Modeling the SARS-CoV-2 nsp1--5'-UTR complex via extended ensemble simulations Nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a 180-residue protein that blocks translation of host mRNAs in SARS-CoV-2-infected cells. Although it is known that SARS-CoV-2s own RNA evades nsp1s host translation shutoff, the molecular mechanism underlying the evasion was poorly understood. We performed an extended ensemble molecular dynamics simulation to investigate the mechanism of the viral RNA evasion. Simulation results showed that the stem loop structure of the SARS-CoV-2 RNA 5-untranslated region (SL1) is recognized by both nsp1s globular region and intrinsically disordered region. The recognition presumably enables selective translation of viral RNAs. Cluster analysis of the binding mode and detailed analysis of the binding poses revealed several residues involved in the SL1 recognition mechanism. The simulation results imply that the nsp1 C-terminal helices are lifted from the 40S ribosome upon the binding of SL1 to nsp1, unblocking translation of the viral RNA.
Cryo-EM structure of the Rhodobaca bogoriensis RC-LH1-PufX dimeric complex at 2.9 A The reaction centre-light harvesting 1 (RC-LH1) complex is essential for converting light into proton motive force in photosynthetic bacteria. RC-LH1 is a monomer in most purple bacteria, but in Rhodobacter species, it is a dimer. Its assembly depends on an accessory polypeptide (PufX) and, ultimately, on photosynthetic growth. To date, knowledge on the RC-LH1-PufX structure, where the dimer has two incomplete  C-shaped antenna rings surrounding an RC, is mainly limited to the model organism Rhodobacter sphaeroides. Here we present a cryo-electron microscopy structure at 2.9 [A] from Rhodobaca bogorensis strain LBB1. RCs are surrounded by 30 antennas and incorporate protein Y and PufX. RCs are stably connected by PufX, which self-interacts, electrostatically attracts cytochrome c2 (cyt c2) and forms extensive networks with co-factors. This structure underlines coordinated energy transfer in a combinatorial manner, providing a basis to describe bacterial photosynthesis within a dimeric photosynthetic apparatus.
Inability to switch from ARID1A-BAF to ARID1B-BAF impairs exit from pluripotency and commitment towards neural crest formation in ARID1B-related neurodevelopmental disorders The BAF complex modulates chromatin accessibility. Specific BAF configurations have functional consequences, and subunit switches are essential for cell differentiation. ARID1B and its paralog ARID1A encode for mutually exclusive BAF subunits. De novo ARID1B haploinsufficient mutations cause a neurodevelopmental disorder spectrum, including Coffin-Siris syndrome, which is characterized by neurological and craniofacial features. Here, we reprogrammed ARID1B+/- Coffin-Siris patient-derived skin fibroblasts into iPSCs and modeled cranial neural crest cell (CNCC) formation. We discovered that ARID1B is active only during the first stage of this process, coinciding with neuroectoderm specification, where it is part of a lineage-specific BAF configuration (ARID1B-BAF), which includes SMARCA4 and nine additional subunits. ARID1B-BAF acts as a gatekeeper, ensuring exit from pluripotency and lineage commitment, by attenuating NANOG, SOX2 and thousands of enhancers directly regulated by these two pluripotency factors at the iPSC stage.

In iPSCs, these enhancers are maintained active by an ARID1A-containing BAF. At the onset of differentiation, cells transition from ARID1A-BAF to ARID1B-BAF, eliciting attenuation of the NANOG/SOX2 networks, and triggering pluripotency exit. Coffin-Siris patient cells fail to perform the ARID1A/ARID1B switch and maintain ARID1A-BAF at pluripotency enhancers throughout all stages of CNCC formation. This leads to a persistent and aberrant SOX2 and NANOG activity, which impairs CNCC formation. In fact, despite showing the typical neural crest signature (TFAP2A+, SOX9+), ARID1B-haploinsufficient CNCCs are also NANOG/OCT4-positive, in stark contrast with the ARID1B-wt CNCCs, which are NANOG/OCT4-negative.

These findings suggest a connection between ARID1B mutations, neuroectoderm formation, and a pathogenic mechanism for Coffin-Siris syndrome. Critical re-evaluation of the slope factor of the operational model of agonism Background and PurposeAlthough being a relative term, agonist efficacy is a cornerstone in the proper assessment of agonist selectivity and signalling bias. The operational model of agonism (OMA) has become successful in the determination of agonist efficacies and ranking them. In 1985, Black et al. introduced the slope factor to the OMA to make it more flexible and allow for fitting steep as well as flat concentration-response curves.

Experimental ApproachFunctional analysis of OMA demonstrates that the slope factor implemented by Black et al. affects relationships among parameters of the OMA, leading to wrong estimates of operational efficacy and affinity of an agonist. In contrast, functional analysis of OMA with Hill coefficient as a slope factor has not this flaw.

Key ResultsFitting of the OMA with Black et al. slope factor to concentration-response curves of several models (homotropic allosteric modulation, substrate inhibition and non-competitive auto-inhibition) gives wrong estimates of operational efficacy and affinity. In contrast, fitting of the OMA modified by the Hill coefficient to concentration-response curves of these several models gives correct estimates of operational efficacy and affinity.

Conclusion and ImplicationsThe OMA modified by the Hill coefficient can be used for correct ranking of agonism and subsequent analysis, like quantification of signalling bias. Conformist social learning inoculates against adverse risk aversion Given the ubiquity of potentially adverse behavioural bias owing to myopic trial-and-error learning, it seems paradoxical that improvements in decision-making performance through conformist social learning, a process widely considered to be bias amplification, still prevail in animal collective behaviour. Here we show, through model analyses and large-scale interactive behavioural experiments with 585 human subjects, that conformist influence can indeed promote favourable risk taking in repeated experience-based decision making, even though many individuals are systematically biased towards adverse risk aversion. Although strong positive feedback conferred by copying the majoritys behaviour could result in unfavourable informational cascades, our differential equation model of collective behavioural dynamics identified a key role for increasing exploration by negative feedback arising when a weak minority influence undermines the inherent behavioural bias. This  collective behavioural rescue, emerging through coordination of positive and negative feedback, highlights a benefit of collective learning in a broader range of environmental conditions than previously assumed and resolves the ostensible paradox of adaptive collective behavioural flexibility under conformist influences. Cell trajectory modeling identifies a primitive trophoblast state defined by BCAM enrichment In early placental development, progenitor cytotrophoblasts (CTBs) differentiate along one of two cellular trajectories: the villous or extravillous pathways. CTBs committed to the villous pathway fuse with neighboring CTBs to form the outer multinucleated syncytiotrophoblast (SCT), while CTBs committed to the extravillous pathway differentiate into invasive extravillous trophoblasts (EVT). Unfortunately, little is known about the processes controlling human CTB progenitor maintenance and differentiation. To address this, we established a single cell RNA sequencing (scRNA-seq) dataset from first trimester placentas to identify cell states important in trophoblast progenitor establishment, renewal, and differentiation. Multiple distinct trophoblast states were identified, representing progenitor CTBs, column CTBs, SCT precursors, and EVT. Lineage trajectory analysis identified a progenitor origin that was reproduced in human trophoblast stem cell organoids. Heightened expression of basal cell adhesion molecule (BCAM) defined this primitive state, where BCAM enrichment or gene silencing resulted in enhanced or diminished organoid growth. Together, this work describes at high-resolution trophoblast heterogeneity within the first trimester, resolves gene networks within human CTB progenitors, and identifies BCAM as a primitive progenitor marker and possible regulator.

Summary StatementLineage trajectory modeling identifies multiple human progenitor trophoblast states and defines trophoblast differentiation kinetics, where BCAM-expressing progenitors demonstrate enhanced regenerative ability. IRI-CCE: A Platform for Evaluating CRISPR-based Base Editing Tools and Its Components Rapid assessment of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated Cas protein)-based genome editing (GE) tools and their components are critical aspects for successful applications in different organisms. In many bacteria, double-stranded breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient non-homologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs capable of converting C-to-T (CBE) and A-to-G (ABE) have been recently developed but yet to be tested in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising different combinations of promoters/terminators. We demonstrate the use of IRI-CEE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e), exhibiting that each independent BE has its specific editing pattern for a given target site and promoter type. Additionally, the IRI-CCE platform offers a rapid way to screen functional gRNAs. In summary, CRISPR-BE components expressed by promoters of different strengths in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and gRNAs. Subjective understanding of actions and emotions requires interaction of the semantic and action observation networks Previous studies have investigated the neural basis of action and emotion perception conveyed by whole body postures and movements. However, subjective understanding of other peoples bodily actions and emotions, raises different issues that are currently not well understood and have so far seldom been investigated. In this 7T fMRI study, we proceeded beyond conventional univariate/multivariate analyses with predefined categories, and examined the representational geometry of subjective action- and emotion-understanding by mapping individual subjective reports with word embeddings. Dimensionality reduction revealed that the representations for perceived action and emotion were high dimensional, each correlated to but were not reducible to the predefined action and emotion categories. Searchlight representational similarity analysis showed that the representations in the left middle superior temporal sulcus and left dorsal premotor cortex corresponded to the subjective action and emotion understanding of the participants. Furthermore, using task-residual functional connectivity and hierarchical clustering, we found that areas in the action observation network and the semantic/default-mode network were functionally connected to these two seed regions, and showed similar representations. Our study provides direct evidence that both networks were concurrently involved in subjective action and emotion understanding. Our approach shows how subjective understanding of action and emotion stimuli can be reliably studied and complement insights based on from predefined stimulus categories. Inter-microscope comparability of dental microwear texture data obtained from different optical profilometers. Dental microwear texture analysis has become a well-established approach for dietary inference and reconstruction of mammals and other tetrapods, both extant and extinct. As the amount of available data grows immensely, researchers could benefit from combining data gathered by others to perform meta-analyses. However, different devices used to capture three-dimensional surface scans for DMTA are known to produce variation even when measuring the same surface. Here we compare DMTA data of 36 guinea pigs that received different diets in a controlled feeding experiment, measured on a confocal disc-scanning and a confocal laser-scanning microscope. We are testing different pre-analysis filtering protocols to mitigate differences. We find inter-microscopes and filter-related differences for the majority of analysed 41 DMTA parameters. Certain microscope-specific filter routines resulted in less differences than other pre-analysis protocols. We further identify DMTA parameters which were stable regardless of microscope or pre-analysis treatment. Overall, the results obtained on both microscopes show the same dietary differentiation between guinea pig feeding groups, which supports that DMTA is a suitable method to obtain repeatable, objective dietary inferences. We finally propose a roadmap to enhance data exchange and inter-lab comparability and collaboration in the future. Swine H1N1 influenza virus variants with enhanced polymerase activity and HA stability promote airborne transmission in ferrets Understanding how animal influenza A viruses (IAVs) acquire airborne transmissibility in humans and ferrets is needed to prepare for and respond to pandemics. Here, we investigated in ferrets the replication and transmission of swine H1N1 isolates P4 and G15, whose majority population had decreased polymerase activity and poor HA stability, respectively. For both isolates, a minor variant was selected and transmitted in ferrets. Polymerase-enhancing variant PA-S321 airborne-transmitted and propagated in one ferret. HA-stabilizing variant HA1-S210 was selected in all G15-inoculated ferrets and was transmitted by contact and airborne routes. With an efficient polymerase and a stable HA, the purified minor variant G15-HA1-S210 had earlier and higher peak titers in inoculated ferrets and was recovered at a higher frequency after airborne transmission than P4 and G15. Overall, HA stabilization played a more prominent role than polymerase enhancement in the replication and transmission of these viruses in ferrets. The results suggest pandemic risk-assessment studies may benefit from deep sequencing to identify minor variants with human-adapted traits.

IMPORTANCEDiverse IAVs circulate in animals, yet few acquire the viral traits needed to start a human pandemic. A stabilized HA and mammalian-adapted polymerase have been shown to promote the adaptation of IAVs to humans and ferrets (the gold-standard model for IAV replication, pathogenicity, and transmissibility). Here, we used swine IAV isolates of the gamma lineage a model to investigate the importance of HA stability and polymerase activity in promoting replication and transmission in ferrets. These are emerging viruses that bind to both -2,6- and -2,3-linked receptors. Using isolates containing mixed populations, a stabilized HA was selected within days in inoculated ferrets. An enhanced polymerase was also selected and propagated after airborne transmission to a ferret. Thus, HA stabilization was a stricter requirement, yet both traits promoted transmissibility. Knowing the viral traits needed for pandemic potential, and the relative importance of each, will help identify emerging viruses of greatest concern. The adenoviral E1B-55k protein present in HEK293 cells mediates abnormal accumulation of key WNT signaling proteins in large cytoplasmic aggregates. HEK293 cells are one of the most widely used cell lines in research and HEK293 cells are frequently used as an in vitro model for studying the WNT signaling pathway. The HEK293 cell line was originally established by transfection of human embryonic kidney cells with sheared adenovirus 5 DNA and it is known that that HEK293 cells stably express the adenoviral E1A and E1B-55k proteins. Here we show that HEK293 cells display an unexpected distribution of key components of the WNT/{beta}-catenin signaling pathway where AXIN1, APC, DVL2 and tankyrase are all co-localized in large spherical cytoplasmic aggregates. The cytoplasmic aggregates are enclosed by a narrow layer of the adenoviral E1B-55k protein. Reduction of E1B-55k protein levels leads to disappearance of the cytoplasmic aggregates thus corroborating an essential role of the E1B-55k protein in mediating the formation of the aggregates. Furthermore, HEK293 cells with reduced E1B-55k protein levels display reduced levels of transcriptional activation of WNT/{beta}-catenin signaling upon stimulation by the Wnt3A agonist. The demonstrated influence of the E1B-55k protein on the cellular localization of WNT/{beta}-catenin signaling components and on transcriptional regulation of WNT/{beta}-catenin signaling asks for caution in the interpretation of data derived from the HEK293 cell line. Synthetic periphyton as a model system to understand species dynamics in complex microbial freshwater communities Phototrophic biofilms, also known as periphyton, are microbial freshwater communities that drive crucial ecological processes in streams and lakes. Gaining a deep mechanistic understanding of the biological processes occurring in natural periphyton remains challenging due to the high complexity and variability of such communities. To address this challenge, we rationally developed a workflow to construct a synthetic community by co-culturing 26 phototrophic species (i.e., diatoms, green algae and cyanobacteria) that were inoculated in a successional sequence to create a periphytic biofilm on glass slides. We show that this community is diverse, stable and highly reproducible in terms of microbial composition, function and 3D spatial structure of the biofilm. We also demonstrate the ability to monitor microbial dynamics at the single species level during periphyton development and how their abundances are impacted by stressors such as increased temperature and a herbicide, singly and in combination. Overall, such a synthetic periphyton, grown under controlled conditions, can be used as a model system for theory testing through targeted manipulation. Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9 CRISPR-mediated genome editing has become a powerful tool for genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve the HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3' end of the MKK2 MAP Kinase gene to generate a BleoR-MKK2 fusion protein. Based on exogenous BleoR expression, the HDR-mediated knock-in efficiency was up to [~]40-fold greater when using a XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared to no HDR enhancement or NHEJ silencing. Furthermore, this corporation of HDR enhancer overexpression and NHEJ repression also resulted in 7-fold fewer CRISPR-induced Insertions and Deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammalians for improving CRISPR-mediated gene knock-in efficiency. Onlinemeta: A Web Server For Meta-Analysis Based On R-shiny Meta-analysis is a common statistical method used to summarize multiple studies that cover the same topic. It can provide less biased results and explain heterogeneity between studies. Although there exists a variety of meta-analysis software, they are rarely both convenient to use and capable of comprehensive analytical functions. As a result, we established a meta-analysis web tool called Meta-Analysis Online (Onlinemeta). Onlinemeta includes both risk bias analysis and meta-analysis. The risk bias analysis tool can produce heatmaps and histograms, whereas the meta-analysis tool can be used to analyze dichotomous variables, single-armed dichotomous variables, continuous variables, single-armed continuous variables, survival data, and diagnostic experiments. In addition, it can be used to generate forest plots, funnel plots, SROC curve, and crosshair plots. Onlinemeta can be found at the following address: https://smuonco.Shinyapps.io/Onlinemeta/. 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. Intron-mediated induction of phenotypic heterogeneity Introns are universally present in the nuclear genomes of eukaryotes1. The budding yeast, an otherwise intron-poor species, preserves two sets of ribosomal protein (RP) genes differing primarily in their introns2-4. Despite recent findings on the role of RP introns under stress and starvation5-7, understanding the contribution of introns to ribosome regulation remains challenging. Here, combining isogrowth profiling8 with single-cell protein measurements9, we found that introns can mediate inducible phenotypic heterogeneity conferring a clear fitness advantage. Osmotic stress leads to bimodal expression of the small ribosomal subunit protein Rps22B, mediated by an intron in the 5 untranslated region of its transcript. The two resulting yeast subpopulations differ in their ability to cope with starvation. Low Rps22B protein levels resulted in prolonged survival under sustained starvation, while high Rps22B levels enabled cells to grow faster after transient starvation. Further, yeast growing at high sugar concentrations - similar to those in ripe grapes - exhibit bimodal Rps22B expression when approaching stationary phase. Differential intron-mediated regulation of RP genes thus provides a way to diversify the population when starvation looms in natural environments. Our findings reveal a new role for introns in inducing phenotypic heterogeneity in changing environments and suggest that duplicated RP genes in yeast contribute to resolving the evolutionary conflict between precise expression control and environmental responsiveness10. Ineffectual AEC1 differentiation from KRT8hi transitional state without fibrosis is associated with fatal COVID-19 ARDS ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis. Synergy of chemotherapy and macrophage depletion leads to B cell-mediated T cell memory activation and durable triple negative breast cancer regression Immunosuppressive elements within the tumor microenvironment such as Tumor Associated Macrophages (TAMs) can present a barrier to successful anti-tumor responses by cytolytic T cells. We employed preclinical syngeneic p53 null mouse models of triple negative breast cancer (TNBC) to develop a treatment regimen that harnessed the immunostimulatory effects of low-dose cyclophosphamide coupled with the pharmacologic inhibition of TAMs using either a small molecule CSF1R inhibitor or an anti-CSF1R antibody. This therapeutic combination was used to successfully treat several highly aggressive TNBC murine mammary tumors and lung metastasis. Using this regimen and single cell RNA sequencing we characterized tumor infiltrating lymphocytes (TILs) including helper T cells and antigen-presenting B cells that were highly enriched in good responders to combination therapy. Using high dimensional imaging techniques, we identified the close spatial localization of B220+ CD86+ activated B cells and CD4+ T cells in tertiary lymphoid structures that were present up to 6 weeks post-treatment in one model that also exhibited long-term tumor regression post-treatment. We also characterized the transcriptional and metabolic heterogeneity of TAMS in these two closely related claudin-low/mesenchymal subtype tumor models with differential treatment responses. A murine TAM signature derived from the T12 model is highly expressed and conserved in human claudin-low breast cancers, and high expression of the T12 signature correlated with reduced overall survival. This T12 tumor TAM signature may help identify human claudin-low breast cancer patients that will benefit from the combination of cyclophosphamide and anti-CSF1R therapy. These studies illustrate the complexity of the tumor immune microenvironment and highlight different immune responses that result from rationale combinations of immunotherapy.

SignificanceA treatment regimen that harnessed the immunostimulatory effects of cyclophosphamide coupled with the inhibition of CSF1R was used to successfully treat several highly aggressive claudin-low TNBC murine mammary tumors and lung metastasis. ABPP-HT* - Deep meets fast for activity-based profiling of deubiquitylating enzymes using advanced DIA mass spectrometry methods Activity-based protein profiling (ABPP) uses a combination of activity-based chemical probes with mass spectrometry (MS) to selectively characterise a particular enzyme or enzyme class. ABPP has proven invaluable for profiling enzymatic inhibitors in drug discovery. When applied to cell extracts and cells, challenging the ABP-enzyme complex formation with a small molecule can simultaneously inform on potency, selectivity, reversibility/binding affinity, permeability, and stability. ABPP can also be applied to pharmacodynamic studies to inform on cellular target engagement within specific organs when applied to in vivo models. Recently, we established separate high depth and high throughput ABPP (ABPP-HT) protocols for the profiling of deubiquitylating enzymes (DUBs). However, the combination of the two, deep and fast, in one method has been elusive. To further increase the sensitivity of the current ABPP-HT workflow we implemented state-of-the-art data-independent acquisition (DIA) and data-dependent acquisition (DDA) MS analysis tools. Hereby, we describe an improved methodology, ABPP-HT* (enhanced high-throughput-compatible activity-based protein profiling), that in combination with DIA MS methods allowed for the consistent profiling of 35-40 DUBs and provided a reduced number of missing values, whilst maintaining a throughput of 100 samples per day. Mutations in coral soma and sperm imply lifelong stem cell renewal and cell lineage selection In many animals, the germline differentiates early in embryogenesis, so only mutations that accumulate in germ cells are inherited by offspring1. Exceptions to this developmental process may indicate that other mechanisms have evolved to limit the effects of deleterious mutation accumulation2. Stony corals are animals that can live for hundreds of years3 and have long been thought to produce gametes from somatic tissue4. To clarify conflicting evidence about germline-soma distinction in corals, we sequenced high coverage, full genomes with technical replicates for parent coral branches and their sperm pools. We identified post-embryonic single nucleotide variants (SNVs) unique to each parent branch, then checked if each SNV was shared by the respective sperm pool: 26% of post-embryonic SNVs were shared by the sperm but 74% were not. We also identified germline SNVs, those that were present in the sperm but not in the parent. These data suggest that self-renewing stem cells in corals differentiate into germ and soma throughout the adult life of the colony, with SNV rates and patterns differing markedly in stem, soma, and germ lineages. In addition to informing the important place in the evolutionary spectrum from non-Weismannian to Weismmanian animals that corals occupy, these insights inform how corals may generate adaptive diversity necessary in the face of global climate change. 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. Population differences in the length and early-life dynamics of telomeres among European pied flycatchers Telomere length and shortening rate are increasingly used as biomarkers for long-term costs in ecological and evolutionary studies because of their relationships with survival and fitness. Both early-life conditions and growth, and later-life stressors can create variation in telomere shortening rate. Studies on between- population telomere length and dynamics are scarce, despite the expectation that populations exposed to varying environmental constraints would present divergent telomere length patterns. The pied flycatcher (Ficedula hypoleuca) is a passerine bird breeding across Eurasia (from Spain to western Siberia) and migrating through the Iberian Peninsula to spend the non-breeding period in sub-Saharan Africa. Thus, different populations show marked differences in migration distance. We studied the large-scale variation of telomere length and early-life dynamics in the pied flycatcher by comparing six European populations across a north- south gradient (Finland, Estonia, England, and Spain) predicting negative effect of migration distance on adult telomere length, and of nestling growth on nestling telomere dynamics. There were clear population differences in telomere length, with English birds from mid-latitudes having the longest telomeres. Telomere length did not thus show consistent latitudinal variation and was not linearly linked to differences in migration distance. Early-life telomere shortening rate tended to vary between populations. Fast growth was associated with shorter telomeres in the early life, but faster nestling growth affected telomeres more negatively in northern than southern populations. While the sources of between-population differences in telomere-related biology remain to be more intensively studied, our study illustrates the need to expand telomere studies at the between-population level. Cell-type specific circadian bioluminescence rhythms in Dbp reporter mice Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre-recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues. "Adopt a Microorganism" methodology enhances the discourse richness of high school students in both hybrid learning and remote education The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. Lymph node swelling combined with temporary effector T cell retention aids T cell response in a model of adaptive immunity Swelling of lymph nodes is commonly observed during the adaptive immune response, yet the impact on T cell trafficking and subsequent immune response is not well known. To better understand the effect of macro-scale alterations, we developed an agent-based model of the lymph node para-cortex, describing T cell trafficking and response to antigen-presenting dendritic cells alongside swelling-induced changes in T cell recruitment and egress, and regulation of expression of egress-modulating T cell receptor Sphingosine-1-phosphate receptor-1. Analysis of effector T cell response under varying swelling conditions showed that swelling consistently aided T cell activation. However, subsequent effector CD8+ T cell production could be reduced if swelling occurred too early in the T cell proliferative phase or if T cell cognate frequency was low due to increased opportunity for T cell exit. Temporarily extending retention of newly differentiated effector T cells, mediated by Sphingosine-1-phosphate receptor-1 expression, mitigated any negative effects of swelling by allowing facilitation of activation to outweigh increased access to exit areas. These results suggest targeting temporary effector T cell retention and egress associated with swelling may offer new ways to modulate effector TC responses in, for example, immuno-suppressed patients and optimisation of vaccine design. Hepatic MIR20B promotes nonalcoholic fatty liver disease by suppressing PPARA BackgroundNon-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation and imbalances in lipid metabolism in the liver. Although nuclear receptors (NRs) play a crucial role in hepatic lipid metabolism, the underlying mechanisms of NR regulation in NAFLD remain largely unclear.

MethodsUsing network analysis and RNA-seq to determine the correlation between NRs and microRNA in human NAFLD patients, we revealed that MIR20B specifically targets PPARA. MIR20B mimic and anti-MIR20B were administered to human HepG2 and Huh-7 cells and mouse primary hepatocytes as well as high fat diet (HFD)- or methionine-deficient diet (MCD)-fed mice to verify the specific function of MIR20B in NAFLD. We tested the inhibition of the therapeutic effect of a PPAR agonist, fenofibrate, by MIR20B and the synergic effect of combination of fenofibrate with anti-MIR20B.

ResultsWe revealed that MIR20B specifically targets PPARA through miRNA regulatory network analysis of nuclear receptor genes in NAFLD. The expression of MIR20B was upregulated in free fatty acid (FA)- treated hepatocytes and the livers of both obesity-induced mice and NAFLD patients. Overexpression of MIR20B significantly increased hepatic lipid accumulation and triglyceride levels. Furthermore, MIR20B significantly reduced FA oxidation and mitochondrial biogenesis by targeting PPARA. In MIR20B-introduced mice, the effect of fenofibrate to ameliorate hepatic steatosis was significantly suppressed. Finally, inhibition of MIR20B significantly increased FA oxidation and uptake, resulting in improved insulin sensitivity and a decrease in NAFLD progression. Moreover, combination of fenofibrate and anti-MIR20B exhibited the synergic effect on improvement of NAFLD in MCD-fed mice.

ConclusionsTaken together, our results demonstrate that the novel MIR20B targets PPARA, plays a significant role in hepatic lipid metabolism, and present an opportunity for the development of novel therapeutics for NAFLD.

FundingThis research was funded by Korea Mouse Phenotyping Project (2016M3A9D5A01952411), the National Research Foundation of Korea (NRF) grant funded by the Korea government (2020R1F1A1061267, 2018R1A5A1024340, NRF-2021R1I1A2041463, 2020R1I1A1A01074940), and the Future-leading Project Research Fund (1.210034.01) of UNIST.