title: A Systematic Evaluation of the Computational Tools for Ligand-receptor-based Cell-Cell Interaction Inference

abstract: Cell-cell interactions (CCIs) are essential for multicellular organisms to coordinate biological processes and functions. Many molecules and signaling processes can mediate CCIs. One classical type of CCI mediator is the interaction between secreted ligands and cell surface receptors, i.e., ligand-receptor (LR) interaction. With the recent development of single-cell technologies, a large amount of single-cell RNA Sequencing (scRNA-Seq) data has become widely available. This data availability motivated the single-cell-resolution study of CCIs, particularly LR-based CCIs. Dozens of computational methods and tools have been developed to predict CCIs by identifying LR-based CCIs. Many of these tools have been theoretically reviewed. However, there is little study on current LR-based CCI prediction tools regarding their performance and running results on public scRNA-Seq datasets. In this work, to fill this gap, we tested and compared nine of the most recent computational tools for LR-based CCI prediction. We used fifteen mouse scRNA-Seq samples that correspond to nearly 100K single cells under different experimental conditions for testing and comparison. Besides briefing the methodology used in these nine tools, we summarized the similarities and differences of these tools in terms of both LR prediction and CCI inference between cell types. We provided insight into using these tools to make meaningful discoveries in understanding cell communications.


title: A Novel Prognosis Model based on Comprehensive Analysis of Pyroptosis-Related Genes in Breast Cancer

abstract:
BackgroundPyroptosis plays a dual role in cancer. It can not only induce chronic tumor necrosis, but also stimulate acute inflammation to enhance immune response. However, the study of cell pyroptosis in Breast Cancer (BC) is still limited.

MethodsA total of 26 pyroptosis-related differential genes were obtained, and PPI network and correlation analysis were demonstrated as well. Besides, 3 pyroptosis-related prognostic genes were collected by the univariate COX analysis. Through these 3 genes, patients were clustered into 2 sub-types by the Consensus clustering, then the EM and XMeans were also carried out to recheck the result. Furthermore, the significant prognosis-related genes were obtained by the Random Forest and LASSO analysis. Based on the above, the corresponding weights were calculated through the univariate and multivariate Cox analysis, and the prognostic model was constructed. In addition, the ROC curve, risk curve, PCA, t-SNE, COX, GO, KEGG and ssGSEA were analyzed, respectively.

ResultsThrough the verification of 3 different clustering algorithms, 2 sub-types of BC were obtained. Furthermore,  ELOVL2,  IGLV6-57,  FGBP1,  HLA-DPB2 with weights of -0.183, -0.101, -0.227 and -0.254 were employed to establish the prognostic model. Validation shows that our model has good effect and the Go KEGG and Immune microenvironment analysis showed the enrichment of the differential genes.

ConclusionOur study identified 4 genes that are closely correlated with the overall survival of BC patients, and the prognostic model can effectively divide patients into high- and low-risk groups, which may have certain guiding significance for prognosis.

category: bioinformatics


title: SARS-CoV-2 mutations affect proteasome processing to alter CD8+ T cell responses

abstract:
Viral CD8+ epitopes are generated by the cellular turnover of viral proteins, predominantly by the proteasome. Mutations located within viral epitopes can result in escape from memory T cells but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two of the most dominant SARS-CoV-2 nucleoprotein CD8+ epitopes, we identified mutations in epitope flanking regions and investigated the contribution of these mutations to antigen processing and T cell activation using SARS-CoV-2 nucleoprotein transduced B cell lines and in vitro proteasomal processing of peptides. We found that decreased NP9-17-B*27:05 CD8+ T cell responses to the NP-Q7K mutation correlated with lower epitope surface expression, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103N/Y mutations flanking the NP9-17-B*27:05 and NP105-113-B*07:02 epitopes, respectively, increased CD8+ T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on antigen processing and presentation, thereby contributing to escape from immunodominant T cell responses. Alternatively, mutations could enhance antigen processing and efficacy of T cell recognition, opening new avenues for improving future vaccine designs.

One Sentence SummaryNatural mutations in the flanking regions of known immunodominant SARS-CoV-2 nucleoprotein epitopes can decrease CD8+ T cell responses leading to partial escape.

category: immunology


title: SARS-CoV-2 and its variants, but not Omicron, induces thymic atrophy and impaired T cell development

abstract: Pathogenic infections cause thymic atrophy, perturb thymic-T cell development and alter immunological response. Previous studies reported dysregulated T cell function and lymphopenia in coronavirus disease-19 (COVID-19) patients. However, immune-pathological changes, in the thymus, post severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection have not been elucidated. Here, we report SARS-CoV-2 infects thymocytes, depletes CD4+CD8+ (double positive; DP) T cell population associated with an increased apoptosis of thymocytes, which leads to severe thymic atrophy in K18-hACE2-Tg mice. CD44+CD25-T cells were found to be enriched in infected thymus, indicating an early arrest in the T cell developmental pathway. Further, Interferon gamma (IFN-{gamma}) was crucial for thymic atrophy, as anti-IFN-{gamma} antibody neutralization rescued the loss of thymic involution. Therapeutic use of remdesivir (prototype anti-viral drug) was also able to rescue thymic atrophy. While Omicron variant of SARS-CoV2 caused marginal thymic atrophy, delta variant of SARS-CoV-2 exhibited most profound thymic atrophy characterized by severely depleted DP T cells. Recently characterized broadly SARS-CoV-2 neutralizing monoclonal antibody P4A2 was able to rescue thymic atrophy and restore thymic developmental pathway of T cells. Together, we provide the first report of SARS-CoV-2 associated thymic atrophy resulting from impaired T cell developmental pathway and also explains dysregulated T cell function in COVID-19.


title: Prostaglandins and calprotectin are genetically and functionally linked to the Inflammatory Bowel Diseases

abstract:
BackgroundGenome wide association studies (GWAS) have identified and validated more than 200 genomic loci associated with the inflammatory bowel disease (IBD), although for most the causal gene remains unknown. Given the importance of myeloid cells in IBD pathogenesis, the current study aimed to uncover the role of genes within IBD genetic loci that are endogenously expressed in this cell lineage.

MethodsThe open reading frames (ORF) of 43 genes from IBD-associated loci were expressed via lentiviral transfer in the THP-1 model of human monocytes and the impact of each of these on the cells transcriptome was analyzed using a novel RNA sequencing-based approach. We used a combination of genetic and pharmacologic approaches to validate our findings in the THP-1 line with further validation in human induced pluripotent stem cell (hiPSC)-derived-monocytes.

ResultsThis functional genomics screen provided evidence that genes in four IBD GWAS loci (PTGIR, ZBTB40, SLC39A11 and NFKB1) are involved in controlling S100A8 and S100A9 genes expression, which encode the two subunits of calprotectin (CP). We demonstrated that increasing PTGIR expression and/or stimulating PTGIR signaling resulted in increased CP expression in THP-1. This was further validated in hiPSC-derived monocytes. Conversely, knocking-down PTGIR endogenous expression and/or inhibiting PTGIR signaling led to decreased CP expression. These analyses were extended to the known IBD gene PTGER4, whereby its specific agonist also led to increased CP expression. Furthermore, we demonstrated that the PTGIR and PTGER4 mediated control of CP expression was dependent on signaling via adenylate cyclase and STAT3. Finally, we demonstrated that LPS-mediated increases in CP expression could be potentiated by agonists of PTGIR and PTGER4, or diminished by their antagonists, with an opposite effect on TNF-alpha expression.

ConclusionOur results support a causal role for the PTGIR, ZBTB40, SLC39A11 and NFKB1 genes in IBD, with all four genes regulating the expression of CP in myeloid cells, as well as an important role for the prostacyclin/prostaglandin biogenesis and signaling pathways in IBD susceptibility and pathogenesis.

Author SummaryCrohns Disease and Ulcerative colitis are the two main types of inflammatory bowel diseases (IBD). These are debilitating chronic inflammatory diseases of the digestive tract. IBD pathogenesis is complex and involves multiple different cell types within the intestinal mucosa. While over 200 regions of the genome have been associated with susceptibility to IBD, for most the causal gene remains to be identified. In the current study we have focused on genes from IBD loci that are endogenously expressed in monocytes or macrophages, given the importance of these cells in IBD pathogenesis. Specifically, we modulated the expression of 43 genes from within validated IBD loci, in a human monocyte/macrophage cell line, and determined the impact of this increased expression on the rest of the transcriptome. We found evidence that four of these genes (PTGIR, ZBTB40, SLC39A11 and NFKB1) control the expression of calprotectin, which is a proinflammatory molecule that is used as a marker of intestinal mucosal inflammation. We then elucidated how prostaglandin signaling via prostaglandin receptors PTGIR and PTGER4, another IBD gene, regulates calprotectin expression. This work provides evidence that all five genes are causal and provide the first link between calprotectin and disease susceptibility.

category: genetics


title: D27-LIKE1 carotenoid isomerase has a preference towards trans/cis and cis/cis conversions in Arabidopsis

abstract: Carotenoids are colourful isoprenoids that contribute to a variety of physiological processes in plants. They also function as biosynthesis precursors of abscisic acid (ABA) and strigolactones (SLs). SL biosynthesis starts with the enzymatic conversion of all-trans-{beta}-carotene to 9-cis-{beta}-carotene by the DWARF27 (D27) isomerase. In Arabidopsis, D27 has two closely related paralogs, D27-LIKE1 and D27-LIKE2 which were predicted to be {beta}-carotene-isomerases. Here we characterised D27-LIKE1 and identified some key aspects of its function. Arabidopsis d27-like1-1 mutant does not display any SL or karrikin-deficient traits, however, it exhibits a substantially higher 9-cis-violaxanthin content. In vitro feeding assays with recombinant D27-LIKE1 revealed that the protein exhibits affinity to all {beta}-carotene isoforms but with an exclusive preference towards trans/cis conversions and the interconversion between 9-cis, 13-cis and 15-cis-{beta}-carotene forms. Feeding experiments with zeaxanthin and violaxanthin isomers revealed that D27-LIKE1 accepts these xanthophylls as substrates. The remarkably higher 9-cis-violaxanthin content of the mutant is accompanied by a slightly higher ABA level. Finally, we presented evidence that D27-LIKE1 mRNA is phloem mobile and D27-LIKE1 is an ancient isomerase with long evolutionary history. In summary, we demonstrated that D27-LIKE1 is a carotenoid isomerase with multi-substrate specificity and has a characteristic preference towards the catalysation of cis/cis interconversion of carotenoids. Therefore, D27-LIKE1 is a potential regulator of carotenoid cis pools and eventually, SL and ABA biosynthesis pathways.

category: plant biology


title: NVX-CoV2373 vaccination induces functional SARS-CoV-2-specific CD4+ and CD8+ T cell responses

abstract: NVX-CoV2373 is an adjuvanted recombinant full-length SARS-CoV-2 spike trimer protein vaccine demonstrated to be protective against COVID-19 in efficacy trials. Here we demonstrate that vaccinated subjects made CD4+ T cell responses after one and two doses of NVX-CoV2373, and a subset of individuals made CD8+ T cell responses. Characterization of the vaccine-elicited CD8+T cells demonstrated IFN{gamma} production. Characterization of the vaccine-elicited CD4+ T cells revealed both circulating T follicular helper cells (cTFH) and TH1 cells (IFN{gamma}, TNF, and IL-2) were detectable within 7 days of the primary immunization. Spike-specific CD4+ T cells were correlated with the magnitude of the later SARS-CoV-2 neutralizing antibody titers, indicating that robust generation of CD4+ T cells, capable of supporting humoral immune responses, may be a key characteristic of NVX-CoV2373 which utilizes Matrix-M adjuvant.


title: Oncogenic metabolic rewiring independent of proliferative control in human mammary epithelial cells

abstract: The use of isotopic tracers and metabolic flux analysis (MFA) has unveiled a number of metabolic pathways differentially activated in cancer cells. To support efforts to design effective metabolic therapies for cancer, we sought to distinguish metabolic behavior in cancer versus normal cells growing at the same rate. To this end, we performed 13C-isotope tracing and MFA in human mammary epithelial cells (HMECs) harboring different combinations of oncogenes. By introducing a new quantity termed metabolic flux intensity, defined as pathway flux divided by specific growth rate, we showed that metabolism is dually controlled by proliferation and oncogenotypes. 13C-MFA further revealed that oxidative pentose phosphate pathway (oxPPP), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) were most enhanced in cancerous HMECs. Drug targeting of these pathways selectively reduced growth in the tumorigenic HMEC line. Our study provides direct evidence that metabolism of cancer cells is different than that of normal proliferating cells.

category: biochemistry


title: Recycling of Polymerase Chain Reaction (PCR) kits

abstract: During the outbreak of the SARS-CoV-2 pandemic, PCR (polymerase chain reaction) kits have been used as a common diagnosing method, with daily worldwide usage in the millions. It is well known that at the beginning of the pandemic there was a shortage of PCR kits. So far, the ecosystem of PCR kit is linear use, that is kits are produced, used one-time, and disposed in biolab wastes. Here we show that, to mitigate the risk of future shortages, it is possible to envision recyclable PCR kits, based on a more sustainable use of nucleic acid resources. A PCR kit is mainly composed of primers, nucleotides, and enzymes. In the case of a positive test, the free nucleotides are polymerized onto the primers to form longer DNA strands. Our approach depolymerizes such strands keeping the primers and regenerating the nucleotides, i.e., returning the nucleic acid materials to the original state. The polymerized long DNA strands are hydrolyzed into nucleotides monophosphates that are then phosphorylated in triphosphates using a method that is a development of a recently published one. We used oligonucleotides with 3-terminal phosphorothioate (PS) backbone modification as non-hydrolysable PCR primers, so to undergo the recycling process unchanged. We have successfully recycled both PCR primers ([~]65% yield for 4-PS modification, and [~]40% yield for 2-PS modification) and nucleotides ([~]75% yield). We demonstrate that the method allows for direct re-use of the PCR kits. We also show that the recycled primers can be isolated and then added to end point or quantitative PCR. This recycling approach provides a new path for circularly reusing PCR nucleic acids.


title: Expression Analysis, Molecular Characterization and Prognostic Evaluation on TMED4 and TMED9 Gene Expression in Glioma

abstract: Here, we utilized a database mining approach to unfold the prognostic and therapeutic potentials of Transmembrane EmP24 Trafficking Protein 4 (TMED4) and 9 (TMED) coding gene expressions in glioma. Both the genes were found to be overexpressed at the mRNA and protein level in low grade glioma (LGG) and glioblastoma multiforme (GBM) tissues including different glioma cell lines. Significant increase in the expression level of these genes with advancing glioma patients age, glioma grades and histological subtypes was observed. Differential and distinct promoter and coding sequence methylation pattern of TMED4 and TMED9 was observed in LGG and GBM tissues that may aid in methylation-sensitive diagnosis of glioma patients. The presence of multiple heterozygous genetic alterations (frequency: 0.4-1.1%) in those genes unveiled their potentials in high-throughput screening of glioma patients. The overexpression of TMED4 and TMED9 genes was associated with poor overall survival (OS) of LGG and GBM patients (HR:>1.6). Association of the expression levels of these genes with different immune cell infiltration levels i.e., B cell and T cell and modulators like CD274 and IL10RB was observed providing assurance in TMED-based diagnostic measure and therapeutic intervention discovery. Furthermore, functional enrichment analysis of the neighbor genes of TMED4 and TMED9 revealed that they are involved in metal ion binding, focal adhesion of cells and protein processing, and the deregulation of these activities are associated with gliomagenesis. Altogether, this study suggests that TMED4 and TMED9 are potential prognostic and therapeutic targets for glioma. However, further laboratory research is warranted.

category: cancer biology


title: NF kappa B Regulator Bcl3 Controls Development and Function of Classical Dendritic Cells in Toxoplasma gondii

abstract:
The atypical I{kappa}B family member Bcl3 associates with p50/NF-{kappa}B1 or p52/NF-{kappa}B2 homodimers in the nucleus, and positively or negatively modulates transcription in a context-dependent manner. In mice lacking Bcl3 globally or specifically in CD11c+ cells, Toxoplasma gondii infection is uniformly fatal and is associated with an impaired Th1 immune response. Since Bcl3 expression in dendritic cells (DC) is pivotal for antigen presentation and since classical DCs (cDC) are major antigen presenting cells, we investigated the role of Bcl3 specifically in cDCs in T. gondii infection in vivo by crossing Zbtb46 cre mice with Bcl3flx/flx mice. The conditional cDC Bcl3 KO was as susceptible to lethal T. gondii infection as the total Bcl3 KO and generated poor Th1 responses. Splenocyte single cell RNA seq in the model revealed defective Bcl3-dependent expression of genes involved in antigen processing. Consistent with this, soluble toxoplasma antigen presentation was impaired in Bcl3-deficient cDCs, and tetramer staining demonstrated defective T. gondii antigen-specific splenic CD4+ and CD8+ T cell responses in infected cDC Bcl3-/- mice. In vitro differentiation of bone marrow progenitors from wildtype and cDC Bcl3-/- mice using Flt3L, NOTCH and IFN-{gamma} stimulation recapitulated the defective Bcl3-dependent cDC antigen-presentation activity observed in vivo. Splenocyte single cell RNA seq also revealed the existence of a unique subpopulation of Zbtb46+LysM+ DC which exhibited Bcl3-dependent expansion after infection. We also detected cDCs coexpressing the monocytic markers CD64 and Ly6C (designated icDC1 and icDC2) mainly in infected spleen, which were less abundant in Bcl3flx/flx Zbtb46 cre mice. Together, our results indicate that Bcl3 in classical DCs is a major determinant of protective T cell responses and survival in T. gondii-infected mice, and shapes DC ontogeny.

Author SummaryDendritic cells initiate immune responses against invading pathogens. As professional antigen presenting cells they process and present antigen via the major histocompatibility complex to T cells and thus activate them. Bcl3, an atypical member of the I{kappa}B family regulates the APC function of dendritic cells. In this study we show that expression of Bcl3 specifically in classical DCs is critical for host protection against a protozoan parasite, Toxoplasma gondii. Host protective proinflammatory mechanisms are compromised in mice deficient in Bcl3 in classical DCs leading to an elevated organ parasite load and eventually death of the infected animals. We also found the emergence of Bcl3-dependent hybrid DCs upon T. gondii infection, which have mixed phenotypic markers from DCs and monocytes. Antigen processing genes are significantly downregulated in Bcl3-deficient cDCs, which may account for defective cross presentation of T. gondii antigens. In an in vitro differentiation model, we showed that development of XCR1+cross presenting cDC1s is critically regulated by Bcl3. Overall, this study reveals the complexity of dendritic cell ontogeny and the role of Bcl3 in classical DC function in the context of Toxoplasma infection.


title: Gαq modulates the energy metabolism of osteoclasts

abstract: The bacterial protein toxin Pasteurella multocida toxin (PMT) mediates RANKL-independent osteoclast differentiation. Although these osteoclasts are small, their resorptive activity is high and destroys the nasal turbinate bones of pigs. Analysis of the proteome of classical and toxin-derived osteoclasts showed that PMT induces the upregulation of metabolic pathways. This includes strong glycolytic activity, increased expression of GLUT1 and upregulation of the mTOR pathway. As OxPhos components are also expressed more efficiently, cells display increased mitochondrial respiration. We found that the heterotrimeric G protein Gq plays a central role in this hypermetabolic cell activation. Gq triggers mitochondrial relocalisation of pSerSTAT3 and an increase in OPA1 expression. Overexpression of Gq in Hoxb8 cells mimicked this hypermetabolic phenotype and resulted in higher glycolytic and mitochondrial activity as well as increased bone resorptive activity. Rheumatoid arthritis patients show an increase in Gnaq expression especially in the synovial fluid, suggesting that Gq is a target of pathophysiological relevance.


title: TLR2 axis on peripheral blood mononuclear cells regulates inflammatory responses to circulating, non-infectious immature dengue virus particles

abstract:
Severe dengue virus (DENV) infection is characterized by exacerbated inflammatory responses that lead to endothelial dysfunction and plasma leakage. We have recently demonstrated that Toll-like receptor 2 (TLR2) on blood monocytes senses DENV infection leading to endothelial activation. Here, we report that non-infectious immature DENV particles, which are released in large numbers by DENV-infected cells, drive endothelial activation via the TLR2 axis. We show that fully immature DENV particles induce a rapid, within 6 hours post-infection, inflammatory response in PBMCs. Furthermore, pharmacological blocking of TLR2/TLR6/CD14 and/or NF-kB prior to exposure of PBMCs to immature DENV reduces the initial production of inter alia TNF- and IL-1{beta} by monocytes and prevents endothelial activation. However, prolonged TLR2 block induces TNF- production and leads to exacerbated endothelial activation, indicating that TLR2-mediated responses play an important role not only in the initiation but also the resolution of inflammation. Altogether, these data indicate that the maturation status of the virus has the potential to influence the kinetics and extent of inflammatory responses during DENV infection.

Author SummarySevere dengue virus (DENV) infection is characterized by endothelial dysfunction leading to vascular permeability and plasma leakage. This is thought to be mediated by the exacerbated production of inflammatory mediators from cells of the innate immune system. We have previously demonstrated that Toll-like receptor 2 (TLR2), a pattern recognition receptor present on the surface of human monocytes, can sense DENV infection leading to the activation of the endothelium. Importantly, a large proportion of DENV particles is immature and are not readily infectious. Here we aimed to elucidate if and how these non-infectious immature DENV particles contribute to systemic inflammation. We evaluated if monocytes sense immature DENV and found that sensing of immature DENV induced early inflammatory responses in PBMCs. Pharmacological inhibition of TLR2/TLR6/CD14 and/or NF-{kappa}B prior to exposure to immature DENV demonstrates that this is the pathway leading to the early production inflammatory mediators and endothelial activation. However, prolonged inhibition of TLR2 induced the production of TNF- and the subsequent activation of the endothelium, suggesting that TLR2-mediated responses play an important tole during both, initiation and resolution of inflammation. We propose that the maturation status of DENV in the human host can influence the extent and kinetics of the inflammatory responses during DENV infection.


title: Acod1 negatively impacts osteoclastogenesis via GPR91-mediated NFATc1 activation

abstract: Immune cells undergo metabolic reprogramming upon exposure to inflammatory stimuli. The immuneresponsive gene 1 (Irg1) encodes aconitate decarboxylase (Acod1), an enzyme that generates itaconate from cis-aconitate in the TCA cycle and is one of the most highly induced genes in macrophages during inflammation. Itaconate inhibits succinate dehydrogenase activity leading to the accumulation of succinate. As the adjustment of energy metabolism also plays an important role during the formation of bone-resorbing osteoclasts, we investigated if Irg1 is regulated during osteoclastogenesis. We show that M-CSF/RANKL treatment induces Irg1 at an early time-point in bone marrow-derived macrophages (BMDM) as well as in the RAW264.7 macrophage cell line. Next, we stably overexpressed Acod1 in RAW264.7 cells. The metabolism of these Acod1 cells shifted towards glycolysis, as indicated by an increase in mTOR activation, subsequent 4EB-P1 phosphorylation and reduced ATP levels. When we investigated the ability of Acod1 cells to differentiate into osteoclasts, we observed a remarkable suppression of osteoclast-associated genes and the number of TRAP-positive, multi-nucleated osteoclasts was greatly reduced but not completely abrogated. Surprisingly, NFATc1 was detectable in nuclear extracts in untreated Acod1 cells leading to residual transcriptional NFAT activity in luciferase assays. This is caused by the elevated levels of succinate in Acod1 cells, as succinate can bind extracellularly to its cognate receptor GPR91 leading to Gq-mediated activation of NFATc1. When we investigated the expression of Gpr91, we found RANKL-mediated induction of Gpr91 to be severely reduced in Acod1 cells and we suggest that GPR91 is a target of RANKL-mediated NFATc1 activation. However, on the protein level, the receptor was still expressed at the cell surface. The observed repression of Gpr91 in Acod1 overexpressing cells was also detected by treatment with octyl-itaconate, showing that this is an itaconate-mediated effect. We hypothesize that the itaconate-mediated increase in succinate levels causes activation of NFATc1 signalling, although the transcriptional activity does not lead to osteoclastogenesis. In the presence of RANKL, these pre-activated cells are slow in switching to RANKL-mediated induction of osteoclast genes, which decreases their ability to differentiate into osteoclasts.


title: Antibiotic-producing Micrococcales govern the microbiome that inhabits the fur of two- and three-toed sloths

abstract: Sloths have a dense coat on which insects, algae, and fungi coexist in a symbiotic relationship. This complex ecosystem requires different levels of control, however, most of these mechanisms remain unknown. We investigated the bacterial communities inhabiting the hair of two- (Choloepus Hoffmani) and three-toed (Bradypus variegatus) sloths and evaluated their potential for producing antibiotic molecules capable of exerting control over the hair microbiota. The analysis of 16S rRNA amplicon sequence variants (ASVs) revealed that the communities in both host species are dominated by Actinobacteriota and Firmicutes. The most abundant genera were Brevibacterium, Kocuria/Rothia, Staphylococcus, Rubrobacter, Nesterenkonia, and Janibacter. In addition, we isolated nine strains of Brevibacterium and Rothia able to produce substances that inhibited the growth of common mammalian pathogens. The analysis of the biosynthetic gene clusters (BCGs) of these nine isolates suggests that the pathogen-inhibitory activity could be mediated by the presence of siderophores, terpenes, beta-lactones, Type III polyketide synthases (T3PKS), ribosomally synthesized, and post-translationally modified peptides (RiPPs), non-alpha poly-amino acids (NAPAA) like e-Polylysin, ectoine or nonribosomal peptides (NRPs). Our data suggest that Micrococcales inhabiting sloth hair could have a role in controlling microbial populations in that habitat, improving our understanding of this highly complex ecosystem.

category: microbiology


title: Ultra-high-throughput microbial single-cell whole genome sequencing for genome-resolved metagenomics

abstract: Microbes exist widely in nature. However, less than 1% of the species of microorganisms have been discovered so far, and more than 99% of microorganisms still remain unknown, which is called microbial dark matter. Unravelling microbial dark matter is not only helpful for exploring the unknown microbial world, but also of great significance for human health research. Ever since a long time ago, due to technical limitations, the microbiome, to a great extent, is still a black box. The traditional population-based metagenomic analysis tools are without single microbial resolution and they are stretched when studying rare and unknown microbial species. Here, we use high-throughput microdroplet technology to achieve the whole genome amplification of microbial single-cell genomes in pico-litter sized microdroplets, and develop subsequent library preparation steps such as indexing of the genome, to achieve the development of ultra-high-throughput microbial single-cell whole genome sequencing for genome-resolved metagenomics, aiming to provide a powerful new weapon for exploring microbial dark matter and other microbiological studies.


title: Allele specific PCR for a major marker of levamisole resistance in Haemonchus contortus

abstract: Haemonchus contortus is a haematophagous parasitic nematode that infects small ruminants and causes significant animal health concerns and economic losses within the livestock industry on a global scale. Treatment primarily depends on broad-spectrum anthelmintics, however, resistance is established or rapidly emerging against all major drug classes. Levamisole (LEV) remains an important treatment option for parasite control, as resistance to LEV is less prevalent than to members of other major classes of anthelmintics. LEV is an acetylcholine receptor (AChR) agonist that, when bound, results in paralysis of the worm. Numerous studies implicated the AChR sub-unit, ACR-8, in LEV sensitivity and in particular, the presence of a truncated acr-8 transcript or a deletion in the acr-8 locus in some resistant isolates. Recently, a single non-synonymous SNP in acr-8 conferring a serine-to-threonine substitution (S168T) was identified that was strongly associated with LEV resistance. Here, we investigate the role of genetic variation at the acr-8 locus in a controlled genetic cross between the LEV susceptible MHco3(ISE) and LEV resistant MHco18(UGA 2004) isolates of H. contortus. Using single worm PCR assays, we found that the presence of S168T was strongly associated with LEV resistance in the parental isolates and F3 progeny of the genetic cross surviving LEV treatment. We developed and optimised an allele-specific PCR assay for the detection of S168T and validated the assay using laboratory isolates and field samples that were phenotyped for LEV resistance. In the LEV-resistant field population, a high proportion (>75%) of L3 encoded the S168T variant, whereas the variant was absent in the susceptible isolates studied. These data further support the potential role of acr-8 S168T in LEV resistance, with the allele-specific PCR providing an important step towards establishing a sensitive molecular diagnostic test for LEV resistance.


title: A new microfluidic device for simultaneous detection of enzyme secretion and elongation of a single hypha

abstract:
Fungal cells perform enzyme secretion and elongation by exocytosis in the apical region. The widespread branching of hyphae and the inability to control environmental conditions over long periods make it difficult to observe and analyze single hyphae with conventional assays. Therefore, although hyphal morphology is closely linked to productivity, no real-time measurements of morphology and exoenzymes have been carried out. In this study, a microfluidic system was developed to compartmentalize a single hypha germinated from a single spore. This allowed detailed observation of a single hypha and provided new insights, such as the fact that enlarged vacuoles inhibit nuclear movement. Furthermore, a cellulase detection assay based on subtle differences in molecular polarity was developed to detect hyphal growth and enzyme secretion in real-time using Trichoderma reesei, a potent cellulase-producing hypha, as a model. When the fluorescence from the detection assay was compared with the GFP fluorescence intensity using a strain fused with cellulase CBHI and GFP, a strong correlation was observed. As T. reesei secretes a series of cellulases, these results prove that the extracellular enzymes can be measured in real time. This microfluidic system has enabled real-time visualization and analysis of cellular heterogeneity, hyphal and enzyme dynamics associated with carbon source exchange, and quantitative dynamics of gene expression. The technology can be applied to a wide range of other biosystems exhibiting similar polar cell growth, from bioenergy production to human health.

IMPORTANCEHyphal morphology and productivity of filamentous fungi are linked by exocytosis. Conventional assay methods make it difficult to observe and analyze single hyphae. Here, a robust and high-performance microfluidic system was developed to compartmentalize single hyphae germinated from a single spore, enabling their long-term observation. Using the potent cellulase-producing fungus Trichoderma reesei, the system made it possible to visualize and analyze cell heterogeneity, hyphae, enzyme dynamics, and quantitative gene expression dynamics associated with carbon source exchange. The technique can be immediately extended to various other biosystems exhibiting similar polar cell growth and is expected to contribute significantly to the elucidation of filamentous fungi production systems.


title: The RNA-RNA interactome between a phage and its satellite virus reveals a small RNA differentially regulates gene expression across both genomes

abstract: Phage satellites exhibit various regulatory mechanisms to manipulate phage gene expression to the benefit of the satellite. While small RNAs (sRNAs) are well documented as regulators of prokaryotic gene expression, they have not been shown to play a regulatory role in satellite-phage conflicts. Vibrio cholerae encodes the phage inducible chromosomal island-like element (PLE), a phage satellite, to defend itself against the lytic phage ICP1. Here we use Hi-GRIL-seq to identify a complex RNA-RNA interactome between PLE and ICP1. Both inter- and intragenome RNA interactions were detected, headlined by the PLE-encoded trans-acting sRNA, SviR. SviR regulates both PLE and ICP1 gene expression uniquely, decreasing translation of ICP1 targets and affecting PLE mRNAs processing. The striking conservation of SviR across all known PLEs suggests the sRNA is deeply rooted in the PLE-ICP1 conflict and implicates sRNAs as unidentified regulators of phage-satellite interactions.


title: Intestinal inflammation reversibly alters the microbiota to drive susceptibility to Clostridioides difficile colonization in a mouse model of colitis

abstract:
Susceptibility to Clostridioides difficile infection (CDI) typically follows the administration of antibiotics. Patients with inflammatory bowel disease (IBD) have increased incidence of CDI, even in the absence of antibiotic treatment. However, the mechanisms underlying this susceptibility are not well understood. To explore the intersection between CDI and IBD, we recently described a mouse model where colitis triggered by the murine gut bacterium, Helicobacter hepaticus, in IL-10-/- mice led to susceptibility to C. difficile colonization without antibiotic administration. The current work disentangles the relative contributions of inflammation and gut microbiota in colonization resistance to C. difficile in this model. We show that inflammation drives changes in microbiota composition, which leads to CDI susceptibility. Decreasing inflammation with an anti-p40 monoclonal antibody promotes a shift of the microbiota back toward a colonization-resistant state. Transferring microbiota from susceptible and resistant mice to germ-free animals transfers the susceptibility phenotype, supporting the primacy of the microbiota in colonization resistance. These findings shine light on the complex interactions between the host, microbiota, and C. difficile in the context of intestinal inflammation, and may form a basis for the development of strategies to prevent or treat CDI in IBD patients.

ImportancePatients with inflammatory bowel disease (IBD) have an increased risk of developing C. difficile infection (CDI), even in the absence of antibiotic treatment. Yet, mechanisms regulating C. difficile colonization in IBD patients remain unclear. Here, we use an antibiotic-independent mouse model to demonstrate that intestinal inflammation alters microbiota composition to permit C. difficile colonization in mice with colitis. Notably, treating inflammation with an anti-p40 monoclonal antibody, a clinically relevant IBD therapeutic, restores microbiota-mediated colonization resistance to the pathogen. Through microbiota transfer experiments in germ-free mice, we confirm that the microbiota shaped in the setting of IBD is the primary driver of susceptibility to C. diffiicile colonization. Collectively, our findings provide insight into CDI pathogenesis in the context of intestinal inflammation, which may inform methods to manage infection in IBD patients. More broadly, this work advances our understanding of mechanisms by which the host-microbiota interface modulates colonization resistance to C. difficile.


title: Molecular Architecture of the Chikungunya Virus Replication Complex

abstract:
All positive-strand (+) RNA viruses assemble membrane-associated replication complexes (RCs) for viral RNA synthesis in virus-infected cells. However, how these multi-component RCs assemble and function in synthesizing, processing, and transporting viral RNAs to the cytosol remains poorly defined. Here, we determined both the structure of the core RNA replicase of chikungunya virus (family Togaviridae) at a near-atomic level and the native RC architecture in its cellular context at the subnanometer resolution, using in vitro reconstitution and in situ electron cryotomography, respectively. Within the core RNA replicase (nsP1+2+4), the viral RNA-dependent RNA polymerase nsP4, in complex with nsP2 helicase-protease, was found to co-fold with the membrane-anchored nsP1 RNA-capping dodecameric ring and is located asymmetrically within nsP1 central pore. This complex forms the minimal core RNA replicase, while the addition of a large cytoplasmic ring next to the C-terminus of nsP1 forms the holo-RNA-RC as observed at the neck of spherules formed in virus-infected cells. These results represent a major conceptual advance in elucidating the molecular mechanisms of RNA virus replication and the principles underlying the molecular architecture of RCs, likely to be shared with many pathogenic (+) RNA viruses. At last, our study will direct the needed development of antiviral therapies targeting RCs of pathogenic viruses.

SummaryCryoEM structure of the chikungunya virus replication complex reveals a multicomponent RNA synthesis nanomachine embedded in the plasma membrane of the host cell.


title: A CRISPR-based instant DNA repositioning system and the early intranuclear life of HSV-1

abstract:
The intranuclear localization of viral DNA genomes in relation to the intranuclear environment plays critical roles in determining virus fate. Recent advances in the application of chromosome conformation capture-based sequencing analysis (3C technologies) have revealed valuable aspects of the spatiotemporal interplay of viral genomes with host chromosomes. However, to elucidate the causal relationship between the subnuclear localization of viral genomes and the pathogenic outcome of the infection, manipulative tools are needed. Instant repositioning of viral DNAs to specific subnuclear compartments amid infection is a powerful approach to synchronize and interrogate this dynamically changing process in space and time. Herein, we report an inducible CRISPR-based two-component platform that relocates extrachromosomal DNA pieces (5 kb to 170 kb) to the nuclear periphery instantly (CRISPR-nuPin). Based on this system, investigations of herpes simplex virus 1 (HSV-1), a prototype member of the human herpesvirus family, revealed unprecedently reported insights into the early intranuclear life of the pathogen: I) Viral genomes tethered to the nuclear periphery upon entry, compared with those in the nuclear interior, were wrapped around histones with increased suppressive modifications and subjected to stronger transcriptional silencing and prominent inhibition. II) Relocating HSV-1 genomes at 1 hour post infection significantly promoted transcription of viral {beta} and {gamma} genes, termed an "Escaping" effect. III) Early accumulation of ICP0 was a sufficient but not necessary condition mediating "Escaping". IV) Subnuclear localization was only critical during early infection. Importantly, the CRISPR-nuPin tactic should be widely applicable to many DNA viruses.

SummaryThe intranuclear localization of viral DNA genomes plays a critical role in determining virus fate. To elucidate the causal relationship between subnuclear localization and the pathogenic outcome of DNA viruses, manipulative tools are needed. Herein, we report an inducible CRISPR-based two-component platform that relocates DNA pieces (5 kb to 170 kb) to the nuclear periphery instantly (CRISPR-nuPin). Utilizing this tactic, we interrogated the early intranuclear life of herpes simplex virus 1 (HSV-1), a prototype of human herpesviruses, in space and time and revealed that I) viral genomes tethered to the nuclear edge upon entry were prone to suppressive histone packaging and severe inhibition. II) Relocating HSV-1 genomes to the nuclear fringe at 1 hour post infection promoted transcription of viral genes ("Escaping"). III) Early accumulation of ICP0 was a sufficient but not necessary condition mediating "Escaping". IV) Subnuclear localization was a critical factor only during early infection.

HighlightsO_LICRISPR-nuPin is an inducible two-component DNA repositioning system
C_LIO_LIIt mediates instant nuclear edging of viral DNA during infection
C_LIO_LIA powerful approach to interrogate DNA viruses in space and time
C_LIO_LIViral DNA at the nuclear periphery upon entry is strongly silenced
C_LI

In briefAn inducible two-component CRISPR-based platform that instantly repositions HSV-1 genomes to the nuclear edge unveils intranuclear space heterogeneity for the incoming viral genomes and dynamic stages of the host-virus interplay during early infection of the pathogen.


title: Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses

abstract: Lagoviruses, family Caliciviridae, are some of the most virulent vertebrate viruses known. They infect leporids, i.e., rabbits (Oryctolagus spp.), hares (Lepus spp.) and cottontails (Sylvilagus spp.) and rapidly kill over 95% of susceptible animals. Pathogenic lagoviruses are hepatotropic and induce a fulminant hepatitis that typically leads to disseminated intravascular coagulation within 24-72 hours of infection. However, the pathophysiological mechanisms governing this extreme phenotype and other aspects of the fundamental biology of these viruses are poorly understood due to a lack of cell culture systems. Here, we report on a robust and reliable ex vivo model for the cultivation of hepatotropic lagoviruses. We show that three rabbit haemorrhagic disease virus (RHDV) variants, RHDV1, RHDV2 and RHDVa-K5, replicate in monolayer cultures derived from rabbit hepatobiliary organoids. Viral replication was demonstrated by a (i) increase in viral RNA levels of greater than one log10 over a 23-hour period, (ii) detection of viral structural and non-structural proteins, and (iii) detection of double-stranded RNA viral replication intermediates. Furthermore, we generated hepatobiliary organoids from a feral cat (Felis domesticus), a wild mouse (Mus musculus) and a European brown hare (Lepus europaeus) and showed that monolayer cultures derived from the cat and mouse organoids were not permissive for lagovirus infection, while those derived from the hare organoids only supported replication of RHDV2, recapitulating the species tropism that has been observed with these viruses. Our organoid culture system will facilitate future studies into the molecular biology of lagoviruses, which will have considerable import for the conservation of endangered leporid species in Europe and North America and the biocontrol of overabundant rabbit populations in Australia and New Zealand.


title: Pneumococcus triggers NFkB degradation in COMMD2 aggresome-like bodies.

abstract: NF-kB driven cellular immunity is essential for both pro- and anti-inflammatory responses to microbes, which makes it one of the most frequently targeted pathways by bacteria during pathogenesis. How NF-kB tunes the epithelial response to Streptococcus pneumoniae across the spectrum of commensal to pathogenic host phenotypic outcomes is not fully understood. In this study, we compare a commensal-like 6B ST90 strain to an invasive TIGR4 isolate and demonstrate that TIGR4 both blunts and antagonizes NF-kB activation. We identified, through comparative mass spectrometry of the p65 interactome, that the 6B ST90 isolate drives a non-canonical NF-kB RelB cascade, whereas TIGR4 induces p65 degradation though aggrephagy. Mechanistically, we show that during TIGR4 challenge a novel interaction of COMMD2 with p65 and p62 is established to mediate degradation of p65. With these results, we establish a role for COMMD2 in negative NF-kB regulation, and present a paradigm for diverging NF-kB responses to pneumococcus. Thus, our studies reveal for the first time a new bacterial pathogenesis mechanism to repress host inflammatory response though COMMD2 mediated turnover of p65.


title: Dietary and Reproductive Traits of Two Brachyuran Crab Species

abstract: Many animals have flexible morphological traits that allow them to succeed in differing circumstances with differing diets available to them. For brachyuran crabs, claw height and gut size are diet-specific and largely reflect foraging strategies, while abdomen width reflects relative levels of fecundity. However, the link between claw size and diet has largely been documented only for primarily carnivorous crabs, while the link between diet and fecundity is strong in herbivorous crabs. We sought to determine the nature of the intraspecific relationship between claw size, dietary habits, and fecundity for two primarily herbivorous crab species, Hemigrapsus sanguineus and Aratus pisonii. Specifically, we examined whether claw size and/or abdomen width can be used as reliable measures of individual diet strategy. To test these hypotheses, we collected crabs and measured the dimensions of their claws, abdomens, and guts. By comparing these dimensions for each individual, we found that strongly predictive relationships do not exist between these traits for the primarily herbivorous species in our study. Thus, identifying external morphological features that can be used to assess diets of primarily herbivorous crabs remains elusive.

category: ecology


title: Wax worm saliva and the enzymes therein are the key to polyethylene degradation by Galleria mellonella

abstract: Plastic degradation by biological systems with re-utilization of the by-products can be the future solution to the global threat of plastic waste accumulation. We report that the saliva of Galleria mellonella larvae (wax worms) is capable of oxidizing and depolymerizing polyethylene (PE), one of the most produced and sturdy polyolefin-derived plastics. This effect is achieved after a few hours exposure at room temperature and physiological conditions (neutral pH). The wax worm saliva can indeed overcome the bottleneck step in PE biodegradation, that is the initial oxidation step. Within the saliva, we identified two enzymes that can reproduce the same effect. This is the first report of enzymes with this capability, opening up the way to new ground-breaking solutions for plastic waste management through bio-recycling/up-cycling.

category: molecular biology


title: Soil water consumption characteristics in rain-fed apple orchards and wheat fields

abstract: Agricultural production in Weibei rain-fed highland, Northwest China, is facing severe drought and water shortages. Here, soil water consumption characteristics in rain-fed orchards and farmlands were explored to ascertain the rationality of planting orchards in Weibei. Soil moisture dynamics was monitored in the 0-150-cm soil profiles of different aged  Red Fuji apple orchards (young: 7 years, mature: 13 years, and old: 22 years), and in long-term cultivated winter wheat fields during the growing season of apple trees. The over-consumption and consumption of soil water were analyzed to evaluate water stress and differential water consumption by distinct vegetation, respectively. Soil desiccation index was used to determine the occurrence of dry soil layers. Generally, there was no water stress in the 0-150-cm orchard soil profiles, while water stress was observed at the 0-70-cm soil depths in the old orchards (mid-June) and farmlands (mid-May-mid-July). Water consumption took place at deeper depths for longer periods in the older orchards than in the younger orchards. Soil desiccation was not observed in the young orchards, while mild desiccation occurred at the 0-80-cm soil depths in the mature and old orchards in mid-June. The desiccation intensity was mild at the 0-60-cm soil depths in mid-April-mid-May, intense at the soil 0-150-cm depths in mid-June, and moderate at the 20-150-cm soil depths in mid-July. In conclusion, conversion from wheat fields to apple orchards could reduce soil water stress, reduce dry soil layers, and mitigate soil desiccation in the rain-fed highland area.


title: Multiple lines of evidence indicate survival of the Ivory-billed Woodpecker in Louisiana

abstract: The history of decline of the Ivory-billed Woodpecker is long, complex, and controversial. The last widely accepted sighting of this species in continental North America was 1944. Reports of Ivory-billed Woodpeckers have continued, yet in 2021 the U.S. Fish and Wildlife Service proposed declaring the species extinct. We draw on 10 years of search effort, and provide trail camera photos and drone videos suggesting the consistent presence of Ivory-billed Woodpeckers at our study site. Data indicate repeated re-use of foraging sites and core habitat. We offer insights into behaviors of the Ivory-billed Woodpecker that contribute to difficulty in finding this species. We discuss results with regard to the value of accumulated evidence, and what repeated observations may indicate for continued survival of this iconic species.


title: Co-Component Signal Transduction Systems: fast-evolving virulence regulation cassettes discovered in enteric bacteria

abstract:
Bacterial signal transduction systems sense changes in the environment and transmit these signals to control cellular responses. The simplest one-component signal transduction systems include an input sensor domain and an output response domain encoded in a single protein chain. Alternately, two-component signal transduction systems transmit signals by phosphorelay between input and output domains from separate proteins. The membrane tethered periplasmic bile acid sensor that activates the Vibrio parahaemolyticus type III secretion system adopts an obligate heterodimer of two proteins encoded by partially overlapping VtrA and VtrC genes. This co-component signal transduction system binds bile acid using a lipocalin-like domain in VtrC and transmits the signal through the membrane to a cytoplasmic DNA-binding transcription factor in VtrA. Using the domain and operon organization of VtrA/VtrC, we identify a fast-evolving superfamily of co-component systems in enteric bacteria. Accurate machine learning-based fold predictions for the candidate co-components support their homology in the twilight zone of rapidly evolving sequence and provide mechanistic hypotheses about previously unrecognized lipid-sensing functions.

Significance statementUsing the domain and operon organization of VtrA/VtrC, combined with fold predictions, we identify new co-component signal transduction systems in enteric bacteria that likely regulate virulence. We observe that the heterodimeric VtrA/VtrC periplasmic bile acid receptor controlling Vibrio parahaemolyticus T3SS2 is a distant homolog of the ToxR/ToxS master regulator of virulence and has evolved beyond confident sequence recognition. Exploiting the newly developed machine learning methods for structure prediction, we observe a VtrC-like lipocalin fold for both the ToxS periplasmic domain and for other detected periplasmic sensor components. This structure prediction supports the divergent evolution of VtrA/VtrC-like co-component signal transduction systems and suggests a role for lipid sensing in regulating virulence in enteric bacteria.

category: evolutionary biology


title: Organisation of gene programs revealed by unsupervised analysis of diverse gene-trait associations

abstract: Genome wide association studies provide statistical measures of gene-trait associations that reveal how genetic variation influences phenotypes. This study develops an unsupervised dimensionality reduction method called UnTANGLeD (Unsupervised Trait Analysis of Networks from Gene Level Data) which organises 16,849 genes into discrete gene programs by measuring the statistical association between genetic variants and 1,393 diverse complex traits. UnTANGLeD reveals 242 gene clusters enriched for protein-protein interactions and highly distinct biological processes governing development, signalling, disease, and homeostasis. We identify diverse gene networks with robust interactions but not associated with known biological processes. Analysis of independent disease traits shows that UnTANGLeD gene clusters are conserved across all complex traits, providing a simple and powerful framework to predict novel gene candidates and programs influencing orthogonal disease phenotypes. Collectively, this study demonstrates that gene programs co-ordinately orchestrating cell functions can be identified without reliance on prior knowledge, providing a method for use in functional annotation, hypothesis generation, machine learning and prediction algorithms, and the interpretation of diverse genomic data.

category: genomics


title: Discovery and Visualization of Age-dependent Patterns in the Diurnal Transcriptome of Drosophila

abstract: Many critical life processes are regulated by input from 24-hour external light/dark cycles, such as metabolism, cellular homeostasis, and detoxification. The circadian clock, which helps coordinate the response to these diurnal light/dark cycles, remains rhythmic across lifespan; however, rhythmic transcript expression is altered during normal aging. To better understand how aging impacts diurnal expression, we present an improved Fourier-based method for detecting and visualizing rhythmicity that is based on the relative power of the 24-hour period compared to other periods (RP24). We apply RP24 to transcript-level expression profiles from the heads of young (5-day) and old (55-day) Drosophila melanogaster, and reveal novel age-dependent rhythmicity changes that may be masked at the gene level. We show that core clock transcripts phase advance during aging, while most rhythmic transcripts phase delay. Transcripts rhythmic only in young flies tend to peak before lights on, while transcripts only rhythmic in old peak after lights on. We show that several pathways including glutathione metabolism, gain or lose coordinated rhythmic expression with age providing insight into possible mechanisms of age-onset neurodegeneration. Remarkably, we find that many pathways show very robust coordinated rhythms across lifespan, highlighting their putative roles in promoting neural health. We investigate statistically enriched transcription factor binding site motifs that may be involved in these rhythmicity changes.

category: systems biology


title: Structure of Importin-4 bound to the H3-H4·ASF1 histone·histone chaperone complex

abstract: Importin-4 is the primary nuclear import receptor of core histones H3 and H4. Importin-4 binds the H3-H4 dimer and histone-chaperone ASF1 prior to nuclear import, but available structures of Importin-4{middle dot}histone tail complexes do not explain how Importin-4 recognizes the biologically relevant heterotrimeric H3-H4{middle dot}ASF1 cargo. Our 3.5 [A] Importin-4{middle dot}H3-H4{middle dot}ASF1 cryo-electron microscopy structure revealed interactions with H3-H4{middle dot}ASF1 different those suggested by previous Importin-H3 tail peptide structures. The N-terminal half of Importin-4 clamps the globular histone domain and the H3 N helix while its C-terminal half binds the H3 N-terminal tail weakly, with negligible tail contribution to binding energy; ASF1 binds H3-H4 without contacting Importin-4. Together, ASF1 and Importin-4 shield nucleosomal interfaces of H3-H4 to chaperone and import it into the nucleus, where Importin-4 undergoes large conformational changes as RanGTP binds to release H3-H4{middle dot}ASF1. This work explains the mechanisms of nuclear import of full-length H3-H4.


title: Distal-less homeobox genes Dlx5/6 regulate Mulllerian duct regression

abstract: Dlx5 and Dlx6 encode distal-less homeodomain transcription factors that are present in the genome as a linked pair at a single locus. Dlx5 and Dlx6 have redundant roles in craniofacial, skeletal, and uterine development. Previously, we performed a transcriptome comparison for anti-Mullerian hormone (AMH)-induced genes expressed in the Mullerian duct mesenchyme of male and female mouse embryos. In that study, we found that Dlx5 transcripts were nearly seven-fold higher in males compared to females and Dlx6 transcripts were found only in males, suggesting they may be AMH-induced genes. Therefore, we investigated the role of Dlx5 and Dlx6 during AMH-induced Mullerian duct regression. We found that Dlx5 was detected in the male Mullerian duct mesenchyme from E14.5 to E16.5. In contrast, in female embryos Dlx5 was detected in the Mullerian duct epithelium. Dlx6 expression in Mullerian duct mesenchyme was restricted to males. Dlx6 expression was not detected in female Mullerian duct mesenchyme or epithelium. Genetic experiments showed that AMH signaling is necessary for Dlx5 and Dlx6 expression. Mullerian duct regression was variable in Dlx5 homozygous mutant males at E16.5, ranging from regression like controls to a block in Mullerian duct regression. In E16.5 Dlx6 homozygous mutants, Mullerian duct tissue persisted primarily in the region adjacent to the testes. In Dlx5-6 double homozygous mutant males Mullerian duct regression was also found to be incomplete but more severe than either single mutant. These studies suggest that Dlx5 and Dlx6 act redundantly to mediate AMH-induced Mullerian duct regression during male differentiation.

category: developmental biology


title: Effects of seasonal changes on the carbon dynamics in mixed coniferous forests

abstract: We characterized seasonal changes in the residual rate and mass-loss rate of litter, as well as the carbon release dynamics of litter and soil, in mixed coniferous forests in the Xiaoxinganling region by conducting a controlled freeze-thaw experiment. The carbon release rate and mass-loss rate of litter at two levels of decomposition (undecomposed and semi-decomposed) were measured during four seasons: the unfrozen season, freeze-thaw season, frozen season, and thaw season. The temperature was higher, the mass-loss rate was faster, and the overall mass-loss rate of litter was higher in the unfrozen season than in the other three seasons; litter organic carbon increased and soil organic carbon increased due to the strong carbon sequestration capacity of plants. The temperature fluctuated above and below 0{degrees}C during the freeze-thaw season, which results in the physical breakage of the undecomposed litter and increases in the mass-loss rate. This leads to increases in the organic carbon of undecomposed litter and decreases in the soil organic carbon of undecomposed litter; the opposite patterns were observed for changes in the organic carbon of semidecomposed litter and soil organic carbon. There was noticeable mass loss of litter during the frozen season, and the rate of mass loss of litter during the thaw season was the lowest. Litter organic carbon decreased and soil organic carbon increased in both seasons. The organic carbon of undecomposed litter was highest in the thaw season, followed by the freeze-thaw season, frozen season, and unfrozen season. The organic carbon of semi-decomposed litter was highest in the frozen season, followed by the thaw season, freeze-thaw season, unfrozen season to freeze-thaw season, frozen season, thaw season, and unfrozen season. After freeze-thaw treatment, the organic carbon in deadfall soil was highest in the unfrozen season, freeze-thaw season, frozen season, thaw season to unfrozen season, frozen season, thaw season, and freeze-thaw season. The findings of this study provide new insights into the material cycling process under freeze-thawing, as well as information on the effect of seasonal freeze-thaws on the forest carbon cycle.


title: Different factors drive community assembly of rare and common ectomycorrhizal fungi

abstract: 1.Understanding what drives community assembly processes and how communities respond to environmental gradients are fundamental goals in community ecology. Ectomycorrhizal fungi support major forest systems across the globe, but the diversity, distribution and environmental controls affecting ectomycorrhizal community composition are unknown in many regions, particularly in the southern hemisphere. Here we investigate the assembly of ectomycorrhizal fungal communities based on eDNA samples from 81 Nothofagus forests across New Zealands South Island. We apply zeta diversity analysis and multi-site generalised dissimilarity modelling (MS-GDM) to investigate assembly patterns and quantify the effects of 43 biotic and environmental variables on community turnover. The zeta diversity MS-GDM framework differentiates between the environmental factors driving turnover of rare and common species, so provides a more complete picture of community dynamics than traditional beta diversity analyses. Results showed that community assembly was dominated by deterministic rather than stochastic processes. Soil variables were important drivers across the full range of rare, intermediate and common species. Ground cover variables, forest patch size and rainfall had greater effects on turnover of rare species, whereas temperature variables and host tree size had greater effects on common species turnover. Applying these methods for the first time to fungi demonstrates that there are distinct differences in the ecological processes affecting different aspects of the ectomycorrhizal community, which has important implications for understanding the functional effects of community responses to environmental change.


title: Inferring bound structure and residue specific contributions to binding energetics in the Intrinsically Disordered Protein, CcdA

abstract: In contrast to globular proteins, much less is known about the mutational effects on the function of Intrinsically Disordered Proteins (IDPs). We employ Yeast Surface Display of a mutant library of the IDP CcdA, coupled to next generation sequencing to rapidly estimate apparent binding affinities of each library member to cognate target, CcdB. This yields insights into sequence-function relationships in disordered CcdA and also enables prediction of the interacting interface residues and the local structural signatures of CcdA in its bound form. We show that the non-interface residue, Gly63 with non-canonical backbone conformation, to be essential for optimal binding to the high affinity site on CcdB. Additionally, this data provides insights into the much-debated role of helicity and disorder in partner binding of IDPs. Relative to globular proteins, in the present case we observe much smaller effects on binding affinity for point mutations, presumably because of the extended binding interface. Based on this exhaustive mutational sensitivity data, a model was developed to predict mutational effects on binding affinity of IDPs forming alpha-helical structures upon binding.

category: biophysics


title: The natural diversity of the yeast proteome reveals chromosome-wide dosage compensation in aneuploids

abstract: Aneuploidy, an imbalance in chromosome copy numbers, causes genetic disorders, and drives cancer progression, drug tolerance, and antimicrobial resistance. While aneuploidy can confer stress resistance, it is not well understood how cells overcome the fitness burden caused by aberrant chromosomal copy numbers. Studies using both systematically generated 1-5 and natural aneuploid yeasts 6-8 triggered an intense debate about the role of dosage compensation, concluding that aneuploidy is transmitted to the transcriptome and proteome without significant buffering at the chromosome-wide level, and is, at least in lab strains, associated with significant fitness costs. Conversely, systematic sequencing and phenotyping of large collections of natural isolates revealed that aneuploidy is frequent and has few - if any - fitness costs in nature 9. To address these discrepant findings at the proteomic level, we developed a platform that yields highly precise proteomic measurements across large numbers of genetically diverse samples, and applied it to natural isolates collected as part of the 1011 genomes project 9. For 613 of the isolates, we were able to match the proteomes to their corresponding transcriptomes and genomes, subsequently quantifying the effect of aneuploidy on gene expression by comparing 95 aneuploid with 518 euploid strains. We find, as in previous studies, that aneuploid gene dosage is not buffered chromosome-wide at the transcriptome level. Importantly, in the proteome, we detect an attenuation of aneuploidy by about 25% below the aneuploid gene dosage in natural yeast isolates. Furthermore, this chromosome-wide dosage compensation is associated with the ubiquitin-proteasome system (UPS), which is expressed at higher levels and has increased activity across natural aneuploid strains. Thus, through systematic exploration of the species-wide diversity of the yeast proteome, we shed light on a long-standing debate about the biology of aneuploids, revealing that aneuploidy tolerance is mediated through chromosome-wide dosage compensation at the proteome level.


title: Learning and Expression of Dopaminergic Reward Prediction Error via Plastic Representations of Time

abstract: Dopamine (DA) releasing neurons in the midbrain learn response patterns that represent reward prediction error (RPE). Typically, models proposing a mechanistic explanation for how dopamine neurons learn to exhibit RPE are based on temporal difference (TD) learning, a machine learning algorithm. However, mechanistic models motivated by TD learning face two significant hurdles. First, TD based models typically require rather unrealistic components, such as long and robust temporal chains of feature specific neurons which tile, a priori, each interval from a given stimulus to a given reward. Secondly, various predictions of TD clash with experimental observations of how RPE evolves over learning. Here, we present a biophysically plausible plastic network model of spiking neurons, that learns RPEs and can replicate results observed in multiple experiments. This model, coined FLEX (Flexibly Learned Errors in Expected Reward), learns feature specific representations of time, allowing for neural representations of stimuli to adjust their timing and relation to rewards in an online manner. Following learning, model dopamine neurons in FLEX report a distribution of response types, as observed experimentally and as used in machine learning. Dopamine firing in our model reflects an RPE before and after learning but not necessarily during learning, allowing the model to reconcile seemingly inconsistent experiments and make unique predictions that contrast those of TD.

category: neuroscience


title: The Neural Response at the Fundamental Frequency of Speech is Modulated by Word-level Acoustic and Linguistic Information

abstract: Spoken language comprehension requires rapid and continuous integration of information, from lower-level acoustic to higher-level linguistic features. Much of this processing occurs in the cerebral cortex. Its neural activity exhibits, for instance, correlates of predictive processing, emerging at delays of a few hundred milliseconds. However, the auditory pathways are also characterized by extensive feedback loops from higher-level cortical areas to lower-level ones as well as to subcortical structures. Early neural activity can therefore be influenced by higher-level cognitive processes, but it remains unclear whether such feedback contributes to linguistic processing. Here, we investigated early speech-evoked neural activity that emerges at the fundamental frequency. We analyzed EEG recordings obtained when subjects listened to a story read by a single speaker. We identified a response tracking the speakers fundamental frequency that occurred at a delay of 11 ms, while another response elicited by the high-frequency modulation of the envelope of higher harmonics exhibited a larger magnitude and longer latency of about 18 ms. Subsequently, we determined the magnitude of these early neural responses for each individual word in the story. We then quantified the context-independent frequency of each word and used a language model to compute context-dependent word surprisal and precision. The word surprisal represented how predictable a word is, given the previous context, and the word precision reflected the confidence about predicting the next word from the past context. We found that the word-level neural responses at the fundamental frequency were predominantly influenced by the acoustic features: the average fundamental frequency and its variability. Amongst the linguistic features, only context-independent word frequency showed a weak but significant modulation of the neural response to the high-frequency envelope modulation. Our results show that the early neural response at the fundamental frequency is already influenced by acoustic as well as linguistic information, suggesting top-down modulation of this neural response.


title: Photoperiod modulates mu-opioid receptor availability in brown adipose tissue

abstract:
Photoperiod drives metabolic activity of brown adipose tissue (BAT), and affects food intake and weight gain in mammals. Sympathetic innervation in BAT controls thermogenesis and facilitates physiological adaption to seasons, but the exact mechanism remains elusive. Previous studies show that the central opioid signaling tunes BAT heating and the brain muopioid receptor (MOR) levels have seasonal patterns. It is hence intriguing to know whether the peripheral MOR signaling shows seasonal variation. Here, we examined the effect of photoperiod on BAT MOR availability using [11C]carfentanil positron emission topography (PET). Adult rats (n = 9) were repeatedly imaged under changing photoperiods which simulates the local seasons. Long photoperiod downregulated MOR availability in BAT, while MOR availability in the muscles was unaffected. We confirmed the expression of MOR in BAT and muscle using immunofluorescence imaging. We conclude that photoperiod causally affects MOR availability in BAT, and sympathetic innervation of BAT may influence thermogenesis via the peripheral MOR system.

Significance of the studyPhotoperiod impacts the metabolic activity of brown adipose tissue (BAT) with the exact mechanism still unclear. The current study shows that photoperiod causally affects the mu-opioid receptor (MOR) levels in BAT, with longer photoperiod leading to lower MOR availability. This possibly indicates down-regulated innervation during bright seasons. Immunofluorescence staining data reveal expression of MOR in both brain and peripheral tissues, drawing attention to the under-investigated peripheral MOR system. Also, the study highlights the feasibility of [11C]carfentanil PET in studying the peripheral MOR signaling.


title: Transcription factors organize into functional groups on the linear genome and in 3D chromatin

abstract: Transcription factors (TFs) and their binding sites have evolved to interact cooperatively or competitively with each other. Here we examine in detail, across multiple cell lines, such cooperation or competition among TFs both in sequential and spatial proximity (using chromatin conformation capture assays) on one hand, and based on both in vivo binding as well as TF binding motifs on the other. We ascertain significantly co-occurring ("attractive") or avoiding ("repulsive") TF pairs using robust randomized models that retain the essential characteristics of the experimental data. Across human cell lines TFs organize into two groups, with intra-group attraction and inter-group repulsion. This is true for both sequential and spatial proximity, as well as for both in vivo binding and motifs. Attractive TF pairs exhibit significantly more physical interactions suggesting an underlying mechanism. The two TF groups differ significantly in their genomic and network properties, as well in their function--while one group regulates housekeeping function, the other potentially regulates lineage-specific functions, that are disrupted in cancer. We also show that weaker binding sites tend to occur in spatially interacting regions of the genome. Our results suggest a complex pattern of spatial cooperativity of TFs that has evolved along with the genome to support housekeeping and lineage-specific functions.


title: Structural and biophysical properties of FopA, a major outer membrane protein of Francisella tularensis

abstract: Francisella tularensis is an extremely infectious pathogen and a category A bioterrorism agent. It causes the highly contagious zoonosis, Tularemia. Currently, FDA approved vaccines against tularemia are unavailable. F. tularensis outer membrane protein A (FopA) is a well-studied virulence determinant and protective antigen against tularemia. It is a major outer membrane protein (Omp) of F. tularensis. However, FopA-based therapeutic intervention is hindered due to lack of complete structural information for membrane localized mature FopA. In our study, we established recombinant expression, monodisperse purification, crystallization and X-ray diffraction ([~]6.5 [A]) of membrane localized mature FopA. Further, we performed bioinformatics and biophysical experiments to unveil its structural organization in the outer membrane. FopA consists of 393 amino acids and has less than 40% sequence identity to known bacterial Omps. Using comprehensive sequence alignments and structure predictions together with existing partial structural information, we propose a two-domain organization for FopA. Circular dichroism spectroscopy and heat modifiability assay confirmed FopA has a {beta}-barrel domain consistent with alphafold2s prediction of an eight stranded {beta}-barrel at the N-terminus. Small angle X-ray scattering (SAXS) and native-polyacrylamide gel electrophoresis revealed FopA purified in detergent micelles is predominantly dimeric. Molecular density derived from SAXS at 31 [A] shows putative dimeric N-terminal {beta}-barrels surrounded by detergent corona and connected to C-terminal domains via flexible linker. Disorder analysis predicts N- and C-terminal domains are interspersed by a long intrinsically disordered region and alphafold2 predicts this region to be largely unstructured. Taken together, we propose a dimeric, two-domain organization of FopA in the outer membrane: the N-terminal {beta}-barrel is membrane embedded, provides dimerization interface and tethers to membrane extrinsic C-terminal domain via long flexible linker. Structure determination of membrane localized mature FopA is essential to understand its role in pathogenesis and develop anti-tularemia therapeutics. Our results pave the way towards it.


title: Beta-cell mass expansion during puberty involves serotonin signaling and determines glucose homeostasis in adulthood

abstract: Puberty is associated with transient insulin resistance that normally recedes at the end of puberty; however, in overweight children insulin resistance persists leading to an increased risk of type 2 diabetes. The mechanisms whereby pancreatic {beta} cells adapt to pubertal insulin resistance, and how they are affected by the metabolic status, have not been investigated. Here we show that puberty is associated with a transient increase in {beta}-cell proliferation in rats and humans of both sexes. In rats, {beta}-cell proliferation correlated with a rise in growth hormone (GH) levels. Serum from pubertal rats and humans promoted {beta}-cell proliferation, suggesting the implication of a circulating factor. In pubertal rat islets, expression of genes of the GH/serotonin (5-HT) pathway underwent changes consistent with proliferative effect. Inhibition of the pro-proliferative 5-HT receptor isoform HTR2b blocked the increase in {beta}-cell proliferation in pubertal islets ex vivo and in vivo. Peri-pubertal metabolic stress blunted {beta}-cell proliferation during puberty and led to altered glucose homeostasis later in life. This study identifies a role of GH/GHR/5-HT/HTR2b signaling in the control of {beta}-cell mass expansion during puberty and a mechanistic link between pubertal obesity and the risk of developing type 2 diabetes.

category: physiology


title: Hemispheric Asymmetries of Individual Differences in Functional Connectivity

abstract: Resting-state fMRI studies have revealed that individuals exhibit stable, functionally meaningful divergences in large-scale network organization. The strongest deviations (called network "variants") have a characteristic spatial distribution, with qualitative evidence from prior reports suggesting that this distribution differs across hemispheres. Hemispheric asymmetries can inform us on the developmental constraints of idiosyncratic regions. Here, we used data from the Human Connectome Project to systematically investigate hemispheric differences in network variants. Variants were significantly larger in the right hemisphere, particularly along the frontal operculum and medial frontal cortex. Variants in the left hemisphere were smaller but slightly more numerous, appearing most commonly around the temporoparietal junction. We investigated how variant asymmetries vary by functional network and how they compare with typical network distributions. For some networks, variants seemingly increase group-average network asymmetries (e.g., the language network is slightly bigger in the left hemisphere and variants appeared more frequently in the ipsilateral hemisphere). For other networks, variants counter the group-average network asymmetries (e.g., the default mode network is slightly bigger in the left hemisphere, but variants were more frequent in the right hemisphere). Intriguingly, left- and right-handers differed in their network variant asymmetries for the cinguloopercular and frontoparietal networks, suggesting that variant asymmetries are relevant to lateralized traits. These findings demonstrate that idiosyncratic aspects of brain organization differ systematically across the hemispheres. We discuss how these asymmetries in brain organization may inform us on developmental constraints of network variants, and how they may relate to functions differentially linked to the two hemispheres.


title: In Silico Improvement of Highly Protective Anti-Malarial Antibodies

abstract: Antibody CIS43 binds Plasmodium falciparum circumsporozoite protein (PfCSP) and protects against malaria, as recently demonstrated clinically. To improve the efficacy of CIS43, we developed an in silico pipeline to optimize the interaction energy of CIS43 to its junctional epitope (peptide 21: PfCSP residues 101-115). Starting from two improved CIS43 variants, recently elicited from a CIS43-germline knock-in mice, single and double amino acid substitutions in the peptide 21-proximal heavy (VH) and light (VL) variable regions were introduced. CIS43-variants, selected on the basis of improved in silico interface and stability energies, showed increased affinity to peptide 21 and superior malaria-protective efficacy. The best designed variant, antibody P3-43, was significantly more protective than its template antibody m43.151, with greater liver-burden protection than the current best-in-class (antibody iGL-CIS43.D3). Crystal structures of improved antibodies revealed atomic-level interactions explaining gains in binding affinity. The reported pipeline provides a powerful in silico approach to improve antibody functionality.


title: Development of a monoclonal antibody to a vibriophage as a proxy for Vibrio cholerae detection

abstract: Cholera is an acute watery diarrheal disease that causes high rates of morbidity and mortality without treatment. Early detection of the etiologic agent of toxigenic Vibrio cholerae is important to mobilize treatment and mitigate outbreaks. Monoclonal antibody (mAb) based rapid diagnostic tests (RDTs) enable early detection in settings without laboratory capacity. However, the odds of an RDT testing positive are reduced by nearly 90% when the common virulent bacteriophage ICP1 is present. We hypothesize that adding a mAb for the common, and specific, virulent bacteriophage ICP1 as a proxy for V. cholerae to an RDT will increase diagnostic sensitivity when virulent ICP1 phage are present. In this study, we used an in-silico approach to identify immunogenic ICP1 protein targets that were conserved across disparate time periods and locations. Specificity of targets to cholera patients with known ICP1 was determined, and specific targets were used to produce mAbs in a murine model. Candidate mAbs to the head protein demonstrated specificity to ICP1 by ELISA and an ICP1 phage neutralization assay. The limit of detection of the final mAb candidate for ICP1 phage particles spiked into cholera stool matrix was 8 x 105 plaque forming units by Western blot analysis. This mAb will be incorporated into a RDT prototype for evaluation in a future diagnostic study to test the guiding hypothesis behind this study.


title: Machine learning improves global models of plant diversity

abstract: Despite the paramount role of plant diversity for ecosystem functioning, biogeochemical cycles, and human welfare, knowledge of its global distribution is incomplete, hampering basic research and biodiversity conservation. Here, we used machine learning (random forests, extreme gradient boosting, neural networks) and conventional statistical methods (generalised linear models, generalised additive models) to model species richness and phylogenetic richness of vascular plants worldwide based on 830 regional plant inventories including c. 300,000 species and predictors of past and present environmental conditions. Machine learning showed an outstanding performance, explaining up to 80.9% of species richness and 83.3% of phylogenetic richness. Current climate and environmental heterogeneity emerged as the primary drivers, while past environmental conditions left only small but detectable imprints on plant diversity. Finally, we combined predictions from multiple modelling techniques (ensemble predictions) to reveal global patterns and centres of plant diversity at multiple resolutions down to 7,774 km2. Our predictive maps provide the most accurate estimates of global plant diversity available to date at grain sizes relevant for conservation and macroecology.


title: The senescence regulator S40 family members from Caragana intermedia and Arabidopsis thaliana inhibit leaf senescence via promoting cytokinins synthesis

abstract:
Leaf senescence is regulated by both endogenous hormones and environmental stimuli in a programmed and concerted way. The members of the S40 family have been reported to play roles in leaf senescence. Here we report that overexpression of an S40 family member from Caragana intermedia, CiS40-11, delayed the leaf senescence. Phylogenetic analysis revealed that the CiS40-11 protein had the highest identity with AtS40-5 and AtS40-6 of A. thaliana. CiS40-11 was highly expressed in leaves and was down-regulated after dark treatment. The subcellular localization analysis showed that CiS40-11 was a cytoplasm-nucleus dual-localized protein. Leaf senescence was delayed in CiS40-11 transgenic A. thaliana or by its transient expression in C. intermedia. Transcriptomic analysis and endogenous hormones assay revealed that CiS40-11 inhibited leaf senescence via promoting the biosynthesis of cytokinins, through blocking AtMYB2 expression in CiS40-11 overexpression lines. Furthermore, in the ats40-5a and ats40-6a mutants, AtMYB2 expression was increased and their leaves exhibited a premature senescence phenotype. Our results show that CiS40-11 (and its orthologs, AtS40-5 and AtS40-6) promoted cytokinin synthesis by inhibiting the expression of MYB2 and releasing its negative regulation on the expression of IPTs to inhabit leaf senescence.

HighlightThe senescence regulator S40 family members CiS40-11, AtS40-5 and AtS40-6 are induced by light and inhibition leaf senescence by promoting cytokinin synthesis.


title: Tissue-Specific Transcriptome Responses to Fusarium Head Blight and Fusarium Root Rot

abstract: Fusarium head blight (FHB) and Fusarium root rot (FRR) are important diseases of small-grained cereals caused by Fusarium species. While host response to FHB has been subject to extensive study, very little is known about response to FRR and the transcriptome responses of FHB and FRR have not been thoroughly compared. Brachypodium distachyon (Bd) is an effective model for investigating host responses to both FHB and FRR. In this study the transcriptome response of Bd to F. graminearum (Fg) infection of heads and roots was investigated. An RNA-seq analysis was performed on both Bd FHB and FRR during the early infection. Additionally, an RNA-seq analysis was performed on in vitro samples of Fg for comparison with Fg gene expression in planta. Differential gene expression and gene-list enrichment analyses were used to compare FHB and FRR transcriptome responses in both Bd and Fg. Differential expression of selected genes was confirmed using RT-qPCR. Most genes associated with receptor signalling, cell-wall modification, oxidative stress metabolism, and cytokinin and auxin biosynthesis and signalling genes were generally upregulated in FHB or were downregulated in FRR. In contrast, Bd genes involved in jasmonic acid and ethylene biosynthesis and signalling, and antimicrobial production were similarly differentially expressed in both tissues in response to infection. A transcriptome analysis of predicted Fg effectors with the same infected material revealed elevated expression of both core tissue- independent genes including cell-wall degradation enzymes and the gene cluster for DON production but also several tissue-dependent genes including those for aurofusarin production and cutin degradation. This evidence suggests that Fg modulates its transcriptome to different tissues of the same host.


title: Prioritizing Candidate eQTL Causal Genes in Arabidopsis using Random Forests

abstract: Expression quantitative trait locus (eQTL) mapping has been widely used to study the genetic regulation of gene expression in Arabidopsis thaliana. As a result, a large amount of eQTL data has been generated for this model plant; however, only a few causal eQTL genes have been identified, and experimental validation is costly and laborious. A prioritization method could help speed up the identification of causal eQTL genes. This study extends the machine-learning-based QTG-Finder2 method for prioritizing candidate causal genes in phenotype QTLs to be used for eQTLs by adding gene structure, protein interaction, and gene expression. Independent validation shows that the new algorithm can prioritize sixteen out of twenty-five potential eQTL causal genes within the 20% rank percentile. Several new features are important in prioritizing causal eQTL genes, including the number of protein-protein interactions, unique domains, and introns. Overall, this study provides a foundation for developing computational methods to prioritize candidate eQTL causal genes. The prediction of all genes is available in the AraQTL workbench (https://www.bioinformatics.nl/AraQTL/) to support the identification of gene expression regulators in Arabidopsis.


title: Identification of YAB11-NGAL1 controlling leaf serrations through enhanced genome-wide association studies of Populus

abstract: Leaf margins are complex plant morphological features and contribute to the diversity of leaf shapes which effect on plant structure, yield and adaptation. Although several regulators of leaf margins have been identified, the genetic basis of natural variation therein has not been fully elucidated. We first profiled two distinct types (serration and smooth) of leaf morphology using the persistent homology mathematical framework (PHMF) in poplar. Combined genome-wide association studies (GWAS) and expression quantitative trait nucleotide (eQTN) mapping to create a module of leaf morphology controlling using data from Populus tomentosa and P. simonii association population, respectively. Natural variation of leaf margins is associated with transcript abundances of YABBY11 (YAB11) in poplar. In P. tomentosa, PtoYAB11 carries premature stop codon (PtoYAB11PSC) resulting in lost its positive regulation in PtoNGAL-1, PtoRBCL, PtoATPA, PtoATPE, and PtoPSBB. Overexpression of PtoYAB11PSC serrated leaf margin, enlarged leaves, promoted photosynthesis and increased biomass. Overexpression of PsiYAB11 in P. tomentosa could rescue leaf margin serration and increase stomatal density and light damage repair ability. In poplar, YAB11-NGAL1is sensitive to environmental conditions and play positive regulator of leaf margin serration. It might be important regulator which bridge environment signaling to leaf morphological plasticity.


title: The multi-BRCT domain protein DDRM2 is required for homologous recombination in plants

abstract:
DNA double-strand breaks (DSBs) are the most toxic DNA damage for cells. Homologous recombination (HR) is a precise DSB repair mechanism as well as a basis for gene targeting using genome-editing techniques. Despite the importance of HR, the HR mechanism in plants is poorly understood. In a genetic screen for DNA Damage Response Mutants (DDRMs), we find that the Arabidopsis ddrm2 mutant is hypersensitive to DSB-inducing reagents. DDRM2 encodes a protein with four BRCA1 C-terminal (BRCT) domains and is highly conserved in plants including the earliest land plant linage, bryophytes. The plant-specific transcription factor SOG1 binds to the promoter of DDRM2 and activates its expression, suggesting that DDRM2 is a direct target of SOG1. In consistence, the expression of DDRM2 is induced by DSBs in a SOG1-dependent manner. Epistasis analysis indicates that DDRM2 functions downstream of SOG1. Similar to the sog1 mutant, the ddrm2 mutant shows dramatically reduced HR efficiency. Our study suggests that the SOG1-DDRM2 module is required for HR, providing new insights into the HR mechanisms in plants and a potential target for improving the efficiency of gene targeting.

One Sentence SummaryA genetic screen in Arabidopsis reveals that the multi-BRCT domain protein DDRM2 is required for homologous recombination and is targeted by the master DNA damage response regulator SOG1.


title: A baseline for the genetic stock identification of Atlantic herring, Clupea harengus, in ICES Divisions 6.a, 7.b-c

abstract: Atlantic herring in ICES Divisions 6.a, 7.b-c comprises at least three populations, distinguished by temporal and spatial differences in spawning, which have until recently been managed as two stocks defined by geographic delineators. Outside of spawning the populations form mixed aggregations, which are the subject of acoustic surveys. The inability to distinguish the populations has prevented the development of separate survey indices and separate stock assessments. A panel of 45 SNPs, derived from whole genome sequencing, were used to genotype 3,480 baseline spawning samples (2014-2021). A temporally stable baseline comprising 2,316 herring from populations known to inhabit Division 6.a was used to develop a genetic assignment method, with a self-assignment accuracy >90%. The long-term temporal stability of the assignment model was validated by assigning archive (2003-2004) baseline samples (270 individuals) with a high level of accuracy. Assignment of non-baseline samples (1,514 individuals) from Division 6.a, 7.b-c indicated previously unrecognised levels of mixing of populations outside of the spawning season. The genetic markers and assignment models presented constitute a  toolbox that can be used for the assignment of herring caught in mixed survey and commercial catches in Division 6.a into their population of origin with a high level of accuracy.

category: zoology


title: Traits of soil bacteria predict plant responses to soil moisture

abstract: Microbes can promote beneficial plant and animal responses to abiotic environments, but the ecological drivers of this benefit remain elusive. Here we investigated byproduct benefits, which occur when traits that increase the fitness of one species provide incidental benefits to another species with no direct cost to the provider species. In experimental mesocosms, microbial traits predicted plant responses to soil moisture such that bacteria with self-beneficial traits in drought increased plant early growth, size at reproduction, and chlorophyll concentration under drought, while bacteria with self-beneficial traits in well-watered environments increased these same plant traits in well-watered environments. Thus, microbial traits that promote microbial success in different soil moisture environments also promote plant success in these same environments. Our results show that the concept of byproduct benefits, originally conceived to explain the evolution of cooperation in pairwise mutualisms, also applies to interactions between plants and non-symbiotic soil microbes.


title: Restoration of high-sensitivity patterned vision in motion with an engineered light-gated G protein-coupled receptor

abstract: Inherited retinal degenerations (IRDs) result in blindness due to apoptotic cell death of rods and cones, but spare other retinal neurons, providing a potential that delivery of a light-activated signaling protein to surviving neurons may restore vision. We previously demonstrated that aspects of vision could be restored by introduction into surviving cells of a G protein-coupled receptor for glutamate (mGluR) bearing a tethered photoswitchable agonist. However, this system, containing one photoswitchable agonist per glutamate binding site, yielded low sensitivity, responding only to visual stimuli at the intensity of bright outdoor light, similar to channelrhodopsins. To increase sensitivity, we designed a multi-branched photoswitch, bearing four light-activatable glutamates for each glutamate binding site. When tethered to a modified mGluR2 expressed in retinal ganglion cells via intravitreal AAV gene delivery, this photoswitch boosted sensitivity by ~100-fold compared to the unbranched (single photo-ligand) photoswitch. This improvement in sensitivity enabled an IRD mouse model (rd1) to perform visually-guided object recognition under incidental room light and pattern recognition using standard LCD computer displays. The restored line pattern differentiation approached the acuity reported for normal mouse vision. Pattern recognition functioned as well as wildtype vision with line patterns moving at speeds of up to 36{degrees}/s. In summary, this two-component chemical-optogenetic approach combines high sensitivity and high acuity with superior motion vision, and, unlike optogenetic gene therapy, can be adjusted for dose, upgraded, as new photoswitches are developed, and discontinued at will.