Guanosine penta- and tetraphosphate [(p)ppGpp] and their adenosine analogs [(p)ppApp] are bacterial second messengers known as alarmones. Members of the RelA-SpoT homolog (RSH) family synthesize (p)ppGpp to mediate the stringent response during nutrient starvation, whereas (p)ppApp synthetases have been identified as bactericidal toxins in diverse contexts including type VI secretion systems, toxin-antitoxin modules, and phages. Although alarmone synthesis has traditionally been viewed as a cytoplasmic process, early studies in Streptomyces suggested the existence of secreted alarmone synthetases. Here, we identify SaEAS, an exported alarmone synthetase (EAS) from Streptomyces albidoflavus, as the long-mysterious source of extracellular alarmone synthetase activity in Streptomyces. SaEAS produces both (p)ppGpp and (p)ppApp at rates exceeding 100,000 molecules per minute and has kinetic properties adapted to low substrate environments. A broader bioinformatic survey reveals ~600 EASs linked to a range of specialized bacterial secretion systems. Characterization of two additional EASs, VpEAS from Vibrio parahaemolyticus and AaEAS from Amycolatopsis azurea, shows that both produce (p)ppGpp exclusively and inhibit bacterial growth when localized to the cytoplasm. These findings challenge the longstanding view of (p)ppGpp as strictly pro-survival and unveil a diverse family of secreted RSH enzymes with potential roles in interbacterial antagonism and environmental signaling.IMPORTANCEAlarmone synthetases are intracellular enzymes that promote bacterial survival by responding to environmental stress. Although extracellular alarmone production has been reported in Streptomyces, the enzymes responsible for this activity remain unknown. Here, we identify hundreds of predicted exported alarmone synthetases (EASs) associated with bacterial protein export pathways. We show that SaEAS, secreted by Streptomyces albidoflavus, efficiently synthesizes (p)ppGpp and (p)ppApp in low substrate extracellular environments. When localized to the cytoplasm, SaEAS and two (p)ppGpp-specific EASs from Vibrio parahaemolyticus and Amycolatopsis azurea rapidly inhibit cell growth. Overall, our findings show that (p)ppGpp is not always a bacteriostatic, pro-survival molecule and suggest that the physiological consequences of alarmone production depend more on context and enzyme kinetics than on alarmone identity.

Unstable atherosclerotic plaques are a major cause of acute cardiovascular events and ischemic stroke. Clinical studies have suggested a link between periodontitis and atherosclerotic plaque progression, but the underlying mechanisms remain unclear. To investigate this, transcriptomic datasets related to periodontitis and atherosclerosis were downloaded from Gene Expression Omnibus. A weighted gene co-expression network analysis was used to identify gene modules associated with periodontitis, and the Limma R package identified differentially expressed genes (DEGs) between unstable and stable plaques. Overlapping genes were defined as periodontitis-related DEGs, followed by functional enrichment analysis and protein–protein interaction network construction. Machine learning methods were used to identify biomarkers for unstable plaques related to periodontitis, which were validated using external datasets. Immune infiltration and single-cell analyses were performed to explore the relationship between biomarkers and immune cells. A total of 161 periodontitis-related DEGs were identified, with the pathway analysis showing associations with immune regulation and collagen matrix degradation. HCK, NCKAP1L, and WAS were identified as biomarkers for unstable plaques, demonstrating a high diagnostic value (AUC: 0.9884, 95% CI: 0.9641–1). Immune infiltration analysis revealed an increase in macrophages within unstable plaques. Single-cell analysis showed HCK expression in macrophages and dendritic cells, while NCKAP1L and WAS were expressed in macrophages, dendritic cells, NK cells, and T cells. Consensus clustering identified three expression patterns within unstable plaques. Our findings were validated in atherosclerotic mouse models with periodontitis. This study provides insights into how periodontitis contributes to plaque instability, supporting diagnosis and intervention in patients with periodontitis.

We synthesized a WNT5A beta-hairpin peptide responsible for the protein's binding to its receptor, Frizzled. Full affinity to Frizzled requires all three disulfides but not an invariant tryptophan that significantly contributes to the buried surface area. Our work opens the avenue to target the Wnt-Fzd interface using synthetic peptides.

BackgroundAccording to some recent observational studies, the gut microbiota influences atherosclerosis via the gut microbiota-artery axis. However, the causal role of the gut microbiota in atherosclerosis remains unclear. Therefore, we used a Mendelian randomization (MR) strategy to try to dissect this causative link.MethodsThe biggest known genome-wide association study (GWAS) (n = 13,266) from the MiBioGen collaboration was used to provide summary data on the gut microbiota for a two-sample MR research. Data on atherosclerosis were obtained from publicly available GWAS data from the FinnGen consortium, including cerebral atherosclerosis (104 cases and 218,688 controls), coronary atherosclerosis (23,363 cases and 187,840 controls), and peripheral atherosclerosis (6631 cases and 162,201 controls). The causal link between gut microbiota and atherosclerosis was investigated using inverse variance weighting, MR-Egger, weighted median, weighted mode, and simple mode approaches, among which inverse variance weighting was the main research method. Cochran’s Q statistic was used to quantify the heterogeneity of instrumental variables (IVs), and the MR Egger intercept test was used to assess the pleiotropy of IVs.ResultsInverse-variance-weighted (IVW) estimation showed that genus Ruminiclostridium 9 had a protective influence on cerebral atherosclerosis (OR = 0.10, 95% CI: 0.01–0.67, P = 0.018), while family Rikenellaceae (OR = 5.39, 95% CI: 1.50–19.37, P = 0.010), family Streptococcaceae (OR = 6.87, 95% CI: 1.60–29.49, P = 0.010), genus Paraprevotella (OR = 2.88, 95% CI: 1.18–7.05, P = 0.021), and genus Streptococcus (OR = 5.26, 95% CI: 1.28–21.61, P = 0.021) had pathogenic effects on cerebral atherosclerosis. For family Acidaminococcaceae (OR = 0.87, 95% CI: 0.76–0.99, P = 0.039), the genus Desulfovibrio (OR = 0.89, 95% CI: 0.80–1.00, P = 0.048), the genus RuminococcaceaeUCG010 (OR = 0.80, 95% CI: 0.69–0.94, P = 0.006), and the Firmicutes phyla (OR = 0.87, 95% CI: 0.77–0.98, P = 0.023) were protective against coronary atherosclerosis. However, the genus Catenibacterium (OR = 1.12, 95% CI: 1.00–1.24, P = 0.049) had a pathogenic effect on coronary atherosclerosis. Finally, class Actinobacteria (OR = 0.83, 95% CI: 0.69–0.99, P = 0.036), family Acidaminococcaceae (OR = 0.76, 95% CI: 0.61–0.94, P = 0.013), genus Coprococcus2 (OR = 0.76, 95% CI: 0.60–0.96, P = 0.022), and genus RuminococcaceaeUCG010 (OR = 0.65, 95% CI: 0.46–0.92, P = 0.013), these four microbiota have a protective effect on peripheral atherosclerosis. However, for the genus Lachnoclostridium (OR = 1.25, 95% CI: 1.01–1.56, P = 0.040) and the genus LachnospiraceaeUCG001 (OR = 1.22, 95% CI: 1.04–1.42, P = 0.016), there is a pathogenic role for peripheral atherosclerosis. No heterogeneity was found for instrumental variables, and no considerable horizontal pleiotropy was observed.ConclusionWe discovered that the presence of probiotics and pathogens in the host is causally associated with atherosclerosis, and atherosclerosis at different sites is causally linked to specific gut microbiota. The specific gut microbiota associated with atherosclerosis identified by Mendelian randomization studies provides precise clinical targets for the treatment of atherosclerosis. In the future, we can further examine the gut microbiota’s therapeutic potential for atherosclerosis if we have a better grasp of the causal relationship between it and atherosclerosis.

Septic cardiomyopathy (SCM) is characterized by an abnormal inflammatory response and increased mortality. The role of efferocytosis in SCM is not well understood. We used integrated multi-omics analysis to explore the clinical and genetic roles of efferocytosis in SCM. We identified six module genes (ATP11C, CD36, CEBPB, MAPK3, MAPKAPK2, PECAM1) strongly associated with SCM, leading to an accurate predictive model. Subgroups defined by EFFscore exhibited distinct clinical features and immune infiltration levels. Survival analysis showed that the C1 subtype with a lower EFFscore had better survival outcomes. scRNA-seq analysis of peripheral blood mononuclear cells (PBMCs) from sepsis patients identified four genes (CEBPB, CD36, PECAM1, MAPKAPK2) associated with high EFFscores, highlighting their role in SCM. Molecular docking confirmed interactions between diagnostic genes and tamibarotene. Experimental validation supported our computational results. In conclusion, our study identifies a novel efferocytosis-related SCM subtype and diagnostic biomarkers, offering new insights for clinical diagnosis and therapy.

When challenged by starvation, bacterial organisms synthesize guanosine pentaphosphate and tetraphosphate, collectively denoted as (p)ppGpp, as second messengers to reprogram metabolism toward slower growth and enhanced stress tolerance. When starvation is alleviated, the RelA-SpoT Homolog (RSH) hydrolases downregulate (p)ppGpp, cleaving the 3'-diphosphate to produce GTP or GDP. Metazoan RSH hydrolases possess phosphatase activity responsible for converting cytoplasmic NADPH to NADH in mammalian cells. Inhibitor development for this family may therefore provide therapies to combat bacterial infection or metabolic dysregulation. Despite the availability of dozens of high-resolution structures, catalytic mechanisms of RSH hydrolases have remained poorly understood. All RSH hydrolases tightly bind a Mn2+ near its active center, which is believed sufficient for hydrolase activity. In contrast to this notion, we demonstrate, using the (p)ppGpp hydrolase SpoT from Acinetobacter baumannii, that a second divalent cation, presumably a Mg2+ under physiological conditions, is required for efficient catalysis. We also show that SpoT preferentially cleaves 3'-diphosphate over 3'-phosphate substrates, likely due to a key coordination between the β-phosphate and the second metal center. Metazoan RSH hydrolase replaces this β-phosphate with the side chain of an aspartate residue, thereby functioning as a phosphatase. We propose a binuclear metallohydrolase model where an invariant ED (Glu-Asp) diad, previously believed to activate the water nucleophile, instead coordinates to a Mg2+ center. The refined molecular and evolutionary blueprint of RSH hydrolases will provide a more reliable foundation for the development of small-molecule inhibitors of this important enzyme family.

In response to starvation, virtually all bacteria pyrophosphorylate the 3&'-hydroxy group of GTP or GDP to produce two messenger nucleotides collectively denoted as (p)ppGpp. Also known as alarmones, (p)ppGpp reprograms bacterial physiology to arrest growth and promote survival. Intriguingly, although cellular concentration of dGTP is two orders of magnitude lower than that of GTP, alarmone synthetases are highly selective against using 2&'-deoxyguanosine (2dG) nucleotides as substrates. We thus hypothesize that production of 2dG alarmone, (p)pp(dG)pp, is highly deleterious, which drives a strong negative selection to exclude 2dG nucleotides from alarmone signaling. In this work, we show that the B. subtilis SasB synthetase prefers GDP over dGDP with 65,000-fold higher kcat/Km, a specificity stricter than RNA polymerase selecting against 2&'-deoxynucleotides. Using comparative chemical proteomics, we found that although most known alarmone-binding proteins in Escherichia coli cannot distinguish ppGpp from pp(dG)pp, hydrolysis of pp(dG)pp by the essential hydrolase, SpoT, is 1,000-fold slower. This inability to degrade 2&'-deoxy-3&'-pyrophosphorylated substrate is a common feature of the alarmone hydrolase family. We further show that SpoT is a binuclear metallopyrophoshohydrolase and that hydrolysis of ppGpp and pp(dG)pp shares the same metal dependence. Our results support a model in which 2&'-OH directly coordinates the Mn2+ at SpoT active center to stabilize the hydrolysis-productive conformation of ppGpp. Taken together, our study reveals a vital role of 2&'-OH in alarmone degradation, provides new insight on the catalytic mechanism of alarmone hydrolases, and leads to the conclusion that 2dG nucleotides must be strictly excluded from alarmone synthesis because bacteria lack the key machinery to down-regulate such products.

Background Until now, few articles have revealed the potential roles of innate lymphoid cells (ILCs) in cardiovascular diseases. However, the infiltration of ILC subsets in ischemic myocardium, the roles of ILC subsets in myocardial infarction (MI) and myocardial ischemia-reperfusion injury (MIRI) and the related cellular and molecular mechanisms have not been described with a sufficient level of detail. Method In the current study, 8-week-old male C57BL/6J mice were divided into three groups: MI, MIRI and sham group. Single-cell sequencing technology was used to perform dimensionality reduction clustering of ILC to analyze the ILC subset landscape at a single-cell resolution, and finally flow cytometry was used to confirm the existence of the new ILC subsets in different disease groups. Results Five ILC subsets were found, including ILC1, ILC2a, ILC2b, ILCdc and ILCt. It is worth noting that ILCdc, ILC2b and ILCt were identified as new ILC subclusters in the heart. The cellular landscapes of ILCs were revealed and signal pathways were predicted. Furthermore, pseudotime trajectory analysis exhibited different ILC statuses and traced related gene expression in normal and ischemic conditions. In addition, we established a ligand–receptor–transcription factor–target gene regulatory network to disclose cell communications among ILC clusters. Moreover, we further revealed the transcriptional features of the ILCdc and ILC2a subsets. Finally, the existence of ILCdc was confirmed by flow cytometry. Conclusion Collectively, by characterizing the spectrums of ILC subclusters, our results provide a new blueprint for understanding ILC subclusters’ roles in myocardial ischemia diseases and further potential treatment targets.

Bacteria produce a variety of nucleotide second messengers to adapt to their surroundings. Although chemically similar, the nucleotides guanosine penta- and tetraphosphate [(p)ppGpp] and adenosine penta- and tetraphosphate [(p)ppApp] have distinct functions in bacteria. (p)ppGpp mediates survival under nutrient-limiting conditions and its intracellular levels are regulated by synthetases and hydrolases belonging to the RelA-SpoT homolog (RSH) family of enzymes. By contrast, (p)ppApp is not known to be involved in nutrient stress responses and is synthesized by RSH-resembling toxins that inhibit the growth of bacterial cells. However, it remains unclear whether there exists a family of hydrolases that specifically act on (p)ppApp to reverse its toxic effects. Here, we present the structure and biochemical characterization of adenosine 3′-pyrophosphohydrolase 1 (Aph1), the founding member of a monofunctional (p)ppApp hydrolase family of enzymes. Our work reveals that Aph1 adopts a histidine-aspartate (HD)-domain fold characteristic of phosphohydrolase metalloenzymes and its activity mitigates the growth inhibitory effects of (p)ppApp-synthesizing toxins. Using an informatic approach, we identify over 2,000 putative (p)ppApp hydrolases that are widely distributed across bacterial phyla and found in diverse genomic contexts, and we demonstrate that 12 representative members hydrolyze ppApp. In addition, our in silico analyses reveal a unique molecular signature that is specific to (p)ppApp hydrolases, and we show that mutation of two residues within this signature broadens the specificity of Aph1 to promiscuously hydrolyze (p)ppGpp in vitro. Overall, our findings indicate that like (p)ppGpp hydrolases, (p)ppApp hydrolases are widespread in bacteria and may play important and underappreciated role(s) in bacterial physiology.

Background Calcific aortic valve disease (CAVD) is a common valve disease with an increasing incidence, but no effective drugs as of yet. With the development of sequencing technology, non-coding RNAs have been found to play roles in many diseases as well as CAVD, but no circRNA/lncRNA–miRNA–mRNA interaction axis has been established. Moreover, valve interstitial cells (VICs) and valvular endothelial cells (VECs) play important roles in CAVD, and CAVD differed between leaflet phenotypes and genders. This work aims to explore the mechanism of circRNA/lncRNA–miRNA–mRNA network in CAVD, and perform subgroup analysis on the important characteristics of CAVD, such as key cells, leaflet phenotypes and genders. Results We identified 158 differentially expressed circRNAs (DEcircRNAs), 397 DElncRNAs, 45 DEmiRNAs and 167 DEmRNAs, and constructed a hsa-circ-0073813/hsa-circ-0027587–hsa-miR-525-5p–SPP1/HMOX1/CD28 network in CAVD after qRT-PCR verification. Additionally, 17 differentially expressed genes (DEGs) in VICs, 9 DEGs in VECs, 7 DEGs between different leaflet phenotypes and 24 DEGs between different genders were identified. Enrichment analysis suggested the potentially important pathways in inflammation and fibro-calcification during the pathogenesis of CAVD, and immune cell patterns in CAVD suggest that M0 macrophages and memory B cells memory were significantly increased, and many genes in immune cells were also differently expressed. Conclusions The circRNA/lncRNA–miRNA–mRNA interaction axis constructed in this work and the DEGs identified between different characteristics of CAVD provide a direction for a deeper understanding of CAVD and provide possible diagnostic markers and treatment targets for CAVD in the future.

Wnt signalling is essential for regulation of embryonic development and adult tissue homeostasis(1-3), and aberrant Wnt signalling is frequently associated with cancers(4). Wnt signalling requires palmitoleoylation on a hairpin 2 motif by the endoplasmic reticulum-resident membrane-bound O-acyltransferase Porcupine(5-7) (PORCN). This modification is indispensable for Wnt binding to its receptor Frizzled, which triggers signalling(8,9). Here we report four cryo-electron microscopy structures of human PORCN: the complex with the palmitoleoyl-coenzyme A (palmitoleoyl-CoA) substrate; the complex with the PORCN inhibitor LGK974, an anti-cancer drug currently in clinical trials(10); the complex with LGK974 and WNT3A hairpin 2 (WNT3Ap); and the complex with a synthetic palmitoleoylated WNT3Ap analogue. The structures reveal that hairpin 2 of WNT3A, which is well conserved in all Wnt ligands, inserts into PORCN from the lumenal side, and the palmitoleoyl-CoA accesses the enzyme from the cytosolic side. The catalytic histidine triggers the transfer of the unsaturated palmitoleoyl group to the target serine on the Wnt hairpin 2, facilitated by the proximity of the two substrates. The inhibitor-bound structure shows that LGK974 occupies the palmitoleoyl-CoA binding site to prevent the reaction. Thus, this work provides a mechanism for Wnt acylation and advances the development of PORCN inhibitors for cancer treatment.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Background: Acute myocardial infarction (AMI) is one of the main fatal diseases of cardiovascular diseases. Circular RNA (circRNA) is a non-coding RNA (ncRNA), which plays a role in cardiovascular disease as a competitive endogenous RNA (ceRNA). However, their role in AMI has not been fully clarified. This study aims to explore the mechanism of circRNA-related ceRNA network in AMI, and to identify the corresponding immune infiltration characteristics. Materials and Methods: The circRNA (GSE160717), miRNA (GSE24548), and mRNA (GSE60993) microarray datasets of AMI were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed circRNAs (DEcircRNAs), miRNAs (DEmiRNAs), and mRNAs (DEmRNAs) were identified by the “limma” package. After integrating the circRNA, miRNA and mRNA interaction, we constructed a circRNA-miRNA-mRNA network. The “clusterProfiler” package and String database were used for functional enrichment analysis and protein-protein interaction (PPI) analysis, respectively. After that, we constructed a circRNA-miRNA-hub gene network and validated the circRNAs and mRNAs using an independent dataset (GSE61144) as well as qRT-PCR. Finally, we used CIBERSORTx database to analyze the immune infiltration characteristics of AMI and the correlation between hub genes and immune cells. Results: Using the “limma” package of the R, 83 DEcircRNAs, 54 DEmiRNAs, and 754 DEmRNAs were identified in the microarray datasets of AMI. Among 83 DEcircRNAs, there are 55 exonic DEcircRNAs. Then, a circRNA-miRNA-mRNA network consists of 21 DEcircRNAs, 11 DEmiRNAs, and 106 DEmRNAs were predicted by the database. After that, 10 hub genes from the PPI network were identified. Then, a new circRNA-miRNA-hub gene network consists of 14 DEcircRNAs, 7 DEmiRNAs, and 9 DEmRNAs was constructed. After that, three key circRNAs (hsa_circ_0009018, hsa_circ_0030569 and hsa_circ_0031017) and three hub genes (BCL6, PTGS2 and PTEN) were identified from the network by qRT-PCR. Finally, immune infiltration analysis showed that hub genes were significantly positively correlated with up-regulated immune cells (neutrophils, macrophages and plasma cells) in AMI. Conclusion: Our study constructed a circRNA-related ceRNA networks in AMI, consists of hsa_circ_0031017/hsa-miR-142-5p/PTEN axis, hsa_circ_0030569/hsa-miR-545/PTGS2 axis and hsa_circ_0009018/hsa-miR-139-3p/BCL6 axis. These three hub genes were significantly positively correlated with up-regulated immune cells (neutrophils, macrophages and plasma cells) in AMI. It helps improve understanding of AMI mechanism and provides future potential therapeutic targets.

With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups of bacteria. Teixobactin, the first antibiotic without detectable resistance that binds lipid II, comes from an uncultured Eleftheria terra, a betaproteobacterium; odilorhabdins, from Xenorhabdus, are broad-spectrum inhibitors of protein synthesis, and darobactins from Photorhabdus target BamA, the essential chaperone of the outer membrane of Gram-negative bacteria. Xenorhabdus and Photorhabdus are symbionts of the nematode gut microbiome and attractive producers of secondary metabolites. Only small portions of their biosynthetic gene clusters (BGC) are expressed in vitro. To access their silent operons, we first separated extracts from a small library of isolates into fractions, resulting in 200-fold concentrated material, and then screened them for antimicrobial activity. This resulted in a hit with selective activity against Escherichia coli, which we identified as a novel natural product antibiotic, 3'-amino 3'-deoxyguanosine (ADG). Mutants resistant to ADG mapped to gsk and gmk, kinases of guanosine. Biochemical analysis shows that ADG is a prodrug that is converted into an active ADG triphosphate (ADG-TP), a mimic of GTP. ADG incorporates into a growing RNA chain, interrupting transcription, and inhibits cell division, apparently by interfering with the GTPase activity of FtsZ. Gsk of the purine salvage pathway, which is the first kinase in the sequential phosphorylation of ADG, is restricted to E. coli and closely related species, explaining the selectivity of the compound. There are probably numerous targets of ADG-TP among GTP-dependent proteins. The discovery of ADG expands our knowledge of prodrugs, which are rare among natural compounds. IMPORTANCE Drug-resistant Gram-negative bacteria have become the major problem driving the antimicrobial resistance crisis. Searching outside the overmined actinomycetes, we focused on Photorhabdus, gut symbionts of enthomopathogenic nematodes that carry up to 40 biosynthetic gene clusters coding for secondary metabolites. Most of these are silent and do not express in vitro. To gain access to silent operons, we first fractionated supernatant from Photorhabdus and then tested 200-fold concentrated material for activity. This resulted in the isolation of a novel antimicrobial, 3'-amino 3'-deoxyguanosine (ADG), active against E. coli. ADG is an analog of guanosine and is converted into an active ADG-TP in the cell. ADG-TP inhibits transcription and probably numerous other GTP-dependent targets, such as FtsZ. Natural product prodrugs have been uncommon; discovery of ADG broadens our knowledge of this type of antibiotic.

In Staphylococcus aureus, virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator (agr) genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and post-translational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1-32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that MroQ, an integral membrane protease recently implicated in the agr response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in agr specifiy groups -I and -II, but not group-III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together in vitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining agr specificity group identity. Collectively, our study adds to the molecular understanding of the agr response and Staphylococcus aureus virulence.

Proteins often accumulate neutral mutations that do not affect current functions but can profoundly influence future mutational possibilities and functions. Understanding such hidden potential has major implications for protein design and evolutionary forecasting but has been limited by a lack of systematic efforts to identify potentiating mutations. Here, through the comprehensive analysis of a bacterial toxin–antitoxin system, we identified all possible single substitutions in the toxin that enable it to tolerate otherwise interface-disrupting mutations in its antitoxin. Strikingly, the majority of enabling mutations in the toxin do not contact and promote tolerance non-specifically to many different antitoxin mutations, despite covariation in homologues occurring primarily between specific pairs of contacting residues across the interface. In addition, the enabling mutations we identified expand future mutational paths that both maintain old toxin–antitoxin interactions and form new ones. These non-specific mutations are missed by widely used covariation and machine learning methods. Identifying such enabling mutations will be critical for ensuring continued binding of therapeutically relevant proteins, such as antibodies, aimed at evolving targets. Enabling mutations do not affect current protein function but affect future mutational possibilities. The authors explore the nature of enabling mutations in a bacterial toxin–antitoxin system.

Bortezomib is a classical proteasome inhibitor and previous researches have reported its roles of anti-oxidation and anti-inflammatory functions in various diseases. However, the role of Bortezomib in myocardial ischemia reperfusion injury (MIRI) is unclear. Thus, our research seeks to reveal the protective effects of Bortezomib pretreatment in the mice model of MIRI. First, by the optimization of Bortezomib concentration and pretreatment timepoints, we found that 0.5 mg/kg Bortezomib pretreatment 2 h before MIRI significantly attenuated pathological damage and neutrophil infiltration. Then we found that pretreatment with Bortezomib obviously increased myocardial systolic function ((left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS)) and decreased infarct size, as well as serum Troponin T levels. Meanwhile, Bortezomib pretreatment also remarkably augmented oxidative stress related protein levels of Superoxide dismutase [Cu-Zn] (SOD1), Catalase (CAT) and Glutathione (GSH), while reactive oxygen species (ROS) contents and Malonaldehyde (MDA) protein level were significantly reduced. Mechanistically, Bortezomib pretreatment significantly promoted nuclear translocation of transcriptional factor nuclear factor erythroid 2-related factor 2(Nrf2) and Heme Oxygenase 1(HO-1) expression. Interestingly, co-treatment with ML-385, a new type and selective Nrf2 inhibitor, counteracted antioxidative effects induced by Bortezomib pretreatment. In conclusion, Bortezomib pretreatment mitigates MIRI by inhibiting oxidative damage which is regulated by Nrf2/HO-1 signaling pathway.

Regulatory T cells (Tregs) have been shown to attenuate the development and progression of atherosclerosis; however, the exact mechanism is still unclear. In our study, Tregs were adoptively transferred into ApoE-/- mice, and type 2 innate lymphoid cells (ILC2s) were expanded by the IL-2/Jes6-1 complex or depleted by anti-CD90.2 mAb in ApoE-/-Rag1-/- mice to study their effects on atherosclerosis. Then, Tregs were cocultured with ILC2s in vitro to analyze ILC2s number and IL-13 production. In vivo, ApoE-/-Rag1-/- mice were treated with activated Tregs with or without anti-CD90.2 mAb to explore whether Tregs reduced atherosclerosis through ILC2s. Finally, neutralizing antibodies and Transwell assay were used to investigate how Tregs regulate ILC2s. Our results show that both Tregs and ILC2s reduce atherosclerosis lesions and macrophage infiltration. Moreover, Tregs effectively expanded the number of ILC2s and increased their production of IL-13 in vivo and in vitro. Furthermore, the reductions in plaque size and macrophage infiltration by Tregs were partly reversed by anti-CD90.2 mAb. Mechanistically, our data reveal that IL-10, TGF-β and cell-cell contacts are required for Tregs-ILC2s regulation. These results show that Tregs may play a partial protective role against atherosclerosis by expanding the number of ILC2s and consequently increasing IL-13 production.

Oxidative stress and subsequent cardiac myocyte apoptosis play central roles in the initiation and progression of myocardial ischemia-reperfusion (I/R) injury. Homeobox transcript antisense intergenic RNA (Hotair) was previously implicated in various heart diseases, yet its role in myocardial I/R injury has not been clearly demonstrated. Mice with cardiac-restricted knockdown or overexpression of Hotair were exposed to I/R surgery. H9c2 cells were cultured and subjected to hypoxia/reoxygenation (H/R) stimulation to further verify the role and underlying mechanisms of Hotair in vitro. Histological examination, molecular detection, and functional parameters were determined in vivo and in vitro. In response to I/R or H/R treatment, Hotair expression was increased in a bromodomain-containing protein 4-dependent manner. Cardiac-restricted knockdown of Hotair exacerbated, whereas Hotair overexpression prevented I/R-induced oxidative stress, cardiac myocyte apoptosis, and cardiac dysfunction. Mechanistically, we observed that Hotair exerted its beneficial effects via activating AMP-activated protein kinase alpha (AMPKα). Further detection revealed that Hotair activated AMPKα through regulating the enhancer of zeste homolog 2/microRNA-451/calcium-binding protein 39 (EZH2/miR-451/Cab39) axis. We provide the evidence that endogenous lncRNA Hotair is an essential negative regulator for oxidative stress and cardiac myocyte apoptosis in myocardial I/R injury, which is dependent on AMPKα activation via the EZH2/miR-451/Cab39 axis.

Objective: To explore the clinical characteristics and prognosis of the new coronavirus 2019-nCoV patients combined with cardiovascular disease （CVD). Methods: A retrospective analysis was performed on 112 COVID-19 patients with CVD admitted to the western district of Union Hospital in Wuhan, from January 20, 2020 to February 15, 2020. They were divided into critical group (ICU, n=16) and general group (n=96) according to the severity of the disease and patients were followed up to the clinical endpoint. The observation indicators included total blood count, C-reactive protein (CRP), arterial blood gas analysis, myocardial injury markers, coagulation function, liver and kidney function, electrolyte, procalcitonin (PCT), B-type natriuretic peptide (BNP), blood lipid, pulmonary CT and pathogen detection. Results: Compared with the general group, the lymphocyte count (0.74 (0.34, 0.94)×109/L vs. 0.99 (0.71, 1.29)×109/L, P=0.03) was extremely lower in the critical group, CRP (106.98 (81.57, 135.76) mg/L vs. 34.34 (9.55,76.54) mg/L, P<0.001) and PCT (0.20 (0.15,0.48) μg/L vs. 0.11 (0.06,0.20) μg/L, P<0.001) were significantly higher in the critical group. The BMI of the critical group was significantly higher than that of the general group (25.5 (23.0, 27.5) kg/m2 vs. 22.0 (20.0, 24.0) kg/m2，P=0.003). Patients were further divided into non-survivor group (17, 15.18%) group and survivor group (95, 84.82%). Among the non-survivors, there were 88.24% (15/17) patients with BMI> 25.0 kg/m2, which was significantly higher than that of survivors (18.95% (18/95), P<0.001). Compared with the survived patients, oxygenation index (130 (102, 415) vs. 434 (410, 444), P<0.001) was significantly lower and lactic acid (1.70 (1.30, 3.00) mmol/L vs. 1.20 (1.10, 1.60) mmol/L, P<0.001) was significantly higher in the non-survivors. There was no significant difference in the proportion of ACEI/ARB medication between the critical group and the general group or between non-survivors and survivors （all P>0.05). Conclusion: COVID-19 patients combined with CVD are associated with a higher risk of mortality. Critical patients are characterized with lower lymphocyte counts. Higher BMI are more often seen in critical patients and non-survivor. ACEI/ARB use does not affect the morbidity and mortality of COVID-19 combined with CVD. Aggravating causes of death include fulminant inflammation, lactic acid accumulation and thrombotic events.