title: Both Prey and Predator Features Determine Predation Risk and Survival of Schooling Prey

abstract: Predation is one of the main evolutionary drivers of social grouping. While it is well appreciated that predation risk is likely not shared equally among individuals within groups, its detailed quantification has remained difficult due to the speed of attacks and the highly-dynamic nature of collective prey response. Here, using high-resolution tracking of solitary predators (Northern pike) hunting schooling fish (golden shiners), we not only provide detailed insights into predator decision-making but show which key spatial and kinematic features of predator and prey influence individuals risk to be targeted and survive attacks. Pike tended to stealthily approach the largest groups, and were often already inside the school when launching their attack, making prey in this frontal "strike zone" the most vulnerable to be targeted. From the preys perspective, those fish in central locations, but relatively far from, and less aligned with, neighbours, were most likely to be targeted. While the majority of attacks (70%) were successful, targeted individuals that did manage to avoid capture exhibited a higher maximum acceleration response just before the attack and were further away from the pike s head. Our results highlight the crucial interplay between predators attack strategy and response of prey in determining predation risk in mobile animal groups.


title: REVOLVER: a low-cost automated protein purifier based on parallel preparative gravity column workflows

abstract:
Protein purification is a ubiquitous operation in biochemistry and life sciences and represents a key step to producing purified proteins for research (understanding how proteins work) and various applications. The need for scalable and parallel protein purification systems keeps growing due to the increase in throughput in the production of recombinant proteins and in the ever-growing scale of biochemistry research. Therefore, automating the process to handle multiple samples in parallel with minimal human intervention is highly desirable; yet only a handful of such tools have been developed, all of which are closed source and expensive. To address this challenge, we present REVOLVER, a 3D-printed programmable and automatic protein purification system based on gravity-column workflows and controlled by Arduino boards that can be built for under $130 USD. REVOLVER completes a full protein purification process with almost no human intervention and yields results equivalent to those obtained by an experienced biochemist when purifying a real-world protein sample. We further present and describe MULTI-VOLVER, a scalable version of the REVOLVER that allows for parallel purification of up to six samples and can be built for under $250 USD. Both systems will be useful to accelerate protein purification and ultimately link them to bio-foundries for protein characterization and engineering.

Specifications Table

O_TBL View this table:
org.highwire.dtl.DTLVardef@13fff66org.highwire.dtl.DTLVardef@98aa2forg.highwire.dtl.DTLVardef@cd2e70org.highwire.dtl.DTLVardef@a3bd86org.highwire.dtl.DTLVardef@1efc67d_HPS_FORMAT_FIGEXP  M_TBL C_TBL

category: biochemistry


title: Structure and dynamics of human disease-complication network

abstract:
A complication is an unanticipated disease arisen following, induced by a disease, a treatment or a procedure. We compile the Human Disease-Complication Network from the medical data and investigate the characteristics of the network. It is observed that the modules of the network are dominated by the classes of diseases. The relations between modules are unveiled in detail. Three nontrivial motifs are identified from the network. We further simulate the dynamics of motifs with the Boolean dynamic model. Each motif represents a specific dynamic behavior, which is potentially functional in the disease system, such as generating temporal progressions and governing the responses to fluctuating external stimuli.

Author summaryAdvances in molecular biology lead to a new discipline of network medicine, investigating human diseases in a networked structure perspective. Recently, clinical records have been introduced to the research of complex networks of diseases. An important available medical dataset that has been overlooked so far is the complications of diseases, which are vital for human beings. We compile the Human Disease-Complication Network, representing the causality between the upstream diseases and their downstream complications. This work not only helps us to comprehend why certain groups of diseases appear collectively, but also provides a new paradigm to investigate the dynamics of disease progression. For clinical applications, the investigation of complications may yield new approaches to disease prevention, diagnosis and treatment.

category: bioinformatics


title: Mechanism Driven Early Stage Identification and Avoidance of Antisense Oligonucleotides Causing TRL9 Mediated Inflammatory Responses in Bjab cells

abstract: Nucleic acid-based PS-ASOs have the potential to activate cellular innate immune responses, and the level of activation can vary quite dramatically with sequence. Minimizing degree of proinflammatory effect is one of the main selection criteria for compounds intended to move into clinical trials. While a recently developed hPBMC-based assay showed excellent ability to detect innate immune active PS-ASOs, which can then be discarded from the developmental process, this assay is highly resource-intensive and easily affected by subject variability. This compelled us to develop of a more convenient high-throughput assay. Here, we describe a new in vitro assay, utilizing a cultured human Bjab cell line, which was developed and validated to identify PS-ASOs that may cause innate immune activation. The assay was calibrated to replicate results from the hPMBC assay. The Bjab assay was designed to be high throughput and more convenient by using RT-qPCR readout of mRNA of the chemokine Ccl22. The Bjab assay was also shown to be highly reproducible and to provide a large dynamic range in determining the immune potential of PS-ASOs via comparison to known benchmark PS-ASO controls that were previously shown to be either safe or inflammatory in clinical trials. In addition, we demonstrate that Bjab cells can be used to provide mechanistic information on PS-ASO-TLR9 dependent innate immune activation.

category: pharmacology and toxicology


title: Unveiling effects of growth conditions on crown architecture and growth potential of Scots pine trees

abstract: Trees adapt to their growing conditions by regulating the sizes of their parts and their relationships. For example, removal or death of adjacent trees increases the growing space and the amount of light received by the remaining trees enabling their crowns to expand. Knowledge about the effects of silvicultural practices on crown size and shape as well as about the quality of branches affecting the shape of a crown is, however, still limited. Thus, the aim was to study the crown structure of individual Scots pine trees in forest stands with varying stem densities due to past forest management practices. Furthermore, we wanted to understand how crown and stem attributes as well as tree growth affects stem area at the height of maximum crown diameter (SAHMC), which could be used as a proxy for tree growth potential. We used terrestrial laser scanning (TLS) to generate attributes characterizing crown size and shape. The results showed that increasing stem density decreased Scots pine crown size. TLS provided more detailed attributes for crown characterization compared to traditional field measurements. Furthermore, decreasing stem density increased SAHMC and strong relationships (Spearman correlations >0.5) were found between SAHMC and crown and stem size as well as stem growth. Thus, this study provided quantitative and more comprehensive characterization of Scots pine crowns and their growth potential.

category: ecology


title: N-glycosylation modulates enzymatic activity of Trypanosoma congolense trans-sialidase

abstract: Trypanosomes cause the devastating disease trypanosomiasis, in which the action of trans-sialidase (TS) enzymes harbored on their surface is a key virulence factor. TS are N-glycosylated, but the biological functions of their glycans has remained elusive. In this study, we investigated the influence of N-glycans on the enzymatic activity and structural stability of TconTS1, a recombinant TS from the African parasite Trypanosoma congolense. The enzyme was expressed in CHO Lec1 cells, which produce high-mannose type N-glycans similar to the TS N-glycosylation pattern in vivo. MALDI-TOF MS data revealed that up to eight putative N-glycosylation sites were glycosylated. N-glycan removal via EndoHf treatment of TconTS1 led to a decrease in substrate affinity relative to the untreated enzyme, but apparently has no impact on the conversion rate. No changes in secondary structure elements of hypoglycosylated TconTS1 were observed in circular dichroism experiments. Molecular dynamics simulations provided evidence for interactions between monosaccharide units of the highly flexible N-glycans and some conserved amino acids located at the catalytic site. These interactions led to conformational changes, possibly enhancing substrate accessibility and enzyme-substrate complex stability. The here-observed modulation of catalytic activity via N-glycans represents a so far unknown structure-function relationship potentially inherent in several members of the TS enzyme family.


title: Genome edited colorectal cancer organoid models reveal distinct microRNA activity patterns across different mutation profiles

abstract: Somatic mutations drive colorectal cancer (CRC) by disrupting gene regulatory mechanisms. Distinct combinations of mutations can result in unique changes to regulatory mechanisms leading to variability in the efficacy of therapeutics. MicroRNAs are important regulators of gene expression, and their activity can be altered by oncogenic mutations. However, it is unknown how distinct combinations of CRC-risk mutations differentially affect microRNAs. Here, using genetically-modified mouse intestinal organoid (enteroid) models, we identify ten different modules of microRNA expression patterns across distinct combinations of mutations common in CRC. We also show that miR-24-3p, which is aberrant in genetically-modified mouse enteroids and human colonoids irrespective of mutational context, is a master regulator of gene expression in CRC. In follow-up experiments, we also demonstrate that miR-24 promotes CRC cell survival. These findings offer insight into the mechanisms that drive inter-tumor heterogeneity and highlight candidate microRNA therapeutic targets for the advancement of precision medicine for CRC.

category: cancer biology


title: Water droplet-in-oil digestion method for single-cell proteomics

abstract: Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold as well as the number of quantified transmembrane proteins compared to an in-solution digestion (ISD) method. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10000 cells using the ISD method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

category: systems biology


title: Dynamic Boolean modelling reveals the influence of energy supply on bacterial efflux pump expression

abstract: Antimicrobial resistance (AMR) is a global health issue. One key factor contributing to AMR is the ability of bacteria to export drugs through efflux pumps, which relies on the ATP-dependent expression and interaction of several controlling genes. Recent studies have shown significant cell-to-cell ATP variability exists within clonal bacterial populations, but the contribution of intrinsic cell-to-cell ATP heterogeneity is generally overlooked in understanding efflux pumps. Here, we consider how ATP variability influences gene regulatory networks controlling expression of efflux pump genes in two bacterial species. We develop and apply a generalisable Boolean modelling framework, developed to incorporate the dependence of gene expression dynamics on available cellular energy supply. Theoretical results show differences in energy availability can cause pronounced downstream heterogeneity in efflux gene expression. Cells with higher energy availability have a superior response to stressors. Further, in the absence of stress, model bacteria develop heterogeneous pulses of efflux pump gene expression which contribute to a sustained sub-population of cells with increased efflux expression activity, potentially conferring a continuous pool of intrinsically resistant bacteria. This modelling approach thus reveals an important source of heterogeneity in cell responses to antimicrobials and sheds light on potentially targetable aspects of efflux pump-related antimicrobial resistance.


title: Tissue Optimisation Strategies for High Quality Ex Vivo Diffusion Imaging

abstract: Ex vivo diffusion imaging can be used to study healthy and pathological tissue microstructure in the rodent brain with microscopic resolution, providing a link between in vivo MRI and ex vivo microscopy techniques. A major challenge for the successful acquisition of ex vivo diffusion imaging data however are changes in the relaxivity and diffusivity of brain tissue following perfusion fixation. In this study we address this question by examining the combined effects of tissue preparation factors that influence image quality, including tissue rehydration time, fixative concentration and contrast agent concentration. We present an optimisation strategy combining these factors to manipulate the T1 and T2 of fixed tissue and maximise signal-to-noise ratio (SNR) efficiency. Applying this strategy in the rat brain resulted in a doubling of SNR and an increase in SNR per unit time by 135% in grey matter and 88% in white matter. This enabled the acquisition of excellent quality high-resolution (78 {micro}m isotropic voxel size) diffusion data in less than 4 days, with a b-value of 4000 s/mm2, 30 diffusion directions and a field of view of 40 x 13 x 18 mm, using a 9.4 Tesla scanner with a standard 39 mm volume coil and a 660 mT/m 114 mm gradient insert. It was also possible to achieve comparable data quality for a standard resolution (150 {micro}m) diffusion dataset in 21/4 hours. In conclusion, the optimisation strategy presented here may be used to improve signal quality, increase spatial resolution and/or allow faster acquisitions in preclinical ex vivo diffusion MRI experiments.

category: neuroscience


title: Polyclonal lymphoid expansion drives paraneoplastic autoimmunity in neuroblastoma

abstract: Neuroblastoma is a lethal childhood solid tumor of developing peripheral nerves. Two percent of children with neuroblastoma develop Opsoclonus Myoclonus Ataxia Syndrome (OMAS), a paraneoplastic disease characterized by cerebellar and brainstem-directed autoimmunity, but typically with outstanding cancer-related outcomes. We compared tumor transcriptomes and tumor infiltrating T- and B-cell repertoires from 38 OMAS subjects with neuroblastoma to 26 non- OMAS associated neuroblastomas. We found greater B- and T-cell infiltration in OMAS- associated tumors compared to controls, but unexpectedly showed that both were polyclonal expansions. Tertiary lymphoid structures (TLS) were enriched in OMAS-associated tumors. We identified significant enrichment of the MHC Class II allele HLA-DOB*01:01 in OMAS patients. OMAS severity scores were associated with the expression of several candidate autoimmune genes. We propose a model in which polyclonal autoreactive B lymphocytes act as antigen presenting cells and drive TLS formation, thereby crucially supporting both sustained polyclonal T-cell-mediated anti-tumor immunity and paraneoplastic OMAS neuropathology.


title: CHARM: COVID-19 Health Action Response for Marines - association of antigen-specific interferon-gamma and IL2 responses with asymptomatic and symptomatic infections after a positive qPCR SARS-CoV-2 test

abstract:
SARS-CoV-2 T cell responses are associated with COVID-19 recovery, and Class I- and Class II-restricted epitopes have been identified in the spike (S), nucleocapsid (N) and membrane (M) proteins and others. This prospective COVID-19 Health Action Response for Marines (CHARM) study enabled assessment of T cell responses in symptomatic and asymptomatic SARS-CoV-2 infected participants.

At enrollment all participants were negative by qPCR; follow-up occurred biweekly and then bimonthly for the next 6 weeks. Study participants who tested positive by qPCR SARS-CoV-2 test were asked to enroll in an immune response sub-study. FluoroSpot interferon-gamma (IFN-{gamma}) and IL2 responses following qPCR-confirmed infection at enrollment (day 0), day 7 and 14 and more than 28 days later were measured using pools of 17mer peptides covering S, N, and M proteins, or CD4+CD8 peptide pools containing predicted epitopes from multiple SARS-CoV-2 antigens.

Among 124 asymptomatic and 105 symptomatic participants, SARS-CoV-2 infection generated IFN-{gamma} responses to the S, N and M proteins that persisted longer in asymptomatic cases. IFN-{gamma} responses were significantly (p=0.001) more frequent to the N pool (51.4%) than the M pool (18.9%) among asymptomatic subjects; however, the difference was not statistically significant (p=0.06) for symptomatic subjects (N pool: 44.4%; M pool: 25.9%). In asymptomatic participants IFN-{gamma} responders to the CD4+CD8 pool responded more frequently to the S pool (55.6%) and N pool (57.1%), than the M pool (7.1%), but symptomatic participants, IFN-{gamma} responses were more frequent to the S pool (75.0%) than N pool (33.3%) and M pool (33.3%). The frequencies of IFN-{gamma} responses to the S and N+M pools peaked 7 days after the positive qPCR test among asymptomatic (S pool: 22.2%; N+M pool: 28.7%) and symptomatic (S pool: 15.3%; N+M pool 21.9%) participants and dropped by >28 days. Magnitudes of post-infection IFN-{gamma} and IL2 responses to the N+M pool were significantly correlated with IFN-{gamma} and IL2 responses to the N and M pools.

These data further support the central role of Th1-biased cell mediated immunity IFN-{gamma} and IL2 responses, particularly to the N protein, in controlling COVID-19 symptoms, and justify T cell-based COVID-19 vaccines that include the N and S proteins.

category: immunology


title: Mucosal and systemic responses to SARS-CoV-2 vaccination in infection naive and experienced individuals

abstract: With much of the world infected with or vaccinated against SARS-CoV-2, understanding the immune responses to the SARS-CoV-2 spike (S) protein in different situations is crucial to controlling the pandemic. We studied the clinical, systemic, mucosal, and cellular responses to two doses of SARS-CoV-2 mRNA vaccines in 62 individuals with and without prior SARS-CoV-2 exposure that were divided into three groups based on serostatus and/or degree of symptoms: Antibody negative, Asymptomatic, and Symptomatic. In the previously SARS-CoV-2-infected (SARS2-infected) Asymptomatic and Symptomatic groups, symptoms related to a recall response were elicited after the first vaccination. Anti-S trimer IgA and IgG levels peaked after 1st vaccination in the SARS2-infected groups, and were higher that the in the SARS2-naive group in the plasma and nasal samples at all time points. Neutralizing antibodies titers were also higher against the WA-1 and B.1.617.2 (Delta) variants of SARS-CoV-2 in the SARS2-infected compared to SARS2-naive vaccinees. After the first vaccination, differences in cellular immunity were not evident between groups, but the AIM+ CD4+ cell response correlated with durability of humoral immunity against the SARS-CoV-2 S protein. In those SARS2-infected, the number of vaccinations needed for protection, the durability, and need for boosters are unknown. However, the lingering differences between the SARS2-infected and SARS2-naive up to 10 months post-vaccination could explain the decreased reinfection rates in the SARS2-infected vaccinees recently reported and suggests that additional strategies (such as boosting of the SARS2-naive vaccinees) are needed to narrow the differences observed between these groups.


title: Isolation and comparative analysis of antibodies that broadly neutralize sarbecoviruses

abstract:
The devastation caused by SARS-CoV-2 has made clear the importance of pandemic preparedness. To address future zoonotic outbreaks due to related viruses in the sarbecovirus subgenus, we identified a human monoclonal antibody, 10-40, that neutralized or bound all sarbecoviruses tested in vitro and protected against SARS-CoV-2 and SARS-CoV in vivo. Comparative studies with other receptor-binding domain (RBD)-directed antibodies showed 10-40 to have the greatest breadth against sarbecoviruses and thus its promise as an agent for pandemic preparedness. Moreover, structural analyses on 10-40 and similar antibodies not only defined an epitope cluster in the inner face of the RBD that is well conserved among sarbecoviruses, but also uncovered a new antibody class with a common CDRH3 motif. Our analyses also suggested that elicitation of this class of antibodies may not be overly difficult, an observation that bodes well for the development of a pan-sarbecovirus vaccine.

One sentence summaryA monoclonal antibody that neutralizes or binds all sarbecoviruses tested and represents a reproducible antibody class.

category: microbiology


title: TopHap: Rapid inference of key phylogenetic structures from common haplotypes in large genome collections with limited diversity

abstract:
MotivationBuilding reliable phylogenies from very large collections of sequences with a limited number of phylogenetically informative sites is challenging because sequencing errors and recurrent/backward mutations interfere with the phylogenetic signal, confounding true evolutionary relationships. Massive global efforts of sequencing genomes and reconstructing the phylogeny of SARS-CoV-2 strains exemplify these difficulties since there are only hundreds of phylogenetically informative sites and millions of genomes. For such datasets, we set out to develop a method for building the phylogenetic tree of genomic haplotypes consisting of positions harboring common variants to improve the signal-to-noise ratio for more accurate phylogenetic inference of resolvable phylogenetic features.

ResultsWe present the TopHap approach that determines spatiotemporally common haplotypes of common variants and builds their phylogeny at a fraction of the computational time of traditional methods. To assess topological robustness, we develop a bootstrap resampling strategy that resamples genomes spatiotemporally. The application of TopHap to build a phylogeny of 68,057 genomes (68KG) produced an evolutionary tree of major SARS-CoV-2 haplotypes. This phylogeny is concordant with the mutation tree inferred using the co-occurrence pattern of mutations and recovers key phylogenetic relationships from more traditional analyses. We also evaluated alternative roots of the SARS-CoV-2 phylogeny and found that the earliest sampled genomes in 2019 likely evolved by four mutations of the most recent common ancestor of all SARS-CoV-2 genomes. An application of TopHap to more than 1 million genomes reconstructed the most comprehensive evolutionary relationships of major variants, which confirmed the 68KG phylogeny and provided evolutionary origins of major variants of concern.

AvailabilityTopHap is available on the web at https://github.com/SayakaMiura/TopHap.

Contacts.kumar@temple.edu


title: Language Models for the Prediction of SARS-CoV-2 Inhibitors

abstract: The COVID-19 pandemic highlights the need for computational tools to automate and accelerate drug design for novel protein targets. We leverage deep learning language models to generate and score drug candidates based on predicted protein binding affinity. We pre-trained a deep learning language model (BERT) on [~]9.6 billion molecules and achieved peak performance of 603 petaflops in mixed precision. Our work reduces pre-training time from days to hours, compared to previous efforts with this architecture, while also increasing the dataset size by nearly an order of magnitude. For scoring, we fine-tuned the language model using an assembled set of thousands of protein targets with binding affinity data and searched for inhibitors of specific protein targets, SARS-CoV-2 Mpro and PLpro. We utilized a genetic algorithm approach for finding optimal candidates using the generation and scoring capabilities of the language model. Our generalizable models accelerate the identification of inhibitors for emerging therapeutic targets.


title: Scanning the RBD-ACE2 molecular interactions in Omicron variant

abstract:
The emergence of new SARS-CoV-2 variants poses a threat to the human population where it is difficult to assess the severity of a particular variant of the virus. Spike protein and specifically its receptor binding domain (RBD) which makes direct interaction with the ACE2 receptor of the human has shown prominent amino acid substitutions in most of the Variants of Concern. Here, by using all-atom molecular dynamics simulations we compare the interaction of Wild-type RBD/ACE2 receptor complex with that of the latest Omicron variant of the virus. We observed a very interesting diversification of the charge, dynamics and energetics of the protein complex formed upon mutations. These results would help us in understanding the molecular basis of binding of the Omicron variant with that of SARS-CoV-2 Wild-type.

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category: biophysics


title: Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift

abstract: The recently emerged SARS-CoV-2 Omicron variant harbors 37 amino acid substitutions in the spike (S) protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody therapeutics. Here, we show that the Omicron RBD binds to human ACE2 with enhanced affinity relative to the Wuhan-Hu-1 RBD and acquires binding to mouse ACE2. Severe reductions of plasma neutralizing activity were observed against Omicron compared to the ancestral pseudovirus for vaccinated and convalescent individuals. Most (26 out of 29) receptor-binding motif (RBM)-directed monoclonal antibodies (mAbs) lost in vitro neutralizing activity against Omicron, with only three mAbs, including the ACE2-mimicking S2K146 mAb1, retaining unaltered potency. Furthermore, a fraction of broadly neutralizing sarbecovirus mAbs recognizing antigenic sites outside the RBM, including sotrovimab2, S2X2593 and S2H974, neutralized Omicron. The magnitude of Omicron-mediated immune evasion and the acquisition of binding to mouse ACE2 mark a major SARS-CoV-2 mutational shift. Broadly neutralizing sarbecovirus mAbs recognizing epitopes conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.


title: High temporal resolution transcriptomic profiling delineates distinct patterns of interferon response following Covid-19 mRNA vaccination and SARS-CoV2 infection

abstract: Knowledge of the mechanisms underpinning the development of protective immunity conferred by mRNA vaccines is fragmentary. Here we investigated responses to COVID-19 mRNA vaccination via ultra-low-volume sampling and high-temporal-resolution transcriptome profiling (23 subjects across 22 timepoints, and with 117 COVID-19 patients used as comparators). There were marked differences in the timing and amplitude of the responses to the priming and booster doses. Notably, we identified two distinct interferon signatures. The first signature (A28/S1) was robustly induced both post-prime and post-boost and in both cases correlated with the subsequent development of antibody responses. In contrast, the second interferon signature (A28/S2) was robustly induced only post-boost, where it coincided with a transient inflammation peak. In COVID19 patients, a distinct phenotype dominated by A28/S2 was associated with longer duration of intensive care. In summary, high-temporal-resolution transcriptomic permitted the identification of post- vaccination phenotypes that are determinants of the course of COVID-19 disease.


title: High temporal resolution systems profiling reveals distinct patterns of interferon response after Covid-19 mRNA vaccination and SARS-CoV2 infection


title: Isolation and Characterization of SARS-CoV-2 strains circulating in Eastern India.

abstract: Emergence of SARS-CoV-2 as a serious pandemic has altered the global socioeconomic dynamics. The wide prevalence, high death counts and rapid emergence of new variants urge for establishment of research infrastructure to facilitate rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, five SARS-CoV2 strains (ILS01, ILS02, ILS03, ILS15 and ILS24) of four different clades (19A, 19B, 20A and 20B) were isolated from patient swab samples collected during the 1st COVID-19 wave in Odisha, India. The viral isolates were adapted to in-vitro cultures and further characterized to identify strain specific variations in viral growth characteristics. All the five isolates showed substantial amount of virus induced CPE however ILS03 belonging to 20A clade displayed highest level of CPE. Time kinetics experiment revealed spike protein expression was evident after 16th hours post infection in all five isolates. ILS03 induced around 90% of cytotoxicity. Further, the susceptibility of various cell lines (human hepatoma cell line (Huh-7), CaCo2 cell line, HEK-293T cells, Vero, Vero-E6, BHK-21, THP-1 cell line and RAW 264.7 cells) were assessed. Surprisingly, it was found that the human monocyte cells THP-1 and murine macrophage cell line RAW 264.7 were permissive to all the SARS-CoV-2 isolates. The neutralization susceptibility of viral isolates to vaccine-induced antibodies was determined using sera from individuals vaccinated in the Government run vaccine drive in India. The micro-neutralization assay suggested that both Covaxin and Covishield vaccines were equally effective (100% neutralization) against all of the isolates. The whole genome sequencing of culture adapted viral isolates and viral genome from patient oropharyngeal swab sample suggested that repetitive passaging of SARS-CoV2 virus in Vero-E6 cells did not lead to emergence of many mutations during the adaptation in cell culture. Phylogenetic analyses revealed that the five isolates clustered to respective clades. The major goal was to isolate and adapt SARS-CoV-2 viruses in in-vitro cell culture with minimal modification to facilitate research activities involved in understanding the molecular virology, host-virus interactions, application of these strains for drug discovery and animal challenge models development which eventually will contribute towards the development of effective and reliable therapeutics.


title: Systematic discovery of receptor-ligand biology by engineered cell entry and single-cell genomics

abstract:
Cells communicate with each other via receptor-ligand interactions on the cell surface. Here we describe a technology for lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to decode receptor specificity. Engineered lentiviral particles displaying specific ligands deliver fluorescent proteins into target cells upon cognate receptor-ligand interaction, without genome integration or transgene transcription. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. We develop an effective presentation strategy to capture interactions between B cell receptor (BCR) and intracellular antigen epitopes. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of TCR-antigen specificities, clonality, cell type, and cell states of individual T cells. ENTER-seq of patient blood samples after CMV infection reveals the viral epitopes that drive human effector memory T cell differentiation and inter-clonal phenotypic diversity that targets the same epitope. ENTER enables systematic discovery of receptor specificity, linkage to cell fates, and cell-specific delivery of gene or protein payloads.

HIGHLIGHTSO_LIENTER displays ligands, deliver cargos, and records receptor specificity.
C_LIO_LIENTER deorphanizes antigen recognition of TCR and BCR.
C_LIO_LIENTER-seq maps TCR specificity, clonality and cell state in single cells.
C_LIO_LIENTER-seq of patient sample decodes antiviral T cell memory.
C_LI

category: genomics


title: Production and secretion of functional full-length SARS-CoV-2 spike protein in Chlamydomonas reinhardtii

abstract: The spike protein is the major protein on the surface of coronaviruses. It is therefore the prominent target of neutralizing antibodies and consequently the antigen of all currently admitted vaccines against SARS-CoV-2. Since it is a 1273-amino acids glycoprotein with 22 N-linked glycans, the production of functional, full-length spike protein was limited to mammalian and insect cells, requiring complex culture media. Here we report the production of full-length SARS-CoV-2 spike protein - lacking the C-terminal membrane anchor - as a secreted protein in the prefusion-stabilized conformation in the unicellular green alga Chlamydomonas reinhardtii. We show that the spike protein is efficiently cleaved at the furin cleavage site during synthesis in the alga and that cleavage is abolished upon mutation of the multi-basic cleavage site. We could enrich the spike protein from culture medium by ammonium sulfate precipitation and demonstrate its functionality based on its interaction with recombinant ACE2 and ACE2 expressed on human 293T cells. Chlamydomonas reinhardtii is a GRAS organism that can be cultivated at low cost in simple media at a large scale, making it an attractive production platform for recombinant spike protein and other biopharmaceuticals in low-income countries.

category: synthetic biology


title: A neuron, microglia, and astrocyte triple coculture model to study Alzheimer disease

abstract:
Glial cells are essential to understand Alzheimers disease (AD) progression, given their role in neuroinflammation and neurodegeneration. There is a need for reliable and easy to manipulate models that allow studying the mechanisms behind neuron and glia communication. Currently available models such as cocultures require complex methodologies and/or might not be affordable for all laboratories. With this in mind, we aimed to establish a straightforward in vitro setting with neurons and glial cells to study AD.

We generated a triple co-culture with neurons, microglia and astrocytes. Immunofluorescence, western blot and ELISA techniques were used to characterize the effects of oligomeric A{beta} (oA{beta}) in this model.

We found that, in the triple co-culture, microglia increased the expression of anti-inflammatory markers Arginase I and TGF-{beta}1, and reduced pro-inflammatory iNOS and IL-1{beta}, compared with microglia alone. Astrocytes reduced expression of pro-inflammatory A1 markers AMIGO2 and C3, and displayed a ramified morphology resembling physiological conditions. Lastly, neurons increased post-synaptic markers, and developed more and longer branches than in individual primary cultures. Addition of oA{beta} in the triple coculture reduced synaptic markers and increased microglial activation, which are hallmarks of AD.

Consequently, we developed a reliable model, where cells better resemble physiological conditions: microglia are less inflammatory, astrocytes are less reactive and neurons display a more mature morphology than in individual primary cultures. Moreover, we were able to recapitulate A{beta}-induced synaptic loss and inflammation. This model emerges as a powerful tool to study neurodegeneration and inflammation in the context of AD and other neurodegenerative diseases.

Table of content image O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

Main pointsO_LIIn our model, microglia and astrocytes are less reactive, and neurons have a more mature morphology than in primary cultures.
C_LIO_LIoA{beta} reduced synaptic markers and increased microglial activation.
C_LIO_LIThis triple co-culture is a reliable tool to study neurodegeneration and gliosis in vitro.
C_LI


title: Identifying long-range synaptic inputs using genetically encoded labels and volume electron microscopy

abstract: Enzymes that facilitate the local deposition of electron dense reaction products have been widely used as labels in electron microscopy (EM). Peroxidases, in particular, can efficiently metabolize 3,3-diaminobenzidine tetrahydrochloride hydrate (DAB) to produce precipitates with high contrast under EM following heavy metal staining, and can be genetically encoded to facilitate the labeling of specific cell-types or organelles. Nevertheless, the peroxidase/DAB method has so far not been reported to work in combination with 3D volume EM techniques (e.g. Serial blockface electron microscopy, SBEM; Focused ion beam electron microscopy, FIBSEM) because the surfactant treatment needed for efficient reagent penetration disrupts tissue ultrastructure and because these methods require the deposition of large amounts of heavy metals that can obscure DAB precipitates. However, a recently described peroxidase with enhanced enzymatic activity (dAPEX2) appears to successfully deposit EM-visible DAB products in thick tissue without surfactant treatment. Here we demonstrate that multiplexed dAPEX2/DAB tagging is compatible with both FIBSEM and SBEM volume EM approaches and use them to map long-range genetically identified synaptic inputs from the anterior cingulate cortex to the periaqueductal gray in the mouse brain.


title: Spatially distributed computation in cortical circuits

abstract:
The traditional view of neural computation in the cerebral cortex holds that sensory neurons are specialized, i.e., selective for certain dimensions of sensory stimuli. This view was challenged by evidence of contextual interactions between stimulus dimensions in which a neurons response to one dimension strongly depends on other dimensions. Here we use methods of mathematical modeling, psychophysics, and electrophysiology to address shortcomings of the traditional view. Using a model of a generic cortical circuit, we begin with the simple demonstration that cortical responses are always distributed among neurons, forming characteristic waveforms, which we call neural waves. When stimulated by patterned stimuli, circuit responses arise by interference of neural waves. Resulting patterns of interference depend on interaction between stimulus dimensions. Comparison of these modeled responses with responses of biological vision makes it clear that the framework of neural wave interference provides a useful alternative to the standard concept of neural computation.

TeaserInvestigating interference of neural waves helps to overcome limitations of the traditional view of cortical computation.


title: Alternations of fecal microbes and lung tissue microbes in Lung Cancer patients is more consistent than that of sputum.

abstract: Microbiome in the human body environment is related to the occurrence of a variety of disease phenotypes. Recent studies discovered that lung is an open organism with in touch of air and microbe in it, and the presence of some microbes in lung cancer tissues proved that there are many microbes in lungs. In this project, we collected lung tissue, feces and sputum from three Bioprojects of NCBI related to lung cancer (LC). Each project contains LC cases and lung normal (LN) controls. Those three projects contain a total of 339 samples of 16s rRNA sequencing data. By analyzing the composition of microbes in the three environments, and predicting their functions we found that compared with sputum, the ecological environment of fecal microbe is closer to tissue microbes in terms of evolutionary relationship, indicating that the impact of feces on tissue microbes is greater than that of the sputum. We used Picrust2 to predict the differential microbe function of lung cancer (LC) and the control (Lung Normal, LN) groups in the three environments, and found that at the microbe genetic level, compared to feces and sputum, sputum and tissues, feces and tissues have more common Differential genes, at the level of differential enzyme genes and differential pathways, feces and tissues have more common differences compared to feces and sputum, sputum and tissues. Our results showed that the similarity of feces and tissue microbiome is closer than the similarity of sputum and feces microbe. Through Spearman correlation analysis based on the relative abundance of predicted pathways and the relative abundance of genus classified by LDA analysis as marker diseases and healthy samples. The results indicated that the activation of marker genus in sputum and feces and significantly changed pathways has an opposite trend, and there are many pathways contributing to glycolysis are correlated with marker genus. Patients with LC has potential to regulate the microbe composition of feces, tissues and sputum by regulating metabolism.


title: The influence of Janthinobacterium sp. strain SLB01 on the cell culture of primmorphs of the sponge Lubomirskia baicalensis

abstract: Sponges (phylum Porifera) are ancient, filtering, multicellular metazoans. Freshwater sponges (Demosponges, Lubomirskiidae) dominate the fauna of the littoral zone of Lake Baikal. Over the last years, there have been mass diseases and death of endemic sponges. Previously, the strain Janthinobacterium sp. SLB01 was isolated from the diseased sponge Lubomirskia baicalensis. The studies of the pathogenicity of the strain Janthinobacterium sp. SLB01 for Baikal sponges has not been carried out. Therefore we infected experimentally in vitro to determine its pathogenicity by the cell culture of the primmorphs with subsequent isolation, sequencing, and analysis of the genomes. The purpose of the study is to show that the strain Janthinobacterium sp. SLB01 isolated from the diseased sponge L. baicalensis is a pathogen for the cell culture of primmorphs. The bacteria from the infected samples of the primmorphs were isolated and identified as strain Janthinobacterium sp. PLB02. A comparative analysis of the genomes of the strains Janthinobacterium sp. SLB01 and Janthinobacterium sp. PLB02 showed that they are practically identical. The genomes of both strains contain genes vioABCDE violacein, flok formation, and strong biofilm, and the type VI secretion system (T6SS), as the primary virulence factor. The bacterial strains Janthinobacterium sp. based on a comparison of complete genomes showed similarity with strain Janthinobacterium lividum MTR. Isolated our strains of Janthinobacterium sp. are pathogens for cell cultures of primmorphs and L. baicalensis sponges. The results of the study will help to expand the understanding of microbial relationships in the development of disease and the death of Baikal sponges.


title: Monosomy X in isogenic human iPSC-derived trophoblast model impacts expression modules preserved in human placenta

abstract:
SUMMARY/ABSTRACTMammalian sex chromosomes encode homologous X/Y gene pairs that were retained on the male Y and escape X chromosome inactivation (XCI) in females. Inferred to reflect X/Y-pair dosage sensitivity, monosomy X is a leading cause of miscarriage in humans with near full penetrance. This phenotype is shared with many other mammals but not the mouse, which offers sophisticated genetic tools to generate sex chromosomal aneuploidy but also tolerates its developmental impact. To address this critical gap, we generated X-monosomic human induced pluripotent stem cells (hiPSCs) alongside otherwise isogenic euploid controls from male and female mosaic samples. Phased genomic variants of these hiPSC panels enable systematic investigation of X/Y dosage-sensitive features using in vitro models of human development.

Here, we demonstrate the utility of these validated hiPSC lines to test how X/Y-linked gene dosage impacts a widely-used model for the human syncytiotrophoblast. While these isogenic panels trigger a GATA2/3 and TFAP2A/C -driven trophoblast gene circuit irrespective of karyotype, differential expression implicates monosomy X in altered levels of placental genes, and in secretion of placental growth factor (PlGF) and human chorionic gonadotropin (hCG). Remarkably, weighted gene co-expression network modules that significantly reflect these changes are also preserved in first-trimester chorionic villi and term placenta. Our results suggest monosomy X may skew trophoblast cell type composition, and that the pseudoautosomal region likely plays a key role in these changes, which may facilitate prioritization of haploinsufficient drivers of 45,X extra-embryonic phenotypes.

category: developmental biology


title: Combining δ13C and δ15N from bone and dentine in marine mammal palaeoecological research: insights from toothed whales

abstract:
Stable carbon ({delta}13C) and nitrogen ({delta}15N) isotope compositions of bone and dentine collagen extracted from museum specimens have been widely used to study the paleoecology of past populations. Due to possible systematic differences in stable isotope values between bone and dentine, dentine values can be transformed into bone-collagen equivalent using a correction factor. Here, we provide correction factors to transform dentine{delta} 13C and{delta} 15N values into bone-collagen equivalent for two toothed whales: narwhal and beluga.

We sampled bone and tooth dentine from the skulls of 11 narwhals and 26 belugas. In narwhals, dentine was sampled from tusk and embedded tooth; in beluga, dentine was sampled from tooth.{delta} 13C and{delta} 15N were measured and intraindividual bone and dentine isotopic compositions were used to calculate correction factors for each species.

We detected differences in{delta} 13C and{delta} 15N. In narwhals, we found (i) lower average{delta} 13C and{delta} 15N in bone compared with dentine; (ii) no difference in dentine{delta} 13C between tusk and embedded tooth. For belugas, we also detected lower{delta} 13C and{delta} 15N in bone compared with tooth dentine. The correction factors provided by the study enable the combined analysis of stable isotope data from bone and dentine in these species.


title: Ecology drives the degree of convergence in the gene expression of extremophile fishes

abstract:
Convergent evolution, where independent lineages evolve similar traits when adapting to similar habitats, is a common phenomenon and testament to the repeatability of evolutionary processes. Still, non-convergence is also common, and a major question is whether apparently idiosyncratic, lineage-specific evolutionary changes are reflective of chance events inherent to evolutionary processes, or whether they are also influenced by deterministic genetic or ecological factors. To address this question, we quantified the degree of convergence in genome-wide patterns of gene expression across lineages of livebearing fishes (family Poeciliidae) that span 40 million years of evolution and have colonized extreme environments in the form of toxic, hydrogen-sulfide-rich springs. We specifically asked whether the degree of convergence across lineage pairs was related to their phylogenetic relatedness or the ecological similarity of the habitats they inhabit. Using phylogenetic comparative analyses, we showed that the degree of convergence was highly variable across lineage pairs residing in sulfide springs. While closely related lineages did not exhibit higher degrees of convergence than distantly related ones, we uncovered a strong relationship between degree of convergence and ecological similarity. Our results indicate that variation in the degree of convergence is not merely noise associated with evolutionary contingency. Rather, cryptic environmental variation that is frequently ignored when we employ reductionist approaches can significantly contribute to adaptive evolution. This study highlights the importance of multivariate approaches that capture the complexities of both selective regimes and organismal design when assessing the roles of determinism and contingency in evolution.

Significance StatementWhen different species adapt to similar environmental conditions, we frequently observe a mix between shared (convergent) and lineage-specific (nonconvergent) evolutionary changes. Shared changes provide evidence for the repeatability and predictability of evolution. However, it remains unclear whether lineage-specific changes are caused by random forces that limit the predictability of evolution, or whether they reflect deterministic processes shaped by unidentified genetic and environmental factors. By analyzing patterns of gene expression across fishes in extreme environments, we show that the degree of convergence between lineages is related to ecology, indicating that lineage-specific evolutionary changes are not just noise caused by random processes. Thus, acknowledging the complexity of nature in empirical research is critical if we want to predict evolution.

category: evolutionary biology


title: Glutathione binding to the plant At Atm3 transporter and implications for the conformational coupling of ABC transporters

abstract: The ATP Binding Cassette (ABC) transporter of mitochondria (Atm) from Arabidopsis thaliana (AtAtm3) has been implicated in the maturation of cytosolic iron-sulfur proteins and heavy metal detoxification, plausibly by exporting glutathione derivatives. Using single-particle cryo-electron microscopy, we have determined structures of AtAtm3 in multiple conformational states. These structures not only provide a structural framework for defining the alternating access transport cycle, but also highlight an unappreciated feature of the glutathione binding site, namely the paucity of cysteine residues that could potentially form inhibitory mixed disulfides with glutathione. Despite extensive efforts, we were unable to prepare the ternary complex of AtAtm3 with bound GSSG and MgATP. A survey of structurally characterized type IV ABC transporters that includes AtAtm3 establishes that while nucleotides are found associated with all conformational states, they are effectively required to stabilize occluded and outward-facing conformations. In contrast, transport substrates have only been observed associated with inward-facing conformations. The absence of structures containing both nucleotide and transport substrate suggests that this ternary complex exists only transiently during the transport cycle.


title: Generative Language Modeling for Antibody Design

abstract: Successful development of monoclonal antibodies (mAbs) for therapeutic applications is hindered by developability issues such as low solubility, low thermal stability, high aggregation, and high immunogenicity. The discovery of more developable mAb candidates relies on high-quality antibody libraries for isolating candidates with desirable properties. We present Immunoglobulin Language Model (IgLM), a deep generative language model for generating synthetic libraries by re-designing variable-length spans of antibody sequences. IgLM formulates anti-body design as an autoregressive sequence generation task based on text-infilling in natural language. We trained IgLM on approximately 558M antibody heavy- and light-chain variable sequences, conditioning on each sequences chain type and species-of-origin. We demonstrate that IgLM can be applied to generate synthetic libraries that may accelerate the discovery of therapeutic antibody candidates.

category: bioengineering


title: Identifying Protein Features and Pathways Responsible for Toxicity using Machine learning, CANDO, and Tox21 datasets: Implications for Predictive Toxicology

abstract: Humans are exposed to numerous compounds daily, some of which have adverse effects on health. Computational approaches for modeling toxicological data in conjunction with machine learning algorithms have gained popularity over the last few years. Machine learning approaches have been used to predict toxicity-related biological activities using chemical structure descriptors. However, toxicity-related proteomic features have not been fully investigated. In this study, we construct a computational pipeline using machine learning models for predicting the most important protein features responsible for the toxicity of compounds taken from the Tox21 dataset that is implemented within the multiscale Computational Analysis of Novel Drug Opportunities (CANDO) therapeutic discovery platform. Tox21 is a highly imbalanced dataset consisting of twelve in vitro assays, seven from the nuclear receptor (NR) signaling pathway and five from the stress response (SR) pathway, for more than 10,000 compounds. For the machine learning model, we employed a random forest with the combination of Synthetic Minority Oversampling Technique (SMOTE) and Edited Nearest Neighbor (ENN) method (SMOTE+ENN), which is a resampling method to balance the activity class distribution. Within the NR and SR pathways, the activity of the aryl hydrocarbon receptor (NR-AhR), and mitochondrial membrane potential (SR-MMP) were two of the top-performing twelve toxicity endpoints with AUCROCs of 0.90 and 0.92, respectively. The top extracted features for evaluating compound toxicity were analyzed for enrichment to highlight the implicated biological pathways and proteins. We validated our enrichment results for the activity of the AhR using a thorough literature search. Our case study showed that the selected enriched pathways and proteins from our computational pipeline are not only correlated with AhR toxicity but also form a cascading upstream/downstream arrangement. Our work elucidate significant relationships between protein and compound interactions computed using CANDO and the associated biological pathways to which the proteins belong for twelve toxicity endpoints. This novel study uses machine learning not only to predict and understand toxicity but also elucidates therapeutic mechanisms at a proteomic level for a variety of toxicity endpoints.


title: AutoMicroED: A semi-automated MicroED processing pipeline

abstract: Despite rapid adaptation of micro-electron diffraction (MicroED) for protein and small molecule structure determination to sub-angstrom resolution, the lack of automation tools for easy MicroED data processing remains a challenge for expanding to the broader scientific community. In particular, automation tools, which are novice user friendly, compatible with heterogenous datasets and can be run in unison with data collection to judge the quality of incoming data (similar to cryosparc LIVE for single particle cryoEM) do not exist. Here, we present AutoMicroED, a cohesive and semi-automatic MicroED data processing pipeline that runs through image conversion, indexing, integration and scaling of data, followed by merging of successful datasets that are pushed through phasing and final structure determination. AutoMicroED is compatible with both small molecule and protein datasets and creates a straightforward and reproducible method to solve single structures from pure samples, or multiple structures from mixed populations. The immediate feedback on data quality, data completeness and more parameters, aids users to identify whether they have collected enough data for their needs. Overall, AutoMicroED permits efficient structure elucidation for both novice and experienced users with comparable results to more laborious manual processing.


title: Mechanistic insights of radiation-induced endothelial senescence impelling glioblastoma genomic instability at relapse

abstract: Despite aggressive clinical protocol, all glioblastoma (GBM) recur at the initial site within the irradiated peritumoral microenvironment. Whereas irradiated microenvironment has been recently proposed to accelerate GBM relapse, molecular and cellular mechanisms remain unknown. Here, using relevant in vitro and in vivo models, we decipher how radiation-induced endothelial senescence drives the emergence of aggressive GBM cells. Secretome (SASP) of radiation-induced senescent (RIS) endothelium enhances genomic instability and intratumoral heterogeneity in irradiated GBM cells. In-depth molecular studies revealed that CXCL5 and CXCL8, from the SASP, activate CXCR2 receptor on tumor cells leading to increased DNA hyper-replication, micronuclei formation and aneuploidy. Importantly, through CXCL5/8-CXCR2 axis activation, this SASP increases GBM aggressiveness in vivo. Both chemokines were detected in relapsing, but not primary, GBM biopsies and positively correlated with worst patient outcome. In conclusion, we identify new molecular and preclinical insights of relapsing GBM aggressiveness where RIS vascular niches fuel aggressive tumor emergence.


title: CRCM5484: A BET- BDII Selective Compound With Differential Anti-Leukemic Drug Potentiation

abstract: Differentially screening the Fr-PPIChem chemical library on the BET BRD4-BDII versus -BDI bromodomains led to the discovery of a BDII selective tetrahydropyridothienopyrimidinone (THPTP)-based compound. Structure-activity relationship (SAR) and hit-to-lead approaches allowed us to develop CRCM5484, a potent inhibitor of BET proteins with a preferential and 475-fold selectivity for the second bromodomain of the BRD3 protein (BRD3-BDII) over its first bromodomain (BRD3-BDI). Its very low activity was demonstrated in various cell-based assays, corresponding with recent data describing other selective BDII compounds. However, screening on a drug sensitivity and resistance-profiling platform revealed its ability to modulate the anti-leukemic activity in combination with various FDA-approved and/or in-development drugs in a cell- and context-dependent differential manner. Altogether, the results confirm the originality of the THPTP molecular mode of action in the BD cavity and its potential as starting scaffold for the development of potent and selective bromodomain inhibitors.


title: CRCM5484: A BET- BDII Selective Compound With Differential Anti-Leukemic Drug Modulation


title: LKB1 Is Physiologically Required for Sleep from Drosophila to the Mouse

abstract: LKB1 is known as a master kinase for 14 kinases related to the adenosine monophosphate (AMP)-activated protein kinase (AMPK). Two of them (SIK3 and AMPK) have previously been implicated in sleep regulation. We generated loss-of-function (LOF) mutants for Lkb1 in both Drosophila and mice. Sleep was reduced in Lkb1-mutant flies and in flies with neuronal deletion of Lkb1. Sleep was reduced in mice after virally mediated reduction of Lkb1 in the brain. Electroencephalography (EEG) analysis showed that non-rapid eye movement (NREM) sleep and sleep need were both reduced in Lkb1-mutant mice. These results indicate that LKB1 plays a physiological role in sleep regulation conserved from flies to mice.


title: A library for fMRI real-time processing systems in python (RTPSpy) with comprehensive online noise reduction, fast and accurate anatomical image processing, and online processing simulation

abstract: Real-time fMRI (rtfMRI)has enormous potential for both mechanistic brain imaging studies or treatment-oriented neuromodulation. However, the adaption of rtfMRI has been limited due to technical difficulties implement an efficient computational framework. Here, we introduce a python library for real-time fMRI (rtfMRI) data processing systems, Real-Time Processing System in python (RTPSpy), to provide building blocks for a custom rtfMRI application with extensive and advanced functionalities. RTPSpy is a library package including 1) a fast, comprehensive, and flexible online fMRI image processing modules comparable to offline denoising, 2) utilities for fast and accurate anatomical image processing to define an anatomical target region, 3) a simulation system of online fMRI processing to optimize a pipeline and target signal calculation, 4) simple interface to an external application for feedback presentation, and 5) a boilerplate graphical user interface (GUI) integrating operations with RTPSpy library. The fast and accurate anatomical image processing utility wraps external tools, including FastSurfer, ANTs, and AFNI, to make tissue segmentation and region of interest masks. We confirmed that the quality of the output masks was comparable with FreeSurfer, and the anatomical image processing could complete in a few minutes. The modular nature of RTPSpy provides the ability to use it for a simulation analysis to optimize a processing pipeline and target signal calculation. We present a sample script for building a real-time processing pipeline and running a simulation using RTPSpy. The library also offers a simple signal exchange mechanism with an external application using a TCP/IP socket. While the main components of the RTPSpy are the library modules, we also provide a GUI class for easy access to the RTPSpy functions. The boilerplate GUI application provided with the package allows users to develop a customized rtfMRI application with minimum scripting labor. The limitations of the package as it relates to environment-specific implementations are discussed. These library components can be customized and can be used in parts. Taken together, RTPSpy is an efficient and adaptable option for developing rtfMRI applications. The package is available from GitHub (https://github.com/mamisaki/RTPSpy) with GPL3 license.


title: Angle-Tuned Coils: Enabling Building Blocks for High Performance and Multisite TMS Systems

abstract: Coordinated whole-brain neural dynamics are essential for proper control of the functionality of different brain systems. Multisite simultaneous or sequential stimulations may provide tools for mechanistic studies of brain functions and the treatment of neuropsychiatric disorders. Conventional circular and figure-8 Transcranial Magnetic Stimulation (TMS) coils occupy a large footprint, and it is difficult to reach desired multiple stimulation locations with close proximity for comprehensive multisite stimulations. These conventional coils, limited by the depth-spread tradeoff rule, also lack the required focality for targeted stimulations. In this work, we propose and demonstrate angle-tuned TMS (AT) coils with an intrinsically reduced footprint with their geometric arrangements of stacking and angle tuning. The stimulation depth can be adjusted with the coil stacking number, and the field spread can be reduced by increasing the tilted wire-wrapping angle of the coils. With either smaller or larger diameter coils than a standard commercial figure-8 coil, we show, theoretically and experimentally, improved field decay rate and field intensity, and the reduced field spread spot size at different stimulation depths. These results indicate that the proposed novel coil establishes a better depth-spread tradeoff curve than the conventional circular and figure-8 coils. This coil design has a simple and single element structure and provides a promising solution for an improved multisite brain stimulation performance and serves as the building block of more complex coils for further depth-spread improvements.


title: A core salivary microbiome shows the high prevalence of bacterial members yet variability across human populations

abstract: Human saliva contains diverse bacterial communities, reflecting human health status, dietary patterns and contributing to variability in the sensory perception of food. Many descriptions of salivary microbiome diversity compare commonalities and differences with reference to a diseased state, but the composition of healthy saliva has not been described. Here, we use a meta-analysis approach to define and explore the core membership of the human salivary microbial community by collecting and re-analysing raw 16S rRNA amplicon sequencing data from 47 studies with 2206 saliva samples. We found 68 core bacterial taxa that were consistently detected. Differences induced by various host intrinsic and behaviour factors, including gender, age, geographic location, tobacco usage, and alcohol consumption, were evident. The core of the salivary microbiome was verified by collecting and analysing saliva in an independent study. These results suggest that the methods used can effectively define a core microbial community in human saliva with high prevalence. The core salivary microbiome demonstrated both stability and variability among populations. Geographic location was identified as the host factor with the largest effect in shaping salivary microbiota. The independent analysis underlined that the impact of geographic variation is likely due to diet.


title: The salivary microbiome shows a high prevalence of core bacterial members yet variability across human populations


title: Stratified microbial communities are highly sensitive towards multiple combined global change factors, revealing antagonistic and synergistic effects

abstract: Microbial communities in many ecosystems are facing a broad range of global change drivers, such as nutrient enrichment, chemical pollution, and temperature change. These drivers can cause changes in the abundance of taxa, the composition of communities, and the properties of ecosystems. While the influence of single drivers is already described in numerous studies, the effect and predictability of multiple drivers changing simultaneously is still poorly understood. In this study, we used 240 highly replicable oxic/anoxic aquatic lab micro-ecosystems and four drivers (fertilizer, glyphosate, metal pollution, antibiotics) in all possible combinations at three different temperatures (20 {degrees}C, 24 {degrees}C, and 28 {degrees}C) to shed light into consequences of multiple drivers on different levels of organization, ranging from species abundance to community and ecosystem parameters. We found (i) that at all levels of ecological organisation, combinations of drivers can change the biological consequence and direction of effect compared to single drivers (ii), that effects of combinations are further modified by temperature, (iii) that a larger number of drivers occurring simultaneously is often quite closely related to their effect size, and (iv) that there is little evidence that any of these effects are associated with the level of ecological organisation of the state variable. These findings suggest that, at least in this experimental ecosystem approximating a stratified aquatic ecosystem, there may be relatively little scope for predicting the effects of combinations of drivers from the effects of individual drivers, or by accounting for the level of ecological organisation in question, though there may be some scope for prediction based on the number of drivers that are occurring simultaneous. A priority, though also a considerable challenge, is to extend such research to consider continuous variation in the magnitude of multiple drivers acting together.


title: Predicting the effects of multiple global change drivers onmicrobial communities remains challenging


title: PIKfyve inhibition reveals a novel role for Inpp4b in the regulation of PtdIns(3)P and lysosome dynamics

abstract:
Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that co-ordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the action of diverse PtdIns kinases and phosphatases. Specific roles for PtdIns in lysosomal function and dynamics are currently unclear and require further investigation. PIKfyve, a lipid kinase which synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome "fusion-fission" cycles dynamics, autophagosome turnover and endocytic cargo delivery. We have recently characterized a role for INPP4B, a PtdIns phosphatase which hydrolyses PtdIns(3,4)P2 to form PtdIns(3)P, in the regulation of lysosomal biogenesis and function. To gain a better understanding of PtdIns homeostasis on lysosomes, we investigated the consequence of disrupting PIKfyve in Inpp4b-deficient mouse embryonic fibroblasts. Surprisingly, simultaneous inhibition of Inpp4b and PIKfyve functions impair lysosome "fission" dynamics and thereby exacerbate lysosome enlargement, inhibit autophagic flux. Further examination into the underlying processes that may explain exaggerated lysosome enlargement revealed elevated levels of lysosome associated PtdIns(3)P as contributing factors that control lysosome morphology in cells where Inpp4b and PIKfyve are disrupted. Overall, our study suggests that lysosomal functions are regulated by Inpp4b, through a paradoxical role in suppressing the induction of PtdIns(3)P production.

SUMMARY STATEMENTIn this study we identify a novel crosstalk between Inpp4b and PIKfyve. This crosstalk regulates lysosome membrane phosphoinositide composition and lysosomal homeostasis. Our data demonstrate that Inpp4b restricts VPS34-dependent induction of PtdIns(3)P levels, which is activated upon apilimod-mediated PIKfyve inhibition. Through this mechanism, Inpp4b contributes to the regulation of lysosomal membrane dynamics and homeostasis.

category: cell biology


title: Transcriptomics reveal stretched human pluripotent stem cell-derived cardiomyocytes as an advantageous hypertrophy model

abstract:
Left ventricular hypertrophy, characterized by hypertrophy of individual cardiomyocytes, is an adaptive response to an increased cardiac workload that eventually leads to heart failure. Previous studies using neonatal rat ventricular myocytes (NRVMs) and animal models have revealed several hypertrophy- and mechanical load-associated genes and signaling pathways. However, these models are not directly applicable to humans. Here, we studied the effect of cyclic mechanical stretch on gene expression of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using RNA sequencing. HiPSC-CMs showed distinct hypertrophic changes in gene expression at the level of individual genes and in biological processes. We also identified several differentially expressed genes that have not been previously associated with cardiomyocyte hypertrophy and thus serve as attractive targets for future studies. When compared to previously published data attained from stretched NRVMs and human embryonic stem cell-derived cardiomyocytes, hiPSC-CMs displayed a smaller number of changes in gene expression, but the differentially expressed genes revealed more pronounced enrichment of hypertrophy-related biological processes and pathways. Overall, these results establish hiPSC-CMs as a valuable in vitro model for studying human cardiomyocyte hypertrophy.

Summary statementDistinct hypertrophic gene expression changes in mechanically stretched human induced pluripotent stem cell-derived cardiomyocytes reveal the utility of these cells as an advantageous in vitro model for mechanical overload-induced hypertrophy.


title: Endothelial-immune crosstalk contributes to vasculopathy in non-alcoholic fatty liver disease

abstract: The top cause of mortality in patients with non-alcoholic fatty liver disease (NAFLD) is cardiovascular complications. However, the mechanisms of NAFLD-associated vasculopathy remain understudied. We developed blood outgrowth endothelial cell (BOEC) models from NAFLD and healthy subjects. NAFLD BOECs exhibited global transcriptional upregulation of chemokine hallmarks and human leukocyte antigens. In mouse models of diet-induced NAFLD, we further confirmed enhanced endothelial expressions of CXCL12 in the aortas and liver vasculatures. To elucidate endothelial-immune crosstalk, we performed immunoprofiling by single-cell analysis, uncovering T cell intensification and potentially T-helper type 1 inflammation in NAFLD patients. Functionally, interference of the CXCL12-CXCR4 axis by small molecule AMD3100 selectively modulated the chemotaxis of patient-derived CD4+ T cells and natural killer cells towards NAFLD BOECs, restoring endothelial barrier integrity. Clinically, we detected three folds more circulating damaged endothelial cells in NAFLD patients than healthy controls. Our work provides insights for modulation of interactions with effector immune subsets to mitigate endothelial injury in NAFLD.


title: PTEN inhibits AMPK to control collective migration

abstract: PTEN is one of the most frequently mutated tumor suppressor gene in cancer. PTEN is generally altered in invasive cancers such as glioblastomas, but its function in collective cell migration and invasion is not fully characterized. Herein, we report that the loss of PTEN increases cell speed during collective migration of non-tumourous cells both in vitro and in vivo. We further show that loss of PTEN promotes LKB1-dependent phosphorylation and activation of the major metabolic regulator AMPK. In turn AMPK increases VASP phosphorylation, reduces VASP localization at cell-cell junctions and decreases the interjunctional transverse actin arcs at the leading front, provoking a weakening of cell-cell contacts and increasing migration speed. Targeting AMPK activity not only slows down PTEN-depleted cells, it also limits PTEN-null glioblastoma cell invasion, opening new opportunities to treat glioblastoma lethal invasiveness.


title: Differentiation signals from glia are fine-tuned to set neuronal numbers during development

abstract: Neural circuit formation and function require that diverse neurons are specified in appropriate numbers. Known strategies for controlling neuronal numbers involve regulating either cell proliferation or survival. We used the Drosophila visual system to probe how neuronal numbers are set. Photoreceptors from the eye-disc induce their target field, the lamina, such that for every unit eye there is a corresponding lamina unit (column). Although each column initially contains ~6 post-mitotic lamina precursors, only 5 differentiate into neurons, called L1-L5; the  extra precursor, which is invariantly positioned above the L5 neuron in each column, undergoes apoptosis. By forcing neuronal differentiation in the lamina, we uncovered that though fated to die, the  extra precursor is specified as an L5. Therefore, two precursors are specified as L5s but only one differentiates during normal development. We showed that a glial population called the outer chiasm giant glia (xgO), which resides below the lamina, secretes multiple ligands to induce L5 differentiation in response to EGF from photoreceptors. The row of precursors nearest to xgO differentiate into L5s and, in turn, antagonise differentiation signalling to prevent the  extra precursors from differentiating, resulting in their death. Thus, an intricate interplay of glial signals and feedback from differentiating neurons defines an invariant and stereotyped pattern of neuronal differentiation and programmed cell death to ensure that lamina columns each contain exactly one L5 neuron. In sum, neuronal numbers can be set by inducing differentiation of a subset of neurons from a larger pool of specified progenitors.


title: Landscape dynamics promoted the evolution of mega-diversity in South American freshwater fishes

abstract:
Landscape dynamics and river network rearrangements are widely thought to shape the diversity of Neotropical freshwater fishes, the most species-rich continental vertebrate fauna on Earth. Yet the effects of hydrogeographic changes on fish dispersal and diversification remain poorly understood. Here we integrate an unprecedented occurrence dataset of 4,967 South American freshwater fish species with a species-dense phylogeny to track the evolutionary processes associated with hydrogeographic events over 100 Ma. Net lineage diversification was heterogeneous through time, across space, and among clades. Three abrupt shifts in diversification rates occurred during the Paleogene (between 63 and 23 Ma) in association with major landscape evolution events, and net diversification accelerated from the Miocene to the Recent (c. 20 - 0 Ma). The Western Amazon exhibited the highest rates of in situ diversification and was also the most important source of species dispersing to other regions. All regional biotic interchanges were associated with documented hydrogeographic events and the formation of biogeographic corridors, including Early Miocene (c. 20 Ma) uplift of the Serra do Mar, and Late Miocene (c. 10 Ma) uplift of the Northern Andes and formation of the modern transcontinental Amazon River. Reciprocal mass dispersal of fishes between the Western and Eastern Amazon coincided with this phase of Andean uplift. The Western Amazon has the highest contemporary levels of species richness and phylogenetic endemism. Our results support the hypothesis that landscape dynamics were constrained by the history of drainage basin connections, strongly affecting the assembly and diversification of basin-wide fish faunas.

Significance StatementDespite progress in mapping geographic distributions and genealogical relationships, scientists have few clear answers about the origins of South American freshwater fishes, the most diverse vertebrate fauna on Earth. Here we used the most complete dataset of geographic distributions and evolutionary relationships of South American fishes to track how the geological history of river dynamics influenced the origin, extinction, and interchange of species over the past 100 Ma. We found abrupt increases of species origination between 66 and 23 Ma, coinciding with repeated uplifts of the Andes. The Western Amazon region served as source of freshwater fishes to other regions, as a place where species tended to persist over longer historical periods, and where species originations occurred with higher frequency.


title: Artificial selection of communities drives the emergence of structured interactions

abstract: Species-rich communities, such as the microbiota or environmental microbial assemblages, provide key functions for human health and ecological resilience. Increasing effort is being dedicated to design experimental protocols for selecting community-level functions of interest. These experiments typically involve selection acting on populations of communities, each of which is composed of multiple species. Numerical explorations allowed to link the evolutionary dynamics to the multiple parameters involved in this complex, multi-scale evolutionary process. However, a comprehensive theoretical understanding of artificial selection of communities is still lacking. Here, we propose a general model for the evolutionary dynamics of species-rich communities, each described by disordered generalized Lotka-Volterra equations, that we study analytically and by numerical simulations. Our results reveal that a generic response to selection for larger total community abundance is the emergence of an isolated eigenvalue of the interaction matrix that can be understood as an effective cross-feeding term. In this way, selection imprints a structure on the community, which results in a global increase of both the level of mutualism and the diversity of interactions. Our approach moreover allows to disentangle the role of intraspecific competition, interspecific interactions symmetry and number of selected communities in the evolutionary process, and can thus be used as a guidance in optimizing artificial selection protocols.


title: Enriching for orthologs increases support for Xenacoelomorpha and Ambulacraria sister relationship

abstract: Conflicting studies place a group of bilaterian invertebrates containing xenoturbellids and acoelomorphs, the Xenacoelomorpha, as either the primary emerging bilaterian phylum, or within Deuterostomia, sister to Ambulacraria. While their placement as sister to the rest of Bilateria supports relatively simple morphology in the ancestral bilaterian, their alternative placement within Deuterostomia suggests a morphologically complex ancestral Bilaterian along with extensive loss of major phenotypic traits in the Xenacoelomorpha. More recently, further studies have brought into question whether Deuterostomia should be considered monophyletic at all. Hidden paralogy presents a major challenge for reconstructing species phylogenies. Here we assess whether hidden paralogy has contributed to the conflict over the placement of Xenacoelomorpha. Our approach assesses previously published datasets, enriching for orthogroups whose gene trees support well resolved clans elsewhere in the animal tree of life. We find that the majority of constituent genes in previously published datasets violate incontestable clans, suggesting that hidden paralogy is rife at this depth. We demonstrate that enrichment for genes with orthologous signal alters the final topology that is inferred, whilst simultaneously improving fit of the model to the data. We discover increased, but ultimately not conclusive, support for the existence of Xenambulacraria in our orthology enriched set of genes. At a time when we are steadily progressing towards sequencing all of life on the planet, we argue that long-standing contentious issues in the tree of life will be resolved using smaller amounts of better quality data that can be modelled adequately.


title: Giant Starship elements mobilize accessory genes in fungal genomes

abstract: Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a novel group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27-393 kb and last shared a common ancestor ca. 400 mya. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize 4 additional Starships whose past and ongoing activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyagers activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first known agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum.


title: Genomic landscape, polymorphism and possible LINE-associated delivery of G-Quadruplex motifs in the bovine genes.

abstract: G-Quadruplex structures are non-B DNA structures that occur in regions carrying short runs of guanines. They are implicated in several biological processes including transcription, translation, replication and telomere maintenance as well as in several pathological conditions like cancer and thus they have gained the attention of the scientific community. The rise of the -omics era significantly affected the G-quadruplex research and the genome-wide characterization of G-Quadruplexes has been rendered a necessary first step towards applying genomics approaches for their study. While in human and several model organisms there is a considerable number of works studying genome-wide the DNA motifs with potential to form G-quadruplexes (G4-motifs), there is a total absence of any similar studies regarding livestock animals. The objectives of the present study were to provide a detailed characterization of the bovine genic G4-motifs distribution and properties and to suggest a possible mechanism for the delivery of G4 motifs in the genes. Our data indicate that the distribution of G4s within bovine genes and the annotation of said genes to Gene Ontology terms are similar to what is already shown for other organisms. By investigating their structural characteristics and polymorphism, it is obvious that the overall stability of the putative quadruplex structures is in line with the current notion in the G4 field. Similarly to human, the bovine G4s are overrepresented in specific LINE repeat elements, the L1_BTs in the case of cattle. We suggest these elements as vehicles for delivery of G4 motifs in the introns of the bovine genes. Lastly, it seems that a basis exists for connecting traits of agricultural importance to the genetic variation of G4 motifs, thus, cattle could become an interesting new model organism for G4-related genetic studies.


title: Prolactin maintains parental responses and alters reproductive axis gene expression, but not courtship behaviors, in both sexes of a biparental bird

abstract: Prolactin, a hormone involved in vertebrate parental care, is hypothesized to inhibit reproductive hypothalamic-pituitary-gonadal (HPG) axis activity during parenting, thus maintaining investment in the current brood as opposed to new reproductive efforts. While prolactin underlies many parental behaviors in birds, its effects on other reproductive behaviors, such as courtship, remain unstudied. How prolactin affects neuropeptide and hormone receptor expression across the avian HPG axis also remains unknown. To address these questions, we administered ovine prolactin (oPRL) or a vehicle control to both sexes in experienced pairs of the biparental rock dove (Columba livia), after nest removal at the end of incubation. We found that oPRL promoted parental responses to novel chicks and stimulated crop growth compared to controls, consistent with other studies. However, we found that neither courtship behaviors, copulation rates nor pair maintenance differed with oPRL treatment. Across the HPG, we found oPRL had little effect on gene expression in hypothalamic nuclei, but increased expression of FSHB and hypothalamic hormone receptor genes in the pituitary. In the gonads, oPRL increased testes size and gonadotropin receptor expression, but did not affect ovarian state or small white follicle gene expression. However, the oviducts of oPRL-treated females were smaller and had lower estrogen receptor expression compared with controls. Our results highlight that some species, especially those that show multiple brooding, may be able to maintain mating behavior despite elevated prolactin. Thus, mechanisms may exist for prolactin to promote investment in parental care without concurrent inhibition of reproductive function or HPG axis activity.

category: animal behavior and cognition


title: Acute and chronic physical activity improve spatial pattern separation in humans

abstract: Physical activity benefits both fitness and cognition. However, its effect on long-term memory is unclear. Successful memory involves not only remembering information over time but also keeping memories distinct and less confusing. The ability to separate similar experiences into distinct memories is one of the main features of episodic memory. In this work, we evaluated the effect of acute and chronic physical activity on a new task to assess spatial pattern separation in a 3D virtual reality environment. We manipulated the load of memory similarity and found that 25 minutes of cycling after encoding - but not before retrieval - was sufficient to improve similar, but not dissimilar memories, 24 hours after encoding. Furthermore, we found that participants who engaged in regular physical activity, but not sedentary subjects, showed memory for the similar condition the next day. Thus, physical activity could be a simple way to improve discrimination of spatial memories in humans.


title: A Fast Lasso-Based Method for Inferring Higher-Order Interactions

abstract: AO_SCPLOWBSTRACTC_SCPLOWLarge-scale genotype-phenotype screens provide a wealth of data for identifying molecular alterations associated with a phenotype. Epistatic effects play an important role in such association studies. For example, siRNA perturbation screens can be used to identify combinatorial gene-silencing effects. In bacteria, epistasis has practical consequences in determining antimicrobial resistance as the genetic background of a strain plays an important role in determining resistance. Recently developed tools scale to human exome-wide screens for pairwise interactions, but none to date have included the possibility of three-way interactions. Expanding upon recent state-of-the art methods, we make a number of improvements to the performance on large-scale data, making consideration of three-way interactions possible. We demonstrate our proposed method, Pint, on both simulated and real data sets, including antibiotic resistance testing and siRNA perturbation screens. Pint outperforms known methods in simulated data, and identifies a number of biologically plausible gene effects in both the antibiotic and siRNA models. For example, we have identified a combination of known tumor suppressor genes that is predicted (using Pint) to cause a significant increase in cell proliferation.


title: ProteinGLUE: A multi-task benchmark suite for self-supervised protein modeling.

abstract:
Self-supervised language modeling is a rapidly developing approach for the analysis of protein sequence data. However, work in this area is heterogeneous and diverse, making comparison of models and methods difficult. Moreover, models are often evaluated only on one or two downstream tasks, making it unclear whether the models capture generally useful properties. We introduce the ProteinGLUE benchmark for the evaluation of protein representations: a set of seven tasks for evaluating learned protein representations. We also offer reference code, and we provide two baseline models with hyperparameters specifically trained for these benchmarks.

Pre-training was done on two tasks, masked symbol prediction and next sentence prediction. We show that pre-training yields higher performance on a variety of downstream tasks such as secondary structure and protein interaction interface prediction, compared to no pre-training. However, the larger base model does not outperform the smaller medium. We expect the ProteinGLUE benchmark dataset introduced here, together with the two baseline pre-trained models and their performance evaluations, to be of great value to the field of protein sequence-based property prediction.

Availabilitycode and datasets from https://github.com/ibivu/protein-glue


title: Native SAD Phasing at Room Temperature

abstract: AO_SCPLOWBSTRACTC_SCPLOWSingle-wavelength anomalous diffraction (SAD) is a routine method for overcoming the phase problem when solving a new macromolecular structure. This technique requires the accurate measurement of intensities to sensitively determine differences across Bijvoet pairs, making it a stringent test for the reliability of a data collection method. Although SAD experiments are commonly conducted at cryogenic temperatures to mitigate the effects of radiation damage, such temperatures can alter the conformational ensemble of the protein crystal and may impede the merging of data from multiple crystals due to non-uniform freezing. Here, we propose a data collection strategy to obtain high-quality data from room temperature samples. To illustrate the strengths of this approach we use native SAD phasing at 6.5 keV to solve four structures of three model systems at 295 K. The resulting datasets allow for automatic phasing and model building, and exhibit alternate conformations that are well-supported by the electron density. The high-redundancy data collection method demonstrated here enables the routine collection of high-quality, room-temperature diffraction to improve the study of protein conformational ensembles.


title: Guiding ATR and PARP inhibitor combinations with chemogenomic screens

abstract: Combinations of inhibitors of Ataxia Telangiectasia- and Rad3-related kinase (ATRi) and poly(ADP-ribose) polymerases (PARPi) synergistically kill tumor cells through modulation of complementary DNA repair pathways, but their tolerability is limited by hematological toxicities. To address this we performed a genome-wide CRISPR/Cas9 screen to identify genetic alterations that hypersensitize cells to a combination of the ATRi RP-3500 with PARPi, including deficiency in RNase H2, RAD51 paralog mutations or the Alternative Lengthening of Telomeres telomere maintenance mechanism. We show that RP-3500 and PARPi combinations kill cells carrying these genetic alterations at doses sub-therapeutic as single agents. We also demonstrate the mechanism of combination hypersensitivity in RNase H2-deficient cells, where we observe an irreversible replication catastrophe, allowing us to design a highly efficacious and tolerable in vivo dosing schedule. Altogether, we present a comprehensive dataset to inform development of ATRi and PARPi combinations and an experimental framework applicable to other drug combination strategies.


title: RANK is an independent biomarker of poor prognosis in estrogen receptor-negative breast cancer and a therapeutic target in patient-derived xenografts

abstract: Despite strong preclinical data, the therapeutic benefit of the RANKL inhibitor denosumab in BC patients, beyond its bone-related effects, is unclear. Here, we investigated the prognostic value of RANK expression and its functionality in human BC. We analyzed RANK and RANKL expression in more than 1500 BC cases (777 being estrogen receptor-negative (ER-)) from four independent cohorts. We confirmed that RANK is more frequently expressed in ER- tumors, but it is also found in a subset of ER+ tumors. In ER- BC, RANK expression was independently associated with poor outcome, especially in postmenopausal patients and those who received adjuvant chemotherapy. Gene expression analyses unraveled distinct biology associated with RANK in relation to ER expression and menopause, and evidenced enhanced RANK activation in ER- postmenopausal tumors, together with regulation of metabolic pathways. Functional studies and transcriptomic analyses in ER- RANK+ patients-derived orthoxenografts demonstrated that activation of RANK signaling pathway promotes tumor cell proliferation and stemness, and regulates multiple biological processes including tumor immune surveillance and metabolism. Our results demonstrate that RANK expression is an independent poor prognosis biomarker in postmenopausal ER- BC patients and support the rational of using RANK pathway inhibitors in combination with chemotherapy in ER- BC.


title: An intrinsic endothelial dysfunction causes cerebral small vessel disease

abstract: Small Vessel Disease (SVD) is the leading cause of vascular dementia, causes a quarter of strokes, and worsens stroke outcomes(1, 2). The disease is characterised by cerebral small vessel and white matter pathology, but the underlying mechanisms are poorly understood. Classically, the microvascular and tissue damage has been considered secondary to extrinsic factors, such as hypertension, consisting of microvessel stiffening, impaired vasoreactivity and blood-brain barrier dysfunction identified in human sporadic SVDs. However, increasing evidence points to an underlying vulnerability to SVD-related brain damage, not just extrinsic factors. Here, in a novel normotensive transgenic rat model where the phospholipase flippase Atp11b is deleted, we show pathological, imaging and behavioural changes typical of those in human sporadic SVD, but that occur without hypertension. These changes are due to an intrinsic endothelial cell dysfunction, identified in vessels of the brain white matter and the retina, with pathological evidence of vasoreactivity and blood-brain barrier deficits, which precipitate a secondary maturation block in oligodendroglia and myelin disruption around the small vessels. This highlights that an intrinsic endothelial dysfunction may underlie vulnerability to human sporadic SVD, providing alternative therapeutic targets to prevent a major cause of stroke and dementia.


title: Spike initiation properties in the axon support high-fidelity signal transmission

abstract: The axon initial segment (AIS) converts graded depolarization into all-or-none spikes that are transmitted by the axon to downstream neurons. Analog-to-digital transduction and digital signal transmission call for distinct spike initiation properties (filters) and those filters should, therefore, differ between the AIS and distal axon. Here we show that unlike the AIS, which spikes repetitively during sustained depolarization, the axon spikes transiently and only if depolarization reaches threshold before KV1 channels activate. Rate of depolarization is critical. This was shown by optogenetically evoking spikes in the distal axon of CA1 pyramidal neurons using different photostimulus waveforms and pharmacological conditions while recording antidromically propagated spikes at the soma, thus circumventing the prohibitive difficulty of patching intact axons. Computational modeling shows that KV1 channels in the axon implement a high-pass filter that is matched to the axial current waveform associated with spike propagation, thus maximizing the signal-to-noise ratio to ensure high-fidelity transmission of spike-based signals.