research or by recommendations by other scientists. In both cases, biases may influence who is identified and ultimately included as an expert. To analyze possible biases in science journalism, we analyzed 22,001 non-research articles published by Nature. We chose to analyze Nature non-research articles since its research articles provide a natural comparator. Our analysis considered two possible sources of disparity: gender and name origin. To explore these sources of disparity, we extracted cited authors names as well as extracted names of quoted speakers. While citations and quotations within a piece do not reflect the entire information-gathering process, they can provide insight into the demographics of visible sources. We then used the extracted names to predict gender and name origin of the cited authors and speakers.

In order to appropriately quantify the level of difference, we must identify a suitable reference set for comparison. We chose first and last authors within primary research articles in Nature and a subset of Springer Nature articles in the same time period as our comparator. In our analysis, we found a skew towards male quotation in Nature science journalism-related articles. However, quotation is trending toward equal representation at a faster rate than first and last authorship in academic publishing. Interestingly, we found that the gender disparity in Nature quotes was column-dependent, with the "Career Features" column reaching gender parity. Our name origin analysis found a significant over-representation of names with predicted Celtic/English origin and under-representation of names with a predicted East Asian origin. This finding was observed both in extracted quotes and journal citations, but dampened in citations. Microbial colonization of tannin-rich tropical plants: interplay between degradability, methane production and tannin disappearance in the rumen AO_SCPLOWBSTRACTC_SCPLOWCondensed tannins in plants are found free and attached to protein and fibre but it is not known whether these fractions influence rumen degradation and microbial colonization. This study explored the rumen degradation of tropical tannins-rich plants and the relationship between condensed tannins fractions disappearance and microbial communities colonising plant particles. Leaves from Calliandra calothyrsus, Gliricidia sepium, and Leucaena leucocephala, pods from Acacia nilotica and the leaves of two agricultural by-products: Manihot esculenta and Musa spp. were incubated in situ in the rumen of dairy cows. An in vitro approach was also used to assess the effects of these plants on rumen fermentation parameters. All plants contained more than 100 g/kg of condensed tannins with a large proportion (32 to 61%) bound to proteins. Calliandra calothyrsus had the highest concentration of condensed tannins at 361 g/kg, whereas Acacia nilotica was particularly rich in hydrolysable tannins (350 g/kg). Hydrolysable and free condensed tannins from all plants completely disappeared after 24 h incubation in the rumen. Disappearance of protein-bound condensed tannins was variable with values ranging from 93% for Gliricidia sepium to 21% for Acacia nilotica. In contrast, fibre-bound condensed tannins disappearance averaged ~82% and did not vary between plants. Disappearance of bound fractions of condensed tannins was not associated with degradability of plant fractions. The presence of tannins interfered with the microbial colonisation of plants. Each plant had distinct bacterial and archaeal communities after 3 and 12 h of incubation in the rumen and distinct protozoal communities at 3 h. Adherent communities in tannin-rich plants had a lower relative abundance of fibrolytic microbes, notably Fibrobacter spp. Whereas, archaea diversity was reduced in high tannin-containing Calliandra calothyrsus and Acacia nilotica at 12 h of incubation. Concurrently, in vitro methane production was lower for Calliandra calothyrsus, Acacia nilotica and Leucaena leucocephala although for the latter total volatile fatty acids production was not affected and was similar to control. Here we show that the total amount of hydrolysable and condensed tannins contained in a plant govern the interaction with rumen microbes affecting degradability and fermentation. The effect of protein- and fibre-bound condensed tannins on degradability is less important. Disrupted Surfaces of Porous Membranes Reduce Nuclear YAP Localization and Enhance Adipogenesis through Morphological Changes The disrupted surface of porous membranes, commonly used in tissue-chip and cellular co-culture systems, is known to weaken cell-substrate interactions. Here, we investigated whether disrupted surfaces of membranes with micron and sub-micron scale pores affect YAP localization and differentiation of adipose-derived stem cells (ADSCs). We found that these substrates reduce YAP nuclear localization through decreased cell spreading, consistent with reduced cell-substrate interactions, and in turn enhance adipogenesis, while decreasing osteogenesis.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/429012v3_ufig1.gif" ALT="Figure 1">
View larger version (82K):
org.highwire.dtl.DTLVardef@5fa0daorg.highwire.dtl.DTLVardef@3981d9org.highwire.dtl.DTLVardef@17bd8f5org.highwire.dtl.DTLVardef@1e0c9e5_HPS_FORMAT_FIGEXP  M_FIG C_FIG C. elegans PEZO-1 is a Mechanosensitive Ion Channel Involved in Food Sensation PIEZO channels are force sensors essential for physiological processes, including baroreception and proprioception. The Caenorhabditis elegans genome encodes an ortholog gene of the Piezo family, pezo-1, which is expressed in several tissues, including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal, whose contraction and relaxation are modulated by mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive ion channel and contributes to pharyngeal function remain unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recording to establish that pezo-1 is expressed in the pharynx, including in a proprioceptive-like neuron, and regulates pharyngeal function. Knockout (KO) and gain-of-function (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, sensing osmolarity, and food mechanical properties. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the G-isoform of pezo-1 (among the longest isoforms) demonstrates that pezo-1 encodes a mechanosensitive channel. Our findings reveal that pezo-1 is a mechanosensitive ion channel that regulates food sensation in worms. Decellularized Normal and Tumor Extracellular Matrix as Scaffold for Cancer Organoid Cultures of Colorectal Peritoneal Metastases Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed.

SignificanceEvidence of the value of ex vivo 3D models obtained by combining patient-derived extracellular matrices depleted of cellular components and organoids to mimic the metastatic niche, to be used as a tool to develop new therapeutic strategies in a biologically relevant context, to personalize treatments and increase their efficacy. A novel 5 prime DNA binding site in RFC facilitates PCNA loading for gap DNA repair RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases {delta} and {gamma}. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures revealing a novel 2nd DNA binding site in RFC that binds a 5' duplex. This 5' DNA site is located in the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. It appears 5' DNA binds after 3' DNA binding to RFC. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. We propose that the 5' site facilitates RFCs PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5' DNA binding domain of Rfc1. We further observe that a 5' end facilitates PCNA loading at an RPA coated 50-nt gap, suggesting a potential role of the RFC 5' DNA site in longer gaps including during lagging strand DNA synthesis. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Elucidation of fibril helix structure responsible for swimming in Spiroplasma using electron microscopy Spiroplasma, known pathogens and commensals of arthropods and plants, are helical-shaped bacteria lacking the peptidoglycan layer. They swim by alternating between left- and right-handed cell helicity, which is driven by an internal structure called the ribbon. This system is unrelated to flagellar motility that is widespread in bacteria. The ribbon comprises the bacterial actin homolog MreB and fibril, the protein specific to Spiroplasma. Here, we isolated the ribbon and its core, the fibril filament, and using electron microscopy, found that the helicity of the ribbon and the cell is linked to the helicity of the fibril. Single particle analysis using the negative-staining method revealed that the three-dimensional structures of the fibril filament comprise a repeated ring structure twisting along the filament axis. The handedness of these structures were verified by the structures reconstituted through tomogram from quick-freeze deep-etch replica electron microscopy. Based on these observations, we propose a scheme for the helicity-switching mechanism in which the twists caused by the conformational changes in the fibril filament are accumulated, transmitted to the ribbon, and then propel the cells by rotating the cell body like a screw.

Significance StatementSpiroplasma are commensal and pathogenic bacteria widespread globally in animals and plants. A few species are also recognized as male-killing bacteria of insects. Their special swimming mechanism is caused by helicity switching, which could be the simplest swimming mechanism, because helicity shift can be caused by contraction and extension of paired filaments. This mechanism has attracted research attention for many years because of the possible application in the field of nano actuators; however, the details of this mechanism remain to be clarified. Here, we propose a model for the swimming mechanism based on the structure of the core of the Spiroplasma ribbon. GPI-anchored FGF directs cytoneme-mediated bidirectional signaling to self-regulate tissue-specific dispersion How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). Here, we show that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and activates target-specific bidirectional FGF-FGFR signaling through cytonemes. FGF-source and recipient ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through CAM-like FGF-FGFR binding. Contact-mediated FGF-FGFR binding induces bidirectional signaling, which, in turn, promotes ASP and source cells to polarize cytonemes toward each other and reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source cell membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion and signaling. Genetic interactions drive heterogeneity in causal variant effect sizes for gene expression and complex traits Despite the growing number of genome-wide association studies (GWAS), it remains unclear to what extent gene-by-gene and gene-by-environment interactions influence complex traits in humans. The magnitude of genetic interactions in complex traits has been difficult to quantify because GWAS are generally underpowered to detect individual interactions of small effect. Here, we develop a method to test for genetic interactions that aggregates information across all trait-associated loci. Specifically, we test whether SNPs in regions of European ancestry shared between European American and admixed African American individuals have the same causal effect sizes. We hypothesize that in African Americans, the presence of genetic interactions will drive the causal effect sizes of SNPs in regions of European ancestry to be more similar to those of SNPs in regions of African ancestry. We apply our method to two traits: gene expression in 296 African Americans and 482 European Americans in the Multi-Ethnic Study of Atherosclerosis (MESA) and low-density lipoprotein cholesterol (LDL-C) in 74K African Americans and 296K European Americans in the Million Veteran Program (MVP). We find significant evidence for genetic interactions in our analysis of gene expression; for LDL-C, we observe a similar point estimate although this is not significant, likely due to lower statistical power. These results suggest that gene-by-gene or gene-by-environment interactions modify the effect sizes of causal variants in human complex traits. MTD: a unique pipeline for host and meta-transcriptome joint and integrative analyses of RNA-seq data RNA-seq data contains not only host transcriptomes but also non-host information that comprises transcripts from active microbiota in the host cells. Therefore, joint and integrative analyses of both host and meta-transcriptome can reveal gene expression of microbial community in a given sample as well as the correlative and interactive dynamics of host response to the microbiome. However, there are no convenient tools that can systemically analyze host-microbiota interactions by simultaneously quantifying host and meta-transcriptome in the same sample at the tissue and the single-cell level, which poses a challenge for interested researchers with a limited expertise in bioinformatics. Here, we developed a software pipeline that can comprehensively and synergistically analyze and correlate the host and meta-transcriptome in a single sample using bulk and single-cell RNA-seq data. This pipeline, named MTD, can extensively identify and quantify microbiome, including viruses, bacteria, protozoa, fungi, plasmids, and vectors in the host cells and correlate the microbiome with the host transcriptome. MTD is easy to install and run, involving only a few lines of simple commands. It empowers researchers with unique genomics insights into host immune responses to microorganisms. Full-likelihood genomic analysis clarifies a complex history of species divergence and introgression: the example of the erato-sara group of Heliconius butterflies AO_SCPLOWBSTRACTC_SCPLOWIntrogressive hybridization plays a key role in adaptive evolution and species diversification in many groups of species. However, frequent hybridization and gene flow between species makes estimation of the species phylogeny and key population parameters challenging. Here, we show that by accounting for phasing and using full-likelihood analysis methods, introgression histories and population parameters can be estimated reliably from whole-genome sequence data. We employ full-likelihood methods under the multispecies coalescent (MSC) model with and without gene flow to analyze the genomic data from six members of the erato-sara clade of Heliconius butterflies and infer the species phylogeny and cross-species introgression events. The methods naturally accommodate random fluctuations in genealogical history across the genome due to deep coalescence. To avoid heterozygote phasing errors in haploid sequences commonly produced by genome assembly methods, we process and compile unphased diploid sequence alignments and use analytical methods to average over uncertainties in heterozygote phase resolution. There is robust evidence for introgression across the genome, both among distantly related species deep in the phylogeny and between sister species in shallow parts of the tree. We obtain chromosome-specific estimates of key population parameters such as introgression directions, times and probabilities, as well as species divergence times and population sizes for modern and ancestral species. We confirm ancestral gene flow between the sara clade and an ancestral population of H. telesiphe, a likely hybrid speciation origin for H. hecalesia, and gene flow between sister species H. erato and H. himera. Inferred introgression among ancestral species also explains the history of two chromosomal inversions deep in the phylogeny of the group. This study illustrates how a full-likelihood approach based on the multispecies coalescent makes it possible to extract rich historical information of species divergence and gene flow from genomic data. Unravelling Rubber Tree Growth by Integrating GWAS and Biological Network-Based Approaches Hevea brasiliensis (rubber tree) is a large tree species of the Euphorbiaceae family with inestimable economic importance. Rubber tree breeding programs currently aim to improve growth and production, and the use of early genotype selection technologies can accelerate such processes, mainly with the incorporation of genomic tools, such as marker-assisted selection (MAS). However, few quantitative trait loci (QTLs) have been used successfully in MAS for complex characteristics. Recent research shows the efficiency of genome-wide association studies (GWAS) for locating QTL regions in different populations. In this way, the integration of GWAS, RNA-sequencing (RNA-Seq) methodologies, coexpression networks and enzyme networks can provide a better understanding of the molecular relationships involved in the definition of the phenotypes of interest, supplying research support for the development of appropriate genomic based strategies for breeding. In this context, this work presents the potential of using combined multiomics to decipher the mechanisms of genotype and phenotype associations involved in the growth of rubber trees. Using GWAS from a genotyping-by-sequencing (GBS) Hevea population, we were able to identify molecular markers in QTL regions with a main effect on rubber tree plant growth under constant water stress. The underlying genes were evaluated and incorporated into a gene coexpression network modelled with an assembled RNA-Seq-based transcriptome of the species, where novel gene relationships were estimated and evaluated through in silico methodologies, including an estimated enzymatic network. From all these analyses, we were able to estimate not only the main genes involved in defining the phenotype but also the interactions between a core of genes related to rubber tree growth at the transcriptional and translational levels. This work was the first to integrate multiomics analysis into the in-depth investigation of rubber tree plant growth, producing useful data for future genetic studies in the species and enhancing the efficiency of the species improvement programs. Nrg1/ErbB Signaling-Mediated Regulation of Fibrosis After Myocardial Infarction RATIONALEAppropriate fibrotic tissue formation after myocardial infarction (MI) is crucial to maintenance of the hearts structure. Reparative or M2-like macrophages play a vital role in post-MI fibrosis by activating cardiac fibroblasts. However, the mechanism by which post-MI cardiac fibrosis is regulated is not fully understood.

OBJECTIVEWe investigated the cellular and molecular mechanisms of post-MI fibrotic tissue formation, especially those related to regulation of cellular senescence and apoptosis.

METHODS AND RESULTSIn vivo and in vitro experiments were used to investigate the molecular and cellular mechanisms through which post-MI fibrosis occurs, with a focus on the role of M2-like macrophages. Microarray analysis revealed that CD206+F4/80+CD11b+ M2-like macrophages collected from mouse hearts on post-MI day 7 showed increased expression of neuregulin 1 (Nrg1). Nrg1 receptor epidermal growth factor receptor ErbB was expressed on cardiac fibroblasts in the infarct area. In cardiac fibroblasts in which hydrogen peroxide-induced senescence, M2-like macrophage-derived Nrg1 suppressed both senescence and apoptosis of the fibroblasts, whereas blockade of ErbB function significantly accelerated. M2-like macrophage-derived Nrg1/ErbB/PI3K/Akt signaling, which was shown to be related to anti-senescence, was activated in damaged cardiac fibroblasts. Interestingly, systemic blockade of ErbB function in MI model mice enhanced senescence and apoptosis of cardiac fibroblasts and exacerbated inflammation. Further, increased accumulation of M2-like macrophages resulted in excessive progression of fibrosis in post-MI murine hearts. The molecular mechanism underlying regulation of fibrotic tissue formation in the infarcted myocardium was shown in part to be attenuation of apoptosis and senescence of cardiac fibroblasts by activation of Nrg1/ErbB/PI3K/Akt signaling.

CONCLUSIONSM2-like macrophage-mediated regulation of Nrg1/ErbB signaling, have a substantial effect on fibrotic tissue formation in the infarcted adult mouse heart, is critical for suppressing the progression of senescence and apoptosis of cardiac fibroblasts. High-throughput submicron-resolution microscopyof entire C. elegans populations under strongimmobilization by cooling cultivation plates Despite its profound impact on biology, most high-resolution in vivo microscopy approaches remain low throughput because current immobilization techniques require significant manual effort. We greatly accelerate imaging of the nematode Caenorhabditis elegans by implementing a simple cooling approach to easily immobilize entire populations directly on their cultivation plates. We optimize and characterize cooling immobilization. Counterintuitively, relatively warmer temperatures immobilize animals significantly more effectively than colder temperatures utilized in prior studies. This enhanced immobilization enables clear submicron-resolution fluorescence imaging, which is challenging to achieve with most current immobilization techniques. We demonstrate 64x magnification 3D imaging and timelapse recording of neurons in adults and embryos without motion blur. Compared to standard azide immobilization, cooling immobilization reduces the animal preparation and recovery time by >98%, significantly increasing experimental speed. By obviating individual animal manipulation, our approach could also empower automated imaging of large C. elegans populations within standard experimental setups and workflows. The obstacles and potential clues of prime editing applications in tomato, a dicot plant Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. Modifying the pegRNA sequences by shortening or introducing mismatches to the primer binding sequences (PBS) in order to reduce their melting temperatures (Tm) did not enhance the PE efficiency at the SlBMP21, SlALC and SlALS1 loci. Our data show challenges of PE approach in tomato, indicating a further improvement of the PE system for the successful applications such as use of improved expression systems. Our work provides an important clue for the successful application of the PE approach in crop improvement. High quality mapping of chromatin at or near the nuclear lamina for small numbers of cells The chromatin associated with the nuclear lamina (NL) is referred to as Lamina-Associated Domains (LADs). While mapping of this feature has been done using various technologies, technical limitations exist for each of the methods. Here, we present an adaptation of the Tyramide-Signal Amplification sequencing (TSA-seq) protocol, which we call chromatin pull down-based TSA-seq (cTSA-seq), that can be used to map chromatin regions at or near the NL from as little as 50,000 cells without using carriers. The cTSA-seq mapped regions are composed of LADs and smaller chromatin regions that fall within the chromatin B-compartment known to be enriched for heterochromatin and be present at the nuclear periphery. As a proof of principle, we used cTSA-seq to map chromatin at or near the assembling NL as cells exit mitosis and progress through early and later G1. Consistent with previous reports, lamin-B1 based cTSA-seq revealed that regions toward the distal ends of chromosomes are near or at the reassembling NL during early G1. The cTSA-seq mapping and analyses revealed similarity between the early G1 chromatin and oncogene-induced senescent cell populations. The cTSA-seq reported here represents a useful method for analyzing chromatin at or near the NL from small numbers of cells. Differential regulation of alternative promoters emerges from unified kinetics of enhancer-promoter interaction Many eukaryotic genes contain alternative promoters with distinct expression patterns. How these promoters are differentially regulated remains elusive. Here, we apply single-molecule imaging to quantify the transcriptional regulation of two alternative promoters (P1 and P2) of the Bicoid (Bcd) target gene hunchback in syncytial blastoderm Drosophila embryos. Contrary to the previous notion that Bcd only activates P2, we find that Bcd activates both promoters via the same two enhancers. P1 activation is less frequent and requires binding of more Bcd molecules than P2 activation. Using a theoretical model to relate promoter activity to enhancer states, we show that the two promoters follow common transcription kinetics driven by sequential Bcd binding at the two enhancers. Bcd binding at either enhancer primarily activates P2, while P1 activation relies more on Bcd binding at both enhancers. These results provide a quantitative framework for understanding the kinetic mechanisms of complex eukaryotic gene regulation. `rbioacc`: an R-package to analyse toxicokinetic data O_LI rbioacc is an  R-package dedicated to the analysis of experimental data collected from bioaccumulation tests during which organisms are exposed to a chemical (exposure phase) and then put into a clean media (depuration phase). Internal concentrations are regularly measured over time all along the experiment.
C_LIO_LI rbioacc provides ready-to-use functions to visualize and fully analyze such data. Under a Bayesian framework, this package fits a generic one-compartment toxicokinetic (TK) model automatically built from the data. It provides TK parameter estimates (appropriate uptake and elimination rates) and bioaccumulation metrics (e.g., BCF, BSAF, BMF). All parameter estimates, bioaccumulation metrics as well as predictions of internal concentrations into organisms are delivered with their uncertainty.
C_LIO_LIBioaccumulation metrics are primarily provided in support of environmental risk assessment, in full compliance with regulatory requirements required to approve marketing applications of chemical substances.
C_LIO_LIThis paper gives brief worked examples of the use of  rbioacc from data collected through standard bioaccumulation tests, and publicly available within the scientific literature. These examples constitute step-by-step user-guides to analyze any new data set, uploaded in the right format.
C_LI Imaging sensory transmission and neuronal plasticity in primary sensory neurons with genetically-encoded voltage indicator, ASAP4.4-Kv Detection of somatosensory inputs requires conversion of external stimuli into electrical signals by activation of primary sensory neurons. The mechanisms by which heterogeneous primary sensory neurons encode different somatosensory inputs remains unclear. In vivo dorsal root ganglia (DRG) imaging using genetically-encoded Ca2+ indicators (GECIs) is currently the best technique for this purpose by providing an unprecedented spatial and populational resolution. It permits the simultaneous imaging of >1800 neurons/DRG in live mice. However, this approach is not ideal given that Ca2+ is a second messenger and has inherently slow response kinetics. In contrast, genetically-encoded voltage indicators (GEVIs) have the potential to track voltage changes in multiple neurons in real time but often lack the brightness and dynamic range required for in vivo use. Here, we used soma-targeted ASAP4.4-Kv, a novel GEVI, to dissect the temporal dynamics of noxious and non-noxious neuronal signals during mechanical, thermal, or chemical stimulation in DRG of live mice. ASAP4.4-Kv is sufficiently bright and fast enough to optically characterize individual neuron coding dynamics. Notably, using ASAP4.4-Kv, we uncovered cell-to-cell electrical synchronization between adjacent DRG neurons and robust dynamic transformations in sensory coding following tissue injury. Finally, we found that a combination of GEVI and GECI imaging empowered in vivo optical studies of sensory signal processing and integration mechanisms with optimal spatiotemporal analysis.

HighlightsO_LIIn vivo ultra fast and sensitive dynamic voltage imaging of peripheral primary sensory neurons by a newly generated genetically-encoded voltage indicator.
C_LIO_LIIdentification of mechanical, thermal, or chemical stimuli-evoked voltage signals with superior temporal resolution.
C_LIO_LISingle-cell detection of changes in sub- and suprathreshold voltage dynamics across different disease conditions.
C_LIO_LICombination of voltage (by ASAP4.4-Kv) and Ca2+ (by Pirt-GCaMP3) signals to facilitate the understanding of signal processing and integration of primary sensory neurons, especially for noxious versus non-noxious sensation.
C_LI Co-infection of Candidatus Piscichlamydia trichopodus (order Chlamydiales) and Henneguya sp (Myxosporea, Myxobolidae) in snakeskin gourami Trichopodus pectoralis (Regan 1910) The present study describes a simultaneous infection of a novel Chlamydia-like organism (CLO) with a Myxozoa parasite, Henneguya sp. in snakeskin gourami Trichopodus pectoralis in Thailand. A new CLO is proposed "Candidatus Piscichlamydia trichopodus" (CPT) based on 16S rRNA phylogenetic analysis. Systemic intracellular CPT infection was confirmed by histological examination, in situ hybridization, PCR assay, and sequencing of 16S rRNA. This novel pathogen belongs to the order Chlamydiales but differs in certain aspects from other species. The histopathological changes associated with CPT infection were different from the typical pathological lesions of epitheliocystis caused by previously known CLO. Unlike other CLO, CPT localized in the connective tissue rather than in the epithelial cells and formed smaller clumps of intracellular bacteria that stained dark blue with hematoxylin. On the other hand, typical myxospores of the genus Henneguya with tails were observed in the gill sections. Infection with Henneguya sp. resulted in extensive destruction of the gill filaments, most likely leading to respiratory distress. Due to the frequency of co-infections and the unavailability of culture methods for CLO and Henneguya sp, it was difficult to determine which pathogens were directly responsible for the associated mortality. However, co-infections may increase the negative impact on the host and the severity of the disease. Given the commercial importance of the snakeskin gourami and its significant aquaculture potential, the findings of this study are important for further studies on disease prevention. Common coupling map advances GPCR-G protein selectivity Two-thirds of human hormones and one-third of clinical drugs act on membrane receptors that couple to G proteins to achieve appropriate functional responses. While G protein transducers from literature are annotated in the Guide to Pharmacology database, two recent large-scale datasets now expand the receptor-G protein  couplome. However, these three datasets differ in scope and reported G protein couplings giving different coverage and conclusions on GPCR-G protein signaling. Here, we report a common coupling map uncovering novel couplings supported by both large-scale studies, the selectivity/promiscuity of GPCRs and G proteins, and how the co-coupling and co-expression of G proteins compare to the families from phylogenetic relationships. The coupling map and insights on GPCR-G protein selectivity will catalyze advances in receptor research and cellular signaling towards the exploitation of G protein signaling bias in design of safer drugs. Mechanisms of damage prevention, signalling, and repair impact disease tolerance The insect gut is frequently exposed to pathogenic threats and must not only clear these potential infections, but also tolerate relatively high microbe loads. In contrast to the mechanisms that eliminate pathogens, we currently know less about the mechanisms of disease tolerance. We investigated how well-described mechanisms that either prevent, signal, control, or repair damage during infection contribute to the phenotype of disease tolerance. We established enteric infections with the bacterial pathogen Pseudomonas entomophila in transgenic lines of Drosophila melanogaster fruit flies affecting dcy (a major component of the peritrophic matrix), upd3 (a cytokine-like molecule), irc (a negative regulator of reactive oxygen species) and egfr1 (epithelial growth factor receptor). Flies lacking dcy experienced the highest susceptibility, while loss of function of either irc or upd3 reduced tolerance in both sexes. The disruption of egfr1 resulted in a severe loss in tolerance in male flies but had no substantial effect on the ability of female flies to tolerate P. entomophila infection, despite carrying greater microbe loads than males. Together, our findings provide evidence for the role of damage limitation mechanisms in disease tolerance and highlight how sex differences in these mechanisms could generate sexual dimorphism in disease tolerance.