title: Progenitor-intrinsic Metabolic Sensing Promotes Hematopoietic Homeostasis

abstract: Hematopoietic homeostasis is maintained by stem and progenitor cells in part by extrinsic feedback cues triggered by mature cell loss. We demonstrate a different mechanism by which hematopoietic progenitors intrinsically anticipate and prevent the loss of mature progeny through metabolic switches. We examined hematopoiesis in mice conditionally deficient in long-chain fatty acid oxidation (carnitine palmitoyltransferase 2, Cpt2), glutaminolysis (glutaminase, Gls), or mitochondrial pyruvate import (mitochondrial pyruvate carrier 2, Mpc2). While genetic ablation of Cpt2 or Gls minimally impacted most blood lineages, deletion of Mpc2 led to a sharp decline in mature myeloid cells. However, MPC2-deficient myeloid cells rapidly recovered due to a transient increase in myeloid progenitor proliferation. Competitive bone marrow chimera and stable isotope tracing experiments demonstrated that this proliferative burst was intrinsic to MPC2-deficient progenitors and accompanied by a metabolic switch to glutaminolysis. Thus, hematopoietic progenitors intrinsically adjust to metabolic perturbations independently of feedback from downstream mature cells to maintain homeostasis.

category: developmental biology


title: STAT3-mediated allelic imbalance of novel genetic variant rs1047643 and B cell specific super-enhancer in association with systemic lupus erythematosus

abstract: Mapping of allelic imbalance (AI) at heterozygous loci has the potential to establish links between genetic risk for disease and biological function. Leveraging multi-omics data for AI analysis and functional annotation, we discovered a novel functional risk variant rs1047643 at 8p23 in association with SLE. This variant displays dynamic AI of chromatin accessibility and allelic expression on FDFT1 gene in B cells with SLE. We further found a B-cell restricted super-enhancer (SE) that physically contacts with this SNP-residing locus, an interaction that also appears specifically in B cells. Quantitative analysis of open chromatin and DNA methylation profiles further demonstrated that the SE exhibits aberrant activity in B cell development with SLE. Functional studies identified that STAT3, a master factor associated with autoimmune diseases, directly regulates both the AI of risk variant and the activity of SE in cultured B cells. Our study reveals that STAT3-mediated SE activity and cis-regulatory effects of SNP rs1047643 at 8p23 locus are associated with B cell deregulation in SLE.

category: genetics


title: Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase

abstract: Efficient, targeted integration of large DNA constructs represent a significant hurdle in genetic engineering for the development of mouse models of human disease and synthetic biology research. To address this, we developed a system for efficient and precise, targeted single-copy integration of large transgenes directly into the zygote using multiple mouse genetic backgrounds. Conventional approaches, such as random transgenesis, CRISPR/Cas9-mediated homology-directed repair (HDR), lentivirus-based insertion, or DNA transposases all have significant limitations. Our strategy uses in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) to efficiently generate precise single-copy integrations of transgenes. This is achieved using a transgene "landing pad" composed of dual heterologous Bxb1 attachment (att) sites in cis, pre-positioned in the Gt(ROSA)26Sor safe harbor locus. Successful RMCE is achieved in att carrier zygotes using donor DNA carrying cognate attachment sites flanking the desired donor transgene microinjected along with Bxb1-integrase mRNA. This approach routinely achieves perfect vector-free integration of donor constructs at efficiencies as high as 43% and has generated transgenic animals containing inserts up to [~]43kb. Furthermore, when coupled with a nanopore-based Cas9-targeted sequencing (nCATS) approach, complete verification of the precise insertion sequence can be achieved. As a proof-of-concept we describe the creation and characterization of C57BL/6J and NSG Krt18-ACE2 transgenic mouse models for SARS-CoV2 research with verified heterozygous N1 animals available for experimental use in [~]4 months. In addition, we created a diverse series of mouse backgrounds carrying a single att site version of the landing pad allele in C57BL/6J, NSG, B6(Cg)-Tyrc-2J/J, FVB/NJ, PWK/PhJ, 129S1/SvImJ, A/J, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, and DBA/2J for rapid transgene insertion. Combined, this system enables predictable, rapid creation of precisely targeted transgenic animals across multiple genetic backgrounds, simplifying characterization, speeding expansion and use.


title: Genome wide profiling of histone H3 lysine 4 methylation during the Chlamydomonas cell cycle reveals stable and dynamic properties of lysine 4 trimethylation at gene promoters and near ubiquitous lysine 4 monomethylation

abstract: Chromatin modifications are key epigenetic regulatory features with roles in various cellular events, yet histone mark identification, gene wide distribution and relationship to gene expression remains understudied in green algae. Histone lysine methylation is regarded as an active chromatin mark in many organisms, and is implicated in mediating active euchromatin. We interrogated the genome wide distribution pattern of mono- and trimethylated H3K4 using Chromatin-Immunoprecipitation followed by deep-sequencing (ChIP-Seq) during key phases of the Chlamydomonas cell cycle: early G1 phase (ZT1) when cells initiate biomass accumulation, S/M phase (ZT13) when cells are undergoing DNA replication and mitosis, and late G0 phase (ZT23) when they are quiescent. Tri-methylated H3K4 was predominantly enriched at TSSs of the majority of protein coding genes (85%). The likelihood of a gene being marked by H3K4me3 correlated with it being transcribed at one or more time points during the cell cycle but not necessarily by continuous active transcription. This finding even applied to early zygotic genes whose expression may be dormant for hundreds or thousands of generations between sexual cycles; but core meiotic genes were completely missing H3K4me3 peaks at their TSS. In addition, bi-directional promoters regulating expression of replication dependent histone genes, had transient H3K4me3 peaks that were present only during S/M phase when their expression peaked. In agreement with biochemical studies, mono-methylated H3K4 was the default state for the vast majority of histones that were outside of TSS and terminator regions of genes. A small fraction of the genome which was depleted of any H3 lysine methylation was enriched for DNA cytosine methylation and the genes within these DNA methylation islands were poorly expressed. Genome wide H3K4me3 ChIP-Seq data will be a valuable resource, facilitating gene structural annotation, as exemplified by our validation of hundreds of long non-coding RNA genes.

category: genomics


title: Quantification of new and archived Diaphorina citri transcriptome data using a chromosomal length D. citri genome assembly reveals the vector's tissue-specific transcriptional response to citrus greening disease

abstract:
Background

Huanglongbing (HLB) is the most serious disease of citrus. HLB is caused by the obligate, intracellular bacterium "Candidatus Liberibacter asiaticus" (CLas). CLas is transmitted by Diaphorina citri, the Asian citrus psyllid. Development of transmission blocking strategies to manage HLB relies on knowledge of CLas-D. citri interactions at the molecular level. Prior transcriptome analyses of CLas-infected and un-infected D. citri point to changes in psyllid biology due to CLas-infection. These studies relied on incomplete versions of the D. citri genome, lacked proper host plant controls, and/or were analyzed using different statistical approaches. Therefore, we used standardized experimental and computational approaches to identify differentially expressed genes in both CLas (+) and CLas (-) D. citri. The comparative analysis utilized the newest chromosomal length D. citri genome assembly Diaci_v3. In this work, we present a quantitative transcriptome analysis of excised heads, salivary glands, midguts and bacteriomes from CLas (+) and CLas (-) insects.

Results

Each organ had unique transcriptome profiles and responses to CLas infection. Though most psyllids were infected with CLas, CLas-derived transcripts were not detected in all organs. By analyzing the midgut dataset using both the Diaci_v1.1 and v3.0 D. citri genomes, we showed that improved genome assembly led to significant and quantifiable differences in RNAseq data interpretation.

Conclusions

Our results support the hypothesis that future transcriptome studies on circulative, vector-borne pathogens should be conducted at the tissue specific level using complete, chromosomal-length genome assemblies for the most accurate understanding of pathogen-induced changes in vector gene expression.


title: Acoustic Force Spectroscopy Reveals Subtle Differences in Cellulose Unbinding Behavior of Carbohydrate-Binding Modules

abstract:
To rationally engineer more efficient cellulolytic enzymes for cellulosic biomass deconstruction into sugars for biofuels production, it is necessary to better understand the complex enzyme-substrate interfacial interactions. Carbohydrate binding modules (CBM) are often associated with microbial surface-tethered cellulosomal or freely secreted cellulase enzymes to increase substrate accessibility. However, it is not well known how CBM recognize, bind, and dissociate from polysaccharide surfaces to facilitate efficient cellulolytic activity due to the lack of mechanistic understanding of CBM-substrate interactions. Our work outlines a general approach to methodically study the unbinding behavior of CBMs from model polysaccharide surfaces using single-molecule force spectroscopy. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and methodology for uniform deposition of insoluble polysaccharides on the AFS chip surfaces is demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surface suggests distinct CBM binding conformations that can also explain the improved cellulolytic activity of cellulase tethered to CBM. Applying established dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree well with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results highlight the limitations of applying classical theory to explain the highly multivalent CBM-cellulose interactions seen at higher cellulose-CBM bond rupture forces (>15pN).

Significance StatementCellulases are multi-modular enzymes produced by numerous microbes that catalyze cellulose hydrolysis into glucose. These enzymes play an important role in global carbon cycling as well as cellulosic biofuels production. CBMs are essential components of cellulolytic enzymes involved in facilitating hydrolysis of polysaccharides by tethered catalytic domains (CD). The subtle interplay between CBM binding and CD activity is poorly understood particularly for heterogeneous reactions at solid-liquid interfaces. Here, we report a highly multiplexed single-molecule force spectroscopy method to study CBM dissociation from cellulose to infer the molecular mechanism governing substrate recognition and dissociation. This approach can be broadly applied to study multivalent protein-polysaccharide binding interactions relevant to other carbohydrates such as starch, chitin, or hyaluronan to engineer efficient biocatalysts.

category: biochemistry


title: A simple immunoassay for extracellular vesicle liquid biopsy in microliters of non-processed plasma

abstract: Extracellular vesicles (EVs), released by most cell types, provide an excellent source of biomarkers in biological fluids. Here we describe a method that, using just a few microliters of patients plasma, identifies tumour markers exposed on EVs. Studying physico-chemical properties of EVs in solution, we demonstrate that they behave as stable colloidal suspensions and therefore, in immunocapture assays, many of them are unable to interact with a stationary functionalised surface. Using flocculation methods, like those used to destabilize colloids, we demonstrate that cationic polymers increase EV {zeta}-potential, diameter, and sedimentation coefficient and thus, allow a more efficient capture on antibody-coated surfaces by both ELISA and bead-assisted flow cytometry. These findings led to optimization of a protocol in microtiter plates allowing effective immunocapture of EVs, directly in plasma without previous ultracentrifugation or other EV enrichment. The method, easily adaptable to any laboratory, has been validated using plasma from lung cancer patients in which the epithelial cell marker EpCAM has been detected on EVs. This high throughput, easy to automate, technology allows screening of large numbers of patients to phenotype tumour markers in circulating EVs, breaking barriers for the validation of proposed EV biomarkers and the discovery of new ones.


title: New Insights into the Mechanisms Used by Inhibitors Targeting Glutamine Metabolism in Cancer Cells

abstract: Many cancer cells become dependent on glutamine metabolism to compensate for glycolysis being uncoupled from the TCA cycle. The mitochondrial enzyme Glutaminase C (GAC) satisfies this  glutamine addiction by catalyzing the first step in glutamine metabolism, making it an attractive drug target. Despite one of the allosteric inhibitors (CB-839) being in clinical trials, none of the drugs targeting GAC are approved for cancer treatment and their mechanism of action is not well understood. A major challenge has been the rational design of better drug candidates: standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 and its analogs fail to explain their potency differences. Here, we address this problem by using an emerging technique, serial room temperature crystallography, which enabled us to observe clear differences between the binding conformations of inhibitors with significantly different potencies. A computational model was developed to further elucidate the molecular basis of inhibitor potency. We then corroborated the results from our modeling efforts by using recently established fluorescence assays that directly read-out inhibitor binding to GAC. Together, these findings provide new insights into the mechanisms used by a major class of allosteric GAC inhibitors and for the future rational design of more potent drug candidates.


title: Single-Round Remodeling of the Active Site of an Artificial Metalloenzyme using an Ultrahigh-Throughput Double Emulsion Screening Assay

abstract: The potential of high-throughput compartmentalization renders droplet microfluidics an attractive tool for directed evolution of enzymes as it permits maintenance of the phenotype-genotype linkage throughout the entire optimization procedure. In particular, water-in-oil-in-water double emulsions droplets (DEs) produced by microfluidics enable the analysis of reaction compartments at ultra-high-throughput using commercially available fluorescence-activated cell sorting (FACS) devices. Here we report a streamlined method applicable for the ultrahigh-throughput screening of an artificial metalloenzyme (ArM), an artificial deallylase (ADAse), in double emulsions. The DE-protocol was validated by screening a four hundred member, double-mutant streptavidin library for the CpRu-catalyzed uncaging of aminocoumarin. The most active variants, identified by next generation sequencing of the sorted DE droplets with highest fluorescent intensity, are in good agreement with 96-well plate screening hits. These findings, thus, pave the way towards the systematic implementation of commercially available FACS for the directed evolution of metalloenzymes making ultrahigh-throughput screening more broadly accessible. The use of microfluidics for the formation of uniform compartments with precise control over reagents and cell encapsulation further facilitates the establishment of highly reliable quantitative assays.


title: Hydroxyl radical footprinting analysis of a human haptoglobin-hemoglobin complex

abstract: Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) - haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. Oxidative modifications were precisely localized on specific residues using a timsTOF Pro mass spectrometer. The data allowed determination of amino acids directly involved in Hb - Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Data are available via ProteomeXchange with identifier PXD021621.


title: Global Epigenetic Analysis Reveals H3K27 Methylation as a Mediator of Double Strand Break Repair

abstract: The majority of cancer patients is treated with ionizing radiation (IR), a relatively safe and effective treatment considered to target tumors by inducing DNA double strand breaks (DSBs). Despite clinical interest in increasing the efficacy of IR by preventing successful DSB repair, few effective radio-adjuvant therapies exist. Extensive literature suggests that chromatin modifiers play a role in the DSB repair and thus may represent a novel class of radiosensitizers. Indeed, chromatin has both local and global impacts on DSB formation, recognition of breaks, checkpoint signaling, recruitment of repair factors, and timely DSB resolution, suggesting that epigenetic deregulation in cancer may impact the efficacy of radiotherapy. Here, using tandem mass spectrometry proteomics to analyze global patterns of histone modification in MCF7 breast cancer cells following IR exposure, we find significant and long-lasting changes to the epigenome. Our results confirm that H3K27 trimethylation (H3K27me3), best known for mediating gene repression and regulating cell fate, increases after IR. H3K27me3 changes rapidly, accumulating at sites of DNA damage. Inhibitors of the Polycomb related complex subunit and H3K27 methyltransferase EZH2 confirm that H3K27me3 is necessary for DNA damage recognition and cell survival after IR. These studies provide an argument for evaluating EZH2 as a radiosensitization target and H3K27me3 as a marker for radiation response in cancer. Proteomic data are available via ProteomeXchange with identifier PXD019388.

category: cancer biology


title: Synergic Renoprotective Effects Of Combined ASC Therapy With RAAS Blockade In Experimental Advanced CKD

abstract: Global prevalence of chronic kidney disease (CKD) has increased considerably in the recent decades. Overactivity of the renin-angiotensin-aldosterone system (RAAS), associated to renal inflammation and fibrosis contribute to its evolution. The treatments currently employed to control CKD progression are limited and mainly based on the pharmacological inhibition of RAAS, associated with diuretics and immunosuppressive drugs. However, this conservative management promotes only partial deceleration of CKD evolution, and does not completely avoid the progression of the disease and the loss of renal function, which motivates the medical and scientific community to investigate new therapeutic approaches to detain renal inflammation / fibrosis and CKD progression. Recent studies have shown the application of mesenchymal stem cells (mSC) to exert beneficial effects on the renal tissue of animals submitted to experimental models of CKD. In this context, the aim of the present study was to evaluate the effects of subcapsular application of adipose tissue-derived mSC (ASC) in rats submitted to the 5/6 renal ablation model, 15 days after the establishment of CKD, when the nephropathy was already severe. We also verify whether ASC associated to Losartan, would promote greater renoprotection when compared to the respective monotherapies. Animals were followed until 30 days of CKD, when body weight, systolic blood pressure, biochemical, histological, immunohistochemical and gene expression analysis were performed. The combination of ASC and Losartan was more effective than Losartan monotherapy in reducing systolic blood pressure and glomerulosclerosis, and also promoted the complete normalization of proteinuria and albuminuria, a significant reduction in renal interstitial macrophage infiltration and downregulation of renal IL-6 gene expression. The beneficial effects of ACS are possibly due to the immunomodulatory and anti-inflammatory role of factors secreted by these cells, modulating the local immune response. Although studies are still required, our results demonstrated that a subcapsular inoculation of ASC, associated with the administration of Losartan, exerted additional renoprotective effect in rats submitted to a severe model of established CKD, when compared to Losartan monotherapy, thus suggesting ASC may be a potential adjuvant to RAAS-blockade therapy currently employed in the conservative management of CKD.

category: physiology


title: Loss of Fgfr1 and Fgfr2 in Scleraxis-lineage cells leads to enlarged bone eminences and attachment cell death

abstract: Tendons and ligaments are structural tissues that attach to bone and are essential for joint mobility and stability in vertebrates. Tendon and ligament attachments (i.e., entheses) are often found at bony protrusions (i.e., eminences), and the shape and size of these protrusions depends on both mechanical forces and cellular cues during growth and development. The formation of tendon eminences also contributes to mechanical leverage for skeletal muscle. Fibroblast growth factor receptor (FGFR) signaling plays a critical role in bone development, and Fgfr1 and Fgfr2 are highly expressed in the perichondrium and periosteum of bone where tendon and ligament attachments can be found. However, the role of FGFR signaling in attachment development and maintenance in the limb remains unknown. In this study, we used transgenic mouse models for combinatorial knockout of Fgfr1 and/or Fgfr2 in tendon/ligament and attachment progenitors using ScxCre and measured eminence size and bone shape in the appendicular skeleton. Conditional deletion of both, but not individual, Fgfr1 and Fgfr2 in Scx progenitors led to enlarged eminences in the postnatal appendicular skeleton and smaller secondary ossification centers in long bones. In addition, Fgfr1 Fgfr2 double conditional knockout mice had more variation in the size of collagen fibrils in tendon, narrowed synovial joint spacing, and increased cell death at sites of ligament attachments, as well as decreased plasticity of mature bone compared to age-matched wildtype littermates. These findings identify a role for FGFR signaling in regulating growth and maintenance of tendon/ligament attachments and the size and shape of bony eminences.


title: A single-cell reference atlas delineates CD4+ T cell subtype-specific adaptation during acute and chronic viral infections

abstract: CD4+ T cells are critical orchestrators of immune responses against a large variety of pathogens, including viruses. The multifaceted roles of CD4+ T cells, including their help to innate cells, CD8+ T and B cells and their support for long-lived immunity rely on a profound functional heterogeneity. While multiple CD4+ T cell subtypes and their key transcriptional regulators have been identified, there is a lack of consistent definition for CD4+ T cell transcriptional states. In addition, the progressive changes affecting CD4+ T cell subtypes during and after immune responses remain poorly defined. Taking advantage of single-cell transcriptomics, efficient computational methods, and robust animal models, we characterize the transcriptional landscape of CD4+ T cells responding to self-resolving and chronic viral infections. We build a comprehensive atlas of virus-specific CD4+ T cells and their evolution over time, and identify six major distinct cell states that are consistently observed in acute and chronic infections. During the course of acute infection, T cell composition progressively changes from effector to memory states, with subtype-specific gene modules and kinetics. Conversely, T cells in persistent infections fail to transition from effector to memory states, and acquire distinct, chronicity-associated transcriptional programs. By single-cell T cell receptor (TCR) sequencing analysis, we characterize the clonal structure of virus-specific CD4+ T cells across individuals and T cell subtypes. We find that virus-specific CD4+ T cell responses are mainly private across individuals and that most T cells differentiate into all subtypes independently of their TCR, in both acute and chronic infections. Finally, we show that our CD4+ T cell atlas can be used as a reference to accurately interpret cell states in external single-cell datasets. Overall, this study describes a previously unappreciated level of adaptation of the transcriptional states of CD4+ T cells responding to viruses and provides a new computational resource for CD4+ T cell analysis, available online at https://spica.unil.ch.

category: immunology


title: A conformational switch controlling the toxicity of the prion protein

abstract: Prion infections cause conformational changes of PrPC and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ("H-latch") altering the flexibility of the 2-3 and {beta}2-2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation, prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is causally linked to prion neurotoxicity.

category: molecular biology


title: DNA metabarcoding Uncover the Diet of Subterranean Rodents in China

abstract:
ObjectiveIn the past, the zokor, which lived in northern China, caused great harm to agriculture and forestry production due to its large and sophisticated diet. Since the rat lives underground for most of its life, researchers know little about its dietary habits. Further understanding of its diet in the field is of important meaning for developing green and sustainable control strategies for the rat.

MethodsLongde County in Liupan Mountain area of Ningxia Hui Autonomous Region was selected as the interest area to capture zokor and investigate the species of habitat plants.We selected chloroplast trnL gene and eukaryotic internal transcription spacer 1 (ITS 1) primers to amplify DNA from the gastric contents of zokor,and then sequenced on Illumina Miseq PE300 platform.

ResultsThe gastric contents of Eospalax smithii (n=16) and E.cansus(n=9) were analyzed by operational taxonomic units (OTU) clustering and amplicon sequence variants(ASVs).The OTU clustering method obtained 2,995 OTUs, and the ASV method obtained 4,657 ASVs. The ASV method was better than the OTU clustering method, and the ASV method was adopted in the subsequent analysis. The food list of 32 families, 80 genera and 154 species was obtained by ASV method after the error was removed. The food composition of zokor was evaluated by relative abundance(%RA) method and frequency of occurrence(%ROO) method. At the Family level, it was found that zokor mainly fed on Asteraceae, Poaceae, Rosaceae, Pinaceae, Brassicaceae, Apiaceae, etc. At the Genus level, they are mainlyEchinops, Littledalea,Artemisia,Picea, Cirsium, Elymus and so on. The diet alpha diversity of E.cansus was slightly higher than that of E.smithii (P > 0.05). The correlation coefficient between Sobs index of alpha diversity and body weight of zokor was -0.382 (P = 0.059). The diet beta diversity proved that most zokors (22/25) clustered together, with low heterogeneity. They fed positively on Calamagrostis, Cirsium, Echinops, Medicago, Sanguisorba and Taraxacum. We found that zokor mainly fed on the roots of perennial herbs(PH), which were rich in water, carbohydrate, fat and protein, which provided an important energy source for its survival.

ConclusionHigh-throughput sequencing(HTS) based DNA metabarcoding technology has well revealed the diet of zokor, which are generalist.

category: ecology


title: Current protein structure predictors do not produce meaningful folding pathways

abstract: Protein structure prediction has long been considered a gateway problem for understanding protein folding. Recent advances in deep learning have achieved unprecedented success at predicting a proteins crystal structure, but whether this achievement relates to a better modelling of the folding process remains an open question. In this work, we compare the pathways generated by state-of-the-art protein structure prediction methods to experimental folding data. The methods considered were AlphaFold 2, RoseTTAFold, trRosetta, RaptorX, DMPfold, EVfold, SAINT2 and Rosetta. We find evidence that their simulated dynamics capture some information about the folding pathwhay, but their predictive ability is worse than a trivial classifier using sequence-agnostic features like chain length. The folding trajectories produced are also uncorrelated with parameters such as intermediate structures and the folding rate constant. These results suggest that recent advances in protein structure prediction do not yet provide an enhanced understanding of the principles underpinning protein folding.

category: bioinformatics


title: Comparison of two injectable anaesthetic protocols in Egyptian fruit bats (Rousettus aegyptiacus) undergoing gonadectomy

abstract: Egyptian fruit bats are experimental animals of increasing interest because they have been identified as a natural reservoir for several emerging zoonotic viruses. For this reason, bats could undergo different experimental procedures that require sedation or anaesthesia. Our aim was to compare the effects of two balanced anaesthetic protocols on sedation, cardiopulmonary variables and recovery in bats undergoing gonadectomy. Twenty bats were randomized into two groups; patients in group DK received intramuscular injection of dexmedetomidine (40 g kg-1) and ketamine (7 mg kg-1), whereas those in group DBM were anaesthetized with intramuscular dexmedetomidine (40 g kg-1), butorphanol (0.3 mg kg-1) and midazolam (0.3 mg kg-1). Time of induction, cardiopulmonary parameters and anaesthetic depth were measured. If anaesthesia plan was considered inadequate, fraction of inspired isoflurane was titrate-to-effect to achieve immobility. At the end of the surgery venous blood gas analysis was performed and intramuscular atipamezole (200 g kg-1) or atipamezole (200 g kg-1) and flumazenil (0.03 mg kg-1) was administered for timed and scored recovery phase. A significantly higher heart rate and peripheral oxygen saturation were recorded in DBM group (p = 0.001; p = 0.003 respectively), while respiratory rate was significantly lower than DK group (p = 0.001). All bats required isoflurane supplementation during surgery with no significant difference. No differences were observed in rectal temperature, induction and recovery times. Sodium and chlorine where significantly higher in DBM group (p = 0.001; p = 0.002 respectively). Recovery scores in group DK were significantly better than in group DBM (p = 0.034). Both protocols induced anaesthesia in Egyptian fruit bats with comparable sedative and cardiorespiratory effect. These drug combinations may be useful for minor procedures in bats, and they could be associated with inhalation anaesthesia in determining and maintaining a surgical anaesthetic plan.

category: zoology


title: Cryo-electron tomography of ASC signalling sites in pyroptotic cells

abstract: Inflammasomes induce cell death in response to infection, chemical entities and cell damage. Human genetics and animal models have identified the NLRP3 inflammasome as a gatekeeper of caspase-1-dependent pyroptosis. NLRP3 activation induces polymerization of ASC into a single, micron-scale perinuclear punctum, where caspase-1 is activated. These puncta have yet to be imaged at sufficient resolution to resolve their ultrastructure. Here, we apply correlative cryo-light microscopy and cryo-electron tomography to visualize ASC/caspase-1 puncta and mitochondrial membrane remodelling within NLRP3-activated cells. The puncta are composed primarily of branched ASC filaments. The N-terminal pyrin domain forms an 8-10 nm-diameter tubular core, which is decorated by the flexibly linked C-terminal caspase recruitment domain. The variable filament density allows ribosomes and trans-Golgi-like vesicles to permeate the network. We propose this provides structural integrity while allowing macromolecules and vesicles to diffuse in or bind with the necessary density, timing and localization.


title: Diversity and distribution of sediment bacteria across an ecological and trophic gradient

abstract: Can we cluster bacterial sediment communities based on lake size, depth, and trophic status? Or, are bacterial microbial communities an emergent property of their geography, integrating regional physical and climatic conditions? Lakes in Minnesota are uniquely situated to address these questions because of their wide geographic range and variability in size and basin land-use. In this study, we selected twenty lakes with varying morphological and chemical properties across four ecological regions of Minnesota. Our objectives were to (i)) evaluate the diversity and spatial variation of the bacterial community at the sediment-water interface and (ii) determine how lake location and watershed land-use impact aqueous chemistry and influence community structure. Our data indicate that sediment communities from similar depth intervals are more likely to cluster by ecological region rather than any individual lake properties (e.g., trophic status, TP concentration, lake depth). However, composition is tied to a given lake, wherein samples from the same core were more alike than samples at similar depths across lakes. Our results illustrate the diversity within lake sediment microbial communities and provide insight into relationships between taxonomy, physicochemical, and geographic properties of north temperate lakes.


title: Large-scale two-photon calcium imaging in freely moving mice

abstract:
We developed a miniaturized two-photon microscope (MINI2P) for fast, high-resolution, multiplane calcium imaging of over 1,000 neurons at a time in freely moving mice. With a microscope weight below 3g and a highly flexible connection cable, MINI2P allowed imaging to proceed with no impediment of behavior in half-hour free-foraging trials compared to untethered, unimplanted animals. The improved cell yield was achieved through a new optical system design featuring an enlarged field of view (FOV) and a new micro-tunable lens with increased z-scanning range and speed that allowed for fast and stable imaging of multiple, interleaved planes as well as 3D functional imaging. A novel technique for successive imaging across multiple, adjacent FOVs enabled recordings from more than 10,000 neurons in the same animal. Large-scale proof-of-principle data were obtained from cell populations in visual cortex, medial entorhinal cortex, and hippocampus, revealing spatial tuning of cells in all areas, including visual cortex.

HighlightsWe developed a lightweight 2-photon miniscope for imaging in freely-foraging mice

Activity can be monitored in volumes of over 1,000 visual or entorhinal-cortex cells

A new z-scanning module allows fast imaging across multiple interleaved planes

Successive imaging from adjacent regions enables imaging from more than 10,000 cells

category: neuroscience


title: Efference information flow during skill acquisition mediates its interaction with medical simulation technology

abstract: Despite substantial progress towards establishing virtual reality (VR) simulators as a replacement for physical ones for skill training, its effect on the brain network during skill acquisition has not been well addressed. In this study, we employed portable optical neuroimaging technology and Granger causality approach to uncover the impact of the two medical simulation technologies on the directed functional brain network of the subjects with two different skill levels. The mobile brain-behavior relantionship was evaluated using functional near-infrared spectroscopy (fNIRS) while right-handed subjects performed well-established fundamentals of laparoscopic surgery (FLS) pattern cutting task. A multiple regression path analysis found that the cognitive-action information flow from the right prefrontal cortex to the supplementary motor area statistically significantly predicted the FLS task performance. Here, the skill level (expert vs novice) affected the cognitive-action information flow from the right prefrontal cortex and the efference copy information flow from the left primary motor cortex via supplementary motor area as hub to the cognitive-perception at the left prefrontal cortex, i.e., the action-preception link. The simulation technology (physical vs VR simulator) affected solely the cognitive-action information flow from the right prefrontal cortex to the left primary motor cortex; however, the interaction between the medical simulation technology) and the skill level affected the efference information flow from the left primary motor cortex to the right prefrontal cortex and from the supplementary motor area to the left prefrontal cortex. These discriminative findings are crucial since our VR simulator had face and construct validity. Therefore, our study highlighted the importance of efference information flow within the framework of the perception-action cycle when comparing medical simulation technology for visuomotor skill acquisition.


title: Amyloid β oligomer selective antibodies for Alzheimers therapeutics and diagnostics

abstract: Improvements have been made in the diagnosis of Alzheimers disease (AD), manifesting mostly in the development of in vivo imaging methods that allow for the detection of pathological changes in AD by MRI and PET scans. Many of these imaging methods, however, use agents that probe amyloid fibrils and plaques - species that do not correlate well with disease progression and are not present at the earliest stages of the disease. Amyloid {beta} oligomers (A{beta}Os), rather, are now widely accepted as the A{beta} species most germane to AD onset and progression. Here we report evidence further supporting the role of A{beta}Os as pathological instigators of AD and introduce promising anti-A{beta}O diagnostic probes capable of distinguishing the 5xFAD mouse model from wild type mice by PET and MRI. In a developmental study, A{beta} oligomers in 5xFAD mice were found to appear at 3 months of age, just prior to the onset of memory dysfunction, and spread as memory worsened. The increase of A{beta}Os is prominent in the subiculum and correlates with concomitant development of reactive astrocytosis. The impact of these A{beta}Os on memory is in harmony with findings that intraventricular injection of synthetic A{beta}Os into wild type mice induced hippocampal dependent memory dysfunction within 24 hours. Compelling support for the conclusion that endogenous A{beta}Os cause memory loss was found in experiments showing that intranasal inoculation of A{beta}O-selective antibodies into 5xFAD mice completely restored memory function, measured 30-40 days post-inoculation. These antibodies, which were modified to give MRI and PET imaging probes, were able to distinguish 5xFAD mice from wild type littermates. These results provide strong support for the role of A{beta}Os in instigating memory loss and salient AD neuropathology, and they demonstrate that A{beta}O selective antibodies have potential both for therapeutics and for diagnostics.


title: The therapeutic and diagnostic potential of amyloid β oligomers (AβOs) selective antibodies to treat Alzheimer's disease


title: Identification of potential proteins and microRNAs in Multiple Sclerosis and Huntington's diseases using in silico methods.

abstract: Neurogenerative diseases like multiple sclerosis, Huntingtons disease are the major roadblocks in the way towards a healthy brain. Neurodegenerative diseases like multiple sclerosis and Huntingtons disease are affected by several factors such as environmental, immunological, genetics, and the worse scenario we can think of is that they are on the rise worldwide. Degenerative diseases specifically target a limited group of neurons at first resulting in the loss of specific functions associated with the specific part of the brain. The early diagnosis of these neurodegenerative diseases is important so that treatments can start from the early stages of these diseases. In this study, we have established a link between Multiple sclerosis and Huntingtons disease, and also we were able to establish the possible microRNAs that were connected to the expression of genes associated with these two diseases. In this present study, we analyzed the microarray datasets obtained from Gene Expression Omnibus and we identified 266 differentially expressed genes tried to identify using in silico methods the Hub genes involved in Multiple sclerosis and Huntingtons disease. After identifying the genes and proteins we tried to identify the microRNAs that are interacting with the Hub genes. In our study, we identified that the protein network has PTPRC, CXCL8, RBM25 proteins that have maximum connectivity. The top Hub genes are then subjected to a database that contains information concerning the microRNAs that are interacting with the Hub proteins as well as with each other. According to our study, the hsa-mir-155-5p has one of the highest degrees in the microRNA network. Our study will be useful in the future for the development of new drug targets for these neurodegenerative diseases.


title: Slow-oscillatory tACS does not modulate human motor cortical response to repeated plasticity paradigms

abstract: Previous history of activity and learning modulates synaptic plasticity and can lead to saturation of synaptic connections. According to the synaptic homeostasis hypothesis, neural oscillations during slow-wave sleep play an important role in restoring plasticity within a functional range. However, it is not known whether slow-wave oscillations - without the concomitant requirement of sleep - play a causal role in human synaptic homeostasis. Here, slow-oscillatory transcranial alternating current stimulation (tACS, 1Hz, 1mA, 18 minutes) was interleaved between two plasticity-inducing interventions: motor learning, and a paradigm known to induce long-term-potentiation-like plasticity in human motor cortex (paired associative stimulation; PAS). The hypothesis tested was that slow-oscillatory tACS would abolish the expected interference between motor learning and PAS, and facilitate plasticity from successive interventions. Thirty-six participants received sham and active fronto-motor tACS in two separate sessions, along with electroencephalography (EEG) recordings. A further 38 participants received tACS through a control (posterior midline) montage. Using neuro-navigated transcranial magnetic stimulation (TMS) over the left motor cortex, motor evoked potentials (MEPs) were recorded throughout the session. Bayesian statistics were used to quantify evidence for or against the hypothesis of an effect of each intervention on MEP amplitude. As expected, there was converging evidence that motor training increased MEPs. Importantly, we found moderate evidence against an effect of active tACS in restoring PAS plasticity, and no evidence of lasting entrainment of slow-oscillations in the EEG. This suggests that, under the conditions tested here, slow-oscillatory tACS does not modulate synaptic homeostasis in the motor system of awake humans.


title: Functional network topography of the medial entorhinal cortex

abstract:
The medial entorhinal cortex (MEC) creates a map of local space, based on the firing patterns of grid, head direction (HD), border, and object-vector (OV) cells. How these cell types are organized anatomically is debated. In-depth analysis of this question requires collection of precise anatomical and activity data across large populations of neurons during unrestrained behavior, which neither electrophysiological nor previous imaging methods fully afford. Here we examined the topographic arrangement of spatially modulated neurons in MEC and adjacent parasubiculum using miniaturized, portable two-photon microscopes, which allow mice to roam freely in open fields. Grid cells exhibited low levels of co-occurrence with OV cells and clustered anatomically, while border, HD and OV cells tended to intermingle. These data suggest that grid-cell networks might be largely distinct from those of border, HD and OV cells and that grid cells exhibit strong coupling among themselves but weaker links to other cell types.

Highlights- Grid and object vector cells show low levels of regional co-occurrence
- Grid cells exhibit the strongest tendency to cluster among all spatial cell types
- Grid cells stay separate from border, head direction and object vector cells
- The territories of grid, head direction and border cells remain stable over weeks


title: Performance of temporal and spatial ICA in identifying and removing low-frequency physiological and motion effects in resting-state fMRI

abstract:
Effective separation of signal from noise (including physiological processes and head motion) is one of the chief challenges for improving the sensitivity and specificity of resting-state fMRI (rs-fMRI) measurements and has a profound impact when these noise sources vary between populations. Independent component analysis (ICA) is an approach for addressing these challenges. Conventionally, due to the lower amount of temporal than spatial information in rs-fMRI data, spatial ICA (sICA) is the method of choice. However, with recent developments in accelerated fMRI acquisitions, the temporal information is becoming enriched to the point that the temporal ICA (tICA) has become more feasible. This is particularly relevant as physiological processes and motion exhibit very different spatial and temporal characteristics when it comes to rs-fMRI applications, leading us to conduct a comparison of the performance of sICA and tICA in addressing these types of noise. In this study, we embrace the novel practice of using theory (simulations) to guide our interpretation of empirical data. We find empirically that sICA can identify more noise-related signal components than tICA. However, on the merit of functional-connectivity results, we find that while sICA is more adept at reducing whole-brain motion effects, tICA performs better in dealing with physiological effects. These interpretations are corroborated by our simulation results. The overall message of this study is that if ICA denoising is to be used for rs-fMRI, there is merit in considering a hybrid approach in which physiological and motion-related noise are each corrected for using their respective best-suited ICA approach.

Impact StatementResting-state fMRI is influenced by low-frequency physiological noise and head motion. Independent component analysis (ICA) is becoming increasingly relied on for reducing these influences, but the utility of spatial and temporal ICA remains unclear. We conducted a comparison of the performance of these two ICA types, using physiological-noise and motion time courses as reference. We found that spatial ICA is more adept at reducing motion effects, while temporal ICA performs better in dealing with physiological effects. We believe these findings provide much-needed clarity on the role of ICA, and recommend using a hybrid of tICA and sICA as a paradigm shift in resting-state fMRI.


title: Distinct trans-placental effects of maternal immune activation by TLR3 and TLR7 agonists: implications for schizophrenia risk

abstract: Exposure to infection in utero predisposes towards psychiatric diseases such as autism, depression and schizophrenia in later life. The mechanisms involved are typically studied by administering mimetics of double-stranded (ds) RNA viral or bacterial infection to pregnant rats or mice. The effect of single-stranded (ss) virus mimetics has been largely ignored, despite evidence linking prenatal ss virus exposure specifically with psychiatric disease. Understanding the effects of gestational ss virus exposure has become even more important with the current SARS-CoV-2 pandemic. In this study, in pregnant mice, we compare directly the effects, on the maternal blood, placenta and the embryonic brain, of maternal administration of ds-virus mimetic poly I:C (to activate toll-like receptor 3, TLR3) and ss-virus mimetic resiquimod (to activate TLR7/8). We find that, 4h after the administration, both poly I:C and resiquimod elevated the levels of IL-6, TNF, and chemokines including CCL2 and CCL5, in maternal plasma. Both agents also increased placental mRNA levels of IL-6 and IL-10, but only resiquimod increased placental TNF mRNA. In foetal brain, poly I:C produced no detectable immune-response-related increases, whereas pronounced increases in cytokine (e.g. Il-6, Tnf) and chemokine (e.g. Ccl2, Ccl5) expression were observed with maternal resiquimod administration. The data show substantial differences between the effect of maternal exposure to a TLR7/8 activator as compared to a TLR3 activator. There are significant implications for future modelling of diseases where maternal ss virus exposure contributes to environmental disease risk in offspring.


title: Distinct ground state and activated state modes of spiking in forebrain neurons

abstract: Neuronal firing patterns have significant spatiotemporal variability with no agreed upon theoretical framework. Using a combined experimental and modeling approach, we found that spike interval statistics can be described by discrete modes of activity. Of these, a "ground state" (GS) mode of low-rate spiking is universal among forebrain excitatory neurons and characterized by irregular spiking at neuron-specific rates. In contrast, "activated state" (AS) modes consist of spiking at characteristic timescales and regularity that are specific to neuron populations in a given region and brain state. The majority of spiking is contributed by GS mode, while neurons can transiently switch to AS spiking in response to stimuli or in coordination with population activity patterns. We hypothesize that GS spiking serves to maintain a persistent backbone of neuronal activity while AS modes support communication functions.


title: Homeostatic regulation of presynaptic NMDA receptor subunit composition modulates action potential driven Ca2+ influx into boutons setting the bandwidth for information transfer

abstract: N-Methyl-D-aspartate receptors (NMDARs) play a pivotal role in both short and long-term plasticity. While the functional role of postsynaptic NMDARs is well established, a framework of presynaptic NMDAR (preNMDAR) function is missing. Differences in subunit composition of preNMDARs are documented at central synapses, raising the possibility that subtype composition plays a role in transmission performance. Here, we use electrophysiological recordings at Schaffer collateral - CA1 synapses and Ca2+ imaging coupled to focal glutamate uncaging at boutons of CA3 pyramidal neurones to reveal two populations of presynaptic NMDARs that contain either the GluN2A or GluN2B subunit. Activation of the GluN2B population decreases action potential (AP)-evoked Ca2+ influx via modulation of small conductance Ca2+-activated K+ channels (SK-channels) while activation of the GluN2A containing population does the opposite. Moreover, the level of functional expression of each receptor population can be homeostatically modified, bidirectionally affecting short-term facilitation during burst firing, thus providing a capacity for a fine adjustment of the presynaptic integration time window and therefore the bandwidth of information transfer.


title: Different DGAT1s show different TAG synthesis abilities and a specific amino acid substitution enhances oil accumulation

abstract:
Triacylglycerols (TAGs) are the major component of plant storage lipids. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the Kennedy pathway, and responsible for plant oil accumulation. We previously found DGAT activity of Vernonia galamensis DGAT1 was distinctively higher than that of Arabidopsis thaliana DGAT1 and soybean DGAT1 in a yeast microsome assay. In this study, the DGAT1 cDNAs of Arabidopsis, Vernonia, soybean, and castor were introduced into Arabidopsis (ecotype Col-0). All Vernonia DGAT1 expressing lines showed a significantly higher oil content (average 49% relative increase compared to the wild type) followed by soybean, and castor. Most Arabidopsis DGAT1 over-expressing lines did not show a significant increase. In addition to these four DGAT1s, sunflower, Jatropha and sesame DGAT1 genes were introduced into the TAG biosynthesis defective yeast mutant (H1246). In the yeast expression culture, DGAT1s from Arabidopsis, castor, and soybean only slightly increased TAG content, however, DGAT1s from Vernonia, sunflower, Jatropha, and sesame remarkably increased TAG content more than 10 times higher than the former three DGAT1s. Three amino acid residues were characteristically common in the latter four DGAT1s. Using soybean DGAT1, these amino acid substitutions by site-directed mutagenesis was performed and analyzed. These substitutions substantially increased the TAG content.

HighlightDGAT1s from several plant species were tested their TAG accumulation promotion in Arabidopsis and yeast. They were divided into high and low function and single amino acid substitution enhanced function

category: plant biology


title: Methodological considerations in analyzing synchronization of resting-state brain networks with the intrinsic electrical rhythm of the stomach: Advantages of weighted phase-locking

abstract:
PurposeTo evaluate the amplitude-weighted phase-locking value (awPLV) as a measure of synchronization of brain resting-state networks (RSNs) with the gastric basal electrical rhythm (BER).

MethodsA recent study combined rsfMRI with concurrent cutaneous electrogastrography (EGG), in a highly-sampled individual who underwent 22 scanning sessions (two 15-minute runs per session) at 3.0 Tesla. After excluding three sessions due to weak EGG signals, 9.5 hours of data remained, from which 18 RSNs were estimated using spatial independent component analysis. Previously, using the phase-locking value (PLV), three of the 18 RSNs were determined to be synchronized with the BER. However, RSN power fluctuations in the gastric frequency band could reduce sensitivity of PLV. Accordingly, the current reanalysis used awPLV to unweight contributions from low power epochs. Mismatched EGG and rsfMRI data (from different days) served as surrogate data; for each RSN, empirical awPLV was compared with chance-level awPLV using a Wilcoxon rank test. P-values were adjusted using with a false discovery rate of 0.05. Additionally, simulations were performed to compare PLV and awPLV error rates under settings with a known ground truth.

ResultsSimulations show high false-negative rates when using PLV, but not awPLV. Reanalysis of the highly-sampled individual data using awPLV indicates that 11 of the 18 RSNs were synchronized with the BER.

ConclusionSimulations indicate that awPLV is a more sensitive measure of stomach/brain synchronization than PLV. Reanalysis results imply communication between the enteric nervous system and brain circuits not typically considered responsive to gastric state or function.


title: Connectome-Based Smoothing (CBS): Concepts, methods, and evaluation

abstract:
Structural connectomes are increasingly mapped at high spatial resolutions comprising many hundreds--if not thousands--of network nodes. However, high-resolution connectomes are particularly susceptible to image registration misalignment, tractography artifacts, and noise, all of which can lead to reductions in connectome accuracy and test-retest reliability. We investigate a network analogue of image smoothing to address these key challenges. Connectome Spatial Smoothing (CSS) involves jointly applying a carefully chosen smoothing kernel to the two endpoints of each tractography streamline, yielding a spatially smoothed connectivity matrix. We develop computationally efficient methods to perform CSS using a matrix congruence transformation and evaluate a range of different smoothing kernel choices on CSS performance. We find that smoothing substantially improves the identifiability, sensitivity, and test-retest reliability of high-resolution connectivity maps, though at a cost of increasing storage burden. For atlas-based connectomes (i.e. low-resolution connectivity maps), we show that CSS marginally improves the statistical power to detect associations between connectivity and cognitive performance, particularly for connectomes mapped using probabilistic tractography. CSS was also found to enable more reliable statistical inference compared to connectomes without any smoothing. We provide recommendations on optimal smoothing kernel parameters for connectomes mapped using both deterministic and probabilistic tractography. We conclude that spatial smoothing is particularly important for the reliability of high-resolution connectomes, but can also provide benefits at lower parcellation resolutions. We hope that our work enables computationally efficient integration of spatial smoothing into established structural connectome mapping pipelines.

HighlightsO_LIWe establish a network equivalent of image smoothing for structural connectomes.
C_LIO_LIConnectome Spatial Smoothing (CSS) improves connectome test-retest reliability, identifiability and sensitivity.
C_LIO_LICSS also facilitates reliable inference and improves power to detect statistical associations.
C_LIO_LIBoth high-resolution and atlas-based connectomes can benefit from CSS.
C_LI


title: Connectome Spatial Smoothing (CSS): concepts, methods, and evaluation


title: Chronic hM4Di-DREADD mediated chemogenetic inhibition of forebrain excitatory neurons in postnatal or juvenile life does not alter adult mood-related behavior

abstract: G-protein coupled receptors (GPCRs) coupled to Gi-signaling, in particular downstream of monoaminergic neurotransmission, are posited to play a key role during developmental epochs (postnatal and juvenile), in shaping the emergence of adult anxio-depressive behaviors and sensorimotor gating. To address the role of Gi-signaling in these developmental windows, we used a CamKII-tTA::TRE hM4Di bigenic mouse line to express the hM4Di-DREADD in forebrain excitatory neurons and enhanced Gi-signaling via chronic administration of the DREADD agonist, CNO in the postnatal (PNCNO: postnatal day 2-14) or juvenile (JCNO: postnatal day 28-40) window. We confirmed that the expression of the HA-tagged hM4Di-DREADD was restricted to CamKII-positive neurons in the forebrain, and administration of CNO in postnatal or juvenile windows evoked inhibition in forebrain circuits of the hippocampus and cortex, as indicated by a decline in expression of the neuronal activity marker, c-fos. hM4Di-DREADD mediated inhibition of CamKII-positive forebrain excitatory neurons in postnatal or juvenile life did not impact the weight profile of mouse pups, and also did not influence the normal ontogeny of sensory reflexes. Further, postnatal or juvenile hM4Di-DREADD mediated inhibition of CamKII-positive forebrain excitatory neurons did not alter anxiety or despair-like behaviors in adulthood, and did not impact sensorimotor gating. Collectively, these results indicate that chemogenetic induction of Gi-signaling in CamKII-positive forebrain excitatory neurons in postnatal and juvenile temporal windows does not appear to impinge on the programming of anxio-depressive behaviors in adulthood.


title: A genetic compensatory mechanism regulated by c-Jun and Mef2d modulates the expression of distinct class IIa HDACs to ensure peripheral nerve myelination and repair

abstract: The class IIa histone-deacetylases (HDACs) have pivotal roles in the development of different tissues. Of this family, Schwann cells express HDAC4, 5 and 7 but not HDCA9. Here we show that a transcription factor regulated genetic compensatory mechanism within this family of proteins, blocks negative regulators of myelination ensuring peripheral nerve developmental myelination and remyelination after injury. Thus, when HDAC4 and 5 are knocked-out from Schwann cells, a c-Jun dependent mechanism induces the compensatory overexpression of HDAC7 permitting, although with a delay, the formation of a myelin sheath. When HDAC4,5 and 7 are simultaneously removed, the Myocyte- specific enhancer-factor d (Mef2d) binds to the promoter and induces the de novo expression of HDAC9, and although several melanocytic- lineage genes are mis- expressed and Remak bundle structure is disrupted, myelination proceeds after a long delay. Thus, our data unveil a finely tuned compensatory mechanism within the class IIa HDAC family, coordinated by distinct transcription factors, that guarantees the ability of Schwann cells to myelinate during development and remyelinate after nerve injury.

category: cell biology


title: Molecular insights into RNA recognition and gene regulation by the TRIM-NHL protein Mei-P26

abstract: The TRIM-NHL protein Meiotic P26 (Mei-P26) acts as a regulator of cell fate in Drosophila. Its activity is critical for ovarian germline stem cell maintenance, differentiation of oocytes and spermatogenesis. Mei-P26 functions as a post-transcriptional regulator of gene expression, however, the molecular details of how its NHL domain selectively recognizes and regulates its mRNA targets have remained elusive. Here, we present the crystal structure of the Mei-P26 NHL domain at 1.6 [A] resolution and identify key amino acids that confer substrate specificity and distinguish Mei-P26 from closely related TRIM-NHL proteins. Furthermore, we identify mRNA targets of Mei-P26 in cultured Drosophila cells and show that Mei-P26 can act as either a repressor or activator of gene expression on different RNA targets. Our work reveals the molecular basis of RNA recognition by Mei-P26 and the fundamental functional differences between otherwise very similar TRIM-NHL proteins.


title: Opening the side exit pores of ClpP by lowering the pH of proteolytic chamber coupled with substrate hydrolysis

abstract: The ClpP serine peptidase is a tetradecameric degradation machine involved in many physiological processes. It becomes a competent ATP-dependent protease with Clp-ATPases. Small chemical compounds, acyldepsipeptides (ADEPs), are known to cause dysregulation and activation of ClpP without ATPases, and have potential as novel antibiotics. Previously, structural studies of ClpP from various species revealed the structural details, conformational changes, and activation mechanism. Although product release by the side exit pores has been proposed, the detailed driving force for product release remains elusive. Here, we report crystal structures of ClpP from Bacillus subtilis (BsClpP) in unforeseen ADEP-bound states. Cryo-electron microscopy structures revealed various conformational states at different pH conditions. To understand the conformational change for product release, we investigated the relationship between substrate hydrolysis and the pH lowering process. Our data, together with previous findings, provide insight into the molecular mechanism of product release by ClpP self-compartmentalizing protease.


title: PCDH7 Promotes Cell Migration by Regulating Myosin Activity

abstract: Cell migration requires spatiotemporally coordinated activities of multicomponent structures including the actomyosin cortex, plasma membrane, adhesion complexes and the polarity proteins. How they function together to drive this complex dynamic process remains an outstanding question. Here, we show that a member of the protocadherin family, PCDH7 displays a polarized localization in migratory cells with a dynamic enrichment at the leading and rear edges. Perturbation of PCDH7 interferes with the migration of nontransformed retinal pigment epithelial cells and invasion of cancer cells. The overexpression of PCDH7 enhances the migration capability of cortical neurons in vivo. PCDH7 interacts with the myosin phosphatase subunits MYPT1 and PP1c{beta} and it enhances the phosphorylation of regulatory light chain and ERM at the leading and rear edges of migratory cells. The chemical inhibition of phosphatase activity recovers migration phenotypes of PCDH7 knockout cells. We propose that PCDH7 regulate phosphorylation thus activity of myosin and ERM at the polarized cortex by quenching myosin phosphatase that results in a higher persistence of migrating cells. Collectively, our study suggests a new mechanism for the spatial coordination of plasma membrane and the cortex during cell migration.


title: Admp regulates tail bending by controlling the intercalation of the ventral epidermis through myosin phosphorylation.

abstract:
The tails transient but pronounced ventral bending is found in many chordate embryos and provides an interesting model of how tissue interactions contribute to the overall shape (Lu et al., 2020). Here, we identify one of the upstream regulators of tail bending. Admp knock-down embryo (Admp MO) completely inhibited ventral tail bending. Admp MO showed specific inhibition of the cell-cell intercalation ventral epidermis. In the early stages of the intercalation, each ventral epidermal cell exhibited a boat-shaped morphology (boat cell) with a narrow apical surface in which phosphorylated myosin (pMLC) accumulated. In the Admp MO, the number of the boat cells reduced and pMLC was observed at the basal side in the Admp MO embryo. Finally, laser cutter experiments showed a pMLC-dependent relaxation of the ventral tail bending, which was observed when each ventral midline epidermal cell was cut along the apico-basal axis. Based on these results, we propose a novel function for Admp, in which Admp controls the pMLC translocation to the apical side of the ventral epidermal cells and leads the tail bending by ventral resistance to the notochord extension.

Summary StatementAdmp is an upstream regulator of the tail bending in the protochordate Ciona tailbud embryo, regulating tissue polarity of the ventral midline epidermis by the translocation of the phosphorylated myosin.


title: Admp regulates tail bending by controlling ventral epidermal cell polarity via phosphorylated myosin translocation.


title: Unique genetic features of the naked mole-rat's THADA gene

abstract: Thyroid Adenoma Associated (THADA) is a protein-coding gene that maps to chromosomal band 2p21 and first has been described as a target of recurrent translocation partner in thyroid tumors. Many genome-wide association studies have revealed an association between THADA and two frequent human diseases, i.e. type 2 diabetes and polycystic ovary syndrome. Nevertheless, the function of its protein is not been completely understood. However, recent evidence suggests that in a Drosophila model THADA can act as a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)-interacting protein which uncouples SERCA from this function. Once being uncoupled, SERCA produces an increased amount of heat without transporting calcium thus triggering nonshivering thermogenesis. This data prompted us to compare human THADA with that of 65 other eutherian mammals. This includes a comparison of THADA of a variety of eutherian mammals with that of the naked-mole rat (Heterocephalus glaber) which is known to display unique features of thermoregulation compared to other mammals. Our analysis revealed five positions where only the naked-mole rat presented differences. These latter positions included four single amino acid substitutions and one unique deletion of six or seven amino acids, respectively, between residues 858 and 859. In future studies these changes will be analyzed further in detail for their functional relevance.


title: Semi-automated analysis of an optical ATP indicator in neural synapses

abstract:
SignificanceThe firefly enzyme luciferase has been used in a wide range of biological assays, including bioluminescence imaging of ATP. The biosensor Syn-ATP utilizes subcellular targeting of luciferase to nerve terminals for optical measurement of ATP in this compartment. Manual analysis of Syn-ATP signals is challenging due to signal heterogeneity and cellular motion in long imaging sessions. Here, we have leveraged machine learning tools to develop a method for analysis of bioluminescence images.

AimOur goal was to create a semi-automated pipeline for analysis of bioluminescence imaging to improve measurements of ATP content in nerve terminals.

ApproachWe developed an image analysis pipeline that applies machine learning toolkits to distinguish neurons from background signals, and excludes neural cell bodies, while also incorporating user input.

ResultsSide-by-side comparison of manual and semi-automated image analysis demonstrated that the latter improves precision and accuracy of ATP measurements.

ConclusionsOur method streamlines data analysis and reduces user-introduced bias, thus enhancing the reproducibility and reliability of quantitative ATP imaging in nerve terminals.


title: Semi-automated analysis of an optical ATP indicator in neurons


title: Copeptin response to acute physical stress during hypohydration: An exploratory secondary analysis

abstract:
PurposePlasma copeptin (a surrogate marker of arginine vasopressin) is increasingly being used as a marker of stress in several research and clinical contexts. However, the response to an acute physical stressor in healthy adults has not yet been tested meaning it is unclear whether copeptin reflects dynamic changes in stress or whether this is moderated by different basal copeptin concentrations and how this relates to other stress hormones.

MethodsSecondary data analysis in a subsample of participants (n = 7; n = 1 woman) who opted-in for muscle biopsies in a randomised crossover study investigating the effects of acute hypohydration (HYPO) versus rehydration (RE) on metabolism.

ResultsPlasma copeptin responded to the muscle biopsy stress stimulus, with a similar magnitude of difference according to basal concentrations during HYPO and RE; however, the peak was higher and concentrations typically took longer to return to baseline during HYPO. Despite large differences in copeptin concentrations, adrenocorticotropin hormone (ACTH) and cortisol showed a similar trends in response to the biopsies, regardless of hydration status.

ConclusionCopeptin responded dynamically to an acute physical stressor (muscle biopsy). HYPO induced higher basal copeptin concentrations compared to RE, and resulted in a higher and prolonged copeptin response. Further research should investigate the mechanisms underlying the lack of differences in ACTH and cortisol according to hydration status.


title: Hippocampal representations switch from errors to predictions during acquisition of predictive associations

abstract: We constantly exploit the statistical regularities in our environment to help guide our perception. The hippocampus has been suggested to play a pivotal role in both learning environmental statistics, as well as exploiting them to generate perceptual predictions. However, it is unclear how the hippocampus balances encoding new predictive associations with the retrieval of existing ones. Here, we present the results of two high resolution human fMRI studies (N=24 for both experiments) directly investigating this. Participants were exposed to auditory cues that predicted the identity of an upcoming visual shape (with 75% validity). Using multivoxel decoding analysis, we found that the hippocampus initially preferentially represented unexpected shapes (i.e., those that violated the cue regularities), but later switched to representing the cue-predicted shape regardless of which was actually presented. These findings demonstrate that the hippocampus in involved both acquiring and exploiting predictive associations, and switches between these modes depending on whether learning is ongoing or complete.


title: HCN2 in cholinergic interneurons of the nucleus accumbens mediates reward response

abstract: Cholinergic interneurons (ChIs) of the nucleus accumbens (NAc) are important for mediating the behavioral response to rewarding stimuli. A major role for these cells is to regulate dopamine (DA) transmission by activating cholinergic receptors at local DAergic nerve terminals. However, the mechanisms that enable cholinergic neurons to enhance DA release in response to reward remain unknown. Here we report that the hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) in NAc ChIs mediates an enhancement in DA signaling in response to rewarding stimuli. The HCN current in NAc ChIs and its modulation by DA, as well as the increase in cholinergic efflux by local cocaine infusion were impaired in mice with deletion of HCN2 in cholinergic cells. Enhancement in the DA efflux and signaling in the NAc in response to rewarding stimuli, as well as cocaine conditioning were also dependent on HCN2 in ChIs. These results provide a mechanistic link between the activity of NAc ChIs and reward encoding.


title: Self-organized emergence of hyaline cartilage in hiPSC-derived multi-tissue organoids

abstract: Despite holding great therapeutic potential, existing protocols for in vitro chondrogenesis and hyaline cartilage production from human induced pluripotent stem cells (hiPSC) are laborious and complex with unclear long-term consequences. Here, we developed a simple xeno- and feeder-free protocol for human hyaline cartilage production in vitro using hydrogel-cultured multi-tissue organoids (MTOs). We investigate gene regulatory networks during spontaneous hiPSC-MTO differentiation using RNA sequencing and bioinformatic analyses. We find the interplays between BMPs and neural FGF pathways are associated with the phenotype transition of MTOs. We recognize TGF-beta/BMP and Wnt signaling likely contribute to the long-term maintenance of MTO cartilage growth and further adoption of articular cartilage development. By comparing the MTO transcriptome with human lower limb chondrocytes, we observe that the expression of chondrocyte-specific genes in MTO shows a strong correlation with fetal lower limb chondrocytes. Collectively, our findings describe the self-organized emergence of hyaline cartilage in MTO, its associated molecular pathways, and its spontaneous adoption of articular cartilage development trajectory.


title: The Siberian wild apple, Malus baccata (L.) Borkh., is an additional contributor to the genomes of cultivated European and Chinese apples

abstract: It is crucial to understand domestication to unravel the evolutionary processes that shape the divergence of populations. Differences in life-history traits have probably led to marked differences in the mode and speed of evolution between trees and annuals, particularly the extent of crop-wild gene flow during domestication. Apple is an iconic tree and major fruit crop grown worldwide. The contribution of wild apple species to the genetic makeup of the cultivated apple genome remains a topic of intense investigations. We used population genomics in combination with SNPs to investigate the contributions of the two known wild apple relatives, Malus sylvestris and Malus sieversii, and a supposed contributor, Malus baccata, to European and Chinese rootstock and dessert genomes, with a focus on the extent of wild-crop gene flow during apple domestication. We showed that the European dessert and rootstock apples form a specific gene pool, whereas the Chinese dessert and rootstock apples were a mixture of three wild gene pools. Coalescent-based inferences and gene flow estimates indicated that M. baccata is an additional contributor to the genome of both European and Chinese cultivated apples through wild-to-crop introgressions. We also confirmed previous results on the contribution of M. sylvestris to the cultivated apple genome, and provided insights into the origin of the apple rootstock. This study further demonstrates the role of gene flow during apple domestication, as seen in other woody perennials, and show that domestication of the apple tree involved several wild apple species.

category: evolutionary biology


title: On the origin and evolution of Drosophila new genes during spermatogenesis

abstract: Origin of functional new genes is a basic biological process that has significant contribution to organismal diversity. Previous studies in both Drosophila and mammals showed that new genes tend to be expressed in testis, and avoid the X chromosome presumably because meiotic sex chromosome inactivation (MSCI). Here we analyze the published single-cell transcriptome data of Drosophila adult testis and find an enrichment of male germline mitotic genes, but an underrepresentation of meiotic genes on the X chromosome. This can be attributed to an excess of autosomal meiotic genes that were derived from their X-linked mitotic progenitors, which provides direct cell-level evidence for MSCI in Drosophila. We reveal that new genes, particularly those produced by retrotransposition, tend to exhibit an expression shift toward late spermatogenesis compared to their parental copies, probably due to the more intensive sperm competition or sexual conflict. Our results dissect the complex factors including the age, the origination mechanisms and the chromosomal locations that influence the new gene origination and evolution in testis, and identify new gene cases that show divergent cell-level expression pattern from their progenitors for future functional studies.


title: Experimental evidence for the gaze-signaling hypothesis: White sclera enhances the visibility of eye gaze direction in humans and chimpanzees

abstract: Hallmark social activities of humans, such as cooperation and cultural learning, involve eye-gaze signaling through joint attentional interaction and ostensive communication. The gaze-signaling and related cooperative-eye hypotheses posit that humans evolved unique external eye morphology, including exposed white sclera (the white of the eye), to enhance the visibility of eye-gaze for conspecifics. However, experimental evidence is still lacking. This study tested the ability of human and chimpanzee participants to detect the eye-gaze directions of human and chimpanzee images in computerized tasks. We varied the level of brightness and size in the stimulus images to examine the robustness of the eye-gaze directional signal against visually challenging conditions. We found that both humans and chimpanzees detected gaze directions of the human eye better than that of the chimpanzee eye, particularly when eye stimuli were darker and smaller. Also, participants of both species detected gaze direction of the chimpanzee eye better when its color was inverted compared to when its color was normal; namely, when the chimpanzee eye has artificial white sclera. White sclera thus enhances the visibility of eye-gaze direction even across species, particularly in visually challenging conditions. Our findings supported but also critically updated the central premises of the gaze-signaling hypothesis.

category: animal behavior and cognition


title: What is unique about the human eye? Comparative image analysis on the external eye morphology of human and nonhuman great apes

abstract: The gaze-signaling hypothesis and the related cooperative-eye hypothesis posit that humans have evolved special external eye morphology, including exposed white sclera (the white of the eye), to enhance the visibility of eye-gaze direction and thereby facilitate conspecific communication through joint-attentional interaction and ostensive communication. However, recent quantitative studies questioned these hypotheses based on new findings that humans are not necessarily unique in certain eye features compared to other great ape species. Therefore, there is currently a heated debate on whether external eye features of humans are distinguished from those of other apes and how such distinguished features contribute to the visibility of eye-gaze direction. This study leveraged updated image analysis techniques to test the uniqueness of human eye features in facial images of great apes. Although many eye features were similar between humans and other species, a key difference was that humans have uniformly white sclera which creates clear visibility of both eye outline and iris -the two essential features contributing to the visibility of eye-gaze direction. We then tested the robustness of the visibility of these features against visual noises such as darkening and distancing and found that both eye features remain detectable in the human eye, while eye outline becomes barely detectable in other species under these visually challenging conditions. Overall, we identified that humans have distinguished external eye morphology among other great apes, which ensures robustness of eye-gaze signal against various visual conditions. Our results support and also critically update the central premises of the gaze-signaling hypothesis.


title: Considering unique, shared, and dominant brain activation in the Visual Word Form Area and Lateral Occipital Cortex: A comparison of separate and combined word reading and picture naming

abstract: Identifying printed words and pictures concurrently is ubiquitous in daily tasks, and so it is important to consider the extent to which reading words and naming pictures may share a cognitive-neurophysiological functional architecture. Two functional magnetic resonance imaging (fMRI) experiments examined whether reading along the left ventral occipitotemporal region (vOT; often referred to as a visual word form area, VWFA) has activation that is overlapping with referent pictures (i.e., both conditions significant and shared, or with one significantly more dominant) or unique (i.e., one condition significant, the other not), and whether picture naming along the right lateral occipital complex (LOC) has overlapping or unique activation relative to referent words. Experiment 1 used familiar regular and exception words (to force lexical reading) and their corresponding pictures in separate naming blocks, and showed dominant activation for pictures in the LOC, and shared activation in the VWFA for exception words and their corresponding pictures (regular words did not elicit significant VWFA activation). Experiment 2 controlled for visual complexity by superimposing the words and pictures and instructing participants to either name the word or the picture, and showed primarily shared activation in the VWFA and LOC regions for both word reading and picture naming, with some dominant activation for pictures in the LOC. Overall, these results highlight the importance of including exception words to force lexical reading when comparing to picture naming, and the significant shared activation in VWFA and LOC serves to challenge specialized models of reading or picture naming.


title: Unique, shared, and dominant brain activation in Visual Word Form Area and Lateral Occipital Complex during reading and picture naming


title: A novel B.1.1.523 SARS-CoV-2 variant that combines many spike mutations linked to immune evasion with current variants of concern

abstract: In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic several variants have emerged that are linked to increased transmissibility and immune evasion. These variants are recognized as variants of concern (VOC). In this study, we describe a B.1.1.523 variant that shares many spike mutations with current VOC. Receptor-binding domain mutations E484K and S494P were observed but also a deletion (position 156-158) in the N-terminal antigenic supersite that is similar to the delta-variant. These mutations are linked to immune evasion in VOC that could lead to less effective vaccines. This variant has been reported in various different countries and continents despite the dominance of B.1.1.7 (alpha) and B.1.617.2 (delta) variant. Furthermore, the B.1.1.523 pangolin lineage as a whole is recognized as a variant under monitoring since 14th of July 2021.

category: microbiology


title: IL13Pred: A method for predicting immunoregulatory cytokine IL-13 inducing peptides for managing COVID-19 severity.

abstract:
Interleukin 13 (IL-13) is an immunoregulatory cytokine that is primarily released by activated T-helper 2 cells. It induces the pathogenesis of many allergic diseases, such as airway hyperresponsiveness, glycoprotein hypersecretion and goblet cell hyperplasia. IL-13 also inhibits tumor immunosurveillance, which leads to carcinogenesis. In recent studies, elevated IL-13 serum levels have been shown in severe COVID-19 patients. Thus it is important to predict IL-13 inducing peptides or regions in a protein for designing safe protein therapeutics particularly immunotherapeutic. This paper describes a method developed for predicting, designing and scanning IL-13 inducing peptides. The dataset used in this study contain experimentally validated 313 IL-13 inducing peptides and 2908 non-inducing homo-sapiens peptides extracted from the immune epitope database (IEDB). We have extracted 95 key features using SVC-L1 technique from the originally generated 9165 features using Pfeature. Further, these key features were ranked based on their prediction ability, and top 10 features were used for building machine learning prediction models. In this study, we have deployed various machine learning techniques to develop models for predicting IL-13 inducing peptides. These models were trained, test and evaluated using five-fold cross-validation techniques; best model were evaluated on independent dataset. Our best model based on XGBoost achieves a maximum AUC of 0.83 and 0.80 on the training and independent dataset, respectively. Our analysis indicate that certain SARS-COV2 variants are more prone to induce IL-13 in COVID-19 patients. A standalone package as well as a web server named  IL-13Pred has been developed for predicting IL-13 inducing peptides (https://webs.iiitd.edu.in/raghava/il13pred/).

Key PointsO_LIInterleukin-13, an immunoregulatory cytokine plays an important role in increasing severity of COVID-19 and other diseases.
C_LIO_LIIL-13Pred is a highly accurate in-silico method developed for predicting the IL-13 inducing peptides/ epitopes.
C_LIO_LIIL-13 inducing peptides are reported in various SARS-CoV2 strains/variants proteins.
C_LIO_LIThis method can be used to detect IL-13 inducing peptides in vaccine candidates.
C_LIO_LIUser friendly web server and standalone software is freely available for IL-13Pred
C_LI

Authors BiographyO_LIShipra Jain is currently working as Ph.D. in Computational Biology from Department of Computational Biology, Indraprastha Institute of Information Technology, New Delhi, India.
C_LIO_LIAnjali Dhall is currently working as Ph.D. in Computational Biology from Department of Computational Biology, Indraprastha Institute of Information Technology, New Delhi, India.
C_LIO_LISumeet Patiyal is currently working as Ph.D. in Computational Biology from Department of Computational Biology, Indraprastha Institute of Information Technology, New Delhi, India.
C_LIO_LIGajendra P. S. Raghava is currently working as Professor and Head of Department of Computational Biology, Indraprastha Institute of Information Technology, New Delhi, India.
C_LI


title: Mechanistic Insights into the Effects of Key Mutations on SARS-CoV-2 RBD-ACE2 Binding

abstract: Some recent SARS-CoV-2 variants appear to have increased transmissibility than the original strain. An underlying mechanism could be the improved ability of the variants to bind receptors on target cells and infect them. In this study, we provide atomic-level insight into the binding of the receptor binding domain (RBD) of the wild-type SARS-CoV-2 spike protein and its single (N501Y), double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants to the human ACE2 receptor. Using extensive all-atom molecular dynamics simulations and advanced free energy calculations, we estimate the associated binding affinities and binding hotspots. We observe significant secondary structural changes in the RBD of the mutants, which lead to different binding affinities. We find higher binding affinities of the double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants than the wild type and the N501Y variant, which could contribute to the higher transmissibility of recent variants containing these mutations.

category: biophysics


title: Gene networks under circadian control exhibit diurnal organization in primate organs

abstract: Mammalian organs are individually controlled by autonomous circadian clocks. At the molecular level, this process is defined by the cyclical co-expression of both core transcription factors and off-target genes across time. While interactions between these molecular clocks are likely necessary for proper homeostasis, these features remain undefined. Here, we utilize integrative analysis of a baboon diurnal transcriptome atlas to characterize the properties of gene networks under circadian control. We found that 53.4% (8,120) of baboon genes are rhythmically expressed body-wide. In addition, >30% of gene-gene interactions exhibit periodic co-expression patterns, with core circadian genes more cyclically co-expressed than others. Moreover, two basic network modes were observed at the systems level: daytime and nighttime mode. Daytime networks were enriched for genes involved in metabolism, while nighttime networks were enriched for genes associated with growth and cellular signaling. A substantial number of diseases only form significant disease modules at either daytime or nighttime. In addition, we found that 216 of 313 genes encoding products that interact with SARS-CoV-2 are rhythmically expressed throughout the body. Importantly, more than 80% of SARS-CoV-2 related genes enriched modules are rhythmically expressed, and have significant network proximities with circadian regulators. Our data suggest that synchronization amongst circadian gene networks is necessary for proper homeostatic functions and circadian regulators have close interactions with SARS-CoV-2 infection.

category: systems biology


title: SARS-CoV-2 preS dTM vaccine booster candidates increase functional antibody responses and cross-neutralization against SARS-CoV-2 variants of concern in non-human primates

abstract:
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that partly evade neutralizing antibodies has raised concerns of reduced vaccine effectiveness and increased infection. We previously demonstrated in preclinical models and in human clinical trials that our SARS-CoV-2 recombinant spike protein vaccine adjuvanted with AS03 (CoV2 preS dTM-AS03) elicits robust neutralizing antibody responses in naive subjects. Here, the objective was to document the potency of various booster vaccine formulations in macaques previously vaccinated with mRNA or protein subunit vaccine candidates.

We show that one booster dose of AS03-adjuvanted CoV2 preS dTM, D614 (parental) or B.1.351 (Beta), in monovalent or bivalent (D614 + B.1.351) formulations, significantly boosted pre-existing neutralizing antibodies and elicited high and stable cross-neutralizing antibodies covering the four known SARS-CoV-2 variants of concern (Alpha, Beta, Gamma and Delta) and, unexpectedly, SARS-CoV-1, in primed macaques. Interestingly, the non-adjuvanted CoV2 preS dTM B.1.351 vaccine formulation also significantly boosted and broadened the neutralizing antibody responses.

Our findings show that these vaccine candidates used as a booster have the potential to offer cross-protection against a broad spectrum of variants. This has important implications for vaccine control of SARS-CoV-2 variants of concern and informs on the benefit of a booster with our vaccine candidates currently under evaluation in phase 2 and 3 clinical trials (NCT04762680 and NCT04904549).


title: A BioID-derived proximity interactome for SARS-CoV-2 proteins

abstract: The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how SARS-CoV-2 viral proteins behave in host cells can reveal underlying mechanisms of pathogenesis and assist in development of antiviral therapies. Here we use BioID to map the SARS-CoV-2 virus-host interactome using human lung cancer derived A549 cells expressing individual SARS-CoV-2 viral proteins. Functional enrichment analyses revealed previously reported and unreported cellular pathways that are in association with SARS-CoV-2 proteins. We have also established a website to host the proteomic data to allow for public access and continued analysis of host-viral protein associations and whole-cell proteomes of cells expressing the viral-BioID fusion proteins. Collectively, these studies provide a valuable resource to potentially uncover novel SARS-CoV-2 biology and inform development of antivirals.


title: Probing differences in gene essentiality between the human and animal adapted lineages of the Mycobacterium tuberculosis complex using TnSeq

abstract: Members of the Mycobacterium tuberculosis complex (MTBC) show distinct host adaptations, preferences and phenotypes despite being >99% identical at the nucleic acid level. Previous studies have explored gene expression changes between the members, however few studies have probed differences in gene essentiality. To better understand the functional impacts of the nucleic acid differences between Mycobacterium bovis and Mycobacterium tuberculosis we used the Mycomar T7 phagemid delivery system to generate whole genome transposon libraries in laboratory strains of both species and compared the essentiality status of genes during growth under identical in vitro conditions. Libraries contained insertions in 54% of possible TA sites in M. bovis and 40% of those present in M. tuberculosis, achieving similar saturation levels to those previously reported for the MTBC. The distributions of essentiality across the functional categories were similar in both species. 527 genes were found to be essential in M. bovis whereas 477 genes were essential in M. tuberculosis and 370 essential genes were common in both species. CRISPRi was successfully utilised in both species to determine the impacts of silencing genes including wag31, a gene involved in peptidoglycan synthesis and Rv2182c/Mb2204c, a gene involved in glycerophospholipid metabolism. We observed species specific differences in the response to gene silencing, with the inhibition of expression of Mb2204c in M. bovis showing significantly less growth impact than silencing its ortholog (Rv2182c) in M. tuberculosis. Given that glycerophospholipid metabolism is a validated pathway for antimicrobials, our observations suggest that target vulnerability in the animal adapted lineages cannot be assumed to be the same as the human counterpart. This is of relevance for zoonotic tuberculosis as it implies that the development of antimicrobials targeting the human adapted lineage might not necessarily be effective against the animal adapted lineage. The generation of a transposon library and the first reported utilisation of CRISPRi in M. bovis will enable the use of these tools to further probe the genetic basis of survival under disease relevant conditions.


title: A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems.

abstract: Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein reversed phase ion pair high performance liquid chromatography (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity analytical techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, an electrophilic impurity derived from the ionizable cationic lipid component is shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidation and subsequent hydrolysis of the tertiary amine. It thus remains critical to ensure robust analytical methods and stringent manufacturing control to ensure mRNA stability and high activity in LNP delivery systems.