Decreased Expression of the Host Long-Noncoding RNA-GM Facilitates Viral Escape by Inhibiting the Kinase activity TBK1 via S-glutathionylation.

Viruses have evolved multiple strategies to evade elimination by the immune system. Here we examined the contribution of host long noncoding RNAs (lncRNAs) in viral immune evasion. By functional screening of lncRNAs whose expression decreased upon viral infection of macrophages, we identified a lncRNA (lncRNA-GM, Gene Symbol: AK189470.1) that promoted type I interferon (IFN-I) production and inhibited viral replication. Deficiency of lncRNA-GM in mice increased susceptibility to viral infection and impaired IFN-I production. Mechanistically, lncRNA-GM bound to glutathione S-transferase M1 (GSTM1) and blocked GSTM1 interaction with the kinase TBK1, reducing GSTM1-mediated S-glutathionylation of TBK1. Decreased S-glutathionylation enhanced TBK1 activity and downstream production of antiviral mediators. Viral infection reprogrammed intracellular glutathione metabolism and furthermore, an oxidized glutathione mimetic could inhibit TBK1 activity and promote viral replication. Our findings reveal regulation of TBK1 by S-glutathionylation and provide insight into the viral mediated metabolic changes that impact innate immunity and viral evasion.

MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis.

Translation inhibition is a major but poorly understood mode of action of microRNAs (miRNAs) in plants and animals. In particular, the subcellular location where this process takes place is unknown. Here, we show that the translation inhibition, but not the mRNA cleavage activity, of Arabidopsis miRNAs requires ALTERED MERISTEM PROGRAM1 (AMP1). AMP1 encodes an integral membrane protein associated with endoplasmic reticulum (ER) and ARGONAUTE1, the miRNA effector and a peripheral ER membrane protein. Large differences in polysome association of miRNA target RNAs are found between wild-type and the amp1 mutant for membrane-bound, but not total, polysomes. This, together with AMP1-independent recruitment of miRNA target transcripts to membrane fractions, shows that miRNAs inhibit the translation of target RNAs on the ER. This study demonstrates that translation inhibition is an important activity of plant miRNAs, reveals the subcellular location of this activity, and uncovers a previously unknown function of the ER.

Genome stability from the perspective of telomere length.

Telomeres and their associated proteins protect the ends of chromosomes to maintain genome stability. Telomeres undergo progressive shortening with each cell division in mammalian somatic cells without telomerase, resulting in genome instability. When telomeres reach a critically short length or are recognized as a damage signal, cells enter a state of senescence, followed by cell cycle arrest, programmed cell death, or immortalization. This review provides an overview of recent advances in the intricate relationship between telomeres and genome instability. Alongside well-established mechanisms such as chromosomal fusion and telomere fusion, we will delve into the perspective on genome stability by examining the role of retrotransposons. Retrotransposons represent an emerging pathway to regulate genome stability through their interactions with telomeres.

INDETERMINATE1 autonomously regulates phosphate homeostasis upstream of the miR399-ZmPHO2 signaling module in maize.

The macronutrient phosphorus is essential for plant growth and development. Plants have evolved multiple strategies to increase the efficiency of phosphate (Pi) acquisition to protect themselves from Pi starvation. However, the crosstalk between Pi homeostasis and plant development remains to be explored. Here, we report that overexpressing microRNA399 (miR399) in maize (Zea mays) is associated with premature senescence after pollination. Knockout of ZmPHO2 (Phosphate 2), a miR399 target, resulted in a similar premature senescence phenotype. Strikingly, we discovered that INDETERMINATE1 (ID1), a floral transition regulator, inhibits the transcription of ZmMIR399 genes by directly binding to their promoters, alleviating the repression of ZmPHO2 by miR399 and ultimately contributing to the maintenance of Pi homeostasis in maize. Unlike ZmMIR399 genes, whose expression is induced by Pi deficiency, ID1 expression was independent of the external inorganic orthophosphate status, indicating that ID1 is an autonomous regulator of Pi homeostasis. Furthermore, we show that ZmPHO2 was under selection during maize domestication and cultivation, resulting in a more sensitive response to Pi starvation in temperate maize than in tropical maize. Our study reveals a direct functional link between Pi-deprivation sensing by the miR399-ZmPHO2 regulatory module and plant developmental regulation by ID1.

Gre factors help Salmonella adapt to oxidative stress by improving transcription elongation and fidelity of metabolic genes.

Detoxification, scavenging, and repair systems embody the archetypical antioxidant defenses of prokaryotic and eukaryotic cells. Metabolic rewiring also aids with the adaptation of bacteria to oxidative stress. Evolutionarily diverse bacteria combat the toxicity of reactive oxygen species (ROS) by actively engaging the stringent response, a stress program that controls many metabolic pathways at the level of transcription initiation via guanosine tetraphosphate and the α-helical DksA protein. Studies herein with Salmonella demonstrate that the interactions of structurally related, but functionally unique, α-helical Gre factors with the secondary channel of RNA polymerase elicit the expression of metabolic signatures that are associated with resistance to oxidative killing. Gre proteins both improve transcriptional fidelity of metabolic genes and resolve pauses in ternary elongation complexes of Embden-Meyerhof-Parnas (EMP) glycolysis and aerobic respiration genes. The Gre-directed utilization of glucose in overflow and aerobic metabolism satisfies the energetic and redox demands of Salmonella, while preventing the occurrence of amino acid bradytrophies. The resolution of transcriptional pauses in EMP glycolysis and aerobic respiration genes by Gre factors safeguards Salmonella from the cytotoxicity of phagocyte NADPH oxidase in the innate host response. In particular, the activation of cytochrome bd protects Salmonella from phagocyte NADPH oxidase-dependent killing by promoting glucose utilization, redox balancing, and energy production. Control of transcription fidelity and elongation by Gre factors represent important points in the regulation of metabolic programs supporting bacterial pathogenesis.

A pneumonia outbreak associated with a new coronavirus of probable bat origin.

Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats1-4. Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans5-7. Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor-angiotensin converting enzyme II (ACE2)-as SARS-CoV.

Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease.

RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.

RAVAR: a curated repository for rare variant-trait associations.

Rare variants contribute significantly to the genetic causes of complex traits, as they can have much larger effects than common variants and account for much of the missing heritability in genome-wide association studies. The emergence of UK Biobank scale datasets and accurate gene-level rare variant-trait association testing methods have dramatically increased the number of rare variant associations that have been detected. However, no systematic collection of these associations has been carried out to date, especially at the gene level. To address the issue, we present the Rare Variant Association Repository (RAVAR), a comprehensive collection of rare variant associations. RAVAR includes 95 047 high-quality rare variant associations (76186 gene-level and 18 861 variant-level associations) for 4429 reported traits which are manually curated from 245 publications. RAVAR is the first resource to collect and curate published rare variant associations in an interactive web interface with integrated visualization, search, and download features. Detailed gene and SNP information are provided for each association, and users can conveniently search for related studies by exploring the EFO tree structure and interactive Manhattan plots. RAVAR could vastly improve the accessibility of rare variant studies. RAVAR is freely available for all users without login requirement at http://www.ravar.bio.

G-quadruplexes on chromosomal DNA negatively regulates topoisomerase 1 activity.

Human DNA topoisomerase 1 (Top1) is a crucial enzyme responsible for alleviating torsional stress on DNA during transcription and replication, thereby maintaining genome stability. Previous researches had found that non-working Top1 interacted extensively with chromosomal DNA in human cells. However, the reason for its retention on chromosomal DNA remained unclear. In this study, we discovered a close association between Top1 and chromosomal DNA, specifically linked to the presence of G-quadruplex (G4) structures. G4 structures, formed during transcription, trap Top1 and hinder its ability to relax neighboring DNAs. Disruption of the Top1-G4 interaction using G4 ligand relieved the inhibitory effect of G4 on Top1 activity, resulting in a further reduction of R-loop levels in cells. Additionally, the activation of Top1 through the use of a G4 ligand enhanced the toxicity of Top1 inhibitors towards cancer cells. Our study uncovers a negative regulation mechanism of human Top1 and highlights a novel pathway for activating Top1.

Dissection of 3D chromosome organization in Streptomyces coelicolor A3(2) leads to biosynthetic gene cluster overexpression.

The soil-dwelling filamentous bacteria, Streptomyces, is widely known for its ability to produce numerous bioactive natural products. Despite many efforts toward their overproduction and reconstitution, our limited understanding of the relationship between the host's chromosome three dimension (3D) structure and the yield of the natural products escaped notice. Here, we report the 3D chromosome organization and its dynamics of the model strain, Streptomyces coelicolor, during the different growth phases. The chromosome undergoes a dramatic global structural change from primary to secondary metabolism, while some biosynthetic gene clusters (BGCs) form special local structures when highly expressed. Strikingly, transcription levels of endogenous genes are found to be highly correlated to the local chromosomal interaction frequency as defined by the value of the frequently interacting regions (FIREs). Following the criterion, an exogenous single reporter gene and even complex BGC can achieve a higher expression after being integrated into the chosen loci, which may represent a unique strategy to activate or enhance the production of natural products based on the local chromosomal 3D organization.

The methylglyoxal pathway is a sink for glutathione in Salmonella experiencing oxidative stress.

Salmonella suffer the cytotoxicity of reactive oxygen species generated by the phagocyte NADPH oxidase in the innate host response. Periplasmic superoxide dismutases, catalases and hydroperoxidases detoxify superoxide and hydrogen peroxide (H2O2) synthesized in the respiratory burst of phagocytic cells. Glutathione also helps Salmonella combat the phagocyte NADPH oxidase; however, the molecular mechanisms by which this low-molecular-weight thiol promotes resistance of Salmonella to oxidative stress are currently unknown. We report herein that Salmonella undergoing oxidative stress transcriptionally and functionally activate the methylglyoxal pathway that branches off from glycolysis. Activation of the methylglyoxal pathway consumes a substantial proportion of the glutathione reducing power in Salmonella following exposure to H2O2. The methylglyoxal pathway enables Salmonella to balance glucose utilization with aerobic respiratory outputs. Salmonella take advantage of the metabolic flexibility associated with the glutathione-consuming methylglyoxal pathway to resist reactive oxygen species generated by the enzymatic activity of the phagocyte NADPH oxidase in macrophages and mice. Taken together, glutathione fosters oxidative stress resistance in Salmonella against the antimicrobial actions of the phagocyte NADPH oxidase by promoting the methylglyoxal pathway, an offshoot metabolic adaptation of glycolysis.

Spike substitution T813S increases Sarbecovirus fusogenicity by enhancing the usage of TMPRSS2.

SARS-CoV Spike (S) protein shares considerable homology with SARS-CoV-2 S, especially in the conserved S2 subunit (S2). S protein mediates coronavirus receptor binding and membrane fusion, and the latter activity can greatly influence coronavirus infection. We observed that SARS-CoV S is less effective in inducing membrane fusion compared with SARS-CoV-2 S. We identify that S813T mutation is sufficient in S2 interfering with the cleavage of SARS-CoV-2 S by TMPRSS2, reducing spike fusogenicity and pseudoparticle entry. Conversely, the mutation of T813S in SARS-CoV S increased fusion ability and viral replication. Our data suggested that residue 813 in the S was critical for the proteolytic activation, and the change from threonine to serine at 813 position might be an evolutionary feature adopted by SARS-2-related viruses. This finding deepened the understanding of Spike fusogenicity and could provide a new perspective for exploring Sarbecovirus' evolution.

MicroRNA1432 regulates rice drought stress tolerance by targeting the CALMODULIN-LIKE2 gene.

Due to climate change, drought has become a major threat to rice (Oryza sativa L.) growth and yield worldwide. Understanding the genetic basis of drought tolerance in rice is therefore of great importance. Here, we identified a microRNA, miR1432, which regulates rice drought tolerance by targeting the CALMODULIN-LIKE2 (OsCaML2) gene. Mutation of MIR1432 or suppression of miR1432 expression significantly impaired seed germination and seedling growth under drought-stress conditions. Molecular analysis demonstrated that miR1432 affected rice drought tolerance by directly targeting OsCaML2, which encodes an EF-hand chiral calcium-binding protein. Overexpression of a miR1432-resistant form of OsCaML2 (OEmCaML2) phenocopied the mir1432 mutant and miR1432 suppression plants. Furthermore, the suppression of miR1432 severely affected the expression of genes involved in responses to stimulation, metabolism and signal transduction, especially the mitogen-activated protein kinase (MAPK) pathway and hormone transduction pathway in rice under drought stress. Thus, our findings show that the miR1432-OsCaML2 module plays an important role in the regulation of rice drought tolerance, suggesting its potential utilization in developing molecular breeding strategies that improve crop drought tolerance.

Altered white matter functional pathways in Alzheimer's disease.

Alzheimer's disease (AD) is associated with functional disruption in gray matter (GM) and structural damage to white matter (WM), but the relationship to functional signal in WM is unknown. We performed the functional connectivity (FC) and graph theory analysis to investigate abnormalities of WM and GM functional networks and corpus callosum among different stages of AD from a publicly available dataset. Compared to the controls, AD group showed significantly decreased FC between the deep WM functional network (WM-FN) and the splenium of corpus callosum, between the sensorimotor/occipital WM-FN and GM visual network, but increased FC between the deep WM-FN and the GM sensorimotor network. In the clinical groups, the global assortativity, modular interaction between occipital WM-FN and visual network, nodal betweenness centrality, degree centrality, and nodal clustering coefficient in WM- and GM-FNs were reduced. However, modular interaction between deep WM-FN and sensorimotor network, and participation coefficients of deep WM-FN and splenium of corpus callosum were increased. These findings revealed the abnormal integration of functional networks in different stages of AD from a novel WM-FNs perspective. The abnormalities of WM functional pathways connect downward to the corpus callosum and upward to the GM are correlated with AD.

Intracellular Pseudomonas aeruginosa within the Airway Epithelium of Cystic Fibrosis Lung Tissues.

Rationale: Pseudomonas aeruginosa is the major bacterial pathogen colonizing the airways of adult patients with cystic fibrosis (CF) and causes chronic infections that persist despite antibiotic therapy. Intracellular bacteria may represent an unrecognized reservoir of bacteria that evade the immune system and antibiotic therapy. Although the ability of P. aeruginosa to invade and survive within epithelial cells has been described in vitro in different epithelial cell models, evidence of this intracellular lifestyle in human lung tissues is currently lacking. Objectives: To detect and characterize intracellular P. aeruginosa in CF airway epithelium from human lung explant tissues. Methods: We sampled lung explant tissues from patients with CF undergoing lung transplantation and non-CF lung donor control tissue. We analyzed lung tissue sections for the presence of intracellular P. aeruginosa using quantitative culture and microscopy, in parallel to histopathology and airway morphometry. Measurements and Main Results: P. aeruginosa was isolated from the lungs of seven patients with CF undergoing lung transplantation. Microscopic assessment revealed the presence of intracellular P. aeruginosa within airway epithelial cells in three of the seven patients analyzed at a varying but low frequency. We observed those events occurring in lung regions with high bacterial burden. Conclusions: This is the first study describing the presence of intracellular P. aeruginosa in CF lung tissues. Although intracellular P. aeruginosa in airway epithelial cells is likely relatively rare, our findings highlight the plausible occurrence of this intracellular bacterial reservoir in chronic CF infections.

LncBook 2.0: integrating human long non-coding RNAs with multi-omics annotations.

LncBook, a comprehensive resource of human long non-coding RNAs (lncRNAs), has been used in a wide range of lncRNA studies across various biological contexts. Here, we present LncBook 2.0 (https://ngdc.cncb.ac.cn/lncbook), with significant updates and enhancements as follows: (i) incorporation of 119 722 new transcripts, 9632 new genes, and gene structure update of 21 305 lncRNAs; (ii) characterization of conservation features of human lncRNA genes across 40 vertebrates; (iii) integration of lncRNA-encoded small proteins; (iv) enrichment of expression and DNA methylation profiles with more biological contexts and (v) identification of lncRNA-protein interactions and improved prediction of lncRNA-miRNA interactions. Collectively, LncBook 2.0 accommodates a high-quality collection of 95 243 lncRNA genes and 323 950 transcripts and incorporates their abundant annotations at different omics levels, thereby enabling users to decipher functional significance of lncRNAs in different biological contexts.

Dot1l cooperates with Npm1 to repress endogenous retrovirus MERVL in embryonic stem cells.

Dot1l is a histone methyltransferase without a SET domain and is responsible for H3K79 methylation, which marks active transcription. In contradiction, Dot1l also participates in silencing gene expression. The target regions and mechanism of Dot1l in repressing transcription remain enigmatic. Here, we show that Dot1l represses endogenous retroviruses in embryonic stem cells (ESCs). Specifically, the absence of Dot1l led to the activation of MERVL, which is a marker of 2-cell-like cells. In addition, Dot1l deletion activated the 2-cell-like state and predisposed ESCs to differentiate into trophectoderm lineage. Transcriptome analysis revealed activation of 2-cell genes and meiotic genes by Dot1l deletion. Mechanistically, Dot1l interacted with and co-localized with Npm1 on MERVL, and depletion of Npm1 similarly augmented MERVL expression. The catalytic activity and AT-hook domain of Dot1l are important to suppress MERVL. Notably, Dot1l-Npm1 restricts MERVL by regulating protein level and deposition of histone H1. Furthermore, Dot1l is critical for Npm1 to efficiently interact with histone H1 and inhibit ubiquitination of H1 whereas Npm1 is essential for Dot1l to interact with MERVL. Altogether, we discover that Dot1l represses MERVL through chaperoning H1 by collaborating with Npm1. Importantly, our findings shed light on the non-canonical transcriptional repressive role of Dot1l in ESCs.

Point mutation P174L of the penA gene endowing ceftazidime resistance to Burkholderia pseudomallei in China.

In a clinical isolate of Burkholderia pseudomallei from Hainan, the association between the emergence of ceftazidime resistance and a novel PenA P174L allele was identified for the first time, providing an understanding of one mechanism by which ceftazidime resistance arises in B. pseudomallei.

Loss of growth differentiation factor 9 causes an arrest of early folliculogenesis in zebrafish-A novel insight into its action mechanism.

Growth differentiation factor 9 (GDF9) was the first oocyte-specific growth factor identified; however, most information about GDF9 functions comes from studies in the mouse model. In this study, we created a mutant for Gdf9 gene (gdf9-/-) in zebrafish using TALEN approach. The loss of Gdf9 caused a complete arrest of follicle development at primary growth (PG) stage. These follicles eventually degenerated, and all mutant females gradually changed to males through sex reversal, which could be prevented by mutation of the male-promoting gene dmrt1. Interestingly, the phenotypes of gdf9-/- could be rescued by simultaneous mutation of inhibin α (inha-/-) but not estradiol treatment, suggesting a potential role for the activin-inhibin system or its signaling pathway in Gdf9 actions. In gdf9-null follicles, the expression of activin ßAa (inhbaa), but not ßAb (inhbab) and ßB (inhbb), decreased dramatically; however, its expression rebounded in the double mutant (gdf9-/-;inha-/-). These results indicate clearly that the activation of PG follicles to enter the secondary growth (SG) requires intrinsic factors from the oocyte, such as Gdf9, which in turn works on the neighboring follicle cells to trigger follicle activation, probably involving activins. In addition, our data also support the view that estrogens are not involved in follicle activation as recently reported.

Accurate positioning of functional residues with robotics-inspired computational protein design.

SignificanceComputational protein design promises to advance applications in medicine and biotechnology by creating proteins with many new and useful functions. However, new functions require the design of specific and often irregular atom-level geometries, which remains a major challenge. Here, we develop computational methods that design and predict local protein geometries with greater accuracy than existing methods. Then, as a proof of concept, we leverage these methods to design new protein conformations in the enzyme ketosteroid isomerase that change the protein's preference for a key functional residue. Our computational methods are openly accessible and can be applied to the design of other intricate geometries customized for new user-defined protein functions.