Relationship between vestibular hair cell loss and deficits in two anti-gravity reflexes in the rat The tail-lift reflex and the air-righting reflex in rats are anti-gravity reflexes that depend on vestibular function. To begin identifying their cellular basis, this study examined the relationship between reflex loss and the graded lesions caused in the vestibular sensory epithelia by varying doses of an ototoxic compound. After ototoxic exposure, we recorded these reflexes using high speed video. The movies were used to obtain objective measures of the reflexes: the minimum angle formed by the nose, the back of the neck and the base of the tail during the tail-lift maneuver and the time to right in the air-righting test. The vestibular sensory epithelia were then collected from the rats and used to estimate the loss of type I (HCI), type II (HCII) and all hair cells (HC) in both central and peripheral parts of the crista, utricle, and saccule. As expected, tail-lift angles decreased, and air-righting times increased, while the numbers of HCs remaining in the epithelia decreased in a dose-dependent manner. The results demonstrated greater sensitivity of HCI compared to HCII to the IDPN ototoxicity, as well as a relative resiliency of the saccule compared to the crista and utricle. Comparing the functional measures with the cell counts, we observed that loss of the tail-lift reflex associates better with HCI than with HCII loss. In contrast, most HCI in the crista and utricle were lost before air-righting times increased. These data suggest that these reflexes depend on the function of non-identical populations of vestibular HCs.
Optogenetic stimulation of lateral hypothalamic orexin/dynorphin inputs in the ventral tegmental area potentiates mesolimbic dopamine neurotransmission and promotes reward-seeking behaviours. Reward and reinforcement processes are critical for survival and propagation of genes. While numerous brain systems underlie these processes, a cardinal role is ascribed to mesolimbic dopamine. However, ventral tegmental area (VTA) dopamine neurons receive complex innervation and various neuromodulatory factors, including input from lateral hypothalamic (LH) orexin/hypocretin neurons which also express and co-release the neuropeptide, dynorphin. Dynorphin in the VTA induces aversive conditioning through the Kappa opioid receptor (KOR) and decreases dopamine when administered intra-VTA. Exogenous application of orexin or orexin 1 receptor (oxR1) antagonists in the VTA bidirectionally modulates dopamine-driven motivation and reward-seeking behaviours, including the attribution of motivational value to primary rewards and associated conditioned stimuli. However, the effect of endogenous stimulation of LH orexin/dynorphin-containing projections to the VTA and the potential contribution of co-released dynorphin on mesolimbic dopamine and reward related processes remains uncharacterised. We combined optogenetic, electrochemical, and behavioural approaches to examine this. We found that optical stimulation of LH orexin/dynorphin inputs in the VTA potentiates mesolimbic dopamine neurotransmission in the nucleus accumbens (NAc) core, produces real time and conditioned place preference, and increases the food cue-directed orientation in a Pavlovian conditioning procedure. LH orexin/dynorphin potentiation of NAc dopamine release and real time place preference was blocked by an oxR1, but not KOR antagonist. Thus, rewarding effects associated with optical stimulation of LH orexin/dynorphin inputs in the VTA are predominantly driven by orexin rather than dynorphin.
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. An mRNA SARS-CoV-2 vaccine employing Charge-Altering Releasable Transporters with a TLR-9 agonist induces neutralizing antibodies and T cell memory The SARS-CoV-2 pandemic has necessitated the rapid development of prophylactic vaccines. Two mRNA vaccines have been approved for emergency use by the FDA and have demonstrated extraordinary effectiveness. The success of these mRNA vaccines establishes the speed of development and therapeutic potential of mRNA. These authorized vaccines encode full-length versions of the SARS-CoV-2 spike protein. They are formulated with Lipid Nanoparticle (LNP) delivery vehicles that have inherent immunostimulatory properties. Different vaccination strategies and alternative mRNA delivery vehicles would be desirable to ensure flexibility of future generations of SARS-CoV-2 vaccines and the development of mRNA vaccines in general.

Here, we report on the development of an alternative mRNA vaccine approach using a delivery vehicle called Charge-Altering Releasable Transporters (CARTs). Using these inherently nonimmunogenic vehicles we can tailor the vaccine immunogenicity by inclusion of co-formulated adjuvants such as oligodeoxynucleotides with CpG motifs (CpG-ODN). Mice vaccinated with the mRNA-CART vaccine developed therapeutically relevant levels of RBD-specific neutralizing antibodies in both the circulation and in the lung bronchial fluids. In addition, vaccination elicited strong and long lasting RBD-specific TH1 T cell responses including CD4+ and CD8+ T cell memory. Host phenology can drive the evolution of intermediate virulence strategies in some parasites AO_SCPLOWBSTRACTC_SCPLOWThe traditional mechanistic trade-offs resulting in a negative correlation between transmission and virulence are the foundation of nearly all current theory on the evolution of parasite virulence. Several ecological factors have been shown to modulate the optimal virulence strategies predicted from mechanistic trade-off models, but these ecological factors have not yet been shown to be sufficient to explain the intermediate virulence strategies observed in any natural system. The timing of seasonal activity, or phenology, is a common factor that influences the types and impact of many ecological interactions but is difficult to incorporate into virulence evolution studies. We develop a mathematical model of a disease system with seasonal host activity to study the evolutionary consequences of host phenology on the virulence of obligate-killer parasite. Results from this model demonstrated that seasonal host activity is sufficient to drive the evolution of intermediate parasite virulence in some types of natural disease systems, even when a traditional mechanistic trade-off between transmission and virulence is not assumed in the modeling framework. The optimal virulence strategy in these systems can be determined by both the duration of the host activity period as well as the variation in the host emergence timing. Parasites with low virulence strategies are favored in environments with long host activity periods and in environments in which hosts emerge synchronously. The results demonstrate that host phenology can be sufficient to select for intermediate optimal virulence strategies, providing an alternative mechanism to account for virulence evolution in some natural systems. Nuclear Progestin Receptor Mediated Linkage of Blood Coagulation and Ovulation Ovulation is a dramatic remodeling process that includes rupture of blood capillaries and clotting, but coagulation is not thought to directly regulate this process. Herein, we report remarkable increases of coagulation factors V (f5, [~]3145-fold) and tissue factor (f3a, [~]120-fold) in zebrafish ovarian follicle cells during ovulation. This increase was mediated through the nuclear progestin receptor (Pgr), which is essential for ovulation in zebrafish, and was totally abolished in ovarian follicular cells from pgr-/- mutants. In addition, promoter activities of f5 and f3a were significantly enhanced by progestin (DHP) via Pgr. Similar regulation of human F5 promoter activity was induced via human PGRB, suggesting a conserved mechanism. Site-directed mutagenesis of the zebrafish f5 promoter further demonstrated a direct regulation of coagulation factors via progestin response elements. Moreover, a stark increase of erythrocytes occurred in capillaries meshed in wildtype preovulatory follicles but was absent in pgr-/- mutants. Interestingly, anticoagulants significantly inhibited ovulation both in vitro and in vivo, respectively. Furthermore, reduced fecundity was observed in f5+/- female zebrafish. Taken together, our study provides plausible evidence for steroid regulation of coagulation factors, and a new hypothesis for blood clotting triggered ovulation in vertebrates. Epigenetic scores for the circulating proteome as tools for disease prediction Protein biomarkers have been identified across many age-related morbidities. However, characterising epigenetic influences could further inform disease predictions. Here, we leverage epigenome-wide data to study links between the DNAm signatures of the circulating proteome and incident diseases. Using data from four cohorts, we trained and tested epigenetic scores (EpiScores) for 953 plasma proteins, identifying 109 scores that explained between 1% and 58% of the variance in protein levels after adjusting for known protein quantitative trait loci (pQTL) genetic effects. By projecting these EpiScores into an independent sample, (Generation Scotland; n=9,537) and relating them to incident morbidities over a follow-up of 14 years, we uncovered 137 EpiScore - disease associations. These associations were largely independent of immune cell proportions, common lifestyle and health factors and biological aging. Notably, we found that our diabetes-associated EpiScores highlighted previous top biomarker associations from proteome-wide assessments of diabetes. These EpiScores for protein levels can therefore be a valuable resource for disease prediction and risk stratification. Characterising ontogeny of quantity discrimination in zebrafish A sense of non-symbolic numerical magnitudes is widespread in the animal kingdom and has been documented in adult zebrafish. Here we investigated the ontogeny of this ability using a group size preference task in juvenile zebrafish. Fish showed group size preference from 21 days post fertilization (dpf) and reliably chose the larger group when presented with discriminations of between 1 vs. 3, 2 vs. 5 and 2 vs. 3 conspecifics but not 2 vs. 4 conspecifics. When the ratio between the number of conspecifics in each group was maintained at 1:2, fish could discriminate between 1 vs. 2 individuals and 3 vs. 6, but again, not when given a choice between 2 vs. 4 individuals. These findings are in agreement with studies in other species suggesting the systems involved in quantity representation do not operate separately from other cognitive mechanisms. Rather they suggest quantity processing in fish may be the result of an interplay between attentional, cognitive and memory-related mechanisms as in humans and other animals. Our results emphasise the potential of the use of zebrafish to explore the genetic and neural processes underlying the ontogeny and function of number cognition. Characterization of divalent cation interactions with AASTY native nanodiscs Amphiphilic copolymers show promise in extracting membrane proteins directly from lipid bilayers into  native nanodiscs. However, many such copolymers are polyanionic and sensitive to divalent cations, limiting their applicability. We characterize the Ca2+ and Mg2+ sensitivity of poly(acrylic acid-co-styrene) (AASTY) copolymers with analytical UV and fluorescent size exclusion chromatography, enabling us to separate signals from nanodiscs, copolymers, and soluble aggregates. We find that divalent cations promote aggregation and precipitation of both free and lipid bound copolymers. We see that excess, free copolymer acts as a  cation sink that protects nanodiscs from Ca2+ induced aggregation. Removal of the free copolymer through dialysis induces aggregation that can be mitigated by KCl. Finally, we find that the nanodisc size is dynamic and dependent on lipid concentration. Our results offer insight to nanodisc behaviour, and can help guide experimental design, aimed at mitigating the shortcomings inherent in negatively charged nanodisc forming copolymers. 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. Relationship between shear wave velocity and muscle activation is inconsistent across different muscle types There is an increasing use of shear wave ultrasound elastography to quantify mechanical properties of muscles under various conditions such as changes muscle length and levels of activation in healthy and pathological muscle. However, little is known about the variability in shear wave velocity among muscles as most studies investigate one specific muscle. The purpose of this study was to determine if the relationship between SWV and muscle activation is consistent across muscles with different architectures: biceps brachii, tibialis anterior, and medial gastrocnemius, All measures were made at matching levels of activation and approximately at the optimal length for each muscle to control for length-dependent changes in the relationship between activation and force or stiffness. We also conducted a control experiment to determine how the passive force within a muscle alters the relationship between muscle activation and shear wave velocity. The relationship between shear wave velocity-squared and activation above 10% MVC differed across muscles, with biceps brachii and medial gastrocnemius showing a lower slope than tibialis anterior. Shear wave velocity-squared also differed between muscles at the shortest length (p<0.001) and the increase in shear wave velocity-squared with muscle lengthening also differed between muscle types (p = 0.005) Muscle-specific differences could not be explained by the architectural features such as pennation angle, fiber length, and physiological cross-sectional area. Our results demonstrate that there is not a unique relationship between muscle activation and shear wave velocity, highlighting the importance of understanding the many factors contributing to shear wave propagation in muscle before elastography can be used to make quantitative comparisons across muscle types. Visualizing cancer-originated acetate uptake through MCT1 in reactive astrocytes demarcates tumor border and extends survival in glioblastoma patients Glioblastoma multiforme (GBM) is a devastating brain tumor with dismal prognosis of only 15-month survival regardless of surgical resection. Here, we report an advanced neuroimaging technique combining 11C-acetate PET and MRI (AcePET), visualizing the boundary beyond the MRI-defined tumor. Targeted biopsy of the regions with increased 11C-acetate uptake revealed the presence of reactive astrocytes with enhanced acetate-transporter MCT1, along with cancer stem cells. Reactive astrogliosis and MCT1-dependent 11C-acetate-uptake were recapitulated in U87MG-orthotopic models. Mechanistically, glycolytic tumor cells release excessive acetate causing reactive astrogliosis, leading to the release of aberrant astrocytic GABA and H2O2, which further down-regulate the neuronal glucose uptake through GLUT3. Clincally, AcePET-guided surgery allows complete tumor resection of infiltrating cancer stem cells and extends the overall survival of patients by 5.25 months compared to conventional MRI-guided surgery. We established a new concept of the metabolic interactions between GBM cells and neighboring neurons through reactive astrocytes and developed AcePET-guided surgery to fight against GBM. Differed cell division angle, position of cell proliferative area, and localization of ANGUSTIFOLIA3 in lateral organs Leaf meristem is a cell proliferative zone present in the lateral organ primordia. In this study, we investigated how the proliferative zone affects the final morphology of the lateral organs. We examined how cell proliferative zones differ in the primordia of planar floral organs and polar auxin transport inhibitor (PATI)-treated leaves from normal foliage leaf primordia of Arabidopsis thaliana with a focus on the spatial accumulation pattern of ANGUSTIFOLIA3 (AN3), a key element for leaf meristem positioning. We found that organ shape changes by PATI treatment were correlated to the angle of the cell division plane relative to the leaf primordia axis in the leaf meristem (cell division angle), but not with leaf-meristem positioning, size of the leaf meristem, or the localization pattern of AN3 protein. In contrast, different shapes between sepals and petals compared with foliage leaves were associated with both altered meristem position associated with altered AN3 expression patterns and different distributions of cell division angles. These results suggest that lateral organ shapes are regulated via two aspects: position of meristem and cell division angles

Summary statementDifferent lateral organs with different morphology possess different properties of meristems; cell division angles, position of cell proliferative area and AN3 localization patterns. Design, Mutate, Screen: High-throughput creation of genetic clocks with different period-amplitude characteristics Directed evolution has become an invaluable tool in protein engineering and has also greatly influenced the construction of synthetic gene circuits. The ability to generate diversity at precise targets for directed evolution approaches has improved vastly, allowing researchers to create large, specific mutant libraries with relative ease. Screening approaches for large mutant libraries have similarly come a long way, especially when the desired behavior can easily be tested for with static, single time-point assays. For more complex gene circuits with dynamic phenotypes that change over time, directed evolution approaches to controlling and tuning circuit behavior have been hindered by the lack of sufficiently high-throughput screening methods to isolate variants with desired characteristics. Here we utilize directed mutagenesis and multiplexed microfluidics to develop a workflow for creating, screening and tuning dynamic gene circuits that operate at the population level. Specifically, we create a mutant library of an existing oscillator, the synchronized lysis circuit, and tune its dynamics while uncovering principles regarding its behavior. Lastly, we utilize this directed evolution workflow to construct a new synchronized genetic oscillator that exhibits robust dynamics over long time scales. 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.

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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 Natural variations in reproductive timing of brood size and X-chromosome nondisjunction in Caenorhabditis elegans Reproductive timing underlies self-sperm production and X-chromosome nondisjunction rate in Caenorhabditis elegans. These traits can be measured as brood size and male-production rate, and brood size decreases and male-production rate increases as worms age. These phenotypic changes occur simultaneously; however, whether these changes share common underlying genetic architectures still remains unclear. To enhance our understanding of reproductive timing in C. elegans, we measured and compared these two reproductive timing-related traits of 96 wild strains during early, late and total reproductive periods. We found that the two traits exhibited natural phenotypic variation with few outliers and a similar reproductive timing pattern as previous reports. The brood size and the male-production rate were not genetically correlated, implying that they might have different genetic architectures and that meiosis quality can be maintained despite increased progeny-production in the late reproductive period caused by more self-sperm. We also identified loci and candidate genetic variants significantly associated with male-production rate in the late and total reproductive periods. Our results provide an insight into life history traits in wild C. elegans strains. An alternative UPF1 isoform drives conditional remodeling of nonsense-mediated mRNA decay The nonsense-mediated mRNA decay (NMD) pathway monitors translation termination to degrade transcripts with premature stop codons and regulate thousands of human genes. Here we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL, enables condition-dependent remodeling of NMD specificity. Previous studies indicate that the extension of a conserved regulatory loop in the UPF1LL helicase core confers a decreased propensity to dissociate from RNA upon ATP hydrolysis relative to the major UPF1 isoform, designated UPF1 SL. Using biochemical and transcriptome-wide approaches, we find that UPF1LL overcomes the protective RNA binding proteins PTBP1 and hnRNP L to preferentially bind and down-regulate long 3UTRs normally shielded from NMD. Unexpectedly, UPF1LL supports induction of NMD on new populations of substrate mRNAs in response to activation of the integrated stress response and impaired translation efficiency. Thus, while canonical NMD is abolished by moderate translational repression, UPF1LL activity is enhanced, providing a mechanism to rapidly rewire NMD specificity in response to cellular stress. A new method for the sampling and preservation of placental specimens in low-resource settings for the identification of P. falciparum and analysis of nucleic acids Collection, preservation, and shipment of histological specimens in low-resource settings is challenging. We present a novel method that achieved excellent preservation of placental specimens from rural Mali by using formalin fixation, ethanol dehydration, and long-term storage in a solar-powered freezer. Sample preservation success was 92%, permitting evaluation of current and past malaria infection, anemia, placental maturity, and inflammation. Using RNAscope(R) hybridization we were able to visualize cell-specific gene expression patterns in the formalin-fixed paraffin-embedded (FFPE) specimens. Additionally, our method entailed mirrored sampling from the two cut faces of a cotyledon, one for the FFPE workflows and the other for storage in RNAlater and RNA-seq. Protein sequence sampling and prediction from structural data The increasing integration between protein engineering and machine learning has led to many interesting results. A problem still to solve is to evaluate the likelihood that a sequence will fold into a target structure. This problem can be also viewed as sequence prediction from a known structure.

In the current work, we propose improvements in the recent architecture of Geometric Vector Perceptrons [1] in order to optimize the sampling of sequences from a known backbone structure. The proposed model differs from the original in that there is: (i) no updating in the vectorial embedding, only in the scalar one, (ii) only one layer of decoding. The first aspect improves the accuracy of the model and reduces the use of memory, the second allows for training of the model with several tasks without incurring data leakage.

We treat the trained classifier as an Energy-Based Model and sample sequences by sampling amino acids in a non-autoreggresive manner in the empty positions of the sequence using energy-guided criteria and followed by random mutation optimization. We improve the median identity of samples from 40.2% to 44.7%.

An additional question worth investigating is whether sampled and original sequences fold into similar structures independent of their identity. We chose proteins in our test set whose sampled sequences show low identity (under 30%) but for which our model predicted favorable energies. We used AlphaFold [2, 3] and observed that the predicted structures for sampled sequences highly resemble the predicted structures for original sequences, with an average TM-score of 0.84. Relative contributions of sex hormones, sex chromosomes, and gonads to sex differences in tissue gene regulation Sex differences in physiology and disease in mammals result from the effects of three classes of factors that are inherently unequal in males and females: reversible (activational) effects of gonadal hormones, permanent (organizational) effects of gonadal hormones, and cell-autonomous effects of sex chromosomes, as well as genes driven by these classes of factors. Often, these factors act together to cause sex differences in specific phenotypes, but the relative contribution of each and the interactions among them remain unclear. Here, we used the Four Core Genotypes (FCG) mouse model with or without hormone replacement to distinguish the effects of each class of sex-biasing factors on transcriptome regulation in liver and adipose tissues. We found that the activational hormone levels have the strongest influence on gene expression, followed by the organizational gonadal sex effect and, lastly, sex chromosomal effect, along with interactions among the three factors. Tissue specificity was prominent, with a major impact of estradiol on adipose tissue gene regulation, and of testosterone on the liver transcriptome. The networks affected by the three sex-biasing factors include development, immunity and metabolism, and tissue-specific regulators were identified for these networks. Furthermore, the genes affected by individual sex-biasing factors and interactions among factors are associated with human disease traits such as coronary artery disease, diabetes, and inflammatory bowel disease. Our study offers a tissue-specific account of the individual and interactive contributions of major sex-biasing factors to gene regulation that have broad impact on systemic metabolic, endocrine, and immune functions. Onset and kinetics of B cell polarization depend on substrate mechanics and antigen concentration The immune synapse is the tight contact zone between a lymphocyte and a cell presenting its cognate antigen. This structure serves as a signaling platform and entails a polarization of intra-cellular components, necessary to the immunological function of the cell. While the surface properties of the presenting cell are known to control the formation of the synapse, their impact on polarization has not yet been studied.

Using functional lipid droplets as tunable artificial presenting cells combined with a microfluidic pairing device, we simultaneously observe synchronized synapses and dynamically quantify polarization patterns of individual B cells. By assessing how the ligand concentration, the surface fluidity and the substrate rigidity impact this polarization, we show that its onset depends on the antigen concentration at the synapse, and that the substrate rigidity controls both its onset and its kinetics. Our experimental system enables a fine phenotyping of monoclonal cell populations based on their synaptic readout.

Significance statementTo fulfill their immune function, B cells acquire antigens presented on the surface of antigen-presenting cells and organize their cytoskeleton and lysosomal traffic towards the contact. The polarization mechanism is evolutionary conserved, making the system interesting both from the immunology and the cell biology point of view. We developed and finely characterized antigen-coated droplets as surrogate presenting cells with a physiological-flow microfluidic cell-pairing chip to explore the B cell lysosome polarization in response to changes in droplet mechanics and antigen concentration. We observed that polarization is sensitive to mechanical properties (responding more strongly but less fast on stiffer substrates) and to the local antigen concentration at the contact. Hence, surprisingly, a late cell response is sensitive to the mechanics of the contact. Early loss of endogenous NAD+ following rotenone treatment leads to Sarm1 induction that is ameliorated by PARP inhibition Sarm1 is an evolutionary conserved innate immune adaptor protein that has emerged as a primary regulator of programmed axonal degeneration over the past decade. In vitro structural insights have revealed that although Sarm1 induces energy depletion by breaking down NAD+, it is also allosterically inhibited by NAD+. However, how NAD+ levels modulate the activation of intracellular Sarm1 has not been elucidated so far. This study focuses on understanding the events leading to Sarm1 activation in both neuronal and non-neuronal cells using the mitochondrial complex I inhibitor rotenone. Here we report the regulation of rotenone-induced cell death by loss of NAD+ that may act as a "biological trigger" of Sarm1 activation. Our study revealed that early loss of endogenous NAD+ levels arising due to PARP1 hyperactivation preceded Sarm1 induction following rotenone treatment. Interestingly, replenishing NAD+ levels by the PARP1 inhibitor, PJ34 restored mitochondrial homeostasis and prevented subsequent Sarm1 activation in rotenone treated cells. These cellular data were further validated in Drosophila melanogaster where a significant reduction in rotenone mediated loss of locomotor abilities and reduced dSarm expression was observed in the flies following PARP inhibition. Taken together, these observations not only uncovers a novel regulation of Sarm1 induction by endogenous NAD+ levels but also point towards an important understanding on how PARP inhibitors could be repurposed in the treatment of mitochondrial complex I deficiency disorders mediated by Sarm1. Anaerobic oxidation of methane in sediments of a nitrate-rich, oligo-mesotrophic boreal lake The identity of electron acceptors in promoting anaerobic oxidation of methane (AOM) in the sediments of boreal lakes is currently unknown. Here, we studied the AOM rate of sediment slurries collected from three profundal stations of a nitrate-rich, oligo-mesotrophic, boreal lake (Lake Paajarvi, Finland), under varying nitrate concentrations using 13C-labelling. Furthermore, vertical profiles of the sediment and porewater geochemistry, and the microbial communities (16S rRNA gene and shotgun metagenomic sequencing) were analyzed. Despite geochemical data indicating that simultaneous consumption of nitrate and methane took place at the sediment layers chosen for incubations, AOM rate was not enhanced by nitrate amendments at either of the stations. AOM rate was much higher at the shallow Station 1 (0.9-6.8 nmol C cm-3 d-1) with high contents of labile phytoplankton-derived organic matter, than at the deeper stations, Station 2 (0-0.3 nmol C cm-3 d-1) and 3 (0-0.2 nmol C cm-3 d-1). Accordingly, a higher relative abundance of methanotrophic archaea (Candidatus Methanoperedens) and bacteria (Methylococcales) were observed in the layers chosen for incubations at Station 1 than at the other stations. Besides nitrate, the geochemical profiles indicated that AOM was potentially coupled with iron or sulfate reduction at all stations. Furthermore, putative nitrite-reducing methanotrophs (Ca. Methylomirabilis) were the most abundant methanotrophs above the incubation layer at Station 2 and 3, which suggests that nitrite reduction also plays a role in driving AOM in the study lake. We conclude that AOM is not uniquely coupled to nitrate reduction in sediments of nitrate-rich, oligo-mesotrophic, boreal lakes. Cluster-specific gene markers enhance Shigella and Enteroinvasive Escherichia coli in silico serotyping Shigella and enteroinvasive Escherichia coli (EIEC) cause human bacillary dysentery with similar invasion mechanisms and share similar physiological, biochemical and genetic characteristics. The ability to differentiate Shigella and EIEC from each other is important for clinical diagnostic and epidemiologic investigations. The existing genetic signatures may not discriminate between Shigella and EIEC. However, phylogenetically, Shigella and EIEC strains are composed of multiple clusters and are different forms of E. coli. In this study, we identified 10 Shigella clusters, 7 EIEC clusters and 53 sporadic types of EIEC by examining over 17,000 publicly available Shigella/EIEC genomes. We compared Shigella and EIEC accessory genomes to identify the cluster-specific gene markers or marker sets for the 17 clusters and 53 sporadic types. The gene markers showed 99.63% accuracy and more than 97.02% specificity. In addition, we developed a freely available in silico serotyping pipeline named Shigella EIEC Cluster Enhanced Serotype Finder (ShigEiFinder) by incorporating the cluster-specific gene markers and established Shigella/EIEC serotype specific O antigen genes and modification genes into typing. ShigEiFinder can process either paired end Illumina sequencing reads or assembled genomes and almost perfectly differentiated Shigella from EIEC with 99.70% and 99.81% cluster assignment accuracy for the assembled genomes and mapped reads respectively. ShigEiFinder was able to serotype over 59 Shigella serotypes and 22 EIEC serotypes and provided a high specificity with 99.40% for assembled genomes and 99.38% for mapped reads for serotyping. The cluster markers and our new serotyping tool, ShigEiFinder (https://github.com/LanLab/ShigEiFinder), will be useful for epidemiologic and diagnostic investigations.

Impact statementThe differentiation of Shigella strains from enteroinvasive E. coli (EIEC) is important for clinical diagnosis and public health epidemiologic investigations. The similarities between Shigella and EIEC strains make this differentiation very difficult as both share common ancestries within E. coli. However, Shigella and EIEC are phylogenetically separated into multiple clusters, making high resolution separation using cluster specific genomic markers possible. In this study, we identified 17 Shigella or EIEC clusters including five that were newly identified through examination of over 17,000 publicly available Shigella and EIEC genomes. We further identified an individual or a set of cluster-specific gene markers for each cluster using comparative genomic analysis. These markers can then be used to classify isolates into clusters and were used to develop an in silico pipeline, ShigEiFinder (https://github.com/LanLab/ShigEiFinder) for accurate differentiation, cluster typing and serotyping of Shigella and EIEC from Illumina sequencing reads or assembled genomes. This study will have broad application from understanding the evolution of Shigella/EIEC to diagnosis and epidemiology.

Data summarySequencing data have been deposited at the National Center for Biotechnology Information under BioProject number PRJNA692536.

RepositoriesRaw sequence data are available from NCBI under the BioProject number PRJNA692536. 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. 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.