Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.

Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.

Gut-innervating nociceptors regulate the intestinal microbiota to promote tissue protection.

Nociceptive pain is a hallmark of many chronic inflammatory conditions including inflammatory bowel diseases (IBDs); however, whether pain-sensing neurons influence intestinal inflammation remains poorly defined. Employing chemogenetic silencing, adenoviral-mediated colon-specific silencing, and pharmacological ablation of TRPV1+ nociceptors, we observed more severe inflammation and defective tissue-protective reparative processes in a murine model of intestinal damage and inflammation. Disrupted nociception led to significant alterations in the intestinal microbiota and a transmissible dysbiosis, while mono-colonization of germ-free mice with Gram+Clostridium spp. promoted intestinal tissue protection through a nociceptor-dependent pathway. Mechanistically, disruption of nociception resulted in decreased levels of substance P, and therapeutic delivery of substance P promoted tissue-protective effects exerted by TRPV1+ nociceptors in a microbiota-dependent manner. Finally, dysregulated nociceptor gene expression was observed in intestinal biopsies from IBD patients. Collectively, these findings indicate an evolutionarily conserved functional link between nociception, the intestinal microbiota, and the restoration of intestinal homeostasis.

Fungal microbiota sustains lasting immune activation of neutrophils and their progenitors in severe COVID-19.

Gastrointestinal fungal dysbiosis is a hallmark of several diseases marked by systemic immune activation. Whether persistent pathobiont colonization during immune alterations and impaired gut barrier function has a durable impact on host immunity is unknown. We found that elevated levels of Candida albicans immunoglobulin G (IgG) antibodies marked patients with severe COVID-19 (sCOVID-19) who had intestinal Candida overgrowth, mycobiota dysbiosis and systemic neutrophilia. Analysis of hematopoietic stem cell progenitors in sCOVID-19 revealed transcriptional changes in antifungal immunity pathways and reprogramming of granulocyte myeloid progenitors (GMPs) for up to a year. Mice colonized with C. albicans patient isolates experienced increased lung neutrophilia and pulmonary NETosis during severe acute respiratory syndrome coronavirus-2 infection, which were partially resolved with antifungal treatment or by interleukin-6 receptor blockade. sCOVID-19 patients treated with tocilizumab experienced sustained reductions in C. albicans IgG antibodies titers and GMP transcriptional changes. These findings suggest that gut fungal pathobionts may contribute to immune activation during inflammatory diseases, offering potential mycobiota-immune therapeutic strategies for sCOVID-19 with prolonged symptoms.

Distinct Processing of lncRNAs Contributes to Non-conserved Functions in Stem Cells.

Long noncoding RNAs (lncRNAs) evolve more rapidly than mRNAs. Whether conserved lncRNAs undergo conserved processing, localization, and function remains unexplored. We report differing subcellular localization of lncRNAs in human and mouse embryonic stem cells (ESCs). A significantly higher fraction of lncRNAs is localized in the cytoplasm of hESCs than in mESCs. This turns out to be important for hESC pluripotency. FAST is a positionally conserved lncRNA but is not conserved in its processing and localization. In hESCs, cytoplasm-localized hFAST binds to the WD40 domain of the E3 ubiquitin ligase ß-TrCP and blocks its interaction with phosphorylated ß-catenin to prevent degradation, leading to activated WNT signaling, required for pluripotency. In contrast, mFast is nuclear retained in mESCs, and its processing is suppressed by the splicing factor PPIE, which is highly expressed in mESCs but not hESCs. These findings reveal that lncRNA processing and localization are previously under-appreciated contributors to the rapid evolution of function.

Nutritional regulation of microbiota-derived metabolites: Implications for immunity and inflammation.

Nutrition profoundly shapes immunity and inflammation across the lifespan of mammals, from pre- and post-natal periods to later life. Emerging insights into diet-microbiota interactions indicate that nutrition has a dominant influence on the composition-and metabolic output-of the intestinal microbiota, which in turn has major consequences for host immunity and inflammation. Here, we discuss recent findings that support the concept that dietary effects on microbiota-derived metabolites potently alter immune responses in health and disease. We discuss how specific dietary components and metabolites can be either pro-inflammatory or anti-inflammatory in a context- and tissue-dependent manner during infection, chronic inflammation, and cancer. Together, these studies emphasize the influence of diet-microbiota crosstalk on immune regulation that will have a significant impact on precision nutrition approaches and therapeutic interventions for managing inflammation, infection, and cancer immunotherapy.

Gene regulation by long non-coding RNAs and its biological functions.

Evidence accumulated over the past decade shows that long non-coding RNAs (lncRNAs) are widely expressed and have key roles in gene regulation. Recent studies have begun to unravel how the biogenesis of lncRNAs is distinct from that of mRNAs and is linked with their specific subcellular localizations and functions. Depending on their localization and their specific interactions with DNA, RNA and proteins, lncRNAs can modulate chromatin function, regulate the assembly and function of membraneless nuclear bodies, alter the stability and translation of cytoplasmic mRNAs and interfere with signalling pathways. Many of these functions ultimately affect gene expression in diverse biological and physiopathological contexts, such as in neuronal disorders, immune responses and cancer. Tissue-specific and condition-specific expression patterns suggest that lncRNAs are potential biomarkers and provide a rationale to target them clinically. In this Review, we discuss the mechanisms of lncRNA biogenesis, localization and functions in transcriptional, post-transcriptional and other modes of gene regulation, and their potential therapeutic applications.

SLERT Regulates DDX21 Rings Associated with Pol I Transcription.

Dysregulated rRNA synthesis by RNA polymerase I (Pol I) is associated with uncontrolled cell proliferation. Here, we report a box H/ACA small nucleolar RNA (snoRNA)-ended long noncoding RNA (lncRNA) that enhances pre-rRNA transcription (SLERT). SLERT requires box H/ACA snoRNAs at both ends for its biogenesis and translocation to the nucleolus. Deletion of SLERT impairs pre-rRNA transcription and rRNA production, leading to decreased tumorigenesis. Mechanistically, SLERT interacts with DEAD-box RNA helicase DDX21 via a 143-nt non-snoRNA sequence. Super-resolution images reveal that DDX21 forms ring-shaped structures surrounding multiple Pol I complexes and suppresses pre-rRNA transcription. Binding by SLERT allosterically alters individual DDX21 molecules, loosens the DDX21 ring, and evicts DDX21 suppression on Pol I transcription. Together, our results reveal an important control of ribosome biogenesis by SLERT lncRNA and its regulatory role in DDX21 ring-shaped arrangements acting on Pol I complexes.

Discovery of Reactive Microbiota-Derived Metabolites that Inhibit Host Proteases.

The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis, we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the National Institutes of Health Human Microbiome Project (NIH HMP) stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics, synthetic biology, and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic potential of the microbiota while avoiding the challenges of cultivating fastidious commensals.

Inulin fibre promotes microbiota-derived bile acids and type 2 inflammation.

Dietary fibres can exert beneficial anti-inflammatory effects through microbially fermented short-chain fatty acid metabolites^1,2, although the immunoregulatory roles of most fibre diets and their microbiota-derived metabolites remain poorly defined. Here, using microbial sequencing and untargeted metabolomics, we show that a diet of inulin fibre alters the composition of the mouse microbiota and the levels of microbiota-derived metabolites, notably bile acids. This metabolomic shift is associated with type 2 inflammation in the intestine and lungs, characterized by IL-33 production, activation of group 2 innate lymphoid cells and eosinophilia. Delivery of cholic acid mimics inulin-induced type 2 inflammation, whereas deletion of the bile acid receptor farnesoid X receptor diminishes the effects of inulin. The effects of inulin are microbiota dependent and were reproduced in mice colonized with human-derived microbiota. Furthermore, genetic deletion of a bile-acid-metabolizing enzyme in one bacterial species abolishes the ability of inulin to trigger type 2 inflammation. Finally, we demonstrate that inulin enhances allergen- and helminth-induced type 2 inflammation. Taken together, these data reveal that dietary inulin fibre triggers microbiota-derived cholic acid and type 2 inflammation at barrier surfaces with implications for understanding the pathophysiology of allergic inflammation, tissue protection and host defence.

Bacterial metabolism of bile acids promotes generation of peripheral regulatory T cells.

Intestinal health relies on the immunosuppressive activity of CD4+ regulatory T (Treg) cells1. Expression of the transcription factor Foxp3 defines this lineage, and can be induced extrathymically by dietary or commensal-derived antigens in a process assisted by a Foxp3 enhancer known as conserved non-coding sequence 1 (CNS1)2-4. Products of microbial fermentation including butyrate facilitate the generation of peripherally induced Treg (pTreg) cells5-7, indicating that metabolites shape the composition of the colonic immune cell population. In addition to dietary components, bacteria modify host-derived molecules, generating a number of biologically active substances. This is epitomized by the bacterial transformation of bile acids, which creates a complex pool of steroids8 with a range of physiological functions9. Here we screened the major species of deconjugated bile acids for their ability to potentiate the differentiation of pTreg cells. We found that the secondary bile acid 3ß-hydroxydeoxycholic acid (isoDCA) increased Foxp3 induction by acting on dendritic cells (DCs) to diminish their immunostimulatory properties. Ablating one receptor, the farnesoid X receptor, in DCs enhanced the generation of Treg cells and imposed a transcriptional profile similar to that induced by isoDCA, suggesting an interaction between this bile acid and nuclear receptor. To investigate isoDCA in vivo, we took a synthetic biology approach and designed minimal microbial consortia containing engineered Bacteroides strains. IsoDCA-producing consortia increased the number of colonic RORγt-expressing Treg cells in a CNS1-dependent manner, suggesting enhanced extrathymic differentiation.

Mechanisms of Long Noncoding RNA Nuclear Retention.

Long noncoding RNAs (lncRNAs) are crucial regulators in diverse cellular contexts and biological processes. The subcellular localization of lncRNAs determines their modes of action. Compared to mRNAs, however, many mRNA-like lncRNAs are preferentially localized to the nucleus where they regulate chromatin organization, transcription, and different nuclear condensates. Recent studies have revealed the complex mechanisms that govern lncRNA nuclear retention. We review current understanding of how the transcription and processing of lncRNAs, motifs within lncRNAs, and trans-factors coordinately contribute to their nuclear retention in mammalian cells.

Compact In Situ Electrochemical NMR with Wireless and Anti-interference Strategy in Multiscenario Applications.

The integration of electrochemistry with nuclear magnetic resonance (NMR) spectroscopy recently offers a powerful approach to understanding oxidative metabolism, detecting reactive intermediates, and predicting biological activities. This combination is particularly effective as electrochemical methods provide excellent mimics of metabolic processes, while NMR spectroscopy offers precise chemical analysis. NMR is already widely utilized in the quality control of pharmaceuticals, foods, and additives and in metabolomic studies. However, the introduction of additional and external connections into the magnet has posed challenges, leading to signal deterioration and limitations in routine measurements. Herein, we report an anti-interference compact in situ electrochemical NMR system (AICISENS). Through a wireless strategy, the compact design allows for the independent and stable operation of electrochemical NMR components with effective interference isolation. Thus, it opens an avenue toward easy integration into in situ platforms, applicable not only to laboratory settings but also to fieldwork. The operability, reliability, and versatility were validated with a series of biomimetic assessments, including measurements of microbial electrochemical systems, functional foods, and simulated drug metabolisms. The robust performance of AICISENS demonstrates its high potential as a powerful analytical tool across diverse applications.

D-arabinose acts as antidepressant by activating the ACSS2-PPARγ/TFEB axis and CRTC1 transcription.

CREB-regulated transcription coactivator 1 (CRTC1), a pivotal synaptonuclear messenger, regulates synaptic plasticity and transmission to prevent depression. Despite exhaustive investigations into CRTC1 mRNA reductions in the depressed mice, the regulatory mechanisms governing its transcription remain elusive. Consequently, exploring rapid but non-toxic CRTC1 inducers at the transcriptional level is important for resisting depression. Here, we demonstrate the potential of D-arabinose, a unique monosaccharide prevalent in edible-medicinal plants, to rapidly enter the brain and induce CRTC1 expression, thereby eliciting rapid-acting and persistent antidepressant responses in chronic restrain stress (CRS)-induced depressed mice. Mechanistically, D-arabinose induces the expressions of peroxisome proliferator-activated receptor gamma (PPARγ) and transcription factor EB (TFEB), thereby activating CRTC1 transcription. Notably, we elucidate the pivotal role of the acetyl-CoA synthetase short-chain family member 2 (ACSS2) as an obligatory mediator for PPARγ and TFEB to potentiate CRTC1 transcription. Furthermore, D-arabinose augments ACSS2-dependent CRTC1 transcription by activating AMPK through lysosomal AXIN-LKB1 pathway. Correspondingly, the hippocampal down-regulations of ACSS2, PPARγ or TFEB alone failed to reverse CRTC1 reductions in CRS-exposure mice, ultimately abolishing the anti-depressant efficacy of D-arabinose. In summary, our study unveils a previously unexplored role of D-arabinose in activating the ACSS2-PPARγ/TFEB-CRTC1 axis, presenting it as a promising avenue for the prevention and treatment of depression.

The SUMO protease SENP3 regulates mitochondrial autophagy mediated by Fis1.

Mitochondria are unavoidably subject to organellar stress resulting from exposure to a range of reactive molecular species. Consequently, cells operate a poorly understood quality control programme of mitophagy to facilitate elimination of dysfunctional mitochondria. Here, we used a model stressor, deferiprone (DFP), to investigate the molecular basis for stress-induced mitophagy. We show that mitochondrial fission 1 protein (Fis1) is required for DFP-induced mitophagy and that Fis1 is SUMOylated at K149, an amino acid residue critical for Fis1 mitochondrial localization. We find that DFP treatment leads to the stabilization of the SUMO protease SENP3, which is mediated by downregulation of the E3 ubiquitin (Ub) ligase CHIP. SENP3 is responsible for Fis1 deSUMOylation and depletion of SENP3 abolishes DFP-induced mitophagy. Furthermore, preventing Fis1 SUMOylation by conservative K149R mutation enhances Fis1 mitochondrial localization. Critically, expressing a Fis1 K149R mutant restores DFP-induced mitophagy in SENP3-depleted cells. Thus, we propose a model in which SENP3-mediated deSUMOylation facilitates Fis1 mitochondrial localization to underpin stress-induced mitophagy.

Dapagliflozin Suppresses Isoprenaline-Induced Cardiac Hypertrophy Through Inhibition of Mitochondrial Fission.

ABSTRACT: Dapagliflozin (DAPA) is a novel oral hypoglycemic agent, and there is increasing evidence that DAPA has a protective effect against cardiovascular disease. The study aimed to investigate how DAPA inhibits cardiac hypertrophy and explore its potential mechanisms. By continuously infusing isoprenaline (ISO) for 2 weeks using a subcutaneous osmotic pump, a cardiac hypertrophic model was established in male C57BL/6 mice. On day 14 after surgery, echocardiography showed that left ventricle mass (LV mass), interventricular septum, left ventricle posterior wall diastole, and left ventricular posterior wall systole were significantly increased, and ejection fraction was decreased compared with control mice. Masson and Wheat Germ Agglutinin staining indicated enhanced myocardial fibrosis and cell morphology compared with control mice. Importantly, these effects were inhibited by DAPA treatment in ISO-induced mice. In H9c2 cells and neonatal rat cardiomyocytes, we found that mitochondrial fragmentation and mitochondrial oxidative stress were significantly augmented in the ISO-induced group. However, DAPA rescued the cardiac hypertrophy in ISO-induced H9c2 cells and neonatal rat cardiomyocytes. Mechanistically, we found that DAPA restored the PIM1 activity in ISO-induced H9c2 cells and subsequent increase in dynamin-associated protein 1 (Drp1) phosphorylation at S616 and decrease in Drp1 phosphorylation at S637 in ISO-induced cells. We found that DAPA mitigated ISO-induced cardiac hypertrophy by suppressing Drp1-mediated mitochondrial fission in a PIM1-dependent fashion.

Expansion of exhausted CD8+ T cells associates with increased pulmonary fungal infection risk in anti-melanoma differentiation associated gene 5 dermatomyositis.

OBJECTIVES: Anti-MDA5+ dermatomyositis was associated with poor prognosis due to the high incidence of rapid progressive interstitial lung disease, pulmonary infection. The aim of this study is to investigate the abundance and clinical relevance of exhaustion markers on peripheral CD8 T cells from patients with idiopathic inflammatory myopathy (IIM). METHODS: Twenty-nine healthy controls (HCs) and 71 patients with IIM were enrolled, including 42 with anti-MDA5+ and 18 with anti-MDA5- dermatomyositis (DM) and 11 with anti-synthetase syndrome (ASS). Flow cytometry was applied to detect PD-1, TIM-3 and LAG-3 in CD8 T cells. The clinical associations of the CD8 T cell exhaustion phenotype in patients with anti-MDA5+ DM were analysed. RESULTS: CD8 T cells from patients with anti-MDA5+ DM showed significantly increased LAG-3, TIM-3 and PD-1 compared to those from patients with anti-MDA5- IIM (18 with anti-MDA5- DM and 11 with ASS) or HCs (adjusted p all < 0.05). CD8 T cells with distinct exhaustion levels were all significantly increased in anti-MDA5+ DM patients compared with HCs (p all < 0.05). Patients with high level of PD-1+ TIM-3+LAG-3+ CD8+ T cells had a significant higher incidence of pulmonary fungal infections but lower counts of CD4+ and CD8+ T cells. ROC analysis revealed that the frequency of PD-1+TIM-3+LAG-3+CD8+ T cell significantly predicted pulmonary fungal infections (area under the curve: 0.828). CONCLUSIONS: CD8 T cells from patients with anti-MDA5+ DM show significant exhausted phenotype, and increased exhausted CD8 T cells were associated with high risk of pulmonary fungal infection.

Medial Protrusio Technique Versus Structural Autologous Bone-Grafting Technique in Total Hip Arthroplasty for Crowe Type II to III Hip Dysplasia.

BACKGROUND: It is unclear whether acetabular reconstruction techniques have any impact on clinical outcomes. This study aimed to determine (1) whether acetabular reconstruction techniques influenced the position of the acetabular cup and (2) whether clinical outcomes based on the acetabular reconstruction techniques differ in patients undergoing total hip arthroplasty (THA) with Crowe II to III developmental dysplasia of the hip. METHODS: This was a retrospective analysis of prospectively collected data from 69 patients (74 hips) who were treated with cementless THA using medial protrusio technique (MPT) or structural autologous bone-grafting technique (SABT). There were 39 patients (41 hips) included in the MPT group and 30 patients (33 hips) in the SABT group. Clinical and radiographic outcomes were evaluated. RESULTS: All patients were followed up for at least 3 years. There were similar results between the 2 groups in terms of blood loss, Harris hip score, leg length discrepancy, cup inclination, cup anteversion, and proportion of cup coverage (P > .05). The operative time was significantly longer in the SABT group compared with the MPT group (P < .001). The postoperative vertical center of rotation was significantly higher in the MPT group compared with the SABT group (P = .001), and postoperative horizontal center of rotation was significantly shallower in the SABT group compared with the MPT group (P < .001). CONCLUSION: The MPT and SABT provide similar clinical and radiographic outcomes in the management of Crowe II to III developmental dysplasia of the hip by cementless THA. However, the MPT has the advantage of a shorter operative time, whereas the SABT is more conducive to placing the acetabular cup in an anatomic position. LEVEL OF EVIDENCE: Level III, Therapeutic, Case-Control Study.

Medium- to long-term clinical efficacy of total hip arthroplasty with structural bone grafting for dysplasia of the hip.

BACKGROUND: Patients with dysplasia of the hip (DDH) have different degrees of bone defects above and outside the acetabulum, and anatomically reconstructing the acetabular centre of rotation is difficult in primary total hip arthroplasty (THA). METHODS: From April 2012 to December 2022, 64 patients (64 hips) with DDH treated with THA with structural bone graft in the superolateral acetabulum were selected. The Oxford hip score(OHS), Barthel index (BI), leg length discrepancy, Wibegr central edge-angle(CE), gluteus medius muscle strength, vertical and horizontal distance of the hip rotation center, coverage rate of the bone graft and complications were used to evaluate the clinical effectiveness of the patients. RESULTS: All patients were followed up for an average of 7.3±1.9 years. The OHS improved significantly after the operation (P<0.001). The postoperative BI was significantly greater than that before operation (P<0.001). The postoperative leg length discrepancy was significantly lower than that before the operation (P<0.001). Postoperative bedside photography revealed that the height and horizontal distance to the hip rotation center were significantly lower after surgery than before surgery (P<0.001). The postoperative CE was significantly greater than that before surgery (P<0.001). No acetabular component loosening or bone graft resorption was found during the postoperative imaging examination. CONCLUSIONS: The use of biological acetabular cup combined with structural bone graft in the superolateral acetabulum in THA for DDH can obtain satisfactory medium and long-term clinical and radiological results.

Identification and Expression Analysis of R2R3-MYB Transcription Factors Associated with Flavonoid Biosynthesis in Panax quinquefolius.

Panax quinquefolius L. is an important medicinal plant, and flavonoids are among its main secondary metabolites. The R2R3-MYB transcription factor plays an irreplaceable role in plant growth, development, and secondary metabolism. In our study, we identified 159 R2R3-MYBs and analyzed their physical and chemical properties in P. quinquefolius. The protein length of 159 PqMYBs varied from 107 to 1050 amino acids. The molecular weight ranged from 12.21 to 116.44 kDa. The isoelectric point was between 4.57 and 10.34. We constructed a phylogenetic tree of P. quinquefolius and Arabidopsis thaliana R2R3-MYB family members, and PqMYB members were divided into 33 subgroups. Transcriptome data analysis showed that the expression patterns of PqMYBs in root, leaf, and flower were significantly different. Following the MeJA treatment of seedlings, five candidate PqMYB genes demonstrated a response. A correlation analysis of PqMYBs and candidate flavonoid pathway genes showed that PqMYB2, PqMYB46, and PqMYB72 had correlation coefficients that were higher than 0.8 with PqCHS, PqANS4, and PqCCoAMT10, respectively. Furthermore, a transient expression assay confirmed that the three PqMYBs were localized in the nucleus. We speculated that these three PqMYBs were related to flavonoid biosynthesis in P. quinquefolius. These results provided a theoretical basis and a new perspective for further understanding the R2R3-MYB gene family and the biosynthesis mechanism of secondary metabolites in P. quinquefolius.