title: Novel Object Detection and Multiplexed Motion Representation in Retinal Bipolar Cells

abstract: Antagonistic interactions between the center and surround receptive field (RF) components lie at the heart of the computations performed in the visual system. Center-surround RFs are thought to enhance responses to spatial contrasts (i.e., edges), but how they contribute to motion processing is unknown. Here, we addressed this question in retinal bipolar cells, the first visual neuron with classic center-surround interactions. We found that bipolar glutamate release emphasizes objects that emerge in the RF; their responses to continuous motion are smaller, slower, and cannot be predicted by signals elicited by stationary stimuli. The alteration in signal dynamics induced by novel objects dwarfs the enhancement of spatial edges and can be explained by priming of RF surround during continuous motion. These findings echo the salience of human visual perception and demonstrate an unappreciated capacity of the center-surround architecture to facilitate novel object detection and multiplexed encoding of distinct sensory modalities.


title: Stable choice coding during changes of mind

abstract: How do we choose the best action in a constantly-changing environment? Many natural decisions unfold in dynamic environments where newer observations carry better information about the present state of the world. Recent work has shown that rats can learn to optimally discount old evidence, updating their provisional decision when the environmental state changes. Provisional decisions are thought to be represented in the Frontal Orienting Fields (FOF), but this has only been tested in static environments where the provisional and final decisions are not easily dissociated. Here, we characterize the representation of accumulated evidence in rat FOF during decision-making in a dynamic environment. We find that FOF encodes evidence throughout decision formation with a temporal gain modulation that rises until the period when the animal may need to act. Using a behavioral model to predict the timing of changes of mind revealed that FOF neurons respond rapidly to these events, representing the new provisional decisions in their firing rates. Our results suggest that the FOF represents provisional decisions even in dynamic, uncertain environments, allowing for rapid motor execution when it is time to act.

category: neuroscience


title: Fc-independent neutralization of SARS-CoV-2 by recombinant human monoclonal antibodies

abstract: The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in vivo neutralization of SARS-CoV-2 is not yet clear. Delineating the role this process plays in antibody-mediated protection will have a great impact on the design of such therapeutics. Here, the Fc of two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, targeting distinct domains of the spike, was engineered to abrogate their Fc-dependent functions. The protective activity of these antibodies was tested against lethal SARS-CoV-2 infections in K18-hACE2 transgenic mice, both before or two days post-exposure in comparison to their original, Fc-active antibodies. Antibody treatment with both Fc-variants similarly rescued the mice from death, reduced viral load and prevented signs of morbidity. In addition, surviving animals developed a significant endogenous immune response towards the virus. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in antibody-mediated protection, which should aid in future design of effective antibody-based therapies.

category: immunology


title: Safety, immunogenicity and protection provided by unadjuvanted and adjuvanted formulations of recombinant plant-derived virus-like particle vaccine candidate for COVID-19 in non-human primates

abstract: Although antivirals are important tools to control the SARS-CoV-2 infection, effective vaccines are essential to control the current pandemic. Plant-derived virus-like particle (VLP) vaccine candidates have previously demonstrated immunogenicity and efficacy against influenza. Here we report the immunogenicity and protection induced in macaques by intramuscular injections of VLP bearing SARS-CoV-2 spike protein (CoVLP) vaccine candidate formulated with or without Adjuvant System 03 (AS03) or cytosine phosphoguanine (CpG) 1018. Although a single dose of unadjuvanted CoVLP vaccine candidate stimulated humoral and cell-mediated immune responses, booster immunization (at 28 days after prime) and adjuvants significantly improved both responses with a higher immunogenicity and protection provided by AS03 adjuvanted CoVLP. Fifteen microgram CoVLP adjuvanted with AS03 induced a balanced IL-2 driven response along with IL-4 expression in CD4 T cells and mobilization of CD4 follicular helper cells (Tfh). Animals were challenged by multiple routes (i.e. intratracheal, intranasal and ocular) with a total viral dose of 106 plaque forming units of SARS-CoV-2. Lower viral replication in nasal swabs and broncho-alveolar lavage (BAL) as well as fewer SARS-CoV-2 infected cells and immune cell infiltrates in the lungs concomitant with reduced levels of pro-inflammatory cytokines and chemotactic factors in BAL were observed in the animals immunized with CoVLP adjuvanted with AS03. No clinical, pathologic or virologic evidences of vaccine associated enhanced disease (VAED) were observed in vaccinated animals. CoVLP adjuvanted with AS03 was therefore selected for vaccine development and clinical trials.


title: SETDB1 regulates microtubule dynamics

abstract: SETDB1 is a methyltransferase responsible for the methylation of histone H3-lysine-9, which is mainly related to heterochromatin formation. SETDB1 is overexpressed in various cancer types and is associated with an aggressive phenotype. In agreement with its activity, it mainly exhibits a nuclear localization; however, in several cell types a cytoplasmic localization was reported. Here we show that a substantial cytoplasmic pool of SETDB1 is colocalized with microtubules. Significantly, silencing of SETDB1 led to faster polymerization and reduced rate of catastrophe events of microtubules in parallel to reduced proliferation rate and slower mitotic kinetics. Interestingly, over-expression of either wild-type or catalytic dead SETDB1 altered microtubule polymerization rate to the same extent, suggesting that SETDB1 affects MT dynamics by a methylation-independent mechanism. Finding interaction between SETDB1 and the tubulin deacetylase HDAC6 and increased tubulin acetylation levels upon silencing of SETDB1 suggest a model in which SETDB1 affects microtubule dynamics by interacting with both microtubules and HDAC6 to enhance tubulin deacetylation. Overall, our results suggest a novel cytoplasmic role for SETDB1 in the regulation of microtubule dynamics.

category: cell biology


title: Distant water industrial fishing in developing countries: A case study of Madagascar

abstract:
As industrial vessels continue to expand in both extractive capacity and spatial range, concerns have grown over foreign industrial fishing occurring within the marine territories of developing countries, both legally and illegally. Madagascars status as a "least developed country", coupled with its high marine biodiversity, makes its waters particularly susceptible to fishing by distant water fishing nations (DWFNs). However, given constraints in management and research, it is unclear how foreign industrial fishing, both legal via foreign agreements and illegal, may impact local marine resources that many coastal communities depend on for food security, cultural meaning and livelihoods. We used satellite-derived fishing effort data from 2012-2020, via Global Fishing Watch, to analyze industrial fishing effort occurring within Madagascars Exclusive Economic Zone (EEZ). We documented 907,643 hours of industrial fishing within the Madagascar EEZ across 277 vessels from 17 different countries. We found that Taiwanease vessels (39.8%) using drifting longlines and Malagasy (17.2% shrimp trawlers were the most prevalent. Fishing effort was highly seasonal (68% of effort between October and February) and increased with higher global fish prices and the Indian Ocean Dipole, which is a measure of regional water temperature cycles. We also found a number of instances (17.6% of the fishing effort for 170,726 total hours) of foreign fishing vessels operating close to shore and within a number of marine protected areas. These results highlight the need for increased transparency surrounding foreign fishing agreements and unauthorized fishing within the waters of developing countries. Increases in industrial fishing effort and encroachment into near-shore areas has the potential to severely threaten current sustainable fisheries management initiatives by conservation organizations and coastal communities.

HighlightsO_LIDistant water fishing nations dominated fishing efforts within Madagascars EEZ.
C_LIO_LILonglining by foreign nations was the dominant fishing mode and increased from October-February.
C_LIO_LIMalagasy vessels focused on trawling for shrimp.
C_LIO_LIFishing effort increased during positive Indian Ocean Dipoles and with higher fish prices.
C_LIO_LIDistant water fishing nations frequently fished close to shore and sometimes within MPAs.
C_LI

category: ecology


title: Insularity forcing on plant persistence strategies in edaphic island systems

abstract:
AimTrait-based approaches are increasingly implemented in island biogeography, providing key insights into the eco-evolutionary dynamics of insular systems. However, what determines persistence of plant species once they have arrived and established in an island remains largely unexplored. Here, we examined links between non-acquisitive persistence strategies and insularity across three terrestrial edaphic island systems, hypothesising that insularity promotes strategies for local persistence.

LocationEurope: Western Carpathians, Moravia, and Cantabrian Range.

Time periodPresent.

Major taxa studiedVascular plants.

MethodsFor each system, we used linear models at the island scale to test whether persistence-related plant trait patterns (average trait values and diversity) depend on three insularity metrics (island size, isolation and target effect). We focused on patterns of edaphic island specialists because, in contrast to matrix-derived species, their presence is confined to the edaphic islands.

ResultsWe found that insularity metrics explained large proportions in the variation of the average and diversity of persistence-related traits of edaphic island specialists. Insularity was associated with a decline in the proportion of island specialists that have clonal abilities, yet it affected trait values of specialists towards enhanced abilities to persist locally (e.g. more extensive lateral spread) while reducing trait variability. Higher degrees of insularity within the systems were translated to stronger effects on functional trait patterns.

Main conclusionsInsularity affects plant species diversity, distribution and forms in terrestrial island-like systems, similarly as it is assumed for true islands. Insularity - measured using a single (island size, isolation) or combined (target effect) predictors - may operate selecting for enhanced and less diverse persistence strategies. Ultimately, this process, which we call insularity forcing, operates as a selective process to promote species ability to avoid local extinction and to persist on terrestrial islands.


title: The FDA-approved drug apremilast suppresses alcohol intake: clinical and pre-clinical validation

abstract: Treatment options for Alcohol Use Disorders (AUD) have minimally advanced since 2004, while the annual deaths and economic toll have become alarmingly high. Bringing potential therapeutics beyond the bench and into the clinic for AUD requires rigorous pharmacological screening across molecular, behavioral, pre-clinical, and clinical studies in neuroscience. The repurposing of FDA-approved compounds is an effective and expedited means of screening pharmacotherapies for AUD. Here, we demonstrate that apremilast, a phosphodiesterase type 4 inhibitor that is FDA approved for psoriasis and psoriatic arthritis, reduces binge-like alcohol intake and behavioral measures of motivation in unique, preclinical genetic risk models for drinking to intoxication and reduces excessive alcohol drinking in models of stress-facilitated drinking and alcohol dependence. In a double blind, placebo-controlled human laboratory study in non-treatment seeking individuals with AUD, apremilast significantly reduced the number of drinks per day. Lastly, using site-directed drug infusions and electrophysiology we determined that apremilast may act by increasing neural activity in the nucleus accumbens, an important alcohol-related brain region, to reduce alcohol intake in mice. These results demonstrate that apremilast reduces excessive alcohol drinking across a spectrum of AUD severity and support its importance as a potential therapeutic for AUD.

category: animal behavior and cognition


title: Genes responsible for avoiding attack of a beetle, relating to the duration of death feigning

abstract: Predator avoidance is an important behavior that affects the degree of adaptation of organisms. We compared the DNA variation of one of the predator-avoidance behaviors, the recently extensively studied "death-feigning behavior," between the long strain bred for feigning death for a long time and the short strain bred for feigning death for a short time. To clarify how the difference in DNA sequences between the long and short strains corresponds to the physiological characteristics of the death-feigning duration at the transcriptome level, we performed comprehensive and comparative analyses of gene variants in Tribolium castaneum strains using DNA-re-sequence. The duration of death feigning involves many gene pathways, including caffeine metabolism, tyrosine metabolism, tryptophan metabolism, metabolism of xenobiotics by cytochrome P450, longevity regulating pathways, and circadian rhythm. Artificial selection based on the duration of death feigning results in the preservation of variants of genes in these pathways in the long strain. When an animal wake up from a near-death experience is closely related to its success in avoiding predation. This study suggests that many metabolic pathways and related genes may be involved in the decision-making process of anti-predator animal behavior by forming a network in addition to the tyrosine metabolic system, including dopamine, revealed in previous studies.


title: Expression of GRINA Correlates with Prognosis in Human Cancers: A Pan-cancer Analysis

abstract: GRINA is an emerging target for cancer therapy. However, the role of GRINA expression and its correlation with cancer patient survival has not been comprehensively studied. Here, we found that mRNA and protein expression of GRINA was upregulated in breast, colon, gastric, and prostate cancers and negatively correlated with patient survival. Also, the upregulation of GRINA expression is associated with hypomethylation of its promoter region. Our GRINA-miRNAs network analysis revealed potential regulatory miRNAs regulating the GRINA expression and its downstream pathways. Next, functional enrichment and pathway analysis of genes commonly co-express with GRINA in breast, colon, gastric, and prostate cancers revealed GRINA regulatory pathways. Concurrently, our upstream regulator analysis revealed possible kinases, transcription factors, and proteins that may potentially regulate GRINA. Overall, this study demonstrates the prognostic significance of GRINA expression and identifies potential regulatory mechanisms, which might have significant implications in targeted therapies for human cancers.

category: cancer biology


title: Mechanical demands of the two-handed hardstyle kettlebell swing performed by an RKC-certified Instructor

abstract:
BackgroundThe effects of hardstyle kettlebell training are increasingly cited in strength and conditioning research, yet reference data from a proficient swing is scarce. The aim of this exploratory study was to investigate the force profile of a two-handed hardstyle swing performed by a Russian Kettlebell Challenge (RKC) instructor.

MethodsThe subject is a 44-year-old male, body mass 75.6 kg, height 173.5 cm, with six years of regular hardstyle training experience. Two-handed hardstyle swings were performed with a series of incremental mass kettlebells (8-68 kg). Ground reaction force (GRFs) was obtained from a floor-mounted force platform. Force-time curves (FTCs), peak force, forward force, rate of force development (RFD) and swing cadence were investigated.

ResultsData revealed the FTC of a proficient swing is highly consistent and dominated by a single force peak (mean SD = 47 N), with a profile that remained largely unchanged to 24 kg. Pearson correlation analysis revealed a very strong positive correlation in peak force with kettlebell mass (r = 0.95), which increased disproportionately from the lightest to heaviest kettlebells; net peak force increased from 8.36 {+/-} 0.75 N.kg-1 (0.85 x BW) to 12.82 {+/-} 0.39 N.kg-1 (1.3x BW). There was a strong negative correlation between RFD and kettlebell mass (r = 0.82) that decreased from 39.2 N.s-1.kg-1 to 21.5 N.s-1.kg-1. There was a very strong positive correlation in forward ground reaction force with kettlebell mass (r = 0.99), expressed as a ratio of vertical ground reaction, that increased from 0.092 (9.2%) to 0.205 (20.5%). Swing cadence exceeded 40 swings per minute (SPM) with all kettlebells.

ConclusionOur findings challenge some of the popular beliefs of the hardstyle kettlebell swing. Consistent with hardstyle practice, and previous kinematic analysis of expert and novice, force-time curves show a characteristic single large force peak, differentiating passive from active shoulder flexion. Ground reaction force did not increase proportionate to kettlebell mass, with a magnitude of forward force smaller than described in practice. These results could be useful for coaches and trainers wanting to improve athletic performance, and healthcare providers using the kettlebell swing for therapeutic purposes. Findings from this study were used to inform the BELL Trial, a pragmatic controlled trial of kettlebell training with older adults. www.anzctr.org.au ACTRN12619001177145.

category: physiology


title: Force profile of the two-handed hardstyle kettlebell swing performed by an RKC-certified Instructor


title: Photoperiodic Flowering Response of Essential Oil, Grain, and Fiber Hemp (Cannabis sativa L.) Cultivars

abstract: Cultivation of hemp (Cannabis sativa L.) in tropical and subtropical regions can be challenging if flowering behavior of a given cultivar is unknown, poorly understood, or not accurately selected for the photoperiod. Identifying cultivars adapted to local environmental conditions is key to optimizing hemp vegetative and flowering performance. We investigated the effects of varying light cycles in regulating extension growth and flowering response of 15 essential oil and 12 fiber/grain hemp cultivars both in indoors and outdoors. Plants were subjected to eleven photoperiods in the controlled rooms ranging from 12 h to 18 h, and natural day length in the field. The critical photoperiod threshold was identified for seven essential oil cultivars and two fiber/grain cultivars.  Cherry Wine-CC,  PUMA-3, and  PUMA-4 had the shortest critical day length between 13 h 45 min and 14 h. The flowering of essential oil cultivars was generally delayed by 1 to 2 d when photoperiod exceeded 13 h compared to 12 h, and flowering was further delayed by 7 to 8 d when photoperiod exceed 14 h. In fiber/grain cultivars, flowering was generally delayed by 1 to 3 d when day length exceeded 14 h. Flowering for most essential oil cultivars was delayed by 5 to 13 d under 14 h photoperiod compared to 13 h 45 min, suggesting a photoperiod difference as little as 15 min can significantly influence the floral initiation of some essential oil cultivars. Cultivars represented by the same name but acquired from different sources can perform differently under the same environmental conditions, suggesting genetic variation among cultivars with the same name. Average days to flower of fiber/grain cultivars was correlated with reported cultivar origin with faster flowering occurring among northern cultivars when compared to southern cultivars. Plant height generally increased as the day length increased in essential oil cultivars but was not affected in fiber/grain cultivars. In addition, civil twilight of approximately 2 {micro}mol{middle dot}m-2{middle dot}s-1 was discovered to be biologically effective in regulating hemp flowering. Collectively, we conclude that most of the essential oil cultivars and some southern fiber/grain cultivars tested express suitable photoperiods for tropical and sub-tropical region cultivation.

category: plant biology


title: A large majority of awake hippocampal sharp-wave ripples feature spatial trajectories with momentum

abstract: During periods of rest, hippocampal place cells feature bursts of activity called sharp-wave ripples (SWRs). Heuristic approaches to their analysis have revealed that a small fraction of SWRs appear to "simulate" trajectories through the environment--called awake hippocampal replay--while the functional role of a majority of these SWRs remains unclear. Applying a novel probabilistic approach to characterize the spatio-temporal dynamics embedded in SWRs, we instead show that almost all SWRs of foraging rodents simulate such trajectories through the environment. Furthermore, these trajectories feature momentum, that is, inertia in their velocities, that mirrors the animals natural movement. This stands in contrast to replay events during sleep which seem to follow Brownian motion without such momentum. Lastly, interpreting the replay trajectories in the context of navigational planning revealed that similar past analyses were biased by the heuristic SWR sub-selection. Overall, our approach provides a more complete characterization of the spatio-temporal dynamics within SWRs, highlights qualitative differences between sleep and awake replay, and ought to support future, more detailed, and less biased analysis of the role of awake replay in navigational planning.


title: Transcriptional landscape of human microglia reveals robust gene expression signatures that implicates age, sex and APOE-related immunometabolic pathway perturbations

abstract: Microglia have fundamental roles in health and disease, however effects of age, sex and genetic factors on human microglia have not been fully explored. We applied bulk and single cell approaches to comprehensively characterize human microglia transcriptomes and their associations with age, sex and APOE. We identified a novel microglial signature, characterized its expression in bulk tissue and single cell microglia transcriptomes. We discovered microglial co-expression network modules associated with age, sex and APOE-{varepsilon}4 that are enriched for lipid and carbohydrate metabolism genes. Integrated analyses of modules with single cell transcriptomes revealed significant overlap between age-associated module genes and both pro-inflammatory and disease-associated microglial clusters. These modules and clusters harbor known neurodegenerative disease genes including APOE, PLCG2 and BIN1. Meta-analyses with published bulk and single cell microglial datasets further supported our findings. Thus, these data represent a well-characterized human microglial transcriptome resource; and highlight age, sex and APOE-related microglial immunometabolism perturbations with potential relevance in neurodegeneration.

category: genetics


title: Height, but not binding epitope, affects the potency of synthetic TCR agonists

abstract:
Under physiological conditions, peptide-MHC (pMHC) molecules can trigger T-cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCR can also be activated by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit non-physiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCR{beta} Fab antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab-DNA ligand efficiently activates TCR as a monomer when membrane-associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for effective TCR triggering and cellular activation by Fab-DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex, but that length of the DNA tether is important. Increasing the intermembrane distance spanned by Fab-DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency, consistent with the kinetic-segregation model of TCR triggering. These results establish design parameters for construction of synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand.

STATEMENT OF SIGNIFICANCEWe report geometric requirements for potent T cell activation by synthetic TCR ligands that mimic biophysical properties of the native pMHC ligand, but have the additional ability to activate polyclonal T cell populations. We find that increasing the space between apposed membranes at TCR binding events decreases ligand potency, but that changing the ligands binding epitope on the TCR has essentially no effect. The observed decrease in potency with increased ligand height is attributed to the longer ligands attenuated ability to trigger TCR at binding events.

category: biophysics


title: Markov state models from hierarchical density-based assignment

abstract: Markov state models (MSMs) have become one of the preferred methods for the analysis and interpretation of molecular dynamics (MD) simulations of conformational transitions in biopolymers. While there is great variation in terms of implementation, a well-defined workflow involving multiple steps is often adopted. Typically, molecular coordinates are first subjected to dimensionality reduction and then clustered into small "microstates", which are subsequently lumped into "macrostates" using the information from the slowest eigenmodes. However, the microstate dynamics is often non-Markovian and long lag times are required to converge the MSM. Here we propose a variation on this typical workflow, taking advantage of hierarchical density-based clustering. When applied to simulation data, this type of clustering separates high population regions of conformational space from others that are rarely visited. In this way, density-based clustering naturally implements assignment of the data based on transitions between metastable states. As a result, the state definition becomes more consistent with the assumption of Markovianity and the timescales of the slow dynamics of the system are recovered more effectively. We present results of this simplified workflow for a model potential and MD simulations of the alanine dipeptide and the FiP35 WW domain.


title: Microinductor-Fused Atomic Force Microscopy Cantilevers for Dynamic Imaging and Multimodal Manipulation

abstract: Recent advances in atomic force microscopy (AFM) imaging and force spectroscopy have demonstrated improvements in rapid acquisition of quantitative data for applications in materials science, surface characterization, and biology. However, conventional AFM technology is limited in detection sensitivity and the ability to excite at off-resonance frequencies restricting broad utility of the technology. Here we demonstrate new AFM cantilevers fabricated with a planar microcoil at the tip region, which can be used to generate or sense highly-localized magnetic fields. Torsion/bending actuation of the cantilevers is accomplished with simple experimental configurations, enabling quantitative and simultaneous mapping of both stiffness and friction at the sample surface with more than one order of magnitude improvement in compositional contrast. Our method is compatible with commercial AFM systems, allowing us to clearly resolve small stiffness and friction variations in copolymer and biological samples that were difficult to detect by conventional AFM methods. In combination with fluorescence microscopy, we also generated localized fields to selectively stimulate and monitor structural changes in viable cells with nm-scale detail. Hybrid AFM cantilevers may be useful to characterize a broad range of complex material surfaces, in addition to combined physical and chemical analyses of single cells and biological microenvironments.


title: Leveraging the mass balances of cellular metabolism to infer absolute concentrations from relative abundance metabolomics data

abstract:
MotivationAs the large-scale study of metabolites and a direct readout of a systems metabolic state, metabolomics has significant appeal as a source of information for many metabolic modelling platforms and other metabolic analysis tools. However, metabolomics data are typically reported in terms of relative abundances, which precluding use with tools where absolute concentrations are necessary. While chemical standards can be used to determine the absolute concentrations of metabolites, they are often time-consuming to run, expensive, or unavailable for many metabolites. A computational framework that can infer absolute concentrations without the use of chemical standards would be highly beneficial to the metabolomics community.

ResultsWe have developed and characterized MetaboPAC, a computational strategy that leverages the mass balances of a system to infer absolute concentrations in metabolomics datasets. MetaboPAC uses a kinetic equations approach and an optimization approach to predict the most likely response factors that describe the relationship between absolute concentrations and their relative abundances. We determined that MetaboPAC performed significantly better than the other approaches assessed on noiseless data when at least 60% of kinetic equations are known a priori. Under the most realistic conditions (low sampling frequency, high noise data), MetaboPAC significantly outperformed other methods in the majority of cases when 100% of the kinetic equations were known. For metabolomics datasets extracted from systems that are well-studied and have partially known kinetic structures, MetaboPAC can provide valuable insight about their absolute concentration profiles.

category: systems biology


title: Effects of Slow Oscillatory Transcranial Alternating Current Stimulation on Motor Cortical Excitability Assessed by Transcranial Magnetic Stimulation

abstract: Converging evidence suggests that transcranial alternating current stimulation (tACS) may entrain endogenous neural oscillations to match the frequency and phase of the exogenously applied current and this entrainment may outlast the stimulation (although only for a few oscillatory cycles following the cessation of stimulation). However, observing entrainment in the electroencephalograph (EEG) during stimulation is extremely difficult due to the presence of complex tACS artefacts. The present study assessed entrainment to slow oscillatory (SO) tACS by measuring motor cortical excitability across different oscillatory phases during (i.e., online) and outlasting (i.e., offline) stimulation. 30 healthy participants received 60 trials of intermittent SO tACS (0.75 Hz; 16s on / off interleaved) at an intensity of 2mA peak-to-peak. Motor cortical excitability was assessed using transcranial magnetic stimulation (TMS) of the hand region of the primary motor cortex (M1HAND) to induce motor evoked potentials (MEPs) in the contralateral thumb. MEPs were acquired at four time-points within each trial - early online, late online, early offline, and late offline - as well as at the start and end of the overall stimulation period (to probe longer-lasting aftereffects of tACS). A significant increase in MEP amplitude was observed from pre-to post-tACS (P = 0.013) and from the first to the last tACS block (P = 0.008). However, no phase-dependent modulation of excitability was observed. Therefore, although SO tACS had a facilitatory effect on motor cortical excitability that outlasted stimulation, there was no evidence supporting entrainment of endogenous oscillations as the underlying mechanism.


title: Improved constraints increase the predictivity and applicability of a linear programming-based dynamic metabolic modeling framework

abstract: Current metabolic modeling tools suffer from a variety of limitations, from scalability to simplifying assumptions, that preclude their use in many applications. We recently created a modeling framework, LK-DFBA, that addresses a key gap: capturing metabolite dynamics and regulation while retaining a potentially scalable linear programming structure. Key to this frameworks success are the linear kinetics and regulatory constraints imposed on the system. However, while the linearity of these constraints reduces computational complexity, it may not accurately capture the behavior of many biochemical systems. Here, we developed three new classes of LK-DFBA constraints to better model interactions between metabolites and the reactions they regulate. We tested these new approaches on several synthetic and biological systems, and also performed the first-ever comparison of LK-DFBA predictions to experimental data. We found that no single constraint approach was optimal across all systems examined, and systems with the same topological structure but different parameters were often best modeled by different types of constraints. However, we did find that the optimal constraint approach was generally robust to local perturbations of the system, indicating that just a single wild-type dataset could allow identification of the ideal constraint for a given system. These results suggest that the availability of multiple constraint approaches will allow LK-DFBA to model a wider range of metabolic systems.


title: Diverse classes of constraints enable broader applicability of a linear programming-based dynamic metabolic modeling framework


title: Identification of translocation inhibitors targeting the type III secretion system of enteropathogenic E. coli

abstract: Infections with enteropathogenic E. coli (EPEC) cause severe diarrhea in children. The non-invasive bacteria adhere to enterocytes of the small intestine and use a type III secretion system (T3SS) to inject effector proteins into host cells to modify and exploit cellular processes in favor of bacterial survival and replication. Several studies have shown that the T3SSs of bacterial pathogens are essential for virulence. Furthermore, the loss of T3SS-mediated effector translocation results in increased immune recognition and clearance of the bacteria. The T3SS is, therefore, considered a promising target for antivirulence strategies and novel therapeutics development. Here, we report the results of a high-throughput screening assay based on the translocation of the EPEC effector protein Tir. Using this assay, we screened more than 13,000 small molecular compounds of six different compound libraries and identified three substances which showed a significant dose-dependent effect on translocation without adverse effects on bacterial or eukaryotic cell viability. Additionally, these substances reduced bacterial binding to host cells, effector-dependent cell detachment and abolished A/E lesion formation without affecting the expression of components of the T3SS or associated effector proteins. Moreover, no effects of the inhibitors on bacterial motility or Shiga-toxin expression were observed. In summary, we have identified three new compounds that strongly inhibit T3SS-mediated translocation of effectors into mammalian cells, which could be valuable as lead substances for treating EPEC and EHEC infections.

category: microbiology


title: A Trifector of New Insights into Ovine Footrot for Infection Drivers, Immune Response and Host Pathogen Interactions

abstract:
Footrot is a polymicrobial infectious disease in sheep causing severe lameness, leading to one of the industrys biggest welfare problems. The complex aetiology of footrot makes in-situ or in-vitro investigations difficult. Computational methods offer a solution to understanding the bacteria involved, how they may interact with the host and ultimately providing a way to identify targets for future hypotheses driven investigative work. Here we present the first combined global analysis of the bacterial community transcripts together with the host immune response in healthy and diseased ovine feet during a natural polymicrobial infection state using metatranscriptomics. The intra tissue and surface bacterial populations and the most abundant bacterial transcriptome were analysed, demonstrating footrot affected skin has a reduced diversity and increased abundances of, not only the causative bacteria Dichelobacter nodosus, but other species such as Mycoplasma fermentans and Porphyromonas asaccharolytica. Host transcriptomics reveals a suppression of biological processes relating to skin barrier function, vascular functions, and immunosurveillance in unhealthy interdigital skin, supported by histological findings that type I collagen (associated with scar tissue formation) is significantly increased in footrot affected interdigital skin comparted to outwardly healthy skin. Finally, we provide some interesting indications of host and pathogen interactions associated with virulence genes and the host spliceosome which could lead to the identification of future therapeutic targets.

Impact StatementLameness in sheep is a global welfare and economic concern and footrot is the leading cause of lameness, affecting up to 70% of flocks in the U.K. Current methods for control of this disease are labour intensive and account for approximately 65% of antibiotic use in sheep farming, whilst preventative vaccines suffer from poor efficacy due to antigen competition. Our limited understanding of cofounders, such as strain variation and polymicrobial nature of infection mean new efficacious, affordable and scalable control measures are not receiving much attention. Here we examine the surface and intracellular bacterial populations and propose potential interactions with the host. Identification of these key bacterial species involved in the initiation and progression of disease and the host immune mechanisms could help form the basis of new therapies.

category: genomics


title: CovidExpress: an interactive portal for intuitive investigation on SARS-CoV-2 related transcriptomes

abstract: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans could cause coronavirus disease 2019 (COVID-19). Since its first discovery in Dec 2019, SARS-CoV-2 has become a global pandemic and caused 3.3 million direct/indirect deaths (2021 May). Amongst the scientific communitys response to COVID-19, data sharing has emerged as an essential aspect of the combat against SARS-CoV-2. Despite the ever-growing studies about SARS-CoV-2 and COVID-19, to date, only a few databases were curated to enable access to gene expression data. Furthermore, these databases curated only a small set of data and do not provide easy access for investigators without computational skills to perform analyses. To fill this gap and advance open-access to the growing gene expression data on this deadly virus, we collected about 1,500 human bulk RNA-seq datasets from publicly available resources, developed a database and visualization tool, named CovidExpress (https://stjudecab.github.io/covidexpress). This open access database will allow research investigators to examine the gene expression in various tissues, cell lines, and their response to SARS-CoV-2 under different experimental conditions, accelerating the understanding of the etiology of this disease to inform the drug and vaccine development. Our integrative analysis of this big dataset highlights a set of commonly regulated genes in SARS-CoV-2 infected lung and Rhinovirus infected nasal tissues, including OASL that were under-studied in COVID-19 related reports. Our results also suggested a potential FURIN positive feedback loop that might explain the evolutional advantage of SARS-CoV-2.

category: bioinformatics


title: Potent neutralization of SARS-CoV-2 variants of concern by an antibody with a unique genetic signature and structural mode of spike recognition

abstract: The emergence of novel SARS-CoV-2 lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of COVID-19. Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the LIBRA-seq technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 showed potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryo-EM structure of 54042-4 in complex with the SARS-CoV-2 spike revealed an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses unique genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings motivate 54042-4 as a lead candidate for clinical development to counteract current and future SARS-CoV-2 VOCs.


title: Durability of mRNA-1273-induced antibodies against SARS-CoV-2 variants

abstract:
SARS-CoV-2 mutations may diminish vaccine-induced protective immune responses, and the durability of such responses has not been previously reported. Here, we present a comprehensive assessment of the impact of variants B.1.1.7, B.1.351, P.1, B.1.429, and B.1.526 on binding, neutralizing, and ACE2-blocking antibodies elicited by the vaccine mRNA-1273 over seven months. Cross-reactive neutralizing responses were rare after a single dose of mRNA-1273. At the peak of response to the second dose, all subjects had robust responses to all variants. Binding and functional antibodies against variants persisted in most subjects, albeit at low levels, for 6 months after the primary series of mRNA-1273. Across all assays, B.1.351 had the greatest impact on antibody recognition, and B.1.1.7 the least. These data complement ongoing studies of clinical protection to inform the potential need for additional boost vaccinations.

One-Sentence SummaryMost mRNA-1273 vaccinated individuals maintained binding and functional antibodies against SARS-CoV-2 variants for 6 months.


title: The Spike Proteins of SARS-CoV-2 B.1.617 and B.1.618 Variants Identified in India Provide Partial Resistance to Vaccine-elicited and Therapeutic Monoclonal Antibodies.

abstract: Highly transmissible SARS-CoV-2 variants recently identified in India designated B.1.617 and B.1.618 have mutations within the spike protein that may contribute to their increased transmissibility and that could potentially result in re-infection or resistance to vaccine-elicited antibody. B.1.617 encodes a spike protein with mutations L452R, E484Q, D614G and P681R while the B.1.618 spike has mutations {Delta}145-146, E484K and D614G. We generated lentiviruses pseudotyped by the variant proteins and determined their resistance to neutralization by convalescent sera, vaccine-elicited antibodies and therapeutic monoclonal antibodies. Viruses with B.1.617 and B.1.618 spike were neutralized with a 2-5-fold decrease in titer by convalescent sera and vaccine-elicited antibodies. The E484Q and E484K versions were neutralized with a 2-4-fold decrease in titer. Virus with the B.1.617 spike protein was neutralized with a 4.7-fold decrease in titer by the Regeneron monoclonal antibody cocktail as a result of the L452R mutation. The modest neutralization resistance of the variant spike proteins to vaccine elicited antibody suggests that current vaccines will remain protective against the B.1.617 and B.1.618 variants.


title: Allicin inhibits SARS-CoV-2 replication and abrogates the antiviral host response in the Calu-3 proteome

abstract: The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic is a major health burden. Volatile garlic organosulfur compounds, such as the thiol-reactive allicin (diallyl thiosulfinate) exert strong antimicrobial activity against various respiratory pathogens. Here, we investigated the antiviral activity of allicin against SARS-CoV-2 in infected Vero E6 and Calu-3 lung cells. Calu-3 cells showed greater allicin tolerance due >4-fold increased GSH levels compared to Vero E6. However, biocompatible allicin doses efficiently inhibited viral replication in both cell lines. Proteome analyses of SARS-CoV-2 infected Calu-3 cells revealed a strong induction of the antiviral interferon-stimulated gene (ISG) signature (e.g. cGAS, Mx1, IFIT, IFIH, IFI16, IFI44, 25OAS and ISG15), pathways of vesicular transport, tight junctions (KIF5A/B/C, OSBPL2, CLTC1, ARHGAP17) and ubiquitin modification (UBE2L3/5), as well as reprogramming of host metabolism, transcription and translation. Allicin abrogated the ISG host response and reverted the host cellular pathways to levels of uninfected Calu-3 cells, confirming the antiviral and immunomodulatory activity of allicin in the host proteome. Thus, biocompatible doses of allicin could be promising for protection of lung cells against SARS-CoV-2.


title: Stabilization of the SARS-CoV-2 Spike receptor-binding domain using deep mutational scanning and structure-based design

abstract: The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40{degrees}C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.


title: Pan-ErbB inhibition protects from SARS-CoV-2 replication, inflammation, and injury

abstract: Effective therapies are needed to combat emerging viruses. Seventeen candidates that rescue cells from SARS-CoV-2-induced lethality and target diverse functions emerged in a screen of 4,413 compounds. Among the hits was lapatinib, an approved inhibitor of the ErbB family of receptor tyrosine kinases. Lapatinib and other pan-ErbB inhibitors suppress replication of SARS-CoV-2 and unrelated viruses with a high barrier to resistance. ErbB4, but not lapatinibs cancer targets ErbB1 and ErbB2, is required for SARS-CoV-2 entry and encephalitis alphavirus infection and is a molecular target mediating lapatinibs antiviral effect. In human lung organoids, lapatinib protects from SARS-CoV-2-induced activation of pathways implicated in non-infectious acute lung injury and fibrosis downstream of ErbBs (p38-MAPK, MEK/ERK, and AKT/mTOR), pro-inflammatory cytokine production, and epithelial barrier injury. These findings reveal regulation of viral infection, inflammation, and lung injury via ErbBs and propose approved candidates to counteract these effects with implications for pandemic coronaviruses and unrelated viruses.


title: Discovery of pan-ErbB inhibitors protecting from SARS-CoV-2 replication, inflammation, and lung injury by a drug repurposing screen


title: South American freshwater fish diversity shaped by Andean uplift since the Late Cretaceous

abstract:
South America is home to the highest freshwater fish biodiversity on Earth1,2. The hotspot of species richness is located in the western Amazon Basin, and richness decreases downstream along the Amazon River towards the mouth at the Atlantic coast (Fig. 1b, c)3,4, which contradicts the positive relationship between stream size and biodiversity that is commonly observed in river systems across the world5,6. We investigate the role of river rerouting events caused by Andean mountain building and repeated episodes of flooding in western Amazonia in shaping the modern-day richness pattern of freshwater fishes in South America. To this end, we combine a reconstruction of river networks following Andean surface uplift since 80 million years ago with a mechanistic biological model simulating dispersal, allopatric speciation and extinction over the dynamic landscape of rivers and lakes. We show that the numerous small river rerouting events in western Amazonia resulting from mountain building produced highly dynamic riverine habitats that caused high diversification rates, shaping the exceptional present-day richness of this region. The history of marine incursions and lakes, including the Miocene Pebas megawetland system in western Amazonia, played a secondary role. This study is a major step towards the understanding of the processes involved in the interactions between the solid Earth, landscapes, and life of extraordinary biodiverse South America.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/444133v1_fig1.gif" ALT="Figure 1">
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category: evolutionary biology


title: Desert fish populations tolerate extreme salinity change to overcome hydrological constraints

abstract: The unstable nature of freshwater ponds in arid landscapes represent a sizable challenge for strictly aquatic organisms, such as fishes. Yet the Arabian Desert, bordering the coastline of the Red Sea, plays host to a species very well adapted to such extreme environments: the Arabian pupfish, Aphanius dispar. In this study, we estimated patterns of hydrological connectivity; population structure and stable isotope for samples of A. dispar living in small, isolated ponds of nearly-freshwater in the Arabian desert and highly saline coastal lagoons along the Red Sea. The genomic and hydrological analyses indicate that populations are largely separated by drainage origin, as fish from desert ponds appear to be transported to coastal lagoons of the Red Sea along ephemeral river systems arising from flash flood events. Further, our study indicates there is an ecological change when being washed from pond environments to coastal waters, due to a significant shift in muscle stable isotopes ratios between both groups. Considering that the genetic breaks are mostly observed between drainage origin, this study suggests that A. dispar can survive large changes in salinity and ecological regimes over small time-scales.


title: Evolved Bmp6 enhancer alleles drive spatial shifts in gene expression during tooth development in sticklebacks

abstract: Mutations in enhancers have been shown to often underlie natural variation but the evolved differences between enhancer activity can be difficult to identify in vivo. Threespine sticklebacks (Gasterosteus aculeatus) are a robust system for studying enhancer evolution due to abundant natural genetic variation, a diversity of evolved phenotypes between ancestral marine and derived freshwater forms, and the tractability of transgenic techniques. Previous work identified a series of polymorphisms within an intronic enhancer of the Bone morphogenetic protein 6 (Bmp6) gene that are associated with evolved tooth gain, a derived increase in freshwater tooth number that arises late in development. Here we use a bicistronic reporter construct containing a genetic insulator and a pair of reciprocal two-color transgenic reporter lines to compare enhancer activity of marine and freshwater alleles of this enhancer. In older fish the two alleles drive partially overlapping expression in both mesenchyme and epithelium of developing teeth, but the freshwater enhancer drives a reduced mesenchymal domain and a larger epithelial domain relative to the marine enhancer. In younger fish these spatial shifts in enhancer activity are less pronounced. Comparing Bmp6 expression by in situ hybridization in developing teeth of marine and freshwater fish reveals similar evolved spatial shifts in gene expression. Together, these data support a model in which the polymorphisms within this enhancer underlie evolved tooth gain by shifting the spatial expression of Bmp6 during tooth development, and provide a general strategy to identify spatial differences in enhancer activity in vivo.


title: Genome and transcriptome analysis of the beet armyworm Spodoptera exigua reveals targets for pest control

abstract:
BackgroundThe genus Spodoptera (Lepidoptera: Noctuidae) includes some of the most infamous insect pests of cultivated plants including Spodoptera frugiperda, Spodoptera litura and Spodoptera exigua. To effectively develop targeted pest control strategies for diverse Spodoptera species, genomic resources are highly desired. To this aim, we provide the genome assembly and developmental transcriptome comprising all major life stages of S. exigua, the beet armyworm. Spodoptera exigua is a polyphagous herbivore that can feed from > 130 host plants including several economically important crops.

ResultsThe 419 Mb beet armyworm genome was sequenced from a female S. exigua pupa. Using a hybrid genome sequencing approach (Nanopore long read data and Illumina short read), a high-quality genome assembly was achieved (N50=1.1 Mb). An official gene set (OGS, 18,477 transcripts) was generated by automatic annotation and by using transcriptomic RNA-seq data sets of 18 S. exigua samples as supporting evidence. In-depth analyses of developmental stage-specific expression in combination with gene tree analyses of identified homologous genes across Lepidoptera genomes revealed potential Spodoptera-specific genes of interest such as mg7 and REPAT46 upregulated during 1st and 3rd instar larval stages for targeted pest-outbreak management.

ConclusionsThe beet armyworm genome sequence and developmental transcriptome covering all major developmental stages provides critical insights into the biology of this devastating polyphagous insect pest species with a worldwide distribution. In addition, comparative genomic analyses across Lepidoptera significantly advance our knowledge to further control other invasive Spodoptera species and reveals potential lineage-specific target genes for pest control strategies.


title: Linear and partially linear models of behavioural trait variation using admixture regression

abstract: Admixture regression methodology exploits the natural experiment of random mating between individuals with different ancestral backgrounds to infer the environmental and genetic components to trait variation across racial and ethnic groups. This paper provides a statistical framework for admixture regression based on the linear polygenic index model and applies it to neuropsychological performance data from the Adolescent Brain Cognitive Development (ABCD) database. We develop and apply a new test of the differential impact of multi-racial identities on trait variation, an orthogonalization procedure for added explanatory variables, and a partially linear semiparametric functional form. We find a statistically significant genetic component to neuropsychological performance differences across racial identities, and find some possible evidence of nonlinearity in the link between admixture and neuropsychological performance scores in the ABCD data.


title: Threonine phosphorylation regulates the molecular assembly and signaling of EGFR in cooperation with membrane lipids

abstract: The cytoplasmic domain of the receptor tyrosine kinases (RTKs) plays roles as a phosphorylation enzyme and a protein scaffold but the allocation of these two functions is not fully understood. We here analyzed assembly of the transmembrane (TM)-juxtamembrane (JM) region of EGFR, one of the best studied species of RTKs, by combining single-pair FRET imaging and a nanodisc technique. The JM domain of EGFR contains a threonine residue (Thr654) that is phosphorylated after ligand association. We observed that the TM-JM peptides of EGFR form anionic lipid-induced dimers and cholesterol-induced oligomers. The two forms involve distinct molecular interactions, with a bias towards oligomer formation upon threonine phosphorylation. We further analyzed the functions and oligomerization of whole EGFR molecules, with or without a substitution of Thr654 to alanine, in living cells. The results suggested an autoregulatory mechanism in which Thr654 phosphorylation causes a switch of the major function of EGFR from kinase activation dimers to scaffolding oligomers.


title: Hydrodynamic flow and concentration gradients in the gut enhance neutral bacterial diversity

abstract:
The gut microbiota features important genetic diversity, and the specific spatial features of the gut may shape evolution within this environment. We investigate the fixation probability of neutral bacterial mutants within a minimal model of the gut that includes hydrodynamic flow and resulting gradients of food and bacterial concentrations. We find that this fixation probability is substantially increased compared to an equivalent well-mixed system, in the regime where the profiles of food and bacterial concentration are strongly spatially-dependent. Fixation probability then becomes independent of total population size. We show that our results can be rationalized by introducing an active population, which consists of those bacteria that are actively consuming food and dividing. The active population size yields an effective population size for neutral mutant fixation probability in the gut.

Significance statementThe human body harbors numerous and diverse bacteria, the vast majority of which are located in the gut. These bacteria can mutate and evolve within the gut, which is their natural environment. This can have important public health implications, e.g. when gut bacteria evolve antibiotic resistance. The gut features specific characteristics, including hydrodynamic flow and resulting gradients of food and bacterial concentrations. How do these characteristics impact the evolution and diversity of gut bacteria? We demonstrate that they can substantially increase the probability that neutral mutants reach high proportions and eventually take over the population. This is because only a fraction of gut bacteria is actively dividing. Thus, the specific environment of the gut enhances neutral bacterial diversity.


title: Calcium signaling in the photodamaged skin

abstract:
BACKGROUNDThe mammalian skin, the bodys largest single organ, is a highly organized tissue that forms an essential barrier against dehydration, pathogens, light and mechanical injury. Damage triggers perturbations of the cytosolic free Ca2+ concentration ([Ca2+]c) that spread from cell to cell (known as intercellular Ca2+ waves) in different epithelia, including epidermis. Ca2+ waves are considered a fundamental mechanism for coordinating multicellular responses, however the mechanisms underlying their propagation in the damaged epidermis are incompletely understood.

AIM OF THE PROJECTTo dissect the molecular components contributing to Ca2+ wave propagation in murine model of epidermal photodamage.

METHODSTo trigger Ca2+ waves, we used intense and focused pulsed laser radiation and targeted a single keratinocyte of the epidermal basal layer in the earlobe skin of live anesthetized mice. To track photodamage-evoked Ca2+ waves, we performed intravital multiphoton microscopy in transgenic mice with ubiquitous expression of the sensitive and selective Ca2+ biosensor GCaMP6s. To dissect the molecular components contributing to Ca2+ wave propagation, we performed in vivo pharmacological interference experiments by intradermal microinjection of different drugs.

EXPERIMENTAL RESULTSThe major effects of drugs that interfere with degradation of extracellular ATP or P2 purinoceptors suggest that Ca2+ waves in the photodamaged epidermis are primarily due to release of ATP from the target cell, whose plasma membrane integrity was compromised by laser irradiation. The limited effect of the Connexin 43 (Cx43) selective inhibitor TAT-Gap19 suggests ATP-dependent ATP release though connexin hemichannels (HCs) plays a minor role, affecting Ca2+ wave propagation only at larger distances, where the concentration of ATP released from the photodamaged cell was reduced by the combined effect of passive diffusion and hydrolysis due to the action of ectonucleotidases. The ineffectiveness of probenecid suggests pannexin channels have no role. As GCaMP6s signals in bystander keratinocytes were augmented by exposure to the Ca2+ chelator EGTA in the extracellular medium, the corresponding transient increments of the [Ca2+]c should be ascribed primarily to Ca2+ release from the ER, downstream of ATP binding to P2Y purinoceptors, with Ca2+ entry through plasma membrane channels playing a comparatively negligible role. The effect of thapsigargin (a well-known inhibitor of SERCA pumps) and carbenoxolone (a recently recognized inhibitor of Ca2+ release through IP3 receptors) support this conclusion.

CONCLUSIONSThe one presented here is an experimental model for accidental skin injury that may also shed light on the widespread medical practice of laser skin resurfacing, used to treat a range of pathologies from photodamage and acne scars to hidradenitis suppurativa and posttraumatic scarring from basal cell carcinoma excision. The results of our experiments support the notion that Ca2+ waves reflect chiefly the sequential activation of bystander keratinocytes by the ATP released through the compromised plasma membrane of the cell hit by laser radiation. We attributed the observed increments of the [Ca2+]c chiefly to signal transduction through purinergic P2Y receptors. Several studies have highlighted fundamental roles of P2Y receptors during inflammatory and infectious diseases, and the initial phase of wound healing involves acute inflammation. In addition, hyaluronan is a major component of the extracellular matrix and its synthesis is rapidly upregulated after tissue wounding via P2Y receptor activation. It is tempting to speculate that response coordination after injury in the epidermis occurs via propagation of the ATP-dependent intercellular Ca2+ waves described in this work.


title: Agrobacterium-Mediated Transient Transformation of Marchantia Liverworts

abstract: Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying GUS genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization.


title: Short 5'UTR enables optimal translation of plant virus tricistronic RNA via leaky scanning

abstract: Regardless of the general model of translation in eukaryotic cells, a number of studies suggested that many of mRNAs encode multiple proteins. Leaky scanning, which supplies ribosomes to downstream open reading frames (ORFs) by read-through of upstream ORFs, is the most major regulatory mechanism to translate polycistronic mRNAs. However, the general regulatory factors controlling leaky scanning and their biological relevance have rarely been elucidated, with exceptions such as the Kozak sequence. Here, we have analyzed the strategy of a plant RNA virus to translate three movement proteins from a single RNA molecule through leaky scanning. The in planta and in vitro results indicate that significantly shorter 5' UTR of the most upstream ORF promotes leaky scanning, potentially finetuning the translation efficiency of the three proteins in a single RNA molecule to optimize viral propagation. Moreover, in plant endogenous mRNAs, we found that shorter UTRs were more frequently observed in uORFs of polycistronic mRNAs. We propose that the promotion of leaky scanning induced by a short 5' UTR (LISH), together with the Kozak sequence, is a conserved gene regulation mechanism not only in viruses but also in eukaryotes.


title: Nitric oxide signalling underlies salicylate-induced increases in neuronal firing in the inferior colliculus: a central mechanism of tinnitus?

abstract:
The tinnitus-inducing agent salicylate reduces cochlear output but causes hyperactivity in higher auditory centres, including the inferior colliculus (the auditory midbrain). Using multi-electrode recording in anaesthetised guinea pigs (Cavia porcellus), we addressed the hypothesis that salicylate-induced hyperactivity in the inferior colliculus involves nitric oxide signalling secondary to increased ascending excitatory input.

In the inferior colliculus, systemic salicylate (200 mg/kg i.p., 0 h) markedly increased spontaneous and sound-driven neuronal firing (3-6 h post drug) with both onset and sustained responses to pure tones being massively increased. Reverse microdialysis of increasing concentrations of salicylate directly into the inferior colliculus (100 {micro}M-10 mM, from 0 h) failed to mimic systemic salicylate. In contrast, it caused a small, transient, increase in sound-driven firing (1 h), followed by a larger sustained decrease in both spontaneous and sound-driven firing (2-5 h). When salicylate was given systemically, reverse microdialysis of the neuronal nitric oxide synthase inhibitor L-methyl arginine into the inferior colliculus (500 mM, 2-6 h) completely blocked the salicylate-induced increase in spontaneous and sound-driven neuronal firing.

Our data indicate that systemic salicylate induces neuronal hyperactivity in the auditory midbrain via a mechanism outside the inferior colliculus, presumably upstream in the auditory pathway; and that the mechanism is ultimately dependent on nitric oxide signalling within the inferior colliculus.

Given that nitric oxide is known to mediate NMDA receptor signalling in the inferior colliculus, we propose that salicylate activates an ascending glutamatergic input to the inferior colliculus and that this is an important mechanism underlying salicylate-induced tinnitus.

Graphical abstractTinnitus is associated with increased activity in the central auditory pathway, an effect replicated by high dose sodium salicylate. Here we show that salicylate-induced increases in neuronal activity in the auditory midbrain are mediated by nitric oxide signalling within this region. Nitric oxide is a key mediator of neuronal responses to NMDA receptor stimulation in this region. Our data support the hypothesis that tinnitus is mediated by increased ascending glutamatergic input to the auditory midbrain.

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title: Serine 182 on RORγt regulates T helper 17 and regulatory T cell functions to resolve inflammation

abstract: Intestine homeostasis is maintained by the delicate balance of Th17 effector cells and Treg cells. Dysregulation of these cell populations contributes to inflammation, tissue damage, and chronic conditions. ROR{gamma}t is essential for the differentiation of Th17 and a subset of Treg (ROR{gamma}t+ Treg) cells involved in intestinal inflammation. ROR{gamma}t belongs to the nuclear receptor family of transcription factors with hinge regions that are highly flexible for co-activator/co-repressor interactions. Serine 182 at the hinge region of ROR{gamma}t is phosphorylated. This study aims to uncover how S182 on ROR{gamma}t contributes to mucosal homeostasis and diseases. We used CRISRP technology to generate a phosphor-null knock-in mutant mouse line (ROR{gamma}tS182A) to assess its role in intestine physiology. scRNA-seq was performed on WT and ROR{gamma}tS182A cohoused littermates to evaluate colonic T cell heterogeneity under steady state and colitis settings. Single-cell transcriptomics revealed that ROR{gamma}tS182 maintains colonic T cell heterogeneity under steady state, without interfering T cell development and differentiation. In inflamed tissues, ROR{gamma}tS182 simultaneously restricts IL-1{beta}-mediated Th17 activities and promotes anti-inflammatory cytokine IL-10 production in LT-like Treg cells. Phospho-null ROR{gamma}tS182A knock-in mice challenged with DSS induced colitis and EAE experienced delayed recovery and exacerbated pathology. The double switch role of ROR{gamma}tS182 is critical in resolving T cell-mediated inflammation and provides a potential therapeutic target to combat autoimmune diseases.


title: RORγt serine 182 tightly regulates T cell heterogeneity to maintain mucosal homeostasis and restrict tissue inflammation


title: Single cell analysis of RA synovial B cells reveals a dynamic spectrum of ectopic lymphoid B cell activation and hypermutation characterized by NR4A nuclear receptor expression

abstract:
Ectopic lymphoid structures (ELS) can develop in rheumatoid arthritis (RA) synovial tissue, but the precise pathways of B cell activation and selection are not well understood. Here, we identified a unique B cell population in the synovium characterized by co-expression of a family of orphan nuclear receptors, NR4A1 (also known as NUR77), NR4A2 (NURR1) and NR4A3 (NOR1), that is highly enriched at both early and late stages of RA. NR4A B cells are rare in healthy peripheral blood, RA blood, and SLE kidney, but share markers with blood transcriptomic signatures that peak during RA disease flare. Using combined single cell transcriptomics and B cell receptor (BCR) sequencing, we demonstrate that NR4A synovial B cells have an activated transcriptomic profile that significantly overlaps with germinal center (GC) light zone (LZ) B cells and an accrual of somatic hypermutation that correlates with loss of naive B cell status. NR4A B cells uniquely co-express lymphotoxin {beta} and IL6, supporting important functions in ELS promotion and pro-inflammatory cytokine production. Further, the presence of shared clones in this activated B cell state and NR4A expressing synovial plasma cells (PC) and the rapid up-regulation with BCR stimulation points to in situ differentiation. Taken together, we identified a dynamic progression of B cell activation in RA synovial ELS, with NR4A transcription factors having an important role in antigen activation and local adaptive immune responses.

One sentence summaryB cells in the rheumatoid arthritis synovium undergo a spectrum of in situ activation, with the NR4A family of transcription factors having an important role in antigen stimulation, local adaptive immunity, and pathological B cell responses.


title: Signal Inhibitory Receptor on Leukocytes-1 recognizes S100 proteins

abstract: Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an inhibitory receptor with a hitherto unknown ligand, and is expressed on human monocytes and neutrophils. SIRL-1 inhibits myeloid effector functions such as reactive oxygen species (ROS) production. We here identify S100 proteins as SIRL-1 ligands. S100 proteins are composed of two calcium-binding domains. Various S100 proteins are damage-associated molecular patterns (DAMPs) released from damaged cells, after which they initiate inflammation by ligating activating receptors on immune cells. We now show that the inhibitory SIRL-1 recognizes individual calcium-binding domains of all tested S100 proteins. Blocking SIRL-1 on human neutrophils enhanced S100 protein S100A6-induced ROS production, showing that S100A6 suppresses neutrophil ROS production via SIRL-1. We conclude that SIRL-1 is an inhibitory receptor recognizing the S100 protein family of DAMPs.


title: GP2-enriched pancreatic progenitors give rise to functional beta cells in vivo and eliminate the risk of teratoma formation

abstract: Human pluripotent stem cell (hPSC)-derived pancreatic progenitors (PPs) can be differentiated into beta-like cells in vitro and in vivo, and therefore have therapeutic potential for type 1 diabetes (T1D) treatment. However, the purity of PPs varies across different hPSC lines, differentiation protocols and laboratories. The uncommitted cells may give rise to non-pancreatic endodermal, mesodermal, or ectodermal derivatives in vivo, hampering the safety of hPSC-derived PPs for clinical applications. Recently, proteomics and transcriptomics analyses identified glycoprotein 2 (GP2) as a PP-specific cell surface marker. The GP2-enriched PPs generate higher percentages of beta-like cells in vitro compared to unsorted and GP2- fractions, but their potential in vivo remains to be elucidated. Here, we demonstrate that the GP2-enriched-PPs give rise to all pancreatic cells in vivo, including functional beta-like cells. Remarkably, GP2 enrichment eliminated the formation of teratoma in vivo. This study establishes that the GP2-enriched PPs represent a safe option for T1D treatment.

category: developmental biology


title: Spatial modeling of tidal and land use impacts on Escherichia coli contamination and fluctuation in Asia's urban river environmental gradient

abstract: In the river, pathogens are the leading cause for rivers to exceed water and health quality standards. The GIS based spatial modeling and analysis were conducted to estimate Escherichia coli (E. coli) contaminated river bodies based on environmental and spatial gradients such as dissolved oxygen, pH, tidal, temperature and current in Asias urban river located in Kapuas river of Kalimantan. The E. coli was sampled from the river mouth up to the upstream land uses dominated by residential. The E. coli contamination was higher in the river mouth and in residential area as well. Likewise, E. coli contamination was higher during the low tide than high tide. During the high tide, the E. coli contamination were significantly affected by temperature and current (r2>0.5). Meanwhile, during the low tide, there were no dominant environmental factors that affect E. coli contamination. Hence, by knowing the spatial model of E. coli contamination driven by tidal, land use and environmental gradients, this paper has contributed to the advance management of water and river system.


title: The quality of dissolved organic matter shapes the biogeography of the active bathypelagic microbiome

abstract: The bathypelagic ocean (1000-4000 m depth) is the largest aquatic biome on Earth but it is still largely unexplored. Due to its prevalent low dissolved organic carbon concentrations, most of the prokaryotic metabolic activity is assumed to be associated to particles. The role of free-living prokaryotes has thus been mostly ignored, except that of some chemolithoautotrophic lineages. Here we used a global bathypelagic survey of size-fractionated metagenomic and 16S (genes and transcripts) data and performed a differential abundance analysis to explore the functional traits of the different prokaryotic life-strategies, their contribution to the active microbiome, and the role that the quality of the dissolved organic matter (DOM) plays in driving this contribution. We found that free-living prokaryotes have limited capacity to uplift their metabolism in response to environmental changes and display comparatively lower growth rates than particle associated prokaryotes, but are responsible for the synthesis of vitamins in the bathypelagic. Furthermore, their contribution to the active prokaryotic microbiome increased towards waters depleted of labile DOM, which represented a large fraction of the tropical and subtropical ocean sampled stations. This points to a relevant yet overlooked role of free-living prokaryotes in DOM cycling in the vast bathypelagic desert.


title: The diversification of Pterocarpus (Leguminosae: Papilionoideae) was influenced by biome-switching and infrequent long-distance dispersal

abstract:
AimPhenotypes which evolved for dispersal over ecological timescales may lead to significant macroevolutionary consequences, such as infrequent long-distance dispersal and diversification in novel biomes. We aimed to reconstruct the phylogenetic history of Pterocarpus (Leguminosae/ Fabaceae) to assess whether seed dispersal phenotypes and biome switching explain the current biogeographical patterns of this group.

LocationPantropical

TaxonThe Pterocarpus clade, particularly Pterocarpus (Leguminosae/Fabaceae)

MethodsWe sequenced ~300 nuclear loci captured using Angiosperms-353, a genomic  bait set for flowering plants, from which we generated a time-calibrated phylogenomic tree. To corroborate this, we also generated a time-calibrated phylogenetic tree from data-mined Sanger-sequencing data. We then collated distribution data and fruit dispersal morphology traits to compare trait-dependent and trait-independent biogeographical models, allowing us to assess whether dispersal traits influenced the spatio-temporal evolution of Pterocarpus. Finally, using the results of these model tests, we estimated the ancestral ranges and biomes of Pterocarpus species to better understand their biogeographical history, and assessed the degree and direction of biome switching over the course of Pterocarpus diversification history.

ResultsWe recovered well-supported phylogenetic relationships within Pterocarpus, within which there were two subclades - one Neotropical and the other Palaeotropical. Our divergence date estimates suggested that Pterocarpus largely diversified from around 12 Ma, during the Miocene.

Trait-dependent biogeographical models were rejected for both range and biome evolution within Pterocarpus, but models parameterising dispersal were supported. Pterocarpus ancestral node shared a range across the new-world and old-world tropics, followed by divergence into two clades, one palaeotropical and one neotropical. Biome switching occurred most frequently into rainforest and grassland.

Main conclusionsOverall, our analyses suggest that Pterocarpus underwent infrequent cross-continental dispersal and establishment into novel biomes. While this was minimally impacted by fruit dispersal syndromes, biome switching precipitated by long-distance dispersal and environmental change have played an important role in diversification within Pterocarpus since the Miocene.


title: Vascular KATP channel structural dynamics reveal regulatory mechanism by Mg-nucleotides

abstract: Vascular tone is dependent on smooth muscle KATP channels comprising pore-forming Kir6.1 and regulatory SUR2B subunits, in which mutations cause Cantu syndrome. Unique among KATP isoforms, they lack spontaneous activity and require Mg-nucleotides for activation. Structural mechanisms underlying these properties are unknown. Here, we determined the first cryoEM structures of vascular KATP channels bound to inhibitory ATP and glibenclamide, which differ informatively from similarly determined pancreatic KATP channel isoform (Kir6.2/SUR1). Unlike SUR1, SUR2B subunits adopt distinct rotational "propeller" and "quatrefoil" geometries surrounding their Kir6.1 core. The previously unseen ED-rich linker connecting the two halves of the SUR-ABC core is observed in a quatrefoil-like conformation. MD simulations reveal MgADP-dependent dynamic tripartite interactions between this linker, SUR2B and Kir6.1. The structures captured implicate a progression of intermediate states between MgADP-free inactivated and MgADP-bound activated conformations wherein the ED-rich linker participates as mobile autoinhibitory domain, suggesting a conformational pathway toward KATP channel activation.

category: biochemistry


title: Condensin-driven loop extrusion on supercoiled DNA

abstract: Condensin, a structural maintenance of chromosomes (SMC) complex, has been shown to be a molecular motor protein that organizes chromosomes by extruding loops of DNA. In cells, such loop extrusion is challenged by many potential conflicts, e.g., the torsional stresses that are generated by other DNA-processing enzymes. It has so far remained unclear how DNA supercoiling affects loop extrusion. Here, we use time-lapse single-molecule imaging to study condensin-driven DNA loop extrusion on supercoiled DNA. We find that condensin binding and DNA looping is stimulated by positive supercoiled DNA where it preferentially binds near the tips of supercoiled plectonemes. Upon loop extrusion, condensin collects all nearby plectonemes into a single supercoiled loop that is highly stable. Atomic force microscopy imaging shows that condensin generates supercoils in the presence of ATP. Our findings provide insight into the topology-regulated loading and formation of supercoiled loops by SMC complexes and clarify the interplay of loop extrusion and supercoiling.


title: Conformational buffering underlies functional selection in intrinsically disordered protein regions

abstract: Many disordered proteins conserve essential functions in the face of extensive sequence variation. This makes it challenging to identify the forces responsible for functional selection. Viruses are robust model systems to investigate functional selection and they take advantage of protein disorder to acquire novel traits. Here, we combine structural and computational biophysics with evolutionary analysis to determine the molecular basis for functional selection in the intrinsically disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, triggering early S-phase entry and disrupting normal cellular proliferation. We show that the ability to outcompete host factors depends on the picomolar binding affinity of E1A for Rb, which is driven by two binding motifs tethered by a hypervariable disordered linker. Binding affinity is determined by the spatial dimensions of the linker, which constrain the relative position of the two binding motifs. Despite substantial sequence variation across evolution, the linker dimensions are finely optimized through compensatory changes in amino acid sequence and sequence length, leading to conserved linker dimensions and maximal affinity. We refer to the mechanism that conserves spatial dimensions despite large-scale variations in sequence as conformational buffering. Conformational buffering explains how variable disordered proteins encode functions and could be a general mechanism for functional selection within disordered protein regions.


title: Effect of charge on protein preferred orientation at the air-water interface in cryo-electron microscopy

abstract: The air-water interface (AWI) tends to absorb proteins and frequently causes preferred orientation problems in cryo-electron microscopy (cryo-EM). Here, we examined cryo-EM data from protein samples frozen with different detergents and found that both anionic and cationic detergents promoted binding of proteins to the AWI. By contrast, nonionic and zwitterionic detergents tended to prevent proteins from attaching to the AWI. This ability was positively associated with the critical micelle concentration of the detergent. The protein orientation distributions with different anionic detergents were similar and resembled that obtained without detergent. By contrast, cationic detergents gave distinct orientation distributions. The AWI is negatively charged and the absorption of cationic detergents to the AWI alters its charge. Our results indicates that proteins absorb to charged interface and the negative charge of the AWI plays an important role in absorbing proteins in the conventional cryo-EM sample preparation. According to these findings, a new method was developed to modify the charge distribution of the AWI by adding a very low concentration of anionic detergent. Using this method, the protein particles exhibited a more evenly distributed orientations and still absorbed to the AWI enabling them embedding in a thin layer of ice, which will benefit the cryo-EM structural determination.


title: Non-canonical Wnt/Ror2 signaling status regulates cell-matrix crosstalk to prompt directional tumor cell invasion and dissemination in breast cancer

abstract: Bidirectional cell-extracellular matrix (ECM) interactions represent fundamental exchanges during tumor progression. We demonstrate the noncanonical Wnt receptor, Ror2, regulates tumor cell-driven matrix remodeling in models of breast cancer. Wnt/Ror2 loss-of-function triggers tumor cell invasion, accompanied by changes in actin cytoskeleton, adhesion, and collagen crosslinking gene expression programs. E-cadherin levels decline upon Ror2 depletion, and spatially, we pinpoint the upregulation and redistribution of 5 and {beta}3 integrins together with the production of fibronectin in areas of invasion. Wnt/{beta}-catenin-dependent and Wnt/Ror2 alternative Wnt signaling appear to regulate distinct functions for tumor cells regarding their ability to modify cell-ECM exchanges during invasion. Furthermore, blocking either integrin or focal adhesion kinase (FAK), a downstream mediator of integrin-mediated signal transduction, abrogates the enhanced migration observed upon Ror2 loss. These results reveal a critical function for the alternative Wnt receptor, Ror2, as a determinant of reciprocal communication between tumor cells and ECM during cancer invasion and metastasis.


title: Non-canonical Wnt/Ror2 signaling regulates cell-matrix crosstalk to prompt directional tumor cell invasion and dissemination in breast cancer


title: Versatile and multiplexed mass spectrometry-based absolute quantification with cell-free-synthesized internal standard peptides

abstract: Preparation of stable isotope-labeled internal standard peptides is crucial for mass spectrometry (MS)-based targeted proteomics. Herein, we developed versatile and multiplexed absolute protein quantification method using MS. A previously developed method based on the cell-free peptide synthesis system, termed MS-based quantification by isotope-labeled cell-free products (MS-QBiC), was improved for multiple peptide synthesis in one-pot reaction. We pluralized the quantification tags used for the quantification of synthesized peptides and thus, made it possible to use cell-free synthesized isotope-labeled peptides as mixtures for the absolute quantification. The improved multiplexed MS-QBiC method was proved to be applied to clarify ribosomal proteins stoichiometry in the ribosomal subunit, one of the largest cellular complexes. The study demonstrates that the developed method enables the preparation of several dozens and even several hundreds of internal standard peptides within a few days for quantification of multiple proteins with only a single-run of MS analysis.

category: synthetic biology


title: Predictive evolution of metabolic phenotypes using model-designed selection niches

abstract: Traits lacking fitness benefit cannot be directly selected for under Darwinian evolution. Thus, features such as metabolite secretion are currently inaccessible to adaptive laboratory evolution. Here, we utilize environment-dependency of trait correlations to enable Darwinian selection of fitness-neutral or costly traits. We use metabolic models to design selection niches and to identify surrogate traits that are genetically correlated with cell fitness in the selection niche but coupled to the desired trait in the target niche. Adaptive evolution in the selection niche and subsequent return to the target niche is thereby predicted to enhance the desired trait. We experimentally validate the theory by evolving Saccharomyces cerevisiae for increased secretion of aroma compounds in wine fermentation. Genomic, transcriptomic, and proteomic changes in the evolved strains confirmed the predicted flux re-routing to aroma biosynthesis. The use of model-designed selection niches facilitates the predictive evolution of fitness-costly traits for ecological and biotechnological applications.


title: An updated suite of viral vectors for in vivo calcium imaging using local and retro-orbital injections

abstract: Calcium imaging using genetically encoded Ca2+ indicators (GECIs) is a widely adopted method to measure neural activity in modern neuroscience. Here, we explore the use of systemically administered viral vectors for brain-wide expression of GECIs, and adapt novel GECIs to optimize signal-to-noise. We show that systemic injections of PHP.eB AAVs to express GECIs is a highly promising technique for imaging neural activity and circumvent the need for transgenic GECI expressing mouse lines. We also establish the use of soma-targeted GECIs that outperform current Ca2+ indicators using both systemic and local virus injections.

category: neuroscience


title: Exceptionally high sequence-level variation in the transcriptome of Plasmodium falciparum

abstract: Single-nucleotide variations in RNA (hereafter referred to simply as SNVs), arising from co- and post-transcriptional phenomena including transcription errors and RNA editing, are well studied in organisms ranging from bacteria to humans. In the malaria parasite Plasmodium falciparum, stage-specific and non-specific gene-expression variations are known to accompany the parasites array of developmental and morphological phenotypes over the course of its complex life cycle. However, the extent, rate and effect of sequence-level variation in the parasites transcriptome are unknown. Here, we report the presence of pervasive, non-specific SNVs in the transcriptome of the P. falciparum. We show that these SNVs cover most of the parasites transcriptome. SNV rates for the P. falciparum lines we assayed, as well as for publicly available P. vivax and P. falciparum clinical isolate datasets were of the order of 10-3 per base, about tenfold higher than rates we calculated for bacterial datasets. These SNVs may reflect an intrinsic transcriptional error rate in the parasite, and RNA editing may be responsible for a subset of them. This seemingly characteristic property of the parasite may have implications for clinical outcomes and the basic biology and evolution of P. falciparum and parasite biology more broadly, and we anticipate that our study will prompt further investigations into the exact sources, consequences and possible adaptive roles of these SNVs.


title: Integrative proteogenomics for differential expression and splicing variation in a DM1 mouse model

abstract:
Dysregulated mRNA splicing is involved in the pathogenesis of many diseases including cancer, neurodegenerative diseases, and muscular dystrophies such as myotonic dystrophy type 1 (DM1). Comprehensive assessment of dysregulated splicing on the transcriptome and proteome level has been methodologically challenging, and thus investigations have often been targeting only few genes.

Here, we performed a large-scale coordinated transcriptomic and proteomic analysis to characterize a DM1 mouse model (HSALR) in comparison to wild-type. Our integrative proteogenomics approach comprised gene- and splicing-level assessments for mRNAs and proteins. It recapitulated many known instances of aberrant mRNA splicing in DM1 and identified new ones. It enabled the design and targeting of splicing-specific peptides and confirmed the translation of known instances of aberrantly spliced disease-related genes (e.g. Atp2a1, Bin1, Ryr1), complemented by novel findings (e.g. Ywhae, Flnc, Svil). Comparative analysis of large-scale mRNA and protein expression data showed quantitative agreement of differentially expressed genes and splicing patterns between disease and wild-type.

We hence propose this work as a suitable blueprint for a robust and scalable integrative proteogenomic strategy geared towards advancing our understanding of splicing-based disorders. With such a strategy, splicing-based biomarker candidates emerge as an attractive and accessible option, as they can be efficiently asserted on the mRNA and protein level in coordinated fashion.

Graphical abstract

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title: The Unconventional Self-Cleavage of Selenoprotein K

abstract: Through known association with other proteins, human selenoprotein K (selenok) is currently implicated in the palmitoylation of proteins, degradation of misfolded proteins, innate immune response, and the life cycle of SARS-CoV-2 virus. However, neither the catalytic function of selenoks selenocysteine (Sec), which, curiously, resides in an intrinsically disordered protein segment nor selenoks specific role in these pathways are known to date. This report casts these questions in a new light as it describes that selenok is able -both in vitro and in vivo- to cleave some of its own peptide bonds. The cleavages not only release selenok segments that contain its reactive Sec, but as the specific cleavage sites were identified, they proved to cluster tightly near sites through which selenok interacts with protein partners. Furthermore, it is shown that selenoks cleavage activity is neither restricted to itself nor promiscuous but selectively extends to at least one of its protein partners. Together, selenoks cleavage ability and its features have all hallmarks of a regulatory mechanism that could play a central role in selenoks associations with other proteins and its cellular functions overall.


title: A contraction-reaction-diffusion model for circular pattern formation in embryogenesis

abstract: Reaction-diffusion models have been widely used to elucidate pattern formation in developmental biology. More recently, they have also been applied in modeling cell fate patterning that mimic early-stage human development events utilizing geometrically confined pluripotent stem cells. However, the traditional reaction-diffusion equations could not satisfactorily explain the concentric ring distributions of various cell types, as they do not yield circular patterns even for circular domains. In previous mathematical models that yield ring patterns, certain conditions that lack biophysical understandings had been considered in the reaction-diffusion models. Here we hypothesize that the circular patterns are the results of the coupling of the mechanobiological factors with the traditional reaction-diffusion model. We propose two types of coupling scenarios: tissue tension-dependent diffusion flux and traction stress-dependent activation of signaling molecules. By coupling reaction-diffusion equations with the elasticity equations, we demonstrate computationally that the contraction-reaction-diffusion model can naturally yield the circular patterns.

category: bioengineering