Continuous Theta Burst Stimulation to the Secondary Visual Cortex at 80% Active Motor Threshold Does Not Impair Central Vision in Humans During a Simple Detection Task Continuous theta burst stimulation (cTBS) is a powerful form of repetitive transcranial magnetic stimulation capable of suppressing cortical excitability for up to 50 min. A growing number of studies have applied cTBS to the visual cortex in human subjects to investigate the neural dynamics of visual processing, but few have specifically examined its effects on central vision, which has crucial implications for safety and inference on downstream cognitive effects. The present study assessed the safety of offline, neuronavigated cTBS to V2 by examining its effects on central vision performance. In this single-blind, randomized sham-controlled, crossover study, 17 healthy adults received cTBS (at 80% active motor threshold) and sham to V2 1-2 weeks apart. Their central vision ([&le;]8{degrees}) was tested at 1-min (T1) and again at 50-min (T50) post-stimulation. Effects of condition (cTBS vs. sham) and time (T1 vs. T50) on accuracy and reaction time were examined using Bayes factor. Bayes factor results suggested that cTBS did not impair stimulus detection over the entire central visual field nor subfields at T1 or T50. Our results offer the first explicit evidence supporting that cTBS applied to V2 does not create blind spots in the central visual field in humans during a simple detection task. Any subtler changes to vision and downstream visual perception should be investigated in future studies.
Direct neural evidence for the contrastive roles of the complementary learning systems in adult acquisition of native vocabulary The Complementary Learning Systems (CLS) theory provides a powerful framework for considering the acquisition, consolidation and generalisation of new knowledge. We tested this proposed neural division of labour in adults through an investigation of the consolidation and long-term retention of newly-learned native vocabulary with post-learning functional neuroimaging. Newly-learned items were compared to two conditions: (i) previously known items to highlight the similarities and differences with established vocabulary; and (ii) unknown/untrained items to provide a control for non-specific perceptual and motor-speech output. Consistent with the CLS, retrieval of newly-learned items was supported by a combination of regions associated with episodic memory (including left hippocampus) and the language-semantic areas that support established vocabulary (left inferior frontal gyrus and left anterior temporal lobe). Furthermore, there was a shifting division of labour across these two networks in line with the items consolidation status; faster naming was associated with more activation of language-semantic areas and lesser activation of episodic memory regions. Hippocampal activity during naming predicted more than half the variation in naming retention six months later.
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.



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org.highwire.dtl.DTLVardef@b92cborg.highwire.dtl.DTLVardef@1d261f7org.highwire.dtl.DTLVardef@11da73eorg.highwire.dtl.DTLVardef@1cef6ca_HPS_FORMAT_FIGEXP  M_FIG C_FIG Glutathione regulates transcriptional activation of iron transporters via S-nitrosylation of bHLH factors to modulate subcellular iron homeostasis While glutathione (GSH) is known to regulate iron (Fe) deficiency response in plants, its involvement in modulating subcellular Fe homeostasis remained elusive. In this study, we report that the GSH depleted mutants, cad2-1 and pad2-1 displayed increased sensitivity to Fe deficiency with significant down-regulation of the vacuolar Fe exporters, AtNRAMP3 and AtNRAMP4 and the chloroplast Fe importer, AtPIC1. Moreover, the pad2-1 mutant accumulated higher Fe content in vacuoles and lower in chloroplasts compared with Col-0 under Fe limited condition. Exogenous GSH treatment could enhance the chloroplast Fe content in Col-0 plants but failed to do so in the nramp3nramp4 double mutant demonstrating the role of GSH in modulating subcellular Fe homeostasis. Pharmacological experiments, mutant analysis and promoter assay revealed that this regulation involved transcriptional activation of the transporter genes by a GSH-GSNO module. The Fe responsive bHLH transcription factors (TFs), AtbHLH29, AtbHLH38 and AtbHLH101 were identified to interact with the promoters of these genes which were in turn activated via S-nitrosylation. Together, the present study delineates the role of GSH-GSNO module in regulating subcellular Fe homeostasis by transcriptional activation of the Fe transporters AtNRAMP3, AtNRAMP4 and AtPIC1 via S-nitrosylation of the bHLH TFs during Fe deficiency.

Summary statementGlutathione regulates subcellular iron homeostasis under iron deficiency via GSNO dependent transcriptional activation of AtNRAMP3, AtNRAMP4 and AtPIC1 genes by S-nitrosylation of the iron responsive bHLH transcription factors, AtbHLH29, AtbHLH38 and AtbHLH101. Characterization of methylation profiles in spontaneous preterm birth placental villous tissue Preterm birth is a global public health crisis which results in significant neonatal and maternal mortality. Yet little is known regarding the molecular mechanisms of idiopathic spontaneous preterm birth, and we have few diagnostic markers for adequate assessment of placental development and function. Previous studies of placental pathology and our transcriptomics studies suggest a role for placental maturity in idiopathic spontaneous preterm birth. It is known that placental DNA methylation changes over gestation. We hypothesized that if placental hypermaturity is present in our samples, we would observe a unique idiopathic spontaneous preterm birth DNA methylation profile potentially driving the gene expression differences we previously identified in our placental samples. Our results indicate the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern suggesting hypermaturity. Only seven significant differentially methylated regions fitting the idiopathic spontaneous preterm birth specific (relative to the controls) profile were identified, indicating unusually high similarity in DNA methylation between idiopathic spontaneous preterm birth and term birth samples. We identified an additional 1,718 significantly methylated regions in our gestational age matched controls were the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern, again indicating a striking level of similarity between the idiopathic spontaneous preterm birth and term birth samples. Pathway analysis of these regions revealed differences in genes within the WNT and Cadherin signaling pathways, both of which are essential in placental development and maturation. Taken together, these data demonstrate that the idiopathic spontaneous preterm birth samples are molecularly more mature than expected given their respective gestational age which likely impacts birth timing. Feasibility of Administering Human Pancreatic Cancer Chemotherapy in a Spontaneous Pancreatic Cancer Mouse Model BackgroundBoth modified FOLFIRINOX (mFFX) and gemcitabine/nab-paclitaxel chemotherapy regimens have been shown to improve clinical outcomes in patients with pancreatic cancer, and are often used interchangeably as the standard of care. Preclinical studies often do not use these regimens, since administering these multiagent approaches can be difficult. In this study, we assessed the feasibility of administering these two chemotherapy regimens in spontaneous pancreatic tumors using KPC mice with the ultimate goal of advancing preclinical studies.

MethodsKPC mice were created by breeding KrasLSL-G12D/+ to Trp53fl/fl;Ptf1Cre/+, resulting in KrasLSL-G12D/+;p53fl/+;Ptf1Cre/+ mice. At 14 weeks of age, mice were palpated for spontaneous tumor growth that was verified using ultrasounds. Mice with tumors under 15 mm in diameter were used. The mice were assigned to one of seven treatment regimens: 1 cycle of mFOLFIRINOX (FFX X1), 2 cycles of mFOLFIRINOX (FFX X2), 1 cycle of mFOLFIRINOX with 40Gy SBRT (FFX SBRT), 1 cycle of gemcitabine/nab-paclitaxel (GEM/AB X1), 2 cycles of gemcitabine/nab-paclitaxel (GEM/AB X2), 2 cycles of gemcitabine/nab-paclitaxel with 40Gy SBRT (GEM/AB SBRT), or no chemotherapy treatment (control).

ResultsIn total, 92 mice were included. The median OS in the FFX X2 group was slightly longer that the median OS in the FFX X1 group (15 days vs 11 days, P=0.003). Mice in the GEM/AB X2 group had longer OS when compared to mice in the GEM/AB X1 group (33.5 vs 13 days, P=0.001). Mice treated with chemotherapy survived longer than untreated control animals (median OS: 6.5 days, P <0.001). Moreover, in mice treated with chemotherapy, mice that received 2 cycles of GEM/AB X2 had the longest survival, while the FFX X1 group had the poorest OS (P <0.001). Lastly, chemotherapy followed by consolidative SBRT trended towards increased local control and survival.

ConclusionsWe demonstrate the utility and feasibility of clinicall relevant mFOLFIRINOX and gemcitabine/nab-paclitaxel in preclinical models of pancreatic cancer. Ultrahigh-Throughput Ambient MS: Direct Analysis at 22 Samples per Second by Infrared Matrix-Assisted Desorption Electrospray Ionization Mass Spectrometry Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) mass spectrometry is an ambient-direct sampling method being developed for high-throughput, label-free, biochemical screening of large-scale compound libraries. Here, we report the development of an ultrahigh-throughput continuous motion IR-MALDESI sampling approach capable of acquiring data at rates up to 22.7 samples per second in a 384-well microtiter plate. At top speed, less than 1% analyte carryover is observed from well-to-well and signal intensity relative standard deviations (RSD) of 11.5% and 20.9% for 3 M 1-hydroxymidazolam and 12 M dextrorphan, respectively, are achieved. The ability to perform parallel kinetics studies on 384 samples with ~30s time resolution using an isocitrate dehydrogenase 1 (IDH1) enzyme assay is shown. Finally, we demonstrate the repeatability and throughput of our approach by measuring 115,200 samples from 300 microtiter plate reads consecutively over 5.54 hours with RSDs under 8.14% for each freshly introduced plate. Taken together, these results demonstrate the use of IR-MALDESI at sample acquisition rates that surpass other currently reported direct sampling mass spectrometry approaches used for high throughput compound screening.

For Table of Contents Only

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org.highwire.dtl.DTLVardef@5f916corg.highwire.dtl.DTLVardef@4473b4org.highwire.dtl.DTLVardef@82e8forg.highwire.dtl.DTLVardef@128b57b_HPS_FORMAT_FIGEXP  M_FIG C_FIG Egress of human cytomegalovirus through multivesicular bodies Human Cytomegalovirus (HCMV) can infect a variety of cell types by using virions of varying glycoprotein compositions. It is still unclear how this diversity is generated, but spatio-temporally separated envelopment and egress pathways might play a role. So far, one egress pathway has been described in which HCMV particles are individually enveloped into small vesicles and are subsequently exocytosed continuously. However, some studies have also found enveloped virus particles inside multivesicular structures but could not link them to productive egress or degradation pathways.

We used a novel 3D-CLEM workflow allowing us to investigate these structures in HCMV morphogenesis and egress at high spatio-temporal resolution. We found that multiple envelopment events occurred at individual vesicles leading to multiviral bodies (MViBs), which subsequently traversed the cytoplasm to release virions as intermittent bulk pulses at the plasma membrane to form extracellular virus accumulations (EVAs). Our data support the existence of a novel bona fide HCMV egress pathway, which opens the gate to evaluate divergent egress pathways in generating virion diversity. 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. A rationalized definition of tumor suppressor microRNAsexcludes miR-34a While several microRNAs (miRNAs) have been proposed to act as tumor suppressors, a consensual definition of tumor suppressing miRNAs is still missing. Similarly to coding genes, we propose that tumor suppressor miRNAs must show evidence of genetic or epigenetic inactivation in cancers, and exhibit an anti-tumorigenic (e.g., anti-proliferative) activity under endogenous expression levels. Here we observe that this definition excludes the most extensively studied tumor suppressor candidate miRNA, miR-34a. In analyzable cancer types, miR-34a does not appear to be down-regulated in primary tumors relatively to normal adjacent tissues. Deletion of miR-34a is occasionally found in human cancers, but it does not seem to be driven by an anti-tumorigenic activity of the miRNA, since it is not observed upon smaller, miR-34a-specific alterations. Its anti-proliferative action was observed upon large, supra-physiological transfection of synthetic miR-34a in cultured cells, and our data indicates that endogenous miR-34a levels do not have such an effect. Our results therefore argue against a general tumor suppressive function for miR-34a, providing an explanation to the lack of efficiency of synthetic miR-34a administration against solid tumors. The Inflammatory Role of Milk Fat Globule Epidermal Growth Factor VIII in Angiotensin II Induced Arterial Remodeling BackgroundAngiotensin II (Ang II) and milk fat globule-epidermal growth factor VIII (MFG-E8) are involved in age-associated arterial remodeling; however, the inflammatory role of MFG-E8 in Ang II associated arterial remodeling with aging remains unknown.

Methods and ResultsIn this study, 30-week-old MFG-E8 knock out (KO) and age-matched wild-type (WT) mice were infused with Ang II or saline. After infusion the with Ang II, the aortic molecular, cellular, and structural remodeling were observed in mice and compared to those infused with saline, but these effects were dependent on the expression of MFG-E8: (1) In the WT mice, Ang II infusion substantially increased intimal-medial thickness, elastic lamina degradation, collagen deposition, and the proliferation of VSMCs; in contrast, in the KO mice, these effects were significantly reduced; (2) In the WT mice, Ang II treatment significantly increased the activation and expression of MMP2, TGF-{beta}1, and its downstream signaling molecule p-SMAD2, and collagen type I production, however, in the KO mice, these molecular effects were significantly reduced; (3) In the WT mice, Ang II treatment increased inflammatory p-NF-{kappa}B p65, MCP1, TNF-, ICAM1, and VCAM1 molecular expression, while conversely, in the KO mice, no significant inflammatory changes were found; (4) Importantly, compared to untreated control mice with a wide range of age from 4-96 weeks, Ang II infused "younger" mice produced an "older" arterial inflammatory phenotype, which was alleviated by MFG-E8 deficiency.

ConclusionsMFG-E8 mediates Ang II associated arterial inflammatory remodeling. Targeting MFG-E8 is a novel molecular approach to curb adverse arterial remodeling during aging and hypertension.

CLINICAL PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABS* Both Ang II and MFG-E8 increases are involved in proinflammatory arterial remodeling mediating the molecular, cellular and tissue events in aging and hypertension.
* MFG-E8 is essential for Ang II induced and age-associated adverse arterial remodeling via the increase of proinflammation, intimal medial thickening, elastin fragmentation, collagen deposition, and VSMC proliferation.


What Are the Clinical Implications?Since MFG-E8 mediates Ang II induced proinflammation in arterial wall remodeling in aging and hypertension, targeting MFG-E8 is a potential molecular approach to curb inflammatory arterial remodeling, maintaining the health of the vascular system during aging and hypertension. Modeling microRNA-driven post-transcriptional regulation by using exon-intron split analysis (EISA) in pigs The contribution of microRNAs (miRNAs) to mRNA regulation has often been explored by post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes of mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc-Gottingen minipigs. Exon-intron split analysis (EISA) of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of upregulated miRNAs also in the fed state. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, EISA provides a valuable view about the regulation of gene expression, complementary to canonical differential expression analyses. A novel method for producing functionalized vesicles that efficiently deliver oligonucleotides in vitro in cancer cells and in vivo in mice Nano-based delivery systems have enhanced our ability to administer and target drugs and macromolecules to their targets. Oligonucleotide drugs have great therapeutic potential but often have off-target effects and stability issues. Therefore, they are often encapsulated in vesicles with targeting ligands such as antibodies (Ab) to deliver their cargo. Herein, we describe a novel, scalable and straightforward approach to producing functionalized vesicles called the "Functionalized Lipid Insertion Method," which differs from the older "Detergent-Dialysis Method." The latter method required excess detergent and extensive dialysis over many hours to produce the functionalized vesicles. With our method, only the functionalized lipid is detergent-solubilized during the engineering of the vesicle. The approach reduces the dialysis time, keeps the vesicles intact while orienting the targeting moieties of the functionalized lipid toward the outside of the vesicle. Pilot in vitro and in vivo experiments was performed to show the feasibility of our method. Dynamic light scattering (DLS) experiments suggested that the original vesicular structure was relatively unperturbed, and the functionalized lipid was inserted externally. Our approach efficiently delivered oligonucleotides and affected the function of liver cancer HepG2 cells. Furthermore, functionalized vesicles achieved targeted delivery of oligonucleotides in mice without inducing a significant innate immune response. The industrial and therapeutic significance and implications of functionalized vesicles produced by our method are also discussed. Additional experiments and analyses are recommended to bring out the full potential of this molecular delivery technology. Rapid global phaseout of animal agriculture has the potential to stabilize greenhouse gas levels for 30 years and offset 68 percent of CO2 emissions 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. Relationship between monomer packing, receptor binding domain pocket status, and pH, in the spike trimer of SARS-CoV-2 variants Existence of a SARS-CoV-2 spike protein trimer form with closer packing between monomers when receptor binding domains (RBDs) are all down, locked as opposed to closed, has been associated with linoleic acid (LA) binding at neutral pH, or can occur at acidic pH in the absence of LA binding. The relationship between degree of closure of the LA binding pocket of the RBD, and monomer burial in the trimer, is examined for a range of spike protein structures, including those with D614G mutation, and that of the Delta variant (which also carries D614G). Some spike protein structures with this aspartic acid mutation show monomer packing approaching that of the locked form (at neutral pH, without LA binding) for two segments, a third (around the RBD) remains less closely packed. Mutations in the RBD are a focus for the Omicron variant spike protein. Structure reports suggest that these mutations are involved in increased RBD-RBD interactions, and also that they could lead to a closing of the LA pocket, both of which could impact on pH-dependence. One potential outcome is that the extent of pH-dependent conformational transitions of the pre-fusion SARS-CoV-2 spike trimer are reduced in the Omicron variant. A cell cycle-dependent GARP-like transcriptional repressor regulates the initiation of differentiation in Giardia lamblia Transcriptional regulation of differentiation is critical for parasitic pathogens to adapt to environmental changes and regulate transmission. In response to encystation stimuli, Giardia lamblia cells shift from G1+S phase of the cell cycle to G2+M. By 2-4 hours, cyst wall proteins are upregulated, indicating that key regulatory steps occur within the first four hours of encystation. However, all characterized transcription factors (TFs) in Giardia have only been investigated at later time points of encystation. How TFs initiate encystation and link it to the cell cycle remains enigmatic. Here, we systematically screened six putative early upregulated TFs for nuclear localization, established their dynamic expression profiles, and determined their functional role in regulating encystation. We found a critical repressor, GLP4 that increases rapidly after 30 min of encystation stimuli and downregulates encystation specific markers, including Cyst Wall Proteins (CWPs) and enzymes in the cyst N-acetyl galactosamine (GalNAc) pathway. Depletion of GLP4 increases cyst production. Importantly, we observe that G2+M cells exhibit higher levels of CWP1 resulting from the activation of MYB2, a TF previously linked to encystation in Giardia. GLP4 upregulation occurs in G1+S cells, suggesting a role in repressing MYB2 and encystation specific genes in the G1+S phase of the cell cycle. Furthermore, we demonstrate that depletion of GLP4 upregulates MYB2 and promotes encystation while overexpression of GLP4 downregulates MYB2 and represses encystation. Together, these results suggest that Giardia employs a dose-dependent transcriptional response that involves the cell cycle regulated repressor GLP4 to orchestrate MYB2 and entry into the encystation pathway.

ImportanceTransition between life cycle stages is a common feature among parasitic pathogens and its regulation must be optimized to balance persistence of infection with transmission. The early transcription factors (TFs) regulating commitment to differentiate are totally unknown in Giardia. In this work, we identified GLP4, a previously uncharacterized GARP-like TF, as an early-acting transcriptional repressor that inhibits G1+S cells from entering the encystation pathway. GLP4 is therefore a key regulator controlling the balance between proliferative growth and terminal differentiation into infective cysts. Nascent alt-protein chemoproteomics reveals a repressor of ribosome biogenesis Many unannotated microproteins and alternative proteins (alt-proteins) have recently been found to be co-encoded with canonical proteins, but few of their functions are known. Motivated by the hypothesis that alt-proteins undergoing active or stress-induced synthesis could play important cellular roles, here, we developed a chemoproteomic pipeline to identify nascent alt-proteins in human cells. We identified 22 actively translated unannotated alt-proteins, one of which is upregulated after DNA damage stress. We further defined MINAS-60 (MIcroprotein that Negatively regulates ASsembly of the pre-60S ribosomal subunit), a nucleolar localized alt-protein co-encoded with human RBM10.Depletion of MINAS-60 increases the amount of the mature 60S ribosomal subunit, consequently upregulating global protein synthesis and cell proliferation by repressing late-stage pre-60S assembly and export of the 60S ribosome subunit to the cytoplasm. Together, these results implicate MINAS-60 as a repressor of ribosome biogenesis, and demonstrate that chemoproteomics can enable generation of functional hypotheses for uncharacterized alt-proteins. Understanding the Spin Crossover Dynamical Effects of the Dioxygen Binding and Activation on HOD Enzyme For the cofactor-free 1-H-3-hydroxy-4-oxoquinaldine-2,4-dioxygenase (HOD), the dioxygen (O2) dependent steps are rate-limiting along with a spin state crossover to the singlet spin state. Here, the primary triplet O2 molecule activation on the 2-methyl-3-hydroxy-4(1H)-quinolone (MHQ) is investigated, and the catalytic role of the intersystem crossing effects is highlighted by directly comparing results from the Born-Oppenheimer dynamics and non-adiabatic surface hopping dynamics. This work confirms non-adiabatic dynamical effects are essential to modulate the O2 activation on the substrate MHQ. The time scale of the equilibration and conversion from triplet to singlet state should be in the range of a few hundreds of femtoseconds. We hope this work provides us a fresh look at the underlying physics of dioxygen activation reactions involving more than one spin state. Extending Bayesian Elo-rating to quantify the steepness of dominance hierarchies The steepness of dominance hierarchies provides information about the degree of competition within animal social groups and is thus an important concept in socioecology. The currently most widely-used metrics to quantify steepness are based on Davids scores (DS) derived from dominance interaction networks. One serious drawback of these DS-based metrics is that they are biased, i.e., network density systematically decreases steepness values. Here, we provide a novel approach to estimate steepness based on Elo-ratings, implemented in a Bayesian framework (STEER: Steepness estimation with Elo-rating). Our new metric has two key advantages. First, STEER is unbiased, precise and more robust to data density than DS-based steepness. Second, it provides explicit probability distributions of the estimated steepness coefficient, which allows uncertainty assessment. In addition, it relies on the same underlying concept and is on the same scale as the original measure, and thus allows comparison to existing published results. We evaluate and validate performance of STEER by means of experimentation on empirical and artificial data sets and compare its performance to that of several other steepness estimators. Our results suggest that STEER provides a considerable improvement over existing methods to estimate dominance hierarchy steepness. We provide an R package EloSteepness to calculate the new steepness measure, and also show an example of using steepness in a comparative analysis. 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. Reconstitution of minimal motility system based on Spiroplasma swimming by expressing two bacterial actins in synthetic minimal bacterium Motility is one of the most important features of life, but its evolutionary origin remains unknown. In this study, we focused on Spiroplasma, commensal, or parasitic bacteria. They swim by switching the helicity of a ribbon-like cytoskeleton that comprises six proteins, each of which evolved from a nucleosidase and bacterial actin called MreB. We expressed these proteins in a synthetic, non-motile minimal bacterium, JCVI-syn3.0B, whose reduced genome was computer-designed and chemically synthesized. The synthetic bacterium exhibited swimming motility with features characteristic of Spiroplasma swimming. Moreover, some combinations of the two proteins produced a helical cell shape and swimming, suggesting that the swimming originated from the differentiation and coupling of bacterial actins, and we also obtained a minimal system for motility of the synthetic bacterium.

One-Sentence SummaryThe minimal system comprised two bacterial actins that provided cell helicity and swimming to the synthetic minimal bacterium. A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2 protects against disease and elicits neutralizing antibodies to wild-type virus and Delta variant Several vaccines have been introduced to combat the coronavirus infectious disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current SARS-CoV-2 vaccines include mRNA-containing lipid nanoparticles or adenoviral vectors that encode the SARS-CoV-2 Spike (S) protein of SARS-CoV-2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS-CoV-2 variants such as the Delta variant. Here, we present a novel, well-characterized SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD). RBD-conjugated outer membrane vesicles (RBD-OMVs) were used to immunize the golden Syrian hamster (Mesocricetus auratus) model of COVID-19. Intranasal immunization resulted in high titers of blood anti-RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild-type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titers in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches. Is a Ring-arranged Vascular Pattern Unique to Stems? A Three Dimensional Reconstruction of Anaxagorea (Annonaceae) Carpel Vasculature and its Evolutionary Implications Elucidating the origin of carpel has been a challenge in botany for a long time. The Unifying Theory suggested that the carpel originate from a composite organ comprising an ovule-bearing shoot and a foliar part enclosing the shoot. A logical inference from this theory is that placenta in angiosperms should have radiosymmetrical vasculature, just like that in a young branch. Anaxagorea is the most basal genus of the primitive angiosperm family, Annonaceae. The conspicuous carpel stipe makes it an ideal material for exploring the carpel vasculature. In this study, serial sections of flower and carpel were delineated in Anaxagorea luzonensis and A. javanica, and a three-dimensional model of the carpel vasculature was reconstructed. The results show that (1) vascular bundles at both the carpel stipe and the ovule/placenta are in a radiosymmetrical pattern, (2) the amphicribral bundles would develop into ring-arranged bundle complex with the carpel maturation, (3) the ovule/placenta bundles were separated from the bundles of the carpel wall, and, (4) all the radiosymmetrical vasculature (including amphicribral bundles and ring-arranged bundle complexes) in the carpel were fed by a larger radiosymmetrical bundle system. These results suggest that the radiosymmetrical pattern of carpel vasculature are in line with the Unifying Theory. 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. 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. Surface-associated antigen induces B-cell permeabilization and lysosome exocytosis facilitating antigen uptake and presentation to T-cells B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently presented to T-cells. Thus, PM permeabilization by surface-associated antigen triggers a lysosome-mediated B-cell resealing response, providing the extracellular hydrolases that facilitate antigen internalization and presentation.