Fast, high-throughput production of improved rabies viral vectors for specific, efficient and versatile transsynaptic retrograde labeling To understand the function of neuronal circuits it is crucial to disentangle the connectivity patterns within the network. However, most tools currently used to explore connectivity have low-throughput, low-selectivity, or limited accessibility. Here, we report the development of an improved packaging system for the production of the highly neurotrophic RVdGenvA-CVS-N2c rabies viral vectors, yielding titers orders of magnitude higher with no background contamination, at a fraction of the production time, while preserving the efficiency of transsynaptic labeling. Along with the production pipeline, we developed suites of "starter" AAV and bicistronic RVdG-CVS-N2c vectors, enabling retrograde labeling from a wide range of neuronal populations, tailored for diverse experimental requirements. We demonstrate the power and flexibility of the new system by uncovering hidden local and distal inhibitory connections in the hippocampal formation and by imaging the functional properties of a cortical microcircuit across weeks. Our novel production pipeline provides a convenient approach to generate new rabies vectors, while our toolkit flexibly and efficiently expands the current capacity to label, manipulate and image the neuronal activity of interconnected neuronal circuits in vitro and in vivo.
Numerical Simulation of Concussive-generated Cortical Spreading Depolarization to Optimize DC-EEG Electrode Spacing for Non-invasive Visual Detection BackgroundCortical Spreading Depolarization (SD) is a propagating depolarization wave of neurons and glial cells in the cerebral gray matter. SD occurs in all forms of severe acute brain injury as documented using invasive detection methods. Based on many experimental studies of mechanical brain deformation and concussion, the occurrence of SDs in human concussion has often been hypothesized. However, this hypothesis cannot be confirmed in humans as SDs can only be detected with invasive detection methods that would require either a craniotomy or a burr hole to be performed on athletes. Typical electroencephalography (EEG) electrodes, placed on the scalp, can detect the possible presence of SD but have not been able to accurately and reliably identify SDs.

MethodsTo explore the possibility of a non-invasive method to resolve this hurdle, we developed a finite element numerical model that simulates scalp voltage changes that are induced by a brain-surface SD. We then compared our simulation results with retrospectively evaluated data in aneurysmal subarachnoid hemorrhage (aSAH) patients from Drenckhahn et al. (Brain 135:853, 2012).

ResultsThe ratio of peak scalp to simulated peak cortical voltage, Vscalp/Vcortex, was 0.0735, whereas the ratio from the retrospectively evaluated data was 0.0316 (0.0221, 0.0527) [median (1st quartile, 3rd quartile), n = 161, p < 0.001, one sample Wilcoxon signed rank test]. These differing values provide validation because their differences can be attributed to differences in shape between concussive- and aSAH-SDs, as well as the inherent limitations in human study voltage measurements. This simulated scalp surface potential was used to design a virtual scalp detection array. Error analysis and visual reconstruction showed that 1 cm is the optimal electrode spacing to visually identify the propagating scalp voltage from a cortical SD. Electrode spacings of 2 cm and above produce distorted images and high errors in the reconstructed image.

ConclusionOur analysis suggests that concussive (and other) SDs can be detected from the scalp, which could confirm SD occurrence in human concussion, provide concussion diagnosis based on an underlying physiological mechanism, and lead to non-invasive SD detection in the setting of severe acute brain injury.
Collective polymerase dynamics emerge from DNA supercoiling during transcription All biological processes ultimately come from physical interactions. The mechanical properties of DNA play a critical role in transcription. RNA polymerase can over or under twist DNA (referred to as DNA supercoiling) when it moves along a gene resulting in mechanical stresses in DNA that impact its own motion and that of other polymerases. For example, when enough supercoiling accumulates, an isolated polymerase halts and transcription stops. DNA supercoiling can also mediate non-local interactions between polymerases that shape gene expression fluctuations. Here, we construct a comprehensive model of transcription that captures how RNA polymerase motion changes the degree of DNA supercoiling which in turn feeds back into the rate at which polymerases are recruited and move along the DNA. Surprisingly, our model predicts that a group of three or more polymerases move together at a constant velocity and sustain their motion (forming what we call a polymeton) whereas one or two polymerases would have halted. We further show that accounting for the impact of DNA supercoiling on both RNA polymerase recruitment and velocity recapitulates empirical observations of gene expression fluctuations. Finally, we propose a mechanical toggle switch whereby interactions between genes are mediated by DNA twisting as opposed to proteins. Understanding the mechanical regulation of gene expression provides new insights into how endogenous genes can interact and informs the design of new forms of engineered interactions.

PACS numbers: 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. 3D Echo Planar Time-resolved Imaging (3D-EPTI) for ultrafast multi-parametric quantitative MRI Multi-parametric quantitative MRI has shown great potential to improve the sensitivity and specificity of clinical diagnosis and to enhance our understanding of complex brain processes, but suffers from long scan time especially at high spatial resolution. To address this long-standing challenge, we introduce a novel approach, termed 3D Echo Planar Time-resolved Imaging (3D-EPTI), which significantly increases the acceleration capacity of MRI sampling, and provides high acquisition efficiency for multi-parametric MRI. This is achieved by exploiting the spatiotemporal correlation of MRI data at multiple timescales through new encoding strategies within and between efficient continuous readouts. Specifically, an optimized spatiotemporal CAIPI encoding within the readouts combined with a radial-block sampling strategy across the readouts enables an acceleration rate of 800 folds in the k-t space. A subspace reconstruction was employed to resolve thousands of artifact-free high-quality multi-contrast images spaced at a time interval of ~1 ms. We have demonstrated the ability of 3D-EPTI to provide robust and repeatable whole-brain simultaneous T1, T2, T2*, PD and B1+ mapping at high isotropic resolution within minutes (e.g., 1-mm isotropic resolution in 3 minutes), and to enable submillimeter multi-parametric imaging to study detailed brain structures.

HighlightsO_LIUltra-fast acquisition for 3D multi-parametric quantitative MRI.
C_LIO_LISimultaneous T1 T2 T2* PD and B1+ mapping.
C_LIO_LI3-minute scan at 1-mm isotropic resolution with whole-brain coverage.
C_LIO_LIMulti-parametric mapping at 700-m isotropic resolution in 10 minutes.
C_LIO_LIRepeatable quantification and cortical-depth analysis.
C_LI ZIKV disrupts placental ultrastructure and drug transporter expression in mice Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome. Fatal neuroinvasion and SARS-CoV-2 tropism in K18-hACE2 mice is partially independent on hACE2 expression Animal models recapitulating distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. The precise mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate pneumonia, rapid clinical decline or death of mice was invariably associated with viral neuroinvasion and direct neuronal injury (including brain and spinal neurons). Neuroinvasion was observed as early as 4 dpi, with virus initially restricted to the olfactory bulb supporting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. No evidence of viremia was detected suggesting neuroinvasion occurs independently of entry across the blood brain barrier. SARS-CoV-2 tropism was not restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), and some ACE2-positive lineages were not associated with the presence of viral antigen (e.g., bronchiolar epithelium and brain capillaries). Detectable ACE2 expression was not observed in neurons, supporting overexpression of ACE2 in the nasal passages and neuroepithelium as more likely determinants of neuroinvasion in the K18-hACE2 model. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis that may have clinical relevance acknowledging the growing body of evidence that suggests COVID-19 may result in long-standing neurologic consequences.

IMPORTANCECOVID-19 is predominantly a respiratory disease caused by SARS-CoV-2 that has infected more than 191 million people with over 4 million fatalities (2021-07-20). The development of animal models recapitulating distinctive features of severe COVID-19 is critical to enhancing our understanding of SARS-CoV-2 pathogenesis and in the evaluation of vaccine and therapeutic efficacy. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. Here, we show lethality of this model is invariably associated with viral neuroinvasion linked with viral replication and assembly. Importantly, pneumonia albeit invariably present was generally moderate with the absence of culturable infectious virus at peak neuroinvasion. The dynamics of viral neuroinvasion and pneumonia were only partially dependent on hACE2. Overall, this study provides an in-depth sequential characterization of the K18-hACE2 model following SARS-CoV-2 infection, highlighting its significance to further study the mechanisms of SARS-CoV-2 neuropathogenesis. Do males know? Evidence-driven rainmaking mating strategy by male bonobos to meet the fertile window of females Female bonobos exhibit prolonged receptivity, one suggested function of which is to lower male mating competition. However, it is questionable whether easier access to receptive females can reduce male-male competition, given the exclusive nature of male reproductive success. We tested whether males could determine the fertile phase of females. We found that ovulation probability predicted male mating effort. High-ranking males copulated with females with higher fertility, and male-male agonistic interactions increased when there were fertile females in the party. When there were multiple females with maximal swelling, males concentrated their mating effort on females with an older infant whose maximal swelling started earlier, and they continued mating efforts until detumescence (rainmaking). These findings suggest that male bonobos distinguish between fertile and non-fertile phases of females and that having more receptive females in the party does not reduce male-male competition for fertile females.

TeaserMales use the rainmakers rule to meet the periovulatory phase of the female bonobo for better reproductive success. The RAG1 Ubiquitin Ligase Domain Enhances Assembly and Selection of T Cell Receptor Genes to Restrain the Autoimmune Hazard of Generating T Cell Receptor Diversity RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte antigen receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. However, this process creates highly self-reactive cells that must be properly selected to suppress autoimmunity. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity in vitro. We report that mice with a mutation that inactivates the RAG1 ubiquitin ligase in vitro exhibit modestly reduced thymic cellularity, decreased assembly and altered repertoires of T cell receptor (TCR) {beta} and  genes in thymocytes, and impaired thymocyte developmental transitions that require the assembly of TCR{beta} or  genes and signaling by their proteins. These RAG1 mutant mice also exhibit less efficient positive selection and superantigen-mediated negative selection of conventional {beta} T cells, 2) impaired differentiation of iNKT lineage {beta} T cells, and 3) CD4+ {beta} T cells with elevated autoimmune potential. Our findings demonstrate that the RAG1 ubiquitin ligase domain functions in vivo to stimulate the assembly and selection of TCR{beta} and TCR genes, thereby establishing replete diversity of {beta} TCRs and {beta} T cell lineages while restraining the inherent autoimmune hazard of generating diverse antigen specificities. An automated workflow for multiplexed single-cell proteomics sample preparation at unprecedented sensitivity. The analysis of single-cell proteomes has recently become a viable complement to transcriptomics and genomics studies. Proteins are the primary driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have lately attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lag. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation at surprising sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE(R), reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1,500 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,600 proteins across 170 multiplexed single cells from two highly similar human cell types. This dedicated loss-less workflow allows distinguishing in vitro co-differentiated cell types of self-organizing cardiac organoids based on indicative markers across 150 single cells. In-depth characterization revealed enhanced cellular motility of cardiac endothelial cells and sarcomere organization in cardiomyocytes. Our versatile and automated sample preparation has not only proven to be easily adaptable but is also sufficiently sensitive to drive biological applications of single-cell proteomics. First detection of herpesvirus and prevalence of mycoplasma infection in free-ranging Hermann s tortoises (Testudo hermanni), and in potential pet vectors AO_SCPLOWBSTRACTC_SCPLOWTwo types of pathogens cause highly contagious upper respiratory tract diseases (URTD) in Chelonians: testudinid herpesviruses (TeHV) and a mycoplasma (Mycoplasma agassizii). In captivity, these infections are frequent and can provoke outbreaks. Pet trade generates international flow of tortoises, often without sanitary checking; individuals intentionally or accidentally released in the wild may spread pathogens. A better understanding of the transmission of infectious agents from captivity to wild tortoises is needed. Many exotic individuals have been introduced in populations of the endangered western Hermanns tortoise (Testudo hermanni hermanni), notably spur-thighed tortoises (Testudo graeca). We assessed the presence of TeHV and mycoplasma in native western Hermanns tortoises and in potential pet vectors in south-eastern France. Using a large sample (N=572 tortoises), this study revealed, by PCR, the worrying presence of herpesvirus in 7 free-ranging individuals (3 sub-populations). Additionally, Mycoplasma agassizii was detected, by PCR, in 15 of the 18 populations sampled with a frequency ranging from 3.4% (1 of 29 tortoises) to 25% (3 of 12 tortoises). Exotic spur-thighed tortoises showed high frequency of mycoplasma infection in captivity (18.2%) and in individuals (50%) found in native Hermanns tortoise sub-populations, suggesting that this species could be a significant vector. The paucity of information of TeHV on European tortoise URTD in natural settings, especially in combination with mycoplasma, prompts for further studies. Indeed, sick tortoises remain concealed and may not be easily detected in the field. Our results indicate a good health for most infected tortoise but it should be screened in the field as well as in captivity. Arg-tRNA synthetase links inflammatory metabolism to RNA splicing and nuclear trafficking via SRRM2 Cells respond to perturbations like inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Here, we found that depletion of arginine during inflammation decreased levels of a nuclear form of arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal protein and nuclear speckle component and that arginine depletion impacted both condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a novel mechanism whereby a tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery. Harnessing changes in open chromatin determined by ATAC-seq to generate insulin-responsive reporter constructs. BackgroundGene regulation is critical for proper cellular function. Next-generation sequencing technology has revealed the presence of regulatory networks that regulate gene expression and essential cellular functions. Studies investigating the epigenome have begun to uncover the complex mechanisms regulating transcription. Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is quickly becoming the assay of choice for many epigenomic investigations. However, whether intervention-mediated changes in accessible chromatin determined by ATAC-seq can be harnessed to generate intervention-inducible reporter constructs has not been systematically assayed.

ResultsWe used the insulin signaling pathway as a model to investigate chromatin regions and gene expression changes using ATAC- and RNA-seq in insulin-treated Drosophila S2 cells. We found correlations between ATAC- and RNA-seq data, especially when stratifying differentially-accessible chromatin regions by annotated feature type. In particular, our data demonstrated a strong correlation between chromatin regions annotated to distal promoters (1-2 kb from the transcription start site) and downstream gene expression. We cloned candidate distal promoter regions upstream of luciferase and demonstrate insulin-inducibility of several of these reporters.

ConclusionsInsulin-induced chromatin accessibility determined by ATAC-seq reveals enhancer regions that drive insulin-inducible reporter gene expression. Recurrent exon-deleting activating mutations in AHR act as drivers of urinary tract cancer Bladder cancer has a high recurrence rate and low survival of advanced stage patients. Few genetic drivers of bladder cancer have thus far been identified. We performed in-depth structural variant analysis on whole-genome sequencing data of 206 metastasized urinary tract cancers. In ~10% of the patients, we identified recurrent in-frame deletions of exons 8 and 9 in the aryl hydrocarbon receptor gene (AHR{Delta}e8-9), which codes for a ligand-activated transcription factor. Pan-cancer analyses show that AHR{Delta}e8-9 is highly specific to urinary tract cancer and mutually exclusive with other bladder cancer drivers. In AHR{Delta}e8-9 the ligand-binding domain is disrupted and we show that this results in ligand-independent AHR-pathway activation. In bladder organoids, AHR{Delta}e8-9 induces a transformed phenotype that is characterized by upregulation of AHR target genes, downregulation of differentiation markers and upregulation of genes associated with stemness and urothelial cancer. Furthermore, AHR{Delta}e8-9 expression results in anchorage independent growth of bladder organoids, indicating tumorigenic potential. DNA-binding deficient AHR{Delta}e8-9 fails to induce transformation, suggesting a role for AHR target genes in the acquisition of the oncogenic phenotype. In conclusion, we show that AHR{Delta}e8-9 is a novel driver of urinary tract cancer and that the AHR pathway could be an interesting therapeutic target. The SLIM1 transcription factor is required for arsenic resistance in Arabidopsis thaliana The transcriptional regulators of arsenic-induced gene expression remain largely unknown. Sulfur assimilation is tightly linked with arsenic detoxification. Here we report that mutant alleles in the SLIM1 transcription factor are substantially more sensitive to arsenic than cadmium. Arsenic treatment caused high levels of oxidative stress in the slim1 mutants, and slim1 alleles were impaired in both thiol and sulfate accumulation. We further found enhanced arsenic accumulation in roots of slim1 mutants. Transcriptome analyses indicate an important role for SLIM1 in arsenic-induced tolerance mechanisms. The present study identifies the SLIM1 transcription factor as an essential component in arsenic tolerance and arsenic-induced gene expression. Our results suggest that the severe arsenic sensitivity of the slim1 mutants is caused by altered redox status. Inflammatory response in hematopoietic stem and progenitor cells triggered by activating SHP2 mutations evokes blood defects Gain-of-function mutations in the protein-tyrosine phosphatase SHP2 are the most frequently occurring mutations in sporadic juvenile myelomonocytic leukemia (JMML) and JMML-like myeloproliferative neoplasm (MPN) associated with Noonan syndrome (NS). Hematopoietic stem and progenitor cells (HSPCs) are the disease propagating cells of JMML. Here, we explored transcriptomes of HSPCs with Shp2 mutations derived from JMML patients and a novel NS zebrafish model. In addition to major NS traits, CRISPR/Cas9 knock-in Shp2D61G mutant zebrafish recapitulated a JMML-like MPN phenotype, including myeloid lineage hyperproliferation, ex vivo growth of myeloid colonies and in vivo transplantability of HSPCs. Single cell mRNA sequencing of HSPCs from Shp2D61G zebrafish embryos and bulk sequencing of HSPCs from JMML patients revealed an overlapping inflammatory gene expression pattern. Strikingly, an anti-inflammatory agent rescued JMML-like MPN in Shp2D61G zebrafish embryos. Our results indicate that a common inflammatory response was triggered in the HSPCs from sporadic JMML patients and syndromic NS zebrafish, which potentiated MPN and may represent a future target for JMML therapies. DENTIST - using long reads for closing assembly gaps at high accuracy BackgroundLong sequencing reads allow increasing contiguity and completeness of fragmented, short-read based genome assemblies by closing assembly gaps, ideally at high accuracy. While several gap closing methods have been developed, these methods often close an assembly gap with sequence that does not accurately represent the true sequence.

FindingsHere, we present DENTIST, a sensitive, highly accurate and automated pipeline method to close gaps in short-read assemblies with long error-prone reads. DENTIST comprehensively determines repetitive assembly regions to identify reliable and unambiguous alignments of long reads to the correct loci, integrates a consensus sequence computation step to obtain a high base accuracy for the inserted sequence, and validates the accuracy of closed gaps. Unlike previous benchmarks, we generated test assemblies that have gaps at the exact positions where real short-read assemblies have gaps. Generating such realistic benchmarks for Drosophila (134 Mb genome), Arabidopsis (119 Mb), hummingbird (1 Gb) and human (3 Gb) and using simulated or real PacBio continuous long reads, we show that DENTIST consistently achieves a substantially higher accuracy compared to previous methods, while having a similar sensitivity.

ConclusionDENTIST provides an accurate approach to improve the contiguity and completeness of fragmented assemblies with long reads. DENTISTs source code including a Snakemake workflow, conda package and Docker container is available at https://github.com/a-ludi/dentist. All test assemblies as a resource for future benchmarking are at https://bds.mpi-cbg.de/hillerlab/DENTIST/. Probing Patterning in Microbial Consortia with picCASO: a Cellular Automaton for Spatial Organisation Microbial consortia exhibit spatial patterning across diverse environments. Since probing the self-organization of natural microbial communities is limited by their inherent complexity, synthetic models have emerged as attractive alternatives. In this study, we develop novel frameworks of bacterial communication and explore the emergent spatiotemporal organization of microbes. Specifically, we build quorum sensing-mediated models of microbial growth that are utilized to characterize the dynamics of communities from arbitrary initial configurations and establish the effectiveness of our communication strategies in coupling the growth rates of microbes. Our simulations indicate that the behavior of quorum sensing-coupled consortia can be most effectively modulated by the rates of secretion of AHLs. Such a mechanism of control enables the construction of desired relative populations of constituent species in spatially organized populations. Our models accurately recapitulate previous experiments that have investigated pattern formation in synthetic multi-cellular systems. Additionally, our software tool enables the easy implementation and analysis of our frameworks for a variety of initial configurations and simplifies the development of sophisticated gene circuits facilitating distributed computing. Overall, we demonstrate the potential of spatial organization as a tunable parameter in synthetic biology by introducing a communication paradigm based on the location and strength of coupling of microbial strains.

Author SummaryInteracting microorganisms that coexist in a given environment tend to have well-defined spatial arrangements. While the emergence of such organization is seen across different microbiomes in nature, it is hitherto not well understood. Decoding the inherent spatial patterning of microbes is constrained by the complexity of their natural habitats. Here, we take advantage of synthetic models of microbial communities to study the dynamics of emergent spatial organization. Our framework of bacterial communication utilizes modular synthetic devices to couple growth rates. In addition to uncovering potential principles of spatial organization, this work enables the construction of complex genetic circuits distributed across communicating strains. Additionally, we have developed a software tool, picCASO, that streamlines the investigation of microbial systems communicating through such frameworks. Trade-offs in biosensor optimization for dynamic pathway engineering Recent progress in synthetic biology allows the construction of dynamic control circuits for metabolic engineering. This technology promises to overcome many challenges encountered in traditional pathway engineering, thanks to their ability to self-regulate gene expression in response to bioreactor perturbations. The central components in these control circuits are metabolite biosensors that read out pathway signals and actuate enzyme expression. However, the construction of metabolite biosensors is a major bottleneck for strain design, and a key challenge is to understand the relation between biosensor dose-response curves and pathway performance. Here we employ multiobjective optimization to quantify performance trade-offs that arise in the design and calibration of metabolite biosensors. Our approach reveals strategies for tuning dose-response curves along an optimal trade-off between production flux and the cost of an increased expression burden on the host. We explore properties of control architectures built in the literature, and identify their advantages and caveats in terms of performance and robustness to growth conditions and leaky promoters. We demonstrate the optimality of a control circuit for glucaric acid production in Escherichia coli, which has been shown to increase titer by 2.5-fold as compared to static designs. Our results lay the groundwork for the automated design of control circuits for pathway engineering, with applications in the food, energy and pharmaceutical sectors. LEA motifs promote desiccation tolerance in vivo BackgroundCells and organisms typically cannot survive in the absence of water. However, there are some notable exceptions, including animals such as nematodes, tardigrades, rotifers, and some arthropods. One class of proteins known to play a role in desiccation resistance is the late embryogenesis abundant (LEA) proteins. These largely disordered proteins protect plants and animals from desiccation. A multitude of studies have characterized stress-protective capabilities of LEA proteins in vitro and in heterologous systems. However, the extent to which LEA proteins exhibit such functions in vivo, in their native contexts in animals, is unclear.

Furthermore, little is known about the distribution of LEA proteins in multicellular organisms or tissue-specific requirements in conferring stress protection.

ResultsTo study the endogenous function of an LEA protein in an animal, we created a true null mutant of C. elegans LEA-1, as well as endogenous fluorescent reporters of the protein. We confirmed that C. elegans lacking LEA-1 are sensitive to desiccation. LEA-1 mutant animals were also sensitive to heat and osmotic stress and were prone to protein aggregation. During desiccation, LEA-1 expression increased and became more widespread throughout the body. LEA-1 was required at high levels in body wall muscle for animals to survive desiccation and osmotic stress. We identified minimal motifs within C. elegans LEA-1 that are sufficient to increase desiccation survival of E. coli. To test whether such motifs are central to LEA-1s in vivo functions, we then replaced the sequence of lea-1 with these minimal motifs and found that C. elegans survived mild desiccation and osmotic stress at the same levels as worms with the full-length protein.

ConclusionsOur results provide insights into the endogenous functions and expression dynamics of an LEA protein in a multicellular animal. The results show that LEA-1 buffers animals from a broad range of stresses. Our identification of LEA motifs that can function in both bacteria and in a multicellular organism suggests the possibility of engineering LEA-1-derived peptides for optimized desiccation protection. Preprints in motion: tracking changes between preprint posting and journal publication during a pandemic Amidst the COVID-19 pandemic, preprints in the biomedical sciences are being posted and accessed at unprecedented rates, drawing widespread attention from the general public, press and policymakers for the first time. This phenomenon has sharpened longstanding questions about the reliability of information shared prior to journal peer review. Does the information shared in preprints typically withstand the scrutiny of peer review, or are conclusions likely to change in the version of record? We assessed preprints from bioRxiv and medRxiv that had been posted and subsequently published in a journal through 30th April 2020, representing the initial phase of the pandemic response. We utilised a combination of automatic and manual annotations to quantify how an article changed between the preprinted and published version. We found that the total number of figure panels and tables changed little between preprint and published articles. Moreover, the conclusions of 7.2% of non-COVID-19-related and 17.2% of COVID-19-related abstracts undergo a discrete change by the time of publication, but the majority of these changes do not qualitatively change the conclusions of the paper. The virucidal effects of 405 nm visible light on SARS-CoV-2 and influenza A virus Germicidal potential of specific wavelengths within the electromagnetic spectrum is an area of growing interest. While ultra-violet (UV) based technologies have shown satisfactory virucidal potential, the photo-toxicity in humans coupled with UV associated polymer degradation limit its use in occupied spaces. Alternatively, longer wavelengths with less irradiation energy such as visible light (405 nm) have largely been explored in the context of bactericidal and fungicidal applications. Such studies indicated that 405 nm mediated inactivation is caused by the absorbance of porphyrins within the organism creating reactive oxygen species which result in free radical damage to its DNA and disruption of cellular functions. The virucidal potential of visible-light based technologies has been largely unexplored and speculated to be ineffective given the lack of porphyrins in viruses. The current study demonstrated increased susceptibility of lipid-enveloped respiratory pathogens of importance such as SARS-CoV-2 (causative agent of COVID-19) as well as the influenza A virus to 405nm, visible light in the absence of exogenous photosensitizers indicating a potential porphyrin-independent alternative mechanism of visible light mediated viral inactivation. These results were obtained using less than expected irradiance levels which are generally safe for humans and commercially achievable. Our results support further exploration of the use of visible light technology for the application of continuous decontamination in occupied areas within hospitals and/or infectious disease laboratories, specifically for the inactivation of respiratory pathogens such as SARS-CoV-2 and Influenza A. The compound of baicalein, wogonin, and oroxylin-A inhibiting EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. The evolution of mechanisms to produce phenotypic heterogeneity in microorganisms In bacteria and other microorganisms, the cells within a population often show extreme phenotypic variation. Different species use different mechanisms to determine how distinct phenotypes are allocated between individuals, including coordinated, random, and genetic determination. However, it is not clear if this diversity in mechanisms is adaptive--arising because different mechanisms are favoured in different environments--or is merely the result of non-adaptive artifacts of evolution. We use theoretical models to analyse the relative advantages of the two dominant mechanisms to divide labour between reproductives and helpers in microorganisms. We show that coordinated specialisation is more likely to evolve over random specialisation in well-mixed groups when: (i) social groups are small; (ii) helping is more "essential"; and (iii) there is a low metabolic cost to coordination. We find analogous results when we allow for spatial structure with a more detailed model of cellular filaments. More generally, this work shows how diversity in the mechanisms to produce phenotypic heterogeneity could have arisen as adaptations to different environments. Transcriptome analysis and connectivity mapping of Cissampelos pareira L. provides molecular links of ESR1 modulation to viral inhibition 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. Ending animal agriculture would stabilize greenhouse gas levels for 30 years and offset 70 percent of CO2 emissions this century Animal agriculture contributes significantly to global warming through ongoing emissions of the potent greenhouse gases methane and nitrous oxide, and displacement of biomass carbon on the land used to support livestock. However, because estimates of the magnitude of the effect of ending animal agriculture often focus on only one factor, the full potential benefit of a more radical change remains underappreciated. Here we quantify the full "climate opportunity cost" of current global livestock production, by modeling the combined, long-term effects of emission reductions and biomass recovery that would be unlocked by a phaseout of animal agriculture. We show that, even in the absence of any other emission reductions, persistent drops in atmospheric methane and nitrous oxide levels, and slower carbon dioxide accumulation, following a phaseout of livestock production would, through the end of the century, have the same cumulative effect on the warming potential of the atmosphere as a 25 gigaton per year reduction in anthropogenic CO2 emissions, providing half of the net emission reductions necessary to limit warming to 2{degrees}C. The magnitude and rapidity of these potential effects should place the reduction or elimination of animal agriculture at the forefront of strategies for averting disastrous climate change.

Significance StatementThe use of animals to produce food has a negative impact on the climate, but the benefits of a global switch to a plant based diet are underappreciated. We show that the global warming impact, through the rest of this century, of eliminating greenhouse gas emissions from livestock and allowing native ecosystems to regrow on the land currently used to house and feed livestock, would be equivalent to a 68% reduction in carbon dioxide emissions. We hope putting clearer numbers on the "climate opportunity cost" of our continued use of animals as food technology will help policymakers and the public properly prioritize dietary change as a climate defense strategy.

Declaration of Conflict of InterestPatrick Brown is the founder and CEO of Impossible Foods, a company developing alternatives to animals in food-production. Michael Eisen is an advisor to Impossible Foods. Both are shareholders in the company and thus stand to benefit financially from reduction of animal agriculture.