Cellular feedback to organotelluranes displays modulation of antioxidant proteins gene expression Organotelluranes RT3 and RT4 are thiol reagents that induce mitochondrial transition pore (MTP) opening in a sensitive and insensitive manner to cyclosporin A. Although RT3 and RT4 promote glutathione depletion, paradoxically, they are also an efficient antioxidant for membrane lipids. These compounds antagonistic effects elicited the challenging question of how the gene expression of antioxidant enzymes would respond to treatment with these compounds. The influence of RT3 and RT4 on antioxidant enzyme expression was investigated in cultured aortic smooth muscle cells (ASMC). RT3 and RT4 promoted disruption of ionic calcium homeostasis, mitochondrial transmembrane potential ({Delta}{Psi}), and cell death in a dose-dependent manner. The cell death mechanisms responded qualitatively to the increase of the organotellurane concentration and changed from apoptosis to necrosis. RT3 and RT4 increased the expression of thioredoxin significantly. RT3 also increased the expression of glutaredoxin and glutathione peroxidase, slightly the catalase expression without significant effects on SOD expression. The results are consistent with GSH and protein thiol depletion and discussed based on the cell toxicity mechanism exhibited by these compounds.



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Medium-throughput zebrafish optogenetic platform identifies deficits in subsequent neural activity following brief early exposure to cannabidiol and Δ-9-tetrahydrocannabinol In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development.
Maternal Western-Style Diet Impairs Bone Marrow Development and Drives a Hyperinflammatory Phenotype in Hematopoietic Stem and Progenitor Cells in Fetal Rhesus Macaques BackgroundMaternal obesity adversely impacts the in utero metabolic environment and offsprings health, but its effect on fetal hematopoiesis and immune cell development remains incompletely understood, particularly in models that resemble human development.

MethodsWe studied gestational day 130-135 fetuses derived from rhesus macaque dams chronically exposed to a high-fat Western-style diet (WSD) or a low-fat control diet. Fetal immune cell phenotypes and fetal bone marrow architecture and hematopoietic stem and progenitor cell (FBM HSPC) function were examined using bone computed tomography, histology, flow cytometry, single-cell RNA-sequencing, and HSPC transplantation assays.

FindingsMaternal WSD induced premature FBM cavity opening and a codominant increase in the number of FBM adipocytes. Furthermore, a maternal WSD induced a proinflammatory transcriptional response in FBM HSPCs. FBM macrophages from the WSD group exhibited heightened proinflammatory responses to toll-like receptor agonist stimulation. Maternal WSD exposure suppressed the expression of genes required for B-cell development and decreased the frequencies of FBM B-cells. Finally, maternal WSD led to poor engraftment of FBM HSPCs in nonlethally irradiated immunodeficient NOD/SCID/IL2r{gamma}-/- mice.

InterpretationsMaternal WSD impairs FBM development, drives a hyperinflammatory phenotype, and induces functional and differentiation impairment in FBM HSPCs in a translationally relevant nonhuman primate model.

FundingNational Institute of Health

RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSMaternal obesity is associated with increased risk of infections and proinflammatory disease in offspring. The translationally-relevant rhesus macaque model was utilized to address the effects of maternal obesogenic diet on fetal hematopoietic and immune cell development.

Added value of this studyWe assessed changes in fetal immune cell phenotypes and fetal hematopoietic stem and progenitor cell function using immunohistochemistry, flow cytometry, single-cell RNA sequencing, and transplantation assays. We determined that chronic consumption of a maternal obesogenic diet induced the development of adipogenic and proinflammatory environments in the fetal bone marrow. Additionally, we detected the impairment in B-cell differentiation program in fetal hematopoietic stem and progenitor cells.

Implications of all the available evidenceThese data demonstrate that maternal obesogenic diet modulates fetal hematopoietic development and could impact the offsprings immune system, including proinflammatory phenotype and a decline in B-cell function. Darwin's small and medium ground finches might have taste preferences, but not for human foods Urbanization is rapidly changing ecological niches. On the inhabited Galapagos Islands, Darwins finches consume human-introduced foods preferentially; however, it remains unclear why. Here we presented pastry with flavour profiles typical of human foods (oily, salty, sweet) to small ground finches (Geospiza fuliginosa) and medium ground finches (Geospiza fortis) to test if latent taste preferences might drive selection of human foods. If human-food flavours were consumed more than a neutral or bitter control only at sites with human foods, then we predicted tastes were acquired after urbanization; however, if no site-differences were found then this would indicate latent taste preferences. Contrary to both predictions, we found little evidence that human-food flavours were preferred compared to control flavours at any site. Instead, finches showed a weak aversion to oily foods, but only at remote (no human foods present) sites. This was further supported by behavioural responses, with beak wiping occurring more often at remote sites after finches tasted flavours associated with human foods. Our results suggest, therefore, that while Darwins finches may have acquired a tolerance to the flavours of human food, latent taste preferences are unlikely to have played a major role in their dietary response to increased urbanization. ksrates: positioning whole-genome duplications relative to speciation events in KS distributions SummaryWe present ksrates, a user-friendly command-line tool to position ancient whole-genome duplication (WGD) events with respect to speciation events in a phylogeny by comparing paralog and ortholog KS distributions derived from genomic or transcriptomic sequences, while adjusting for substitution rate differences among the lineages involved.

Availability and implementationksrates is implemented in Python 3 and as a Nextflow pipeline. The source code, Singularity and Docker containers, documentation and tutorial are available via https://github.com/VIB-PSB/ksrates.

Contactsteven.maere@ugent.vib.be, rolf.lohaus@ugent.vib.be The extracellular loops of OmpA control the slow rate of in vitro folding Outer membrane proteins are all beta barrels and these barrels have a variety of well-documented loop conformations. Here we test the effect of three different loop types on outer membrane protein A (OmpA) folding. We designed twelve 5-residue loops and experimentally tested the effect of replacing the long loops of outer membrane protein OmpA with the designed loops. Our studies succeeded in creating the smallest known outer membrane barrel. We find that significant changes in OmpA loops do not have a strong overall effect on OmpA folding. However, when decomposing folding into a fast rate and a slow rate we find that changes in loops strongly affect the slow rate of OmpA folding. Extracellular loop types with higher levels of hydrogen bonds had more instances of increasing the slow folding rate and extracellular loop types with low levels of hydrogen bonds had more instances of decreasing the slow folding rate. Having the slow rate affected by loop composition is consistent with the slow rate being associated with the insertion step of outer membrane protein folding. 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. Sablefish (Anoplopoma fimbria Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions. Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression BackgroundChimeric antigen receptor (CAR) T cells traditionally harbor viral vector-based sequences that encode the CAR transgene in the genome. These T cell products have yet to show consistent anti-tumor activity in patients with solid tumors. Further, viral vector manufacturing is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures.

MethodsAnti-GD2 CAR T cells were generated using CRISPR/Cas9 within nine days using recombinant Cas9 protein and nucleic acids, without any viral vectors or animal components. The CAR was specifically targeted to the T Cell Receptor Alpha Constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, scRNA-seq, spectral cytometry, and ELISA assays. Functionality was evaluated in vivo in an NSG xenograft neuroblastoma model.

ResultsIn comparison to traditional retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory phenotype, and low tonic signaling prior to infusion arising in part from the the knockout of the TCR. Upon exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibited specific cytotoxicity against GD2+ cells in vitro and induced solid tumor regression in vivo, with robust homing, persistence, and low exhaustion against a human neuroblastoma xenograft model.

ConclusionsThis proof-of-principle study leveraging virus-free genome editing technology could enable flexible manufacturing of clinically relevant, high-quality CAR T cells to treat cancers, including solid tumors. Maternal microbiota Bifidobacterium promotes placental morphogenesis, nutrient transport and fetal growth in mice The gut microbiota plays a central role in regulating host metabolism. However, while substantial progress has been made in discerning how the microbiota influences host functions post birth and beyond, little is known about how key members of the maternal gut microbiota can influence feto-placental growth. Here, using germ-free and specific-pathogen-free mice, we demonstrate that the bacterium Bifidobacterium breve UCC2003 modulates maternal body adaptations, placental structure and nutrient transporter capacity, with implications for fetal metabolism and growth. The effects of B. breve UCC2003 on feto-placental growth are mediated, in part, by changes in the maternal and placental metabolome (i.e. acetate and carnitine). Histological analysis of the placenta confirmed that Bifidobacterium modifies placental structure via changes in Igf2P0, Dlk1, Mapk1 and Mapk14 expression. Additionally, B. breve UCC2003, acting through Slc2a1 and Fatp1-4 transporters, was shown to restore fetal glycaemia and fetal growth in association with changes in the fetal hepatic transcriptome. This study provides knowledge towards a novel and safe therapeutic strategy for treating pregnancy disorders via modulation of the maternal gut microbiota. Robust retrieval of data stored in DNA by de Bruijn graph-based de novo strand assembly DNA data storage is a rapidly developing technology with great potential due to its high density, long-term durability, and low maintenance cost. The major technical challenges include various errors, such as the strand breaks, rearrangements, and indels that frequently arise during DNA synthesis, amplification, sequencing, and preservation. Through a de novo assembly strategy, we developed an algorithm based on the de Bruijn graph and greedy path search (DBGPS) to address these issues. DBGPS shows distinct advantages in handling DNA breaks, rearrangements, and indels. The robustness of DBGPS is demonstrated by accelerated aging, multiple independent data retrievals, deep error-prone PCR, and large data scale simulations. Remarkably, 6.8 MB of data can be retrieved accurately from a seriously corrupted sample that has been treated at 70 {degrees}C for 70 days. With DBGPS, we were able to achieve a logical density of 1.30 bits/cycle and a physical density of 295 PB/g.

One-Sentence SummaryA de Bruijn graph-based de novo assembly algorithm for DNA data storage enables fast and robust data readouts even with DNA samples that have been severely corrupted. Arabidopsis AtPME2 has a pH-dependent processivity and control cell wall mechanical properties Pectin methylesterases (PMEs) modify homogalacturonans (HG) chemistry and play a key role in regulating primary cell wall mechanical properties. How PME activity can fine-tune pectin structure in the growing plant has remained elusive. Here we report on the Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum suitable for detecting recombinant and native enzyme independent of species source. At neutral pH AtPME2 is preferentially active on pectins with a degree of 55-70% methylesterification and can be inhibited by PME inhibitor protein (PMEI). We show that the mode of action for AtPME2 can switch from full processivity (at pH 8), creating large blocks of unmethylated galacturonic acid, to low processivity (at pH 5) and relate these observations to the differences in electrostatic potential of the protein at acidic and alkaline pH. To assess the role of AtPME2 in development, we characterized two knock-out lines. We show that in the context of acidified apoplast, low-processive demethylesterification by AtPME2 can loosen the cell wall, with consequent increase in cell elongation and etiolated hypocotyl length. Our study brings insights into how the pH-dependent regulation by PME activity could affect pectin structure and associated cell wall mechanical properties in expansion.

One sentence summaryThe processivity of AtPME2, a pectin methylesterase that fine-tunes cell wall pectins is modulated by pH in vitro and impacts the mechanical properties of the wall, affecting development in planta. The balance between toxic versus nontoxic microRNAs determines platinum sensitivity in ovarian cancer Numerous micro(mi)RNAs (short noncoding RNAs that negatively regulate gene expression) have been linked to platinum (Pt) sensitivity and resistance in ovarian cancer (OC). miRNA activity occurs when the guide strand of the miRNA, with its seed sequence (pos. 2-7/8), is loaded into the RNA induced silencing complex (RISC) and targets complementary short seed matches in the 3 untranslated region of mRNAs. Toxic seeds, targeting genes critical for cancer cell survival, have been found in tumor suppressive miRNAs. Many si- and shRNAs can also kill cancer cells via toxic seeds, the most toxic carrying G-rich 6mer seed sequences. We now show that treatment of OC cells with Pt leads to an increase in RISC-bound miRNAs carrying toxic 6mer seeds and a decrease in miRNAs with nontoxic seeds. Pt-resistant cells did not exhibit this toxicity shift but retained sensitivity to cell death mediated by siRNAs carrying toxic 6mer seeds. Analysis of RISC-bound miRNAs in OC patients revealed that the ratio between miRNAs with toxic versus miRNAs with nontoxic seeds was predictive of treatment outcome. Application of the 6mer seed toxicity concept to cancer relevant miRNAs provides a new framework for understanding and predicting cancer therapy responses. Network-level modeling of stimulation-induced functional connectivity change: An optogenetic study in non-human primate cortex As aberrant network-level functional connectivity underlies a variety of neural disorders, the ability to induce targeted functional reorganization would be a profound development towards therapies for neural disorders. Brain stimulation has been shown to alter large-scale network-wide functional connectivity, but the mapping from stimulation to the modification is unclear. Here, we leverage advances in neural interfaces, interpretable machine learning, and graph theory to arrive at a model which accurately predicts stimulation-induced network-wide functional reorganization. The model jointly considers the stimulation protocol and the cortical network structure, departing from the standard approach which only considers the stimulation protocol. We validate our approach in the primary sensorimotor cortex of non-human primates using paired optogenetic stimulation through a large-scale optogenetic interface. We observe that the stimulation protocol only predicts a small portion of the induced functional connectivity changes while the network structure predicts much more, indicating that the underlying network is the primary mediator of the response to stimulation. We extract the relationships linking the stimulation and network characteristics to the functional connectivity changes and observe that the mappings diverge over frequency bands and successive stimulations. Finally, we uncover shared processes governing real-time and longer-term effects of stimulation. Our framework represents a paradigm shift for targeted neural stimulation and can be used to interrogate, improve, and develop stimulation-based interventions for neural disorders.

TeaserBrain stimulation rewires the brain, but the pre-existing network structure of the brain controls the rewiring. Genetic coupling of life-history and aerobic performance in Atlantic salmon A better understanding of the genetic and phenotypic architecture underlying life-history variation is a longstanding aim in biology. Theories suggest energy metabolism determines life-history variation by modulating resource acquisition and allocation trade-offs, but the genetic underpinnings of the relationship and its dependence on ecological conditions have rarely been demonstrated. The strong genetic determination of age-at-maturity by two unlinked genomic regions (vgll3 and six6) makes Atlantic salmon (Salmo salar) an ideal model to address these questions. Using more than 250 juveniles in common garden conditions, we quantified the covariation between metabolic phenotypes -standard and maximum metabolic rates (SMR and MMR), and aerobic scope (AS) - and the life-history genomic regions and tested if food availability modulates the relationships. We found that the early maturation genotype in vgll3 was associated with higher MMR and consequently AS. Additionally, MMR exhibited physiological epistasis; it was decreased when late maturation genotypes co-occurred in both genomic regions. Contrary to our expectation, the life-history genotypes had no effects on SMR. Further, food availability had no effect on the genetic covariation, suggesting a lack of genotype-by-environment interactions. Our results provide insights on the key organismal processes that link energy use at the juvenile-stage to age-at-maturity, indicating potential mechanisms by which metabolism and life-history can coevolve. Decoding regulatory specificity of human ribosomal proteins Human ribosomes, made of around 80 ribosomal proteins (RPs) and four ribosomal RNAs, have long been thought as uniform passive protein-making factories with little regulatory function. Recently, accumulating evidence showed heterogeneity of RP composition in ribosomes responsible for regulating gene expression in development and tumorigenesis. However, a comprehensive understanding of regulatory spectrum of RPs is still lacking. In this study, we conducted a systematic survey of regulatory specificity of human RPs on global gene expression. We quantified translational and transcriptional changes of gene expression upon deficiency of 75 RPs, including 44 from the large subunit (60S) and 31 from the small subunit (40S), by ribosomal profiling and RNA sequencing analysis. We showed that RP deficiency induced diverse expression changes, particularly at the translational level. RPs were subjected to co-translational regulation under ribosomal stress where deficiency of the 60S and the 40S RPs had opposite effects on the two subunits. RP deficiency perturbed expression of genes related to cell cycle, cellular metabolism, signal transduction and development. Deficiency of RPs from the 60S led to a greater repression effect on cell growth than that from the 40S by perturbing P53 signaling and cell cycle pathways. To demonstrate functional specificity of RPs, we showed that RPS8 deficiency stimulated cellular apoptosis and RPL13 or RPL18 deficiency promoted cellular senescence. We also showed that RPL11 and RPL15 played important roles in retina development and angiogenesis, respectively. Overall, our study demonstrated a widespread regulatory role of RPs in controlling cellular activity, providing an important resource which can offer novel insights into ribosome regulation in human diseases and cancer. 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. Substrate specialization in microbes is driven by biochemical constraints of dynamic flux sensing Central carbon metabolism is highly conserved across microbial species, but can catalyze very different pathways depending on the organism and their ecological niche. Here, we study the dynamic re-organization of central metabolism after switches between the two major opposing pathway configurations of central carbon metabolism, glycolysis and gluconeogenesis in Escherichia coli, Pseudomonas aeruginosa and Pseudomonas putida. We combined growth dynamics and dynamic changes of intracellular metabolite levels with a coarse-grained model that integrates fluxes, regulation, protein synthesis and growth and uncovered fundamental limitations of the regulatory network: after nutrient shifts, metabolite concentrations collapse to their equilibrium, rendering the cell unable to sense which direction the flux is supposed to flow through the metabolic network. The cell can partially alleviate this by picking a preferred direction of regulation at the expense of increasing lag times in the opposite direction. Moreover, decreasing both lag times simultaneously comes at the cost of reduced growth rate or higher futile cycling between metabolic enzymes. These three trade-offs can explain why microorganisms specialize for either glycolytic or gluconeogenic substrates and can help elucidate the complex growth patterns exhibited by different microbial species.

Graphical synopsis O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

Standfirst textMicrobes face a series of fundamental trade-offs that limit their ability to optimize simultaneously for both glycolytic and gluconeogenic growth.

Bullet pointsO_LILag times between glycolysis and gluconeogenesis show asymmetry in many microbes: A long lag in one direction, but a short lag in the other.
C_LIO_LILong lag times are caused by an inability to sense fluxes after nutrient shifts.
C_LIO_LIWith existing regulation, lag time asymmetry can only be overcome by reducing either growth rate or increasing futile cycling in metabolism.
C_LI ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intranasal hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lungs. RNA-Seq analyses illustrated that the model involves an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. Intranasal hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mice lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.

AUTHOR SUMMARYSARS-CoV-2 uses the human ACE2 (hACE2) receptor to infect cells, but cannot infect mice because the virus cannot bind mouse ACE2 (mACE2). We use an ACE2-lentivirus system in vitro to identify four key amino acids in mACE2 that explain why SARS-CoV-2 cannot infect mice. hACE2-lentivirus was used to express hACE2 in mouse lungs in vivo, with the inflammatory responses after SARS-CoV-2 infection similar to those seen in human COVID-19. Genetically modified mice were used to show that type I and III interferon signaling is required for the inflammatory responses. We also show that the hACE2-lentivirus mouse model can be used to test vaccines. Overall this paper demonstrates that our hACE2-lentivirus system has multiple applications in SARS-CoV-2 and COVID-19 research. Population structure analysis and laboratory monitoring of Shigella with a standardised core-genome multilocus sequence typing scheme The laboratory surveillance of bacillary dysentery is based on a standardised Shigella typing scheme that classifies Shigella strains into four serogroups and more than 50 serotypes on the basis of biochemical tests and lipopolysaccharide O-antigen serotyping. Real-time genomic surveillance of Shigella infections has been implemented in several countries, but without the use of a standardised typing scheme. We studied over 4,000 reference strains and clinical isolates of Shigella, covering all serotypes, with both the current serotyping scheme and the standardised EnteroBase core-genome multilocus sequence typing scheme (cgMLST). The Shigella genomes were grouped into eight phylogenetically distinct clusters, within the E. coli species. The cgMLST hierarchical clustering (HC) analysis at different levels of resolution (HC2000 to HC400) recognised the natural groupings for Shigella. By contrast, the serotyping scheme was affected by horizontal gene transfer, leading to a conflation of genetically unrelated Shigella strains and a separation of genetically related strains. The use of this cgMLST scheme will enhance the laboratory surveillance of Shigella infections. LPS-TLR4 pathway exaggerates alcohol-induced acute-on-chronic liver failure via provoking NETs formation Backgrounds: Intrahepatic infiltration of neutrophils is a character of alcoholic acute-on-chronic liver failure (AACLF) and neutrophil extracellular traps (NETs) are an important strategy for neutrophils to fix and kill invading microorganisms. Intestinal bacteria and the gut-liver axis have been thought to play a key role in many liver diseases also including AACLF. However, whether NETs appear in AACLF and play a role in AACLF is still unsure. Methods: WT, NE KO, and TLR4 KO mice were used to build the AACLF model, and the intestinal bacteria were eliminated at the same time and LPS was given. Then the formation of NETs and AACLF related markers were detected. Results: The serum MPO-DNA and LPS concentration was increased in AACLF patients and a correlation was revealed between these two indexes. More intrahepatic NETs formed in AACLF mice by testing MPO-DNA, Cit H3, and NE. These markers decreased with gut detergent and restored markers with gut detergent plus LPS supplement. While NETs formation failed to change with gut microbiome or combine LPS supplement in TLR4 KO mice. As we tested AACLF related characters, liver injury, intrahepatic fat deposition, inflammation, and fibrosis alleviated with depletion of NE. These related marks were also attenuated with gut sterilization by antibiotics and recovered with combined treatment with antibiotics plus LPS. But the liver injury, intrahepatic fat, fibro deposition, and liver inflammation-related markers did show a significant difference in TLR4 KO mice when they received the same treatment. Conclusion: Intestinal-derived LPS promotes NETs formation in AACLF through the TLR4 pathway and further accelerates the AACLF process by NETs. Live Analysis of Position Effect Variegation (PEV) in Drosophila Reveals Different Modes of Action for HP1a and Su(var)3-9 AO_SCPLOWBSTRACTC_SCPLOWPosition Effect Variegation (PEV) results from the juxtaposition of euchromatic and heterochromatic components of eukaryotic genomes, silencing genes near the new euchromatin/heterochromatin junctions. Silencing is itself heritable through S phase, giving rise to distinctive random patterns of cell clones expressing the genes intermixed with clones in which the genes are silenced. Much of what we know about epigenetic inheritance in the soma stems from work on PEV aimed at identifying the components of the silencing machinery and its mechanism of inheritance. The roles of two central gene activities - the Su(var)3-9-encoded histone H3-Lysine-9 methyltransferase and the Su(var)205-encoded methyl-H3-Lysine-9 binding protein HP1a - have been inferred from terminal phenotypes, leaving considerable gaps in understanding how PEV behaves through development. Here, we investigate the PEV phenotypes of Su(var)3-9 and Su(var)205 mutations in live developing tissues. We discovered that mutation in Su(var)205 compromises the initial establishment of PEV in early embryogenesis. Later gains of heterochromatin-induced gene silencing are possible, but are unstable and lost rapidly. In contrast, a strain with mutation in Su(var)3-9 exhibits robust silencing early in development, but fails to maintain it through subsequent cell divisions. Our analyses show that while the terminal phenotypes of these mutations may appear identical, they have arrived at them through different developmental trajectories. We discuss how our findings expand and clarify existing models for epigenetic inheritance of heterochromatin-induced gene silencing.

SO_SCPLOWIGNIFICANCEC_SCPLOWCurrent concepts of epigenetic inheritance are exemplified by Position Effect Variegation, a phenomenon whereby heterochromatin can repress genes in clonal cell lineages. Heterochromatin is required for genome protection as it silences toxic transposable elements and prevents instability of repeat sequences. Histone H3 modified by methylation of Lysine-9 and HP1a are critical components of heterochromatin. Using live cell analysis of PEV in mutants in strains with mutations in Su(var)3-9, which encodes the histone methyltransferase, and Su(var)205, which encodes HP1a, we describe an unexpected dynamism in PEV, challenging current models of epigenetics, and revealing unexpectedly different modes of action of these two fundamental components of heterochromatin. Trait-based responses to land use and canopy dynamics modify long-term diversity changes in forest understories AimLand use is the foremost cause of global biodiversity decline, but species do not respond equally to land-use practices. Instead, it is suggested that responses vary with species traits, but long-term data on the trait-mediated effects of land-use on communities is scarce. Here we study how forest understory communities have been affected by two land-use practices during 4-5 decades, and whether changes in plant diversity are related to changes in functional composition.

LocationFinland

Time period1968-2019

Major taxa studiedVascular plants

MethodsWe resurveyed 245 vegetation plots in boreal herb-rich forest understories, and used hierarchical Bayesian linear models to relate changes in diversity, species composition, average plant size, and leaf economic traits to reindeer abundance, forest management intensity, and changes in climate, canopy cover and composition.

ResultsIntensively managed forests decreased in species richness and had increased turnover, but management did not affect functional composition. Increased reindeer densities corresponded with increased leaf dry matter content, evenness and diversity, and decreased height and specific leaf area. Successional development in the canopy was associated with increased specific leaf area and decreased leaf dry matter content and height in the understory over the study period. Effects of reindeer abundance and canopy density on diversity were partially mediated by vegetation height, which had a negative relationship with evenness. Observed changes in climate had no discernible effect on any variable.

Main conclusionsFunctional traits are useful in connecting vegetation changes to the mechanisms that drive them, and provide unique information compared to turnover and diversity metrics. These trait-dependent selection effects could inform whether species benefit or suffer from land use changes and explain observed biodiversity changes under global change. The C. elegans gonadal sheath Sh1 cells extend asymmetrically over a differentiating germ cell population in the proliferative zone The C. elegans adult hermaphrodite germ line is surrounded by a thin tube formed by somatic sheath cells that support germ cells as they mature from the stem-like mitotic state through meiosis, gametogenesis and ovulation. Recently, we discovered that the distal-most Sh1 sheath cells associate with mitotic germ cells as they exit the niche. Here we report that these distal sheath-associated germ cells differentiate first in animals with temperature-sensitive mutations affecting germ cell state, and stem-like germ cells are maintained distal to the Sh1 boundary. We analyze several markers of the distal sheath, which is best visualized with endogenously tagged membrane proteins, as overexpressed fluorescent proteins fail to localize to distal membrane processes and can cause gonad morphology defects. However, such reagents with highly variable expression can be used to determine the relative positions of the two Sh1 cells, one of which often extends further distal than the other. Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using HDX-MS, we observed dynamic behavior of the major five-fold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

TeaserCryo-EM and HDX-MS analysis of an encapsulin nanocompartment shows that the pores at the five-fold icosahedral vertex of the shell are flexible. Increased PHOSPHO1 expression mediates cortical bone mineral density in renal osteodystrophy Patients with advanced chronic kidney disease (CKD) often present with skeletal abnormalities; a condition known as renal osteodystrophy (ROD). While Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are recognized to be critical for bone mineralization, their role in the etiology of ROD is unclear. To address this, ROD was induced in both wild-type and Phospho1 knockout (P1KO) mice using dietary adenine supplementation. The mice presented with hyperphosphatemia, hyperparathyroidism, and elevated levels of FGF23 and bone turnover markers. In particular, we noted that in CKD mice, bone mineral density (BMD) was increased in cortical bone (p < 0.05) but decreased in trabecular bone (p < 0.05). These changes were accompanied by decreased TNAP (p < 0.01) and increased PHOSPHO1 (p < 0.001) expression in wild-type CKD bones. In P1KO CKD mice, the cortical BMD phenotype was rescued, suggesting that the increased cortical BMD of CKD mice was driven by increased PHOSPHO1 expression. Other structural parameters were also improved in P1KO CKD mice. We further investigated the driver of the mineralization defects, by studying the effects of FGF23, PTH, and phosphate administration on PHOSPHO1 and TNAP expression by primary murine osteoblasts. We found both PHOSPHO1 and TNAP expression to be down-regulated in response to phosphate and PTH. While matrix mineralization was increased with phosphate (Pi), it decreased with PTH and FGF23 had no effect. The in vitro data suggest that the TNAP reduction in CKD-MBD is driven by the hyperphosphatemia and/or hyperparathyroidism noted in these mice, while the higher PHOSPHO1 expression may be a compensatory mechanism in an attempt to protect the bone from hypomineralization. We propose that increased PHOSPHO1 expression in ROD may contribute to the disordered skeletal mineralization characteristic of this progressive disorder.