title: The genome of the poecilogonous annelid Streblospio benedicti

abstract: Streblospio benedicti is a common marine annelid that has become an important model for developmental evolution. It is the only known example of poecilogony, where two distinct developmental modes occur within a single species, that is due to a heritable difference in egg size. The dimorphic developmental programs and life-histories exhibited in this species depend on differences within the genome, making it an optimal model for understanding the genomic basis of developmental divergence. Studies using S. benedicti have begun to uncover the genetic and genomic principles that underlie developmental uncoupling, but until now they have been limited by the lack of availability of genomic tools. Here we present an annotated chromosomal-level genome assembly of S. benedicti generated from a combination of Illumina reads, Nanopore long reads, Chicago and Hi-C chromatin interaction sequencing, and a genetic map from experimental crosses. At 701.4 Mb, the S. benedicti genome is the largest annelid genome to date that has been assembled to chromosomal scaffolds, yet it does not show evidence of extensive gene family expansion, but rather longer intergenic regions. The complete genome of S. benedicti is valuable for functional genomic analyses of development and evolution, as well as phylogenetic comparison within the Annelida and the Lophotrochozoa. Despite having two developmental modes, there is no evidence of genome duplication or substantial gene number expansions. Instead, lineage specific repeats account for much of the expansion of this genome compared to other annelids.


title: Ambulacrarian insulin-related peptides and their putative receptors suggest how insulin and similar peptides may have evolved from Insulin-like Growth Factor

abstract:
BackgroundSome Insulin/IGF-related peptides (irps) stimulate a receptor tyrosine kinase (RTK) that transfers the extracellular hormonal signal into an intracellular response. Other irps, such as relaxin, do not use an RTK, but a G-protein coupled receptor (GPCR). This is unusual since evolutionarily related hormones typically either use the same or paralogous receptors. In arthropods three different irps, i.e. arthropod IGF, gonadulin and Drosophila insulin-like peptide 7 (dilp7), likely evolved from a gene triplication, as in several species genes encoding these three peptides are located next to one another on the same chromosomal fragment. These arthropod irps have homologs in vertebrates, which suggests that the initial gene triplication was perhaps already present in the last common ancestor of deuterostomes and protostomes. It would be interesting to know whether this is indeed so and how insulin might be related to this trio of irps.

MethodologyGenes encoding irps as well as their putative receptors were identified in genomes and transcriptomes from echinoderms and hemichordates.

ResultsA similar triplet of genes coding for irps is also found in some ambulacrarians. Two of these are orthologs of arthropod IGF and dilp7 and the third is likely a gonadulin ortholog. In echinoderms two novel irps emerged, gonad stimulating substance (GSS) and multinsulin, likely from gene duplications of the IGF and dilp7-like genes respectively. The structures of GSS diverged considerably from IGF, which would suggest they use different receptors than IGF, but no novel irp receptors evolved. If IGF and GSS use different receptors and the evolution of GSS from a gene duplication of IGF is not associated with the appearance of a novel receptor while irps are known to use two different types of receptors, it seems to suggest that the ancestor of GSS and IGF might have acted on both types of receptors while one or both of its descendants act on only one. There are three ambulacrarian GPCRs that have amino acid sequences suggestive of being irp GPCRs, two of these are orthologs of the gonadulin and dilp7 receptors. This suggests that the third might be an IGF receptor, and that by deduction GSS only acts on the RTK. The evolution of GSS from IGF may represent a pattern, where IGF gene duplications lead to novel genes coding for shorter peptides that activate an RTK. It is likely this is how insulin and the insect neuroendocrine irps evolved independently from IGF.

ConclusionThe local gene triplication described from arthropods that yielded three genes encoding irps was already present in the last common ancestor of protostomes and deuterostomes. It seems plausible that irps, such as those produced by neuroendocrine cells in the brain of insects and echinoderm GSS evolved independently from IGF and thus are not true orthologs, but the result of convergent evolution.

category: zoology


title: Viral neuroinvasion and neurotropism without neuronal damage in the hACE2 mouse model of COVID-19

abstract: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) not only affects the respiratory tract but also causes neurological symptoms such as loss of smell and taste, headache, fatigue or severe cerebrovascular complications. Using transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) we investigated the spatiotemporal distribution and pathomorphological features in the CNS following intranasal infection with SARS-CoV-2 variants, also after prior influenza A virus infection. Apart from Omicron, we found all variants to frequently spread to and within the CNS. Infection was restricted to neurons and appeared to spread from the olfactory bulb mainly in basally orientated regions in the brain and into the spinal cord, independent of ACE2 expression and without evidence of neuronal cell death, axonal damage or demyelination. However, microglial activation, microgliosis and a mild macrophage and T cell dominated inflammatory response was consistently observed, accompanied by apoptotic death of endothelial, microglial and immune cells, without their apparent infection. Microgliosis and immune cell apoptosis indicate a potential role of microglia for pathogenesis and viral effect in COVID-19 and possible impairment of neurological functions, especially in long COVID. These data may also be informative for the selection of therapeutic candidates, and broadly support investigation of agents with adequate penetration into relevant regions of the CNS.

category: pathology


title: Neuroinvasion and neurotropism by SARS-CoV-2 variants in the K18-hACE2 mouse


title: A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model

abstract: TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly generate human monoclonal antibodies. After immunizing these mice against the spike protein of SARS-CoV-2, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralized SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of neutralizing antibodies binds to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2 induced weight loss. Thus, we report two clusters of potent non-competing SARS-CoV-2 neutralizing antibodies providing potential candidates for therapy and prophylaxis of COVID-19. The study further supports the use of transgenic animals with human immunoglobulin gene repertoires in pandemic preparedness initiatives.

category: immunology


title: Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2

abstract:
SARS-CoV-2 infection in immunocompromised individuals is associated with prolonged virus shedding and the evolution of viral variants. Rapamycin and its analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA-approved as mTOR inhibitors in clinical settings such as cancer and autoimmunity. Rapalog use is commonly associated with increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increases susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. By identifying one rapalog (ridaforolimus) that is less active in this regard, we demonstrate that rapalogs promote Spike-mediated entry into cells by triggering the degradation of IFITM2 and IFITM3 via an endolysosomal remodeling program known as microautophagy. Rapalogs that promote virus entry inhibit the mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitates its nuclear translocation and triggers microautophagy. In rodent models of infection, injection of rapamycin prior to and after virus exposure resulted in elevated SARS-CoV-2 replication and exacerbated viral disease, while ridaforolimus had milder effects. Overall, our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating a lysosome-mediated suppression of intrinsic immunity.

SignificanceRapamycin is an immunosuppressant used in humans to treat cancer, autoimmunity, and other disease states. Here, we show that rapamycin and related compounds promote the first step of the SARS-CoV-2 infection cycle--entry into cells--by disarming cell-intrinsic immune defenses. We outline the molecular basis for this effect by identifying a rapamycin derivative that is inactive, laying the foundation for improved mTOR inhibitors that do not suppress intrinsic immunity. We find that rapamycin analogs that promote SARS-CoV-2 entry are those that activate TFEB, a transcription factor that triggers the degradation of antiviral membrane proteins inside of cells. Finally, rapamycin administration to rodents prior to SARS-CoV-2 challenge results in enhanced viral disease, revealing that its use in humans may increase susceptibility to infection.

category: microbiology


title: MVD-Fuse: Detection of White Matter Degeneration via Multi-View Learning of Diffusion Microstructure

abstract: Detecting neuro-degenerative disorders in early-stage and asymptomatic patients is challenging. Diffusion MRI (dMRI) has shown great success in generating biomarkers for cellular organization at the microscale level using complex biophysical models, but there has never been a consensus on a clinically usable standard model. Here, we propose a new framework (MVD-Fuse) to integrate measures of diverse diffusion models to detect alterations of white matter microstructure. The spatial maps generated by each measure are considered as a different diffusion representation (view), the fusion of these views being used to detect differences between clinically distinct groups. We investigate three different strategies for performing intermediate fusion: neural networks (NN), multiple kernel learning (MKL) and multi-view boosting (MVB). As a proof of concept, we applied MVD-Fuse to a classification of premanifest Huntingtons disease (pre-HD) individuals and healthy controls in the TRACK-ON cohort. Our results indicate that the MVD-Fuse boosts predictive power, especially with MKL (0.90 AUC vs 0.85 with the best single diffusion measure). Overall, our results suggest that an improved characterization of pathological brain microstructure can be obtained by combining various measures from multiple diffusion models.

category: neuroscience


title: Glycine attenuates impairments of stimulus-evokedgamma oscillation in the ketaminemodel of schizophrenia

abstract: Although a substantial number of studies suggests some clinical benefit concerning negative symptoms in schizophrenia through the modulation of NMDA-receptor function, none of these approaches achieved clinical approval. Given the large body of evidence concerning glutamatergic dysfunction in a subgroup of patients, biomarkers to identify those with a relevant clinical benefit through glutamatergic modulation are urgently needed. A similar reduction of the early auditory evoked gamma-band response (aeGBR) as found in schizophrenia patients can be observed in healthy subjects in the ketamine-model, which addresses the putative excitation / inhibition (E/I) imbalance of the diseases. Moreover, this change in gamma-band oscillations can be related to the emergence of negative symptoms. Accordingly, this study investigated whether glycine-related increases of the aeGBR accompany an improvement concerning negative symptoms in the ketamine-model. The impact of subanesthetic ketamine doses and the pretreatment with glycine was examined in twenty-four healthy male participants while performing a cognitively demanding aeGBR paradigm with 64-channel electroencephalography. Negative Symptoms were assessed through the Positive and Negative Syndrome Scale (PANSS). Ketamine alone caused a reduction of the aeGBR amplitude associated with more pronounced negative symptoms compared to placebo. Pretreatment with glycine attenuated both, the ketamine-induced alterations of the aeGBR amplitude and the increased PANSS negative scores in glycine-responders, classified based on relative aeGBR increase. Thus, we propose that the aeGBR represents a possible biomarker for negative symptoms in schizophrenia related to insufficient glutamatergic neurotransmission. This would allow to identify patients with negative symptoms, who might benefit from glutamatergic treatment.


title: Interplay between nucleoid-associated proteins and transcription factors in controlling specialized metabolism in Streptomyces

abstract:
Lsr2 is a small nucleoid-associated protein found throughout the actinobacteria. Lsr2 functions similarly to the well-studied H-NS, in that it preferentially binds AT-rich sequences and represses gene expression. In Streptomyces venezuelae, Lsr2 represses the expression of many specialized metabolic clusters, including the chloramphenicol antibiotic biosynthetic gene cluster, and deleting lsr2 leads to significant upregulation of chloramphenicol cluster expression. We show here that Lsr2 likely exerts its repressive effects on the chloramphenicol cluster by polymerizing along the chromosome, and by bridging sites within and adjacent to the chloramphenicol cluster. CmlR is a known activator of the chloramphenicol cluster, but expression of its associated gene is not upregulated in an lsr2 mutant strain. We demonstrate that CmlR is essential for chloramphenicol production, and further reveal that CmlR functions to  counter-silence Lsr2s repressive effects by recruiting RNA polymerase and enhancing transcription, with RNA polymerase effectively clearing bound Lsr2 from the chloramphenicol cluster DNA. Our results provide insight into the interplay between opposing regulatory proteins that govern antibiotic production in S. venezuelae, which could be exploited to maximize the production of bioactive natural products in other systems.

IMPORTANCESpecialized metabolic clusters in Streptomyces are the source of many clinically-prescribed antibiotics. However, many clusters are not expressed in the laboratory due to repression by the nucleoid-associated protein Lsr2. Understanding how Lsr2 represses cluster expression, and how repression can be alleviated, are key to accessing the metabolic potential of these bacteria. Using the chloramphenicol biosynthetic cluster from Streptomyces venezuelae as a model, we explored the mechanistic basis underlying Lsr2-mediated repression, and activation by the pathway-specific regulator CmlR. Lsr2 polymerized along the chromosome and bridged binding sites located within and outside of the cluster, promoting repression. Conversely, CmlR was essential for chloramphenicol production, and further functioned to counter-silence Lsr2 repression by recruiting RNA polymerase and promoting transcription, ultimately removing Lsr2 polymers from the chromosome. Manipulating the activity of both regulators led to >130x increase in chloramphenicol levels, suggesting that combinatorial regulatory strategies can be powerful tools for maximizing natural product yields.


title: Exploration of clinical breakpoint of Danofloxacin for Glaesserella parasuis in plasma and in PELF

abstract:
BackgroundTo establish the clinical breakpoint (CBP) of danofloxacin to G. parasuis, three cutoff values, including epidemiological cutoff value (ECV), pharmacodynamic cutoff value (COPD) and clinical cutoff value (COCL), was obtained in the present study.

MethodsThe ECV was calculated using ECOFFinder base on MIC distribution of 347 G. parasuis collected from disease pigs. The COPD was established base on in vivo and ex vivo pharmacokinetic (PK) - pharmacodynamic (PD) modeling of danofloxacin both in plasma and pulmonary epithelial lining fluid (PELF) using Hill formula and Monte Carlo analysis. The COCL was established based on the relationship between possibility of cure (POC) and MIC in the clinical trials using "WindoW" approach, nonlinear regression and CART analysis.

ResultsThe MIC50 and MIC90 of danofloxacin against 347 G. parasuis were 2 g/mL and 8 g/mL, respectively. The ECV value was set up as 8 g/mL using ECOFFinder. Concentration-time curve of danofloxacin indicated a two-compartment model for PK analysis. The PK parameters of the maximum concentration (Cmax) and area under concentration-time curve (AUC) in PELF were 3.67 {+/-} 0.25 g/mL and 24.28 {+/-} 2.70 h{middle dot}g/mL, higher than those in plasma (0.67 {+/-} 0.01g/mL and 4.47 {+/-} 0.51 h{middle dot}g/mL). The peak time (Tmax) in plasma was 0.23 {+/-} 0.07 h, shorter than that in PELF (1.61 {+/-} 0.15 h). The COPD in plasma and PELF were 0.125 g/mL and 0.5 g/mL, respectively. The COCL calculated by WindoW approach, nonlinear regression and CART analysis were 0.125[~]4 g/mL, 0.428 g/mL and 0.56 g/mL, respectively. The 0.5 g/mL was selected as eligible COCL. The ECV is much higher than the COPD and COCL, and the clinical breakpoint based on data in plasma was large different with that of in PELF.

ConclusionsOur study firstly established three cutoff values of danofloxacin against G. parasuis. It suggested that epidemiological danofloxacin-resistant G. parasuis may lead to the ineffective treatment by danofloxacin.

ImportanceG. parasuis, a gram-negative respiratory pathogen, can colonize in the upper respiratory tract in swine and cause Glassers disease. As the abuse of antibiotics, antimicrobial resistant G. parasuis emerged in different degrees, which brought serious threat to global economy and public health. Danofloxacin in quinolones are one of the best choices for treatment of G. parasuis infection, because of their strong bactericidal activity and good absorption into blood and great distribution in the lung. However, the clinical breakpoint (CBP) for danofloxacin against G. parasuis had not yet been established by clinical laboratory of standard Institute (CLSI) and European Commission of antimicrobial susceptibility testing (EUCAST). Our study firstly established three cutoff values of danofloxacin against G. parasuis. It suggested that epidemiological danofloxacin-resistant G. parasuis may lead to the ineffective treatment by danofloxacin.


title: Defective satellite DNA clustering into chromocenters underlies hybrid incompatibility in Drosophila

abstract: Although rapid evolution of pericentromeric satellite DNA repeats is theorized to promote hybrid incompatibility (HI)(1-4), how divergent repeats affect hybrid cells remains poorly understood. Recently, we demonstrated that sequence-specific DNA-binding proteins cluster satellite DNA from multiple chromosomes into  chromocenters, thereby bundling chromosomes to maintain the entire genome in a single nucleus(5, 6). Here we show that ineffective clustering of divergent satellite DNA in the cells of Drosophila hybrids results in chromocenter disruption, associated micronuclei formation and tissue atrophy. We further demonstrate that previously identified HI factors trigger chromocenter disruption and micronuclei in hybrids, linking their function to a conserved cellular process. Together, we propose a unifying framework that explains how the widely observed satellite DNA divergence between closely related species can cause reproductive isolation.

category: cell biology


title: The Expression of Angiopoietin-1 and -2 in the Osteogenesis of Mesenchymal Stem Cells

abstract:
ObjectiveThe objectives of this study are to clarify whether rat bone marrow derived Mesenchymal stem cells (MSCs) express Ang1 and Ang2 and their expression in the process of osteogenesis in vitro.

Material and MethodsMSCs were cultured from rat tibia bone marrow cells and the hemopoietic stem cells were deplete by consistently replacement of the culture medium. The MSCs were induced osteogenesis with mineralization conditional medium and Immunohistochemical and immunofluorescent staining were performed to assess the expression of Ang1 and Ang2.

ResultsThe method used to expand rat MSCs in vitro was applicable, and the cell morphology is spindle-like shape that is consistent with the privous reports. The immunohistochemical staining results showed that both Ang1 and Ang2 were expressed by rat MSCs. Both Ang1 and Ang2 were up-regulated in the process of osteogenesis of rat MSCs.

ConclusionRat MSCs express both Ang1 and Ang2 which might play critical roles in the osteogenesis in vitro.


title: Stronger net selection on males across animals

abstract:
Sexual selection is considered the major driver for the evolution of manifold sex differences. However, the eco-evolutionary dynamics of sexual selection and their role for a populations adaptive potential to respond to environmental change have only recently been explored. Theory predicts that sexual selection promotes adaptation at a low demographic cost only if net selection is stronger on males compared to females. We used a comparative approach to show that net selection is indeed stronger in males in species prone to intense sexual selection. Given that both sexes share the vast majority of their genes, our findings corroborate the notion that the genome is often confronted with a more stressful environment when expressed in males. Collectively, our study supports a long-standing key assumption required for sexual selection to bolster adaptation, and intense sexual selection may therefore enable some species to track environmental change more efficiently.

One sentence summaryComparative study finds support for stronger net selection in males.

category: evolutionary biology


title: Validation and Application of a Protocol for the Extraction and Quantitative Analysis of Sphingomyelin in Erythrocyte Membranes of Patients with NAFLD

abstract: A set of constituents of the erythrocyte membrane lipidome has been proposed to serve as biomarkers for liver disease and acute coronary syndrome. In erythrocytes, sphingomyelin hydrolysis provides ceramide, a signaling lipid necessary for phosphatidylserine exposure and eryptosis. Phosphatidylserine exposure further amplifies hepatic inflammation and fibrosis during non-alcoholic fatty liver disease (NAFLD). In this study, we developed and applied a quantitative TLC for erythrocyte membrane sphingomyelin of NAFLD patients. We also compared 10 extraction methods for the isolation of sphingomyelin from erythrocytes. For quantitative TLC, lipids were separated in Silica gel 60 F254 using a mixture of chloroform/methanol/acetic acid/water (60/50/1/4) (v/v/v/v). The separated lipids were stained in a chamber containing iodine, and the intensity of each of the primary colors (red, green, blue) and the sum of the Red plus Green colors (R+G) was analyzed. The method was linear over a wide range of concentrations, presented acceptable precision (inter-day CV(%) 0.34, 0.006 and 0.44 for 2.5, 5.0 and 10 g, respectively), good accuracy (recovery range 85.2-97.1%), and excellent limit of detection (0.137 g/spot) and limit of quantification (0.41 g/spot). Using this quantitation method, we compared various lipid extraction methods and found that lipid extraction with methanol led to higher yield of erythrocyte sphingomyelin (135.35{+/-}1.04% recovery, compared to the Folch method). Application of these methods showed that erythrocytes from NAFLD patients (9 men, 15 women, 57.95{+/-}11.11 years old) contained statistically significantly less sphingomyelin (829.82{+/-}511.60 vs 1892.08{+/-}606.25 g/ml of packed erythrocytes) compared to healthy controls (4 men, 6 women, 39.3{+/-}15.55 years old).

category: biochemistry


title: Decoding microbiome and protein family linkage to improve protein structure prediction

abstract: Information extracted from microbiome sequences through deep-learning techniques can significantly improve protein structure and function modeling. However, the model training and metagenome search were largely blind with low efficiency. Built on 4.25 billion microbiome sequences from four major biomes (Gut, Lake, Soil and Fermentor), we proposed a MetaSource model to decode the inherent link of microbial niches with protein homologous families. Large-scale protein family folding experiments showed that a targeted approach using predicted biomes significantly outperform combined metagenome datasets in both speed of MSA collection and accuracy of deep-learning structure assembly. These results revealed the important link of biomes with protein families and provided a useful bluebook to guide future microbiome sequence database and modeling development for protein structure and function prediction.

category: bioinformatics


title: Separation of cohorts on the basis of bacterial type IV conjugation systems identified from metagenomic assemblies

abstract:
Conjugation enables the exchange of genetic elements throughout environments, including the human gut microbiome. Conjugative elements can carry and transfer clinically relevant metabolic pathways which makes precise identification of these systems in metagenomic samples clinically important. Here, we outline two distinct methods to identify conjugative systems in the human gut microbiome. We first show that conjugative systems exhibit strong population and age-level stratification. Additionally, we find that the total relative abundance of all conjugative systems present in a sample is not an informative metric to use, regardless of the method of identifying the systems. Finally, we demonstrate that the majority of assembled conjugative systems are not included within metagenomic bins, and that only a small proportion of the binned conjugative systems are included in "high-quality" metagenomic bins. Our findings highlight that conjugative systems differ between general North Americans and a cohort of North American pre-term infants, revealing a potential use as an age-related biomarker. Furthermore, conjugative systems can distinguish between other geographical-based cohorts. Our findings emphasize the need to identify and analyze conjugative systems outside of standard metagenomic binning pipelines.

ImportanceThe human gut microbiome is increasingly being associated with human health outcomes through shotgun metagenomic sequencing. The usual approach of metagenomic-level analyses is to bin assembled sequences into approximations of bacterial genomes and perform further investigations on the resultant bins. Here, we show that type IV conjugative systems differ between age and geographically-based cohorts and that these systems are systematically excluded by binning algorithms. We suggest that analysis of type IV conjugative systems should be added to the current metagenomic analysis approaches as they contain much information that could explain differences between cohorts beyond those we investigated.


title: Cholesterol promotes both head group visibility and clustering of PI(4,5)P2 driving unconventional secretion of Fibroblast Growth Factor 2

abstract: Fibroblast Growth Factor 2 (FGF2) is a cell survival factor involved in tumor-induced angiogenesis. FGF2 is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here we demonstrate that both PI(4,5)P2-dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further reveal cholesterol to enhance FGF2 binding to PI(4,5)P2-containing lipid bilayers in a fully reconstituted system. Based on extensive atomistic molecular dynamics simulations and membrane tension experiments, we propose cholesterol to modulate FGF2 binding to PI(4,5)P2 by (i) increasing head group visibility of PI(4,5)P2 on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P2 molecules triggering FGF2 oligomerization and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 towards the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2.

category: biophysics


title: Age-dependent decrease of hepatic geranylgeranoic acid and its oral supplementation prevents spontaneous hepatoma in C3H/HeN mice

abstract:
AimGeranylgeranoic acid (GGA) has been developed as a preventive agent against second primary hepatoma. Recently, GGA was reported to induce cell death in human hepatoma cells via TLR4-mediated pyroptosis. We have reported that GGA is enzymatically biosynthesized from mevalonic acid in human hepatoma-derived cells and that endogenous GGA is found in most organs of rats. In addition, we found that upregulation of endogenous GGA levels by zaragozic acid A (ZAA) induced cell death in human hepatoma-derived cells. Therefore, we investigated the age-related changes of hepatic GGA and the possibility of suppressing hepatocarcinogenesis by GGA supplementation using male C3H/HeN mice that spontaneously develop hepatoma.

MethodsWe measured endogenous GGA and mRNA of MAOB, a key enzyme of GGA biosynthesis, in the liver of male C3H/HeN mice aged 6-93 weeks. We also tried suppressing spontaneous hepatocarcinogenesis by a single administration of GGA to C3H/HeN mice.

ResultsHepatic GGA content and Maob mRNA expression level age-dependently decreased in male C3H/HeN mice, some of which produced spontaneous hepatoma in 2 years. A single oral administration of GGA at 11 months of age significantly prevented hepatoma in terms of the number and weight of tumors per mouse at 23 months. Oral supplementation with GGA or geranylgeraniol significantly increased endogenous hepatic GGA contents dose-dependently, and ZAA dramatically upregulated hepatic GGA.

ConclusionIn this study, we found an age-dependent decrease in hepatic endogenous GGA in male C3H/HeN mice and efficient prevention of spontaneous hepatoma by a single administration of GGA at 11 months of age.

category: cancer biology


title: The bundle sheath of rice is conditioned to play an active role in water transport as well as sulfur assimilation and jasmonic acid synthesis

abstract:
Leaves comprise multiple cell types but our knowledge of the patterns of gene expression that underpin their functional specialization is fragmentary. Our understanding and ability to undertake rational redesign of these cells is therefore limited. We aimed to identify genes associated with the incompletely understood bundle sheath of C3 plants, which represents a key target associated with engineering traits such as C4 photosynthesis into rice. To better understand veins, bundle sheath and mesophyll cells of rice we used laser capture microdissection followed by deep sequencing. Gene expression of the mesophyll is conditioned to allow coenzyme metabolism and redox homeostasis as well as photosynthesis. In contrast, the bundle sheath is specialized in water transport, sulphur assimilation and jasmonic acid biosynthesis. Despite the small chloroplast compartment of bundle sheath cells, substantial photosynthesis gene expression was detected. These patterns of gene expression were not associated with presence/absence of particular transcription factors in each cell type, but rather gradients in expression across the leaf. Comparative analysis with C3 Arabidopsis identified a small gene-set preferentially expressed in bundle sheath cells of both species. This included genes encoding transcription factors from fourteen orthogroups, and proteins allowing water transport, sulphate assimilation and jasmonic acid synthesis. The most parsimonious explanation for our findings is that bundle sheath cells from the last common ancestor of rice and Arabidopsis was specialized in this manner, and since the species diverged these patterns of gene expression have been maintained.

Significance statementThe role of bundle sheath cells in C4 species have been studied intensively but this is not the case in leaves that use the ancestral C3 pathway. Here, we show that gene expression in the bundle sheath of rice is specialized to allow sulphate and nitrate reduction, water transport and jasmonate synthesis, and comparative analysis with Arabidopsis indicates ancient roles for bundle sheath cells in water transport, sulphur and jasmonate synthesis.

category: plant biology


title: Major effect loci for plant size before onset of nitrogen fixation allow accurate prediction of yield in white clover

abstract: White clover is an agriculturally important forage legume grown throughout temperate regions as a mixed clover-grass crop. It is typically cultivated with low nitrogen input, making yield dependent on nitrogen fixation by rhizobia in root nodules. Here, we investigate the effects of clover and rhizobium genetic variation by monitoring plant growth and quantifying dry matter yield of 704 combinations of 145 clover and 169 rhizobium genotypes. We find no significant effect of rhizobium variation. In contrast, we can predict yield based on a few white clover markers strongly associated with plant size prior to nitrogen fixation, and the prediction accuracy for polycross offspring yield is remarkably high. Several of the markers are located near a homolog of Arabidopsis thaliana GIGANTUS 1, which regulates growth rate and biomass accumulation. Our work provides fundamental insight into the genetics of white clover yield and identifies specific candidate genes as breeding targets.


title: TDP-43 regulates GAD1 mRNA splicing and GABA signaling in Drosophila brains

abstract: Alterations in the function of the RNA-binding protein TDP-43 is largely associated with the pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease of the human motor system that leads to motoneurons degeneration and reduced life expectancy by molecular mechanisms not well known. Regarding to that, we found that the expression levels of the glutamic acid decarboxylase enzyme (GAD1), responsible to convert glutamate to {gamma}-aminobutyric acid (GABA), were downregulated in TBPH-null flies and motoneurons derived from ALS patients carrying mutations in TDP-43 suggesting that defects in the regulation of GAD1 may lead to neurodegeneration by affecting neurotransmitter balance. In this study, we observed that TBPH was required to regulate GAD1 pre-mRNA splicing and GABA levels in Drosophila brains. Interestingly, we discovered that pharmacological treatments aimed to modulate GABA neurotransmission were able to revert locomotion deficiencies in TBPH-minus flies revealing novel mechanisms and therapeutic strategies in ALS.


title: Causal roles of prefrontal cortex during spontaneous perceptual switching are determined by brain state dynamics

abstract:
The prefrontal cortex (PFC) is thought to orchestrate cognitive dynamics. However, in tests of bistable visual perception, no direct evidence supporting such presumable causal roles of the PFC has been reported. Here, using a novel brain-state-dependent neural stimulation system, we found that three PFC regions--right frontal eye fields and anterior/posterior dorsolateral PFCs (a/pDLPFCs)--have causal effects on perceptual dynamics but the behavioural effects are detectable only when we modulated the PFC activity in brain-state-/state-history-dependent manners. Also, we revealed that the brain-dynamics-dependent behavioural causality is underpinned by transient changes in the brain state dynamics, and such neural changes are determined by structural transformations of hypothetical energy landscapes. Moreover, we identified different functions of the three PFC areas: in particular, we found that aDLPFC enhances the integration of the two PFC-active brain states, whereas pDLPFC promotes the diversity between them. This work resolves the controversy over the PFC roles in spontaneous perceptual switching and underlines brain state dynamics in fine investigations of brain-behaviour causality.

Impact statementPrefrontal causal roles are changing during bistable visual perception, which was determined by large-scale brain state dynamics and attributable to hypothetical energy landscapes that underpin the brain state dynamics.


title: Neuroprotection in early stages of Alzheimers Disease is promoted by Transthyretin angiogenic properties

abstract: While still controversial, it has been demonstrated that vascular defects can precede the onset of the other AD hallmarks features, making it an important therapeutic target. Given that the protein transthyretin (TTR) has been established as neuroprotective in AD, here we investigated the influence of TTR in the vasculature. AD transgenic mice with TTR genetic reduction, AD/TTR+/-, exhibited a thicker BM in brain microvessels and decreased vessel length than animals with normal TTR levels, AD/TTR+/+. Further in vivo investigation, using the chick chorioallantoic membrane (CAM) assay, revealed that TTR is a pro-angiogenic molecule. Also, TTR increased the expression of key angiogenic molecules, by endothelial cells under tube formation conditions. We showed that TTR reduction leads to a thicker BM in AD mice than in NT animals, strengthening the idea that TTR is a neuroprotective protein. We also studied the effect of TTR tetrameric stabilization on BM thickness, showing that AD mice treated with iododiflunisal (IDIF) displayed a significant reduction of BM thickness and increased vessel length when compared to non-treated littermates. Our in vivo results show the involvement of TTR in angiogenesis, particularly as a modulator of vascular alterations occurring in AD. Since TTR is decreased early in AD, its tetrameric stabilization can represent a therapeutic avenue for the early treatment of AD through the maintenance of the vascular structure.

category: molecular biology


title: Chronic toxicity of three neonicotinoid insecticides and their mixture on two daphniid species: Daphnia magna and Ceriodaphnia dubia

abstract:
Neonicotinoid insecticides represent nearly a quarter of the global market and are widely used in agriculture but also for lawn, garden care, and pest control. They are highly water-soluble, persistent in soil, and may enter the aquatic compartment via spray drift, runoff, or leaching, and contribute to downstream aquatic toxicity. Although insects appear to be the most sensitive group to neonicotinoids, other groups, such as crustaceans and birds, may also be affected. Furthermore, most studies focus on single-insecticide exposure and very little is known concerning the impact of neonicotinoid mixtures on aquatic invertebrates. The present study was designed to test potential toxicological effects of an environmentally relevant mixture of imidacloprid, clothianidin, and thiamethoxam on populations of Ceriodaphnia dubia and Daphnia magna under controlled conditions. Chronic toxicity tests were conducted in the laboratory, and survival and reproduction were measured for both species under exposure to nominal concentrations of imidacloprid (0.256 {micro}g/L), clothianidin (3.11 {micro}g/L), thiamethoxam (1.49 {micro}g/L), and a mixture of the three compounds at the same concentrations of the individual compounds. The neonicotinoids did not affect the survival of C. dubia and D. magna founders. Reproduction of C. dubia was affected only by the mixture. All three individual insecticides as well as the mixture caused a significant reduction in the reproduction of D. magna. Our results highlight the complexity of pesticide toxicity and show that traditional toxicological approaches such as acute mortality studies, especially tests with single compounds, can underestimate negative impacts that occur in the environment.

HighlightsO_LINeonicotinoids are currently the most frequently used insecticides worldwide.
C_LIO_LIAn environmentally relevant mixture of three neonicotinoids was evaluated on two daphniid species.
C_LIO_LIThe mixture negatively affected the reproduction of C. dubia and Daphnia magna.
C_LIO_LITraditional toxicological approaches with single compounds may underestimate the effects occurring in the environment at low concentrations.
C_LI

category: ecology


title: How the Zebra got its Rump Stripes: Salience at Distance and in Motion

abstract:
Zebras stripes cannot protect them from predators, Darwin concluded, and current consensus tends to support his view1,2. In principle, stripes could support crypsis or aposematism, could dazzle, confuse or disrupt predators perception3-8, yet no such effects are manifest in predator-prey interactions9-11. Instead, narrow stripes covering zebras head, neck, limbs and flanks are an effective deterrent to tabanids12, vectors for equine disease13,14. Accordingly, while other potential benefits, e.g., thermoregulation15,16 and intraspecific communication17, cannot be excluded, zebra stripes likely evolved primarily to deter parasites18-20. Rump stripes, however, do not fit this, or any extant view. Typically horizontal and broader in sub-species with width variation, they are ill-suited to crypsis or parasite-deterrence12 and vary with hyaena threat18, perhaps shaped by an additional selective pressure. We observed that rump (and rear-flank) stripes remain highly conspicuous when viewed in motion or at distance, while other stripes do not. To study this striking effect, we filtered images of zebra to simulate acuity limitations in lion and hyaena photopic and mesopic vision. For mountain zebra and plains zebra without shadow striping, rump stripes were the most conspicuous image regions according to computational salience models, corroborated by human observers judgements of maximally attention-capturing image locations, which were strongly biased toward the rear. By hijacking exogenous attention mechanisms to force predator attention to the rear, salient rump stripes confer benefits to zebra, estimated here in pursuit simulations. Benefits of rump stripe salience may counteract anti-parasite benefits and costs of conspicuity to shape rump and shadow stripe variation.

1 HighlightsO_LIZebra stripes likely evolved to deter biting flies, but rump stripes are ill-suited to this.
C_LIO_LIRump-stripes remain highly conspicuous when viewed at distance or in motion.
C_LIO_LIComputational models and human observers judge rump stripes are most salient stripes.
C_LIO_LISalient rump stripes drive predator attention to rear, hindering capture by predators.
C_LIO_LIObserve this striking effect in moving zebra at: viscog.psychol.cam.ac.uk/resources-and-downloads
C_LI


title: Oxford Nanopore and Bionano Genomics technologies evaluation for plant structural variation detection.

abstract:
BackgroundStructural Variations (SVs) are very diverse genomic rearrangements. In the past, their detection was restricted to cytological approaches, then to NGS read size and partitionned assemblies. Due to the current capabilities of technologies such as long read sequencing and optical mapping, larger SVs detection are becoming more and more accessible.

This study proposes a comparison in SVs detection and characterization from long-read sequencing obtained with the MinION device developed by Oxford Nanopore Technologies and from optical mapping produced by the Saphyr device commercialized by Bionano Genomics. The genomes of the two Arabidopsis thaliana ecotypes Columbia-0 (Col-0) and Landsberg erecta 1 (Ler-1) were chosen to guide the use of one or the other technology.

ResultsWe described the SVs detected from the alignment of the best ONT assembly and DLE-1 optical maps of A. thaliana Ler-1 on the public reference Col-0 TAIR10.1. After filtering, 1 184 and 591 Ler-1 SVs were retained from ONT and BioNano technologies respectively. A total of 948 Ler-1 ONT SVs (80.1%) corresponded to 563 Bionano SVs (95.3%) leading to 563 common locations in both technologies. The specific locations were scrutinized to assess improvement in SV detection by either technology. The ONT SVs were mostly detected near TE and gene features, and resistance genes seemed particularly impacted.

ConclusionsStructural variations linked to ONT sequencing error were removed and false positives limited, with high quality Bionano SVs being conserved. When compared with the Col-0 TAIR10.1 reference, most of detected SVs were found in same locations. ONT assembly sequence leads to more specific SVs than Bionano one, the later being more efficient to characterize large SVs. Even if both technologies are obvious complementary approaches, ONT data appears to be more adapted to large scale populations study, while Bionano performs better in improving assembly and describing specificity of a genome compared to a reference.

category: genomics


title: Bioelectricity generation from live marine photosynthetic macroalgae

abstract: Conversion of solar energy into electrical current by photosynthetic organisms has the potential to produce clean energy. Previously reported bio-photoelectrochemical cells (BPECs) have utilized unicellular photosynthetic microorganisms. In this study, we describe for the first time BPECs that utilize intact live marine macroalgae (seaweeds) in natural seawater or saline buffer or natural seawater. The BPECs produce electrical currents from of >50mA/cm2, from both light-dependent (photosynthesis) and light independent processes. These values are significantly greater than the current densities that have been reported for single-cell microorganisms. The photocurrent is inhibited by the Photosystem II inhibitor DCMU, indicating that the source of light-driven electrons is from water oxidation via NADPH and other reduced molecules. We show here that intact seaweed cultures can be used in a large-scale BPEC containing seawater that produces bias-free photocurrent. The ability to produce bioelectricity from intact seaweeds may pave the way to future development of a low-cost energy technology using BPECs.

category: bioengineering


title: Florigen revisited: proteins of the FT/CETS/PEBP/PKIP/YbhB family may be the enzymes of small molecule metabolism

abstract: Flowering signals are sensed in plant leaves and transmitted to the shoot apical meristems, where the formation of flowers is initiated. Searches for a diffusible hormone-like signaling entity ("florigen") went on for many decades, until in the 1990s a product of plant gene FT was identified as the key component of florigen, based on genetic evidence and protein localization studies. Sequence homologs of FT protein are found throughout prokaryotes and eukaryotes; some eukaryotic family members appear to bind phospholipids or interact with the components of the signal transduction cascades. We studied molecular features of the FT homologs in prokaryotes and analyzed their genome context, to find tentative evidence connecting the bacterial family members with small molecule metabolism, often involving sugar- or ribonucleoside-containing substrates. Most FT homologs share a constellation of five charged residues, three of which, i.e., two histidines and an aspartic acid, circumfere the rim of a well-defined cavity on the protein surface. We argue that this conserved feature is more likely to be an enzymatic active center than a catalytically inactive ligand-binding site. We propose that most of FT-related proteins are enzymes operating on small diffusible molecules, which may constitute an overlooked essential ingredient of the florigen signal.


title: Effect of RNA on morphology and dynamics of membraneless organelles.

abstract: Membraneless organelles (MLOs) are spatiotemporally regulated structures that concentrate multi-valent proteins or RNA, often in response to stress. The proteins enriched within MLOs are often classified as high-valency "scaffolds" or low valency "clients", with the former being associated with a phase-separation promoting role. In this study, we employ a minimal model for P-body components, with a defined protein-protein interaction network, to study their phase-separation at biologically realistic low protein concentrations. Without RNA multivalent proteins can assemble into solid-like clusters only in the regime of high concentration and stable interactions. RNA molecules promote cluster formation in an RNA-length dependent manner, even in the regime of weak interactions and low protein volume fraction. Our simulations reveal that long RNA chains act as super-scaffolds that stabilize large RNA-protein clusters by recruiting low-valency proteins within them while also ensuring functional "liquid-like" turnover of components. Our results suggest that RNA-mediated phase separation could be a plausible mechanism for spatiotemporally regulated phase-separation in the cell.


title: Whole Chloroplast Genomes reveals the uniqueness of Bolivian native cacao (Theobroma cacao) from the northern part of Bolivia

abstract: We report the complete chloroplast sequences of two varieties of Theobroma cacao collected in the Bolivian Amazonia using Next-Generation Sequencing. Comparisons made between these two chloroplast genomes and the Belizean reference plastid genome identified 19 and 22 nucleotide variants. The phylogenetic analysis reported three main T. cacao clades belonging to the Forastero, Criollo and Trinitario groups. The Bolivian Native Cacao varieties were located inside the Trinitario group forming their unique branch. The Bolivian Native Cacao branch reveals a possible new subpopulation different from the well-characterized T. cacao subpopulations. The phylogenetic trees showed that the relationships among the T. cacao varieties were consistent with their geographical locations placing the Cacao Center of Origin in Western Amazon. The data presented here will contribute to the usage of ultrabarcoding to distinguish different T. cacao varieties and to identify native cacaos from introduced cacaos. Thus helping in the conservation of local native varieties of T. cacao.


title: Controlling gene expression in mammalian cells using multiplexed conditional guide RNAs for Cas12a

abstract: Spatiotemporal control of the activity of Cas proteins is of considerable interest for both basic research and therapeutics. Only few mechanisms have been demonstrated for regulating the activity of guide RNAs (gRNAs) for Cas12a in mammalian cells, however, and combining and compactly integrating multiple control instances on single transcripts has not been possible so far. Here, we show that conditional processing of the 3 tail is a viable general approach towards switchable Pol II-transcribed Cas12a gRNAs that can activate gene expression in mammalian cells in an input-dependent manner. Processing of the 3 tail can be achieved using microRNA and short hairpin RNA as inputs, via a guanine-responsive ribozyme, and also using an RNA strand displacement mechanism. We further show that Cas12a along with several independently switchable gRNAs can be integrated on a single transcript using stabilizing RNA triplexes, providing a route towards compact Cas12a-based gene regulation constructs with multi-input switching capabilities.

category: synthetic biology


title: Statistical learning of frequent distractor locations in visual search involves regional signal suppression in early visual cortex

abstract: Observers can learn the locations where salient distractors appear frequently to reduce potential interference - an effect attributed to better suppression of distractors at frequent locations. But how distractor suppression is implemented in the visual cortex and frontoparietal attention networks remains unclear. We used fMRI and a regional distractor-location learning paradigm (Sauter et al. 2018, 2020) with two types of distractors defined in either the same (orientation) or a different (colour) dimension to the target to investigate this issue. fMRI results showed that BOLD signals in early visual cortex were significantly reduced for distractors (as well as targets) occurring at the frequent versus rare locations, mirroring behavioural patterns. This reduction was more robust with same-dimension distractors. Crucially, behavioural interference was correlated with distractor-evoked visual activity only for same- (but not different-) dimension distractors. Moreover, with different- (but not same-) dimension distractors, a colour-processing area within the fusiform gyrus was activated more when a colour distractor was present versus absent and with a distractor occurring at a rare versus frequent location. These results support statistical learning of frequent distractor locations involving regional suppression in the early visual cortex and point to differential neural mechanisms of distractor handling with different-versus same-dimension distractors.


title: Adaptive subsets limit the anti-tumoral NK-cell activity in Hepatocellular Carcinoma

abstract:
Background and AimsHepatocellular carcinoma (HCC) is a global health burden with increasing incidence, poor prognosis and limited therapeutic options. Natural killer (NK) cells exhibit potent anti-tumoral activity and therefore represent potential targets for immunotherapeutic approaches in HCC treatment. However, the human NK-cell repertoire is highly diverse including conventional and adaptive NK cells that differ in phenotype and effector function. Adaptive NK-cell frequencies are increased in association with HCMV (human cytomegalovirus) seropositivity that is also common in HCC patients. In this study, we aimed to gain a better understanding of the NK-cell repertoire and the associated anti-tumoral activity in HCC patients.

MethodsIn-depth phenotypic and functional flow-cytometry analyses of the HCMV-associated NK cell-repertoire obtained from 57 HCC patients, 33 liver cirrhosis patients and 36 healthy donors (HD).

ResultsFirst, adaptive subsets are present in all three cohorts with conserved characteristics in patients with liver cirrhosis and HCC. Second, adaptive NK cells can be isolated from HCC tissue however lack features of tissue-residency and thus probably represent circulating/infiltrating lymphocytes. Third, the anti-tumoral activity by adaptive NK cells is reduced compared to conventional NK-cell subsets, also in HCC. Lastly, frequencies of adaptive NK cells were increased in patients suffering from Hepatitis B virus-associated HCC providing a link between etiology and the NK-cell repertoire in HCC.

ConclusionAdaptive NK cells limit the anti-tumoral activitity of NK cells in HCC, especially in association with HBV infection that is accompanied by an expansion of this NK cell subset.

Lay summaryIn patients with liver cancer (HCC), a subset of natural killer cells, so called adaptive NK cells show a diminished anti-tumoral activity compared to other, called conventional NK cells. Adaptive NK cells are expanded in patients with HCC associated to Hepatitis B virus infection. Thus, presence of adaptive NK cells contributes to the immune escape in HCC.


title: Assessing the efficacy of antibiotic treatment for the creation of axenic earthworms

abstract: Earthworms are an integral part of soil ecosystems, especially for their role in soil functions such as organic matter (OM) decomposition and nutrient cycling. Earthworms and microorganisms are interdependent, and a considerable portion of the contribution earthworms make to influencing OM fate is through interactions with microorganisms. However, the importance of the earthworm-associated microbiome is not fully understood, because it is difficult to separate the direct influence of the earthworms from the indirect influence of their microbiome. Here, we evaluated an antibiotic-based procedure for producing axenic ecologically-contrasting earthworm species (E. fetida, L. terrestris, A. chlorotica) as the first step towards soil studies aimed at understanding the importance of the earthworm microbiome for host health and function. Individual earthworms were exposed to antibiotics: cycloheximide (150 g ml-1), ampicillin (100 g ml-1), ciprofloxacin (50 g ml-1), nalidixic acid (50 g ml-1), and gentamicin (50 g ml-1) either singly or in a cocktail via culture (96 h) in a semi-solid agar carrier. Compared to the non-antibiotic treated control, the cocktail (for all three species) and ciprofloxacin (for E. fetida and A. chlorotica) treatments significantly reduced (P<0.05) culturable microbial abundance on nutrient agar and potato dextrose agar. The microbial counts were reduced to below detection (<50 CFU individual-1) for E. fetida and A. chlorotica receiving the cocktail. Illumina 16S rDNA amplicon sequence analysis of culturable L. terrestris -associated bacteria showed that antibiotic treatment influenced community composition revealing putative sensitive (Comomonas, Kosakonia and Sphingobacterium) and insensitive (Aeromonas, Pseudochrobactrum) taxa. Overall, we report a rapid, with minimal earthworm-handling, process of creating  axenic E. fetida and A. chlorotica individuals or L. terrestris with a suppressed microbiome as a tool to be used in future ecological studies of earthworm microbial interactions affecting host health and function.


title: Assessment of the Viscoelastic Mechanical Properties of the Porcine Optic Nerve Head using Micromechanical Testing and Finite Element Modeling

abstract: Optic nerve head (ONH) biomechanics is centrally involved in the pathogenesis of glaucoma, a blinding ocular condition often characterized by elevation and fluctuation of the intraocular pressure and resulting loads on the ONH. Further, tissue viscoelasticity is expected to strongly influence the mechanical response of the ONH to mechanical loading, yet the viscoelastic mechanical properties of the ONH remain unknown. To determine these properties, we conducted micromechanical testing on porcine ONH tissue samples, coupled with finite element modeling based on a mixture model consisting of a biphasic material with a viscoelastic solid matrix. Our results provide a detailed description of the viscoelastic properties of the porcine ONH at each of its four anatomical quadrants (i.e., nasal, superior, temporal, and inferior). We showed that the ONHs viscoelastic mechanical response can be explained by a dual mechanism of fluid flow and solid matrix viscoelasticity, as is common in other soft tissues. We obtained porcine ONH properties as follows: matrix Youngs modulus E=1.895 [1.056,2 .391] kPa (median [min., max.]), Poissons ratio{nu} =0.142 [0.060,0 .312], kinetic time-constant{tau} =214 [89,921] sec, and hydraulic permeability k=3.854 x 10-1 [3.457 x 10-2,9.994 x 10-1] mm4/(N sec). These values can be used to design and fabricate physiologically appropriate ex vivo test environments (e.g., 3D cell culture) to further understand glaucoma pathophysiology.


title: Photon-free (s)CMOS camera characterization for artifact reduction in high- and super-resolution microscopy

abstract: Modern implementations of widefield fluorescence microscopy often rely on sCMOS cameras, but this camera architecture inherently features pixel-to-pixel variations. Such variations lead to image artifacts and render quantitative image interpretation difficult. Although a variety of algorithmic corrections exists, they require a thorough characterization of the camera, which typically is not easy to access or perform. Here, we developed a fully automated pipeline for camera characterization based solely on thermally generated signal, and implemented it in the popular open-source software Micro-Manager and ImageJ/Fiji. Besides supplying the conventional camera maps of noise, offset and gain, our pipeline also gives access to dark current and thermal noise as functions of the exposure time. This allowed us to avoid structural bias in single-molecule localization microscopy (SMLM), which without correction is substantial even for scientific-grade, cooled cameras. In addition, our approach enables high-quality 3D super-resolution as well as live-cell time-lapse microscopy with cheap, industry-grade cameras. As our approach for camera characterization does not require any user interventions or additional hardware implementations, numerous correction algorithms demanding camera characterization become easily applicable.


title: Transmission of vibrations in buzz-pollinated plant species with disparate floral morphologies

abstract: In buzz-pollinated plants, bees apply vibrations produced by their thoracic muscles to the flower, causing pollen release from anthers, often through small apical pores. During floral buzzing, bees grasp one or more anthers with their mandibles, and vibrations are transmitted to the focal anther(s), adjacent anthers, and the whole flower. Because pollen release depends on the vibrations experienced by the anther, the transmission of vibrations through flowers with different morphologies may determine patterns of release, affecting both bee foraging and plant fitness. Anther morphology and intra-floral arrangement varies widely among buzz-pollinated plants. Here, we compare the transmission of vibrations among focal and non-focal anthers in four species with contrasting anther morphologies: Cyclamen persicum (Primulaceae), Exacum affine (Gentianaceae), Solanum dulcamara and S. houstonii (Solanaceae). We used a mechanical transducer to apply bee-like artificial vibrations to focal anthers, and simultaneously measured the vibration frequency and displacement amplitude at the tips of focal and non-focal anthers using high-speed video analysis (6,000 frames per second). In flowers in which anthers are tightly held together (C. persicum and S. dulcamara), vibrations in focal and non-focal anthers are indistinguishable in both frequency and displacement amplitude. In contrast, flowers with loosely arranged anthers (E. affine) including those in which stamens are morphologically differentiated within the same flower (heterantherous S. houstonii), show the same frequency but higher displacement amplitude in non-focal anthers compared to focal anthers. Our results suggest that stamen arrangement affects vibration transmission with potential consequences for pollen release and bee behaviour.


title: Rigidifying a de novo enzyme increases activity and induces a negative activation heat capacity

abstract: Conformational sampling profoundly impacts the overall activity and temperature dependence of enzymes. Peroxidases have emerged as versatile platforms for high value biocatalysis owing to their broad palette of potential biotransformations. Here, we explore the role of conformational sampling in mediating a de novo peroxidases activity. We demonstrate that 2,2,2-triflouoroethanol (TFE) affects the equilibrium of enzyme conformational states, tending towards a more globally rigid structure. This is correlated with increases both stability and activity. Notably, these effects are concomitant with the emergence of curvature in the temperature-activity profile, trading off activity gains at ambient temperature with losses at high temperatures. We apply macromolecular rate theory (MMRT) to understand enzyme temperature dependence data. These data point to an increase in protein rigidity associated with a difference in the distribution of protein dynamics between the ground and transition state. We compare the thermodynamics of the de novo enzyme activity to those of a natural peroxidase, horseradish peroxidase. We find that the native enzyme resembles the rigidified de novo enzyme in terms of the thermodynamics of enzyme catalysis and the putative distribution of protein dynamics between the ground and transition state. The addition of TFE apparently causes C45 to behave more like the natural enzyme. Our data suggest robust, generic strategies for improving biocatalytic activity by manipulating protein rigidity; for functional de novo protein catalysts in particular, this can provide more enzyme-like catalysts without further rational engineering, computational redesign or directed evolution.


title: Using embedded alginate microparticles to tune the properties of in situ forming poly(N-isopropylacrylamide)-graft-chondroitin sulfate bioadhesive hydrogels for replacement and repair of the nucleus pulposus of the intervertebral disc

abstract: Low back pain (LBP) is a major public health issue associated with degeneration of the intervertebral disc (IVD). The early stages of degeneration are characterized by the dehydration of the central, gelatinous portion of the IVD, the nucleus pulposus (NP). One possible treatment approach is to replace the NP in the early stages of IVD degeneration with a hydrogel that restores healthy biomechanics while supporting tissue regeneration. The present study evaluates a novel thermosensitive hydrogel based on poly(N-isopropylacrylamide-graft-chondroitin sulfate) (PNIPAAM-g-CS) for NP replacement. The hypothesis was tested that the addition of freeze-dried, calcium crosslinked alginate microparticles (MPs) to aqueous solutions of PNIPAAm-g-CS would enable tuning of the rheological properties of the injectable solution, as well as the bioadhesive and mechanical properties of the thermally precipitated composite gel. Further, we hypothesized that the composite would support encapsulated cell viability and differentiation. Structure-material property relationships were evaluated by varying MP concentration and diameter. The addition of high concentrations (50 mg/mL) of small MPs (20 {+/-} 6 {micro}m) resulted in the greatest improvement in injectability, compressive mechanical properties, and bioadhesive strength of PNIPAAm-g-CS. This combination of PNIPAAM-g-CS and alginate MPs supported the survival, proliferation, and differentiation of adipose derived mesenchymal stem cells (ADMSCs) towards an NP-like phenotype in the presence of soluble GDF-6. When implanted ex vivo into the intradiscal cavity of degenerated porcine IVDs, the formulation restored the compressive and neutral zone (NZ) stiffnesses to intact values and resisted expulsion under lateral bending. Overall, results indicate the potential of the hydrogel composite to serve as a scaffold for supporting NP regeneration. This work uniquely demonstrates that encapsulation of re-hydrating polysaccharide-based MPs may be an effective method for improving key functional properties of in situ forming hydrogels for orthopaedic tissue engineering applications.


title: Comparative analysis of molecular representations in prediction of cancer drug combination synergy and sensitivity

abstract:
AO_SCPLOWBSTRACTC_SCPLOWApplication of machine and deep learning methods in drug discovery and cancer research has gained a considerable amount of attention in the past years. As the field grows, it becomes crucial to systematically evaluate the performance of novel computational solutions in relation to established techniques. To this end we compare rule-based and data-driven molecular representations in prediction of drug combination sensitivity and drug synergy scores using standardized results of 14 throughput screening studies, comprising 64 200 unique combinations of 4 153 molecules tested in 112 cancer cell lines. We evaluate the clustering performance of molecular representations and quantify their similarity by adapting the Centered Kernel Alignment metric. Our work demonstrates that to identify an optimal molecular representation type it is necessary to supplement quantitative benchmark results with qualitative considerations, such as model interpretability and robustness, which may vary between and throughout preclinical drug development projects.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/439299v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1bc4509org.highwire.dtl.DTLVardef@1586640org.highwire.dtl.DTLVardef@a11129org.highwire.dtl.DTLVardef@6dc93b_HPS_FORMAT_FIGEXP  M_FIG C_FIG


title: Comparative analysis of molecular representations in prediction of drug combination effects


title: Oncogenic PKA signaling stabilizes MYC oncoproteins via an aurora kinase A-dependent mechanism

abstract: Genetic alterations that activate protein kinase A (PKA) signaling are found across many tumor types, but their downstream oncogenic mechanisms are poorly understood. We used global phosphoproteomics and kinome activity profiling to map the conserved signaling outputs driven by diverse genetic changes that activate PKA in human cancer, including melanoma and fibrolamellar carcinoma (FLC). We define two consensus networks of effectors downstream of PKA in cancer models. One is centered on RAS/MAPK components, and a second involves Aurora Kinase A (AURKA). We find that AURKA stabilizes c-MYC and n-MYC protein levels and expression programs in PKA-dependent tumor models, in part via a positive feedback loop mediated by the oncogenic kinase PIM2. This process can be suppressed by conformation-disrupting AURKA inhibitors such as CD-532. Our findings elucidate two independent mechanisms of PKA-driven tumor cell growth and offer insight into drug targets for study in FLC and other PKA-dependent malignancies.


title: Dynamic reconfiguration of macaque brain networks during free-viewing of natural scenes

abstract: Natural vision involves the activation of a wide range of higher-level regions processing objects, motion, faces and actions. Here, we pursue a data-driven approach to explore how higher-level visual processes relate to the underlying structural and functional connectivity. Using a free-viewing paradigm in four awake rhesus macaque monkeys, we investigate how different visual scenes change functional connectivity. Additionally, we explore how such functional connectivity, as measured through fMRI, is related to the structural connectivity, as measured through diffusion weighted imaging. At first, we evaluate the consistency of the elicited free-viewing pattern using standard analytical techniques. We also evaluate the underlying structural connectivity via diffusion data by tracking white matter bundle projections from the visual cortex. We then reconstruct free-viewing and structural networks and quantify their properties. Centrality measures over the entire fMRI time-series revealed a consistent functional network engaged during free-viewing that included widespread hub regions across frontal (FEF, 46v), parietal (LIP, Tpt), and occipitotemporal cortex (MT, V4 and TE) among others. Interestingly, a small number of highly-weighted and long-length inter-hemispheric connections indicated the presence of long-range integrative properties during free-viewing. We hypothesized that during free-viewing, networks had the capacity to change their local and distal connections depending on the on-going changes in visual scenes. To capture these network dynamics, we depart from the static modular architecture of the structural networks and demonstrate that hubs in free-viewing networks reorganize according to the presence of objects, motion, and faces in the movie scenes indicating poly-functional properties. Lastly, we compare each NHP subject network and observe high consistency between individuals across the same network type with closer correspondence between structural networks (e.g., diffusion based and those partially assembled from tract-tracing). In summary, our network analyses revealed ongoing changes in large-scale functional organization present during free-viewing in the macaque monkey and highlight the advantages of multi-contrast imaging in awake monkeys for investigating dynamical processes in visual cognition. To further promote the use of naturalistic free-viewing paradigms and increase the development of macaque neuroimaging resources, we share our datasets in the PRIME-DE consortium.


title: Constitutively active SARM1 variants found in ALS patients induce neuropathy

abstract: In response to injury, neurons activate a program of organized axon self-destruction initiated by the NAD+ hydrolase SARM1. In healthy neurons SARM1 is autoinhibited, but single amino acid changes can abolish autoinhibition leading to constitutively-active SARM1 enzymes that promote degeneration when expressed in cultured neurons. To investigate whether naturally-occurring human variants might similarly disrupt SARM1 autoinhibition and potentially contribute to risk for neurodegenerative disease, we assayed the enzymatic activity of 29 rare SARM1 alleles identified among 8,507 amyotrophic lateral sclerosis (ALS) patients. Ten missense variants or small in-frame deletions exhibit constitutive NADase activity, including more than half of those that are unique to the ALS patients or that occur in multiple patients. Expression of these constitutively active ALS-associated SARM1 alleles in cultured dorsal root ganglion (DRG) neurons is pro-degenerative and cytotoxic. Intrathecal injection of an AAV expressing the common SARM1 reference allele is innocuous to mice, but a construct harboring SARM1V184G, the constitutively active variant found most frequently in the ALS patients, causes axon loss, motor dysfunction, and sustained neuroinflammation. These results implicate rare hypermorphic SARM1 alleles as candidate genetic risk factors for ALS and other neurodegenerative conditions.


title: A multimodal 3D neuro-microphysiological system with neurite-trapping microelectrodes

abstract: Three-dimensional cell technologies as pre-clinical models are emerging tools for mimicking the structural and functional complexity of the nervous system. The accurate exploration of phenotypes in engineered 3D neuronal cultures, however, demands morphological, molecular and especially functional measurements. Particularly crucial is measurement of electrical activity of individual neurons with millisecond resolution. Current techniques rely on customized electrophysiological recording set-ups, characterized by limited throughput and poor integration with other readout modalities. Here we describe a novel approach, using multiwell glass microfluidic microelectrode arrays, allowing non-invasive electrical recording from engineered 3D neural tissues. We demonstrate parallelized studies with reference compounds, calcium imaging and optogenetic stimulation. Additionally, we show how microplate compatibility allows automated handling and high-content analysis of human induced pluripotent stem cell-derived neurons. This microphysiological platform opens up new avenues for high-throughput studies on the functional, morphological and molecular details of neurological diseases and their potential treatment by therapeutic compounds.


title: Mindfulness Training Alters Resting-State EEG Dynamics in Novice Practitioners via Mindful Breathing and Body-scan

abstract: Mindfulness-based stress reduction (MBSR) has been proven to improve mental health and quality of life. This study examined how mindfulness training and various types of mindfulness practices altered brain activity. Specifically, the spectral powers of scalp electroencephalography (EEG) of the MBSR group who underwent an 8-week mindfulness training--including mindful breathing and body-scan--were evaluated and compared with those of the waitlist controls. Empirical results indicated that the long-term mindfulness intervention effect significantly elevated the resting-state beta powers and reduced resting-state delta powers in both practices; such changes were not observed in the waitlist control. Compared with mindful breathing, body-scanning resulted in an overall decline in EEG spectral powers at both delta and gamma bands among trained participants. Together with our preliminary data of expert mediators, the aforementioned spectral changes were salient after intervention, but mitigated along with expertise. Additionally, after receiving training, the MBSR groups mindfulness and emotion regulation levels improved significantly, which were correlated with the EEG spectral changes in the theta, alpha, and low-beta bands. This study elaborated the neurophysiological correlates of mindfulness practices, suggesting that MBSR might function as a unique internal processing that involves increased vigilant capability and induces alterations similar to other cognitive training.


title: SARS-CoV-2 spike protein expressing epithelial cells promotes senescence associated secretory phenotype in endothelial cells and increased inflammatory response

abstract: Increased mortality in COVID-19 often associates with thrombotic and microvascular complications. We have recently shown that SARS-CoV-2 spike protein promotes inflammatory cytokine IL-6/IL-6R induced trans-signaling responses which modulate MCP-1 expression in human endothelial cells. MCP-1 is secreted as a major component of the senescence associated secretory phenotype (SASP). Virus infected or Spike transfected human pulmonary epithelial cells (A549) exhibited an increase in senescence related marker proteins. TMNK; as a representative human endothelial cell line, when exposed to cell culture supernatant derived from A549 cells expressing SARS-CoV-2 spike protein (Spike CM) exhibited a senescence phenotype with enhanced p16, p21, and SA-{beta}-galactosidase expression. Inhibition of IL-6 trans-signaling by Tocilizumab, prior to exposure of supernatant to endothelial cells, inhibited p16 and p21 induction. Likewise, inhibition of receptor signaling by Zanabrutinib or Brd4 function by AZD5153 also led to limited induction of p16 expression. Senescence lead to an enhanced level of adhesion molecule, ICAM-1 and VCAM-1 in human endothelial cells, and TPH1 attachment by in vitro assay. Inhibition of senescence or SASP function prevented ICAM/VCAM expression and leukocyte attachment. We also observed an increase in oxidative stress in A549 spike transfected and endothelial cells exposed to Spike CM. ROS generation in TMNK was reduced after treatment with the IL-6 specific inhibitor Tociliximab, and with the specific inhibitors Zanabrutinib and AZD5153. Taken together, we identified that the exposure of human endothelial cells to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers leading to enhanced leukocyte adhesion with coronary blockade potential.


title: Potential transmission chains of variant B.1.1.7 and co-mutations of SARS-CoV-2

abstract: The presence of SARS-CoV-2 mutants, including the emerging variant B.1.1.7, has raised great concerns in terms of pathogenesis, transmission, and immune escape. Characterizing SARS-CoV-2 mutations, evolution, and effects on infectivity and pathogenicity is crucial to the design of antibody therapies and surveillance strategies. Here we analyzed 454,443 SARS-CoV-2 spike genes/proteins and 14,427 whole-genome sequences. We demonstrated that the early variant B.1.1.7 may not have evolved spontaneously in the United Kingdom or within human populations. Our extensive analyses suggested that Canidae, Mustelidae or Felidae, especially the Canidae family (for example, dog) could be a possible host of the direct progenitor of variant B.1.1.7. An alternative hypothesis is that the variant was simply yet to be sampled. Notably, the SARS-CoV-2 whole genome represents a large number of potential co-mutations with very strong statistical significances (p value<E- 44). In addition, we used an experimental SARS-CoV-2 reporter replicon system to introduce the dominant co-mutations NSP12_c14408t, 5UTR_c241t, and NSP3_c3037t into the viral genome, and to monitor the effect of the mutations on viral replication. Our experimental results demonstrated that the co-mutations significantly attenuated the viral replication. The study provides valuable clues for discovering the transmission chains of variant B.1.1.7 and understanding the evolutionary process of SARS-CoV-2.


title: Homo-harringtonine (HHT) - A highly effective drug against coronaviruses and the potential for large-scale clinical applications

abstract: In the search for treatment schemes of COVID-19, we start by examining the general weakness of coronaviruses and then identify approved drugs attacking that weakness. The approach, if successful, should identify drugs with a specific mechanism that is at least as effective as the best drugs proposed and are ready for clinical trials. All coronaviruses translate their non-structural proteins ([~]16) in concatenation, resulting in a very large super-protein. Homo-harringtonine (HHT), which has been approved for the treatment of leukemia, blocks protein elongation very effectively. Hence, HHT can repress the replication of many coronaviruses at the nano-molar concentration. In two mouse models, HHT clears SARS-CoV-2 in 3 days, especially by nasal dripping of 40 ug per day. We also use dogs to confirm the safety of HHT delivered by nebulization. The nebulization scheme could be ready for large-scale applications at the onset of the next epidemics. For the current COVID-19, a clinical trial has been approved by the Ditan hospital of Beijing but could not be implemented for want of patients. The protocol is available to qualified medical facilities.


title: A gene regulatory network for neural induction

abstract: During early vertebrate development, signals from a special region of the embryo, the organizer, can re-direct the fate of non-neural ectoderm cells to form a complete, patterned nervous system. This is called neural induction and has generally been imagined as a single signalling event, causing a switch of fate. Here we undertake a comprehensive analysis, in very fine time-course, of the events following exposure of ectoderm to the organizer. Using transcriptomics and epigenomics we generate a Gene Regulatory Network comprising 175 transcriptional regulators and 5,614 predicted interactions between them, with fine temporal dynamics from initial exposure to the signals to expression of mature neural plate markers. Using in situ hybridization, single-cell RNA-sequencing and reporter assays we show that neural induction by a grafted organizer closely resembles normal neural plate development. The study is accompanied by a comprehensive resource including information about conservation of the predicted enhancers in different vertebrate systems.

category: developmental biology


title: Oligopaint DNA FISH as a tool for investigating meiotic chromosome dynamics in the silkworm, Bombyx mori

abstract:
Accurate chromosome segregation during meiosis is essential for reproductive success. Yet, many fundamental aspects of meiosis remain unclear, including the mechanisms regulating homolog pairing across species. This gap is partially due to our inability to visualize individual chromosomes during meiosis. Here, we employ Oligopaint FISH to investigate homolog pairing and compaction of meiotic chromosomes in a classical model system, the silkworm Bombyx mori. Our Oligopaint design combines multiplexed barcoding with secondary oligo labeling for high flexibility and low cost. These studies illustrate that Oligopaints are highly specific in whole-mount gonads and on meiotic chromosome spreads. We show that meiotic pairing is robust in both males and female meiosis. Additionally, we show that meiotic bivalent formation in B. mori males is highly similar to bivalent formation in C. elegans, with both of these pathways ultimately resulting in the pairing of chromosome ends with non-paired ends facing the spindle pole and microtubule recruitment independent of the centromere-specifying factor CENP-A.

Authors SummaryMeiosis is the specialized cell division occurring exclusively in ovaries and testes to produce egg and sperm cells, respectively. The accurate distribution of chromosomes (the genetic material) during this process is essential to prevent infertility/sterility and developmental disorders in offspring. As researchers are specifically unable to study the mechanisms regulating meiosis in depth in humans, identifying broadly conserved aspects of meiotic chromosome segregation is essential for making accurate inferences about human biology. Here, we use a sophisticated chromosome painting approach called Oligopaints to visualize and study chromosomes during meiosis in the silkworm, Bombyx mori. We illustrate that Oligopaints are highly specific in B. mori and demonstrate how Oligopaints can be used to study the dynamics of meiotic chromosomes in diverse species.

category: genetics


title: Subgroup-specific gene expression profiles and mixed epistasis in chronic lymphocytic leukemia

abstract: Despite the extensive catalogue of recurrent mutations in chronic lymphocytic leukaemia (CLL), the diverse molecular driving events and the resulting range of disease phenotypes remain incompletely understood. To study the molecular heterogeneity of CLL, we performed RNA-sequencing on 184 CLL patient samples. Unsupervised analysis revealed two major independent axes of gene expression variation: the first one aligned with the mutational status of the immunoglobulin heavy variable (IGHV) genes, and concomitantly, with the three-group stratification of CLL by global DNA methylation pattern, and affected biological functions including B- and T-cell receptor signaling. The second one aligned with trisomy 12 status and affected chemokine signaling. Furthermore, we searched for differentially expressed genes associated with gene mutations and copy-number aberrations and detected strong signatures for TP53, BRAF and SF3B1, as well as for del(11)(q22.3), del(17)(p13) and del(13)(q14) beyond the dosage effect. We discovered strong non-additive effects (i.e., genetic interactions, or epistasis) of IGHV mutation status and trisomy 12 on multiple phenotypes, including the expression of 893 genes. Multiple types of epistasis were observed, including synergy, buffering, suppression and inversion. Our study reveals previously underappreciated gene expression signatures for (epi)genomic variants in CLL and the presence of epistasis between them. The findings will serve as a reference for a functional resolution of CLL molecular heterogeneity.


title: Nucleosome patterns in four plant pathogenic fungi with contrasted genome structures

abstract: AO_SCPLOWBSTRACTC_SCPLOWFungal pathogens represent a serious threat towards agriculture, health, and environment. Control of fungal diseases on crops necessitates a global understanding of fungal pathogenicity determinants and their expression during infection. Genomes of phytopathogenic fungi are often compartmentalized: the core genome contains housekeeping genes whereas the fast-evolving genome mainly contains transposable elements and species-specific genes. In this study, we analysed nucleosome landscapes of four phytopathogenic fungi with contrasted genome organizations to describe and compare nucleosome repartition patterns in relation with genome structure and gene expression level. We combined MNase-seq and RNA-seq analyses to concomitantly map nucleosome-rich and transcriptionally active regions during fungal growth in axenic culture; we developed the MNase-seq Tool Suite (MSTS) to analyse and visualise data obtained from MNase-seq experiments in combination with other genomic data and notably RNA-seq expression data. We observed different characteristics of nucleosome profiles between species, as well as between genomic regions within the same species. We further linked nucleosome repartition and gene expression. Our findings support that nucleosome positioning and occupancies are subjected to evolution, in relation with underlying genome sequence modifications. Understanding genomic organization and its role in expression regulation is the next gear to understand complex cellular mechanisms and their evolution.


title: High-resolution structure and dynamics of mitochondrial complex I - insights into the proton pumping mechanism

abstract: Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a 1 MDa membrane protein complex with a central role in energy metabolism. Redox-driven proton translocation by complex I contributes substantially to the proton motive force that drives ATP synthase. Several structures of complex I from bacteria and mitochondria have been determined but its catalytic mechanism has remained controversial. We here present the cryo-EM structure of complex I from Yarrowia lipolytica at 2.1 [A] resolution, which reveals the positions of more than 1600 protein-bound water molecules, of which [~]100 are located in putative proton translocation pathways. Another structure of the same complex under steady-state activity conditions at 3.4 [A] resolution indicates conformational transitions that we associate with proton injection into the central hydrophilic axis. By combining high-resolution structural data with site-directed mutagenesis and large-scale molecular dynamics simulations, we define details of the proton translocation pathways, and offer new insights into the redox-coupled proton pumping mechanism of complex I.


title: Dynamics of switching at stall reveals nonequilibrium mechanism in the allosteric regulation of the bacterial flagellar switch

abstract: Behavior of the bacterial flagellar motor depends sensitively on the external loads it drives. Motor switching, which provides the basis for the run-and-tumble behavior of flagellated bacteria, has been studied for motors under zero to high loads, revealing a non-equilibrium effect that is proportional to the motor torque. However, behavior of the motor switching at stall (with maximum torque) remains unclear. An extrapolation from previous studies would suggest maximum non-equilibrium effect for motor switching at stall. Here, we stalled the motor using optical tweezers and studied the motor switching with a high time resolution of about 2 ms. Surprisingly, our results showed exponentially distributed counterclockwise (CCW) and clockwise (CW) intervals, indicating that motor switching at stall is probably an equilibrium process. Combined with previous experiments at other loads, our result suggested that the non-equilibrium effect in motor switching arises from the asymmetry of the torque generation in the CCW and CW directions. By including this non-equilibrium effect in the general Ising-type conformation spread model of the flagellar switch, we consistently explained the motor switching over the whole range of load conditions. We expect to see similar mechanism of non-equilibrium regulation in other molecular machines.


title: Spatial genomics maps the structure, character and evolution of cancer clones

abstract: Subclonality is a universal feature of cancers yet how clones grow, are spatially organised, differ phenotypically or influence clinical outcome is unclear. To address this, we developed base specific in situ sequencing (BaSISS). In fixed tissues, transcripts harbouring clone-defining mutations are detected, converted into quantitative clone maps and characterised through multi-layered data integration. Applied to 8 samples from key stages of breast cancer progression BaSISS localised 1.42 million genotype informative transcripts across 4.9cm2 of tissue. Microscopic clonal topographies are shaped by resident tissue architectures. Distinct transcriptional, histological and immunological features distinguish coexistent genetic clones. Spatial lineage tracing temporally orders clone features associated with the emergence of aggressive clinical traits. These results highlight the pivotal role of spatial genomics in deciphering the mechanisms underlying cancer progression.


title: Gasdermin D mediates inflammation-induced defects in reverse cholesterol transport and promotes atherosclerosis.

abstract:
Activation of inflammasomes, such as Nlrp3 and Aim2, can exacerbate atherosclerosis in mice and humans. Gasdermin D (GsdmD) serves as a final executor of inflammasome activity, by generating membrane pores for the release of mature Interleukin-1beta (IL-1{beta}). Inflammation dampens reverse cholesterol transport (RCT) and promotes atherogenesis, while anti-IL-1{beta} antibodies were shown to reduce cardiovascular disease in humans. Though Nlrp3/AIM2 and IL-1{beta} nexus is an emerging atherogenic pathway, the direct role of GsdmD in atherosclerosis is not yet fully clear. Here, we used in-vivo Nlrp3 inflammasome activation to show that the GsdmD-/- mice release ~80% less IL-1{beta} vs WT mice. The GsdmD-/- macrophages were more resistant to Nlrp3 inflammasome mediated reduction in cholesterol efflux, showing ~26% decrease vs. ~60% reduction in WT macrophages. GsdmD expression in macrophages exacerbated foam cell formation in an IL-1{beta} dependent fashion. The GsdmD-/- mice were resistant to Nlrp3 inflammasome mediated defect in RCT, with ~32% reduction in plasma RCT vs. ~ 57% reduction in WT mice, ~ 17% reduction in RCT to liver vs. 42% in WT mice, and ~ 37% decrease in RCT to feces vs. ~ 61% in WT mice. The LDLr anti-sense oligonucleotides (ASO) induced hyperlipidemic mouse model showed the role of GsdmD in promoting atherosclerosis. The GsdmD-/- mice exhibit ~42% decreased atherosclerotic lesion area in females and ~33% decreased lesion area in males vs. WT mice. The atherosclerotic plaque-bearing sections stained positive for the cleaved N-terminal fragment of GsdmD, indicating cleavage of GsdmD in atherosclerotic plaques. Our data show that GsdmD mediates inflammation-induced defects in RCT and promotes atherosclerosis.

SummaryGsdmD mediates inflammation-induced defects in RCT and promotes atherosclerosis.

category: pathology


title: A crosstalk between E2F1 and GLP-1 signaling pathways modulates insulin secretion

abstract: Compromised {beta}-cell function contributes to type 2 diabetes (T2D) development. The glucagon like peptide 1 (Glp-1) has emerged as a hormone with broad pharmacological potential toward T2D treatment, notably by improving {beta}-cell functions. Recent data have shown that the transcription factor E2f1, besides its role as a cell cycle regulator, is involved in glucose homeostasis by modulating {beta}-cell mass, function and identity. Here, we demonstrate a crosstalk between the E2F1, phosphorylation of retinoblastoma protein (pRb) and Glp-1 signaling pathways. We found that {beta}-cell specific E2f1 deficient mice (E2f1{beta}-/-) presented with impaired glucose homeostasis and decreased glucose stimulated-insulin secretion mediated by exendin 4 (i.e., GLP1R agonist), which were associated with decreased expression of Glp1r encoding Glp-1 receptor (GLP1R) in E2f1{beta}-/- pancreatic islets. Decreasing E2F1 transcriptional activity with an E2F inhibitor in islets from nondiabetic humans decreased GLP1R levels and blunted the incretin effect of exendin 4 on insulin secretion. Conversely, overexpressing E2f1 in pancreatic {beta} cells increased Glp1r expression associated with enhanced insulin secretion mediated by GLP1R agonist. Interestingly, kinome analysis of mouse islets demonstrated that an acute treatment with exendin 4 increased pRb phosphorylation and subsequent E2f1 transcriptional activity. This study suggests a molecular crosstalk between the E2F1/pRb and GLP1R signaling pathways that modulates insulin secretion and glucose homeostasis.

category: physiology


title: Isolation of the side population from adult neurogenic niches enriches for endothelial cells

abstract: In stem cell research, DNA-binding dyes offer the ability to purify live stem cells using flow cytometry as they form a low-fluorescence side population due to the activity of ABC transporters. Adult neural stem cells exist within the lateral ventricle and dentate gyrus of the adult brain yet the ability of DNA-binding dyes to identify these adult stem cells as side populations remain untested. The following experiments utilize the efflux of a DNA-binding dye, Vyrbant DyeCycle Violet (DCV), to isolate bona fide side populations in the adult mouse dentate gyrus and SVZ and test their sensitivity to ABC transporter inhibitors. A distinct side population was found in both the adult lateral ventricle and dentate gyrus using DCV fluorescence and forward scatter instead of the conventional dual fluorescence approach. These side populations responded strongly to inhibition with the ABC transporter antagonists, verapamil and fumitremorgin C. The cells in the side population were identified as cerebrovascular endothelial cells characterized by their expression of CD31. These findings, therefore, suggest that the side population analysis provides an efficient method to purify endothelial cells, but not adult neural stem cells.

category: neuroscience