TNFSF14+ natural killer cells prevent spontaneous abortion by restricting leucine-mediated decidual stromal cell senescence.

In preparation for a potential pregnancy, the endometrium of the uterus changes into a temporary structure called the decidua. Senescent decidual stromal cells (DSCs) are enriched in the decidua during decidualization, but the underlying mechanisms of this process remain unclear. Here, we performed single-cell RNA transcriptomics on ESCs and DSCs and found that cell senescence during decidualization is accompanied by increased levels of the branched-chain amino acid (BCAA) transporter SLC3A2. Depletion of leucine, one of the branched-chain amino acids, from cultured media decreased senescence, while high leucine diet resulted in increased senescence and high rates of embryo loss in mice. BCAAs induced senescence in DSCs via the p38 MAPK pathway. In contrast, TNFSF14+ decidual natural killer (dNK) cells were found to inhibit DSC senescence by interacting with its ligand TNFRSF14. As in mice fed high-leucine diets, both mice with NK cell depletion and Tnfrsf14-deficient mice with excessive uterine senescence experienced adverse pregnancy outcomes. Further, we found excessive uterine senescence, SLC3A2-mediated BCAA intake, and insufficient TNFRSF14 expression in the decidua of patients with recurrent spontaneous abortion. In summary, this study suggests that dNK cells maintain senescence homeostasis of DSCs via TNFSF14/TNFRSF14, providing a potential therapeutic strategy to prevent DSC senescence-associated spontaneous abortion.

MOV10 recruits DCP2 to decap human LINE-1 RNA by forming large cytoplasmic granules with phase separation properties.

Long interspersed element 1 (LINE-1) is the only active autonomous mobile element in the human genome. Its transposition can exert deleterious effects on the structure and function of the host genome and cause sporadic genetic diseases. Tight control of LINE-1 mobilization by the host is crucial for genetic stability. In this study, we report that MOV10 recruits the main decapping enzyme DCP2 to LINE-1 RNA and forms a complex of MOV10, DCP2, and LINE-1 RNP, exhibiting liquid-liquid phase separation (LLPS) properties. DCP2 cooperates with MOV10 to decap LINE-1 RNA, which causes degradation of LINE-1 RNA and thus reduces LINE-1 retrotransposition. We here identify DCP2 as one of the key effector proteins determining LINE-1 replication, and elucidate an LLPS mechanism that facilitates the anti-LINE-1 action of MOV10 and DCP2.

Proteogenomic characterization of cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a highly heterogeneous cancer with limited understanding and few effective therapeutic approaches. We aimed at providing a proteogenomic CCA characterization to inform biological processes and treatment vulnerabilities. APPROACH AND RESULTS: Integrative genomic analysis with functional validation uncovered biological perturbations downstream of driver events including DPCR1 , RBM47 mutations, SH3BGRL2 copy number alterations, and FGFR2 fusions in CCA. Proteomic clustering identified three subtypes with distinct clinical outcomes, molecular features, and potential therapeutics. Phosphoproteomics characterized targetable kinases in CCA, suggesting strategies for effective treatment with CDK and MAPK inhibitors. Patients with CCA with HBV infection showed increased antigen processing and presentation (APC) and T cell infiltration, conferring a favorable prognosis compared with those without HBV infection. The characterization of extrahepatic CCA recommended the feasible application of vascular endothelial-derived growth factor inhibitors. Multiomics profiling presented distinctive molecular characteristics of the large bile duct and the small bile duct of intrahepatic CCA. The immune landscape further revealed diverse tumor immune microenvironments, suggesting immune subtypes C1 and C5 might benefit from immune checkpoint therapy. TCN1 was identified as a potential CCA prognostic biomarker, promoting cell growth by enhancing vitamin B12 metabolism. CONCLUSIONS: We characterized the proteogenomic landscape of 217 CCAs with 197 paired normal adjacent tissues and identified their subtypes and potential therapeutic targets. The multiomics analyses with other databases and some functional validations have indicated strategies regarding the clinical, biological, and therapeutic approaches to the management of CCA.

N6-Methyladenosine-mediated phase separation suppresses NOTCH1 expression and promotes mitochondrial fission in diabetic cardiac fibrosis.

BACKGROUND: N6-methyladenosine (m6A) modification of messenger RNA (mRNA) is crucial for liquid-liquid phase separation in mammals. Increasing evidence indicates that liquid-liquid phase separation in proteins and RNAs affects diabetic cardiomyopathy. However, the molecular mechanism by which m6A-mediated phase separation regulates diabetic cardiac fibrosis remains elusive. METHODS: Leptin receptor-deficient mice (db/db), cardiac fibroblast-specific Notch1 conditional knockout (POSTN-Cre × Notch1flox/flox) mice, and Cre mice were used to induce diabetic cardiac fibrosis. Adeno-associated virus 9 carrying cardiac fibroblast-specific periostin (Postn) promoter-driven small hairpin RNA targeting Alkbh5, Ythdf2, or Notch1, and the phase separation inhibitor 1,6-hexanediol were administered to investigate their roles in diabetic cardiac fibrosis. Histological and biochemical analyses were performed to determine how Alkbh5 and Ythdf2 regulate Notch1 expression in diabetic cardiac fibrosis. NOTCH1 was reconstituted in ALKBH5- and YTHDF2-deficient cardiac fibroblasts and mouse hearts to study its effects on mitochondrial fission and diabetic cardiac fibrosis. Heart tissue samples from patients with diabetic cardiomyopathy were used to validate our findings. RESULTS: In mice with diabetic cardiac fibrosis, decreased Notch1 expression was accompanied by high m6A mRNA levels and mitochondrial fission. Fibroblast-specific deletion of Notch1 enhanced mitochondrial fission and cardiac fibroblast proliferation and induced diabetic cardiac fibrosis in mice. Notch1 downregulation was associated with Alkbh5-mediated m6A demethylation in the 3'UTR of Notch1 mRNA and elevated m6A mRNA levels. These elevated m6A levels in Notch1 mRNA markedly enhanced YTHDF2 phase separation, increased the recognition of m6A residues in Notch1 mRNA by YTHDF2, and induced Notch1 degradation. Conversely, epitranscriptomic downregulation rescues Notch1 expression, resulting in the opposite effects. Human heart tissues from patients with diabetic cardiomyopathy were used to validate the findings in mice with diabetic cardiac fibrosis. CONCLUSIONS: We identified a novel epitranscriptomic mechanism by which m6A-mediated phase separation suppresses Notch1 expression, thereby promoting mitochondrial fission in diabetic cardiac fibrosis. Our findings provide new insights for the development of novel treatment approaches for patients with diabetic cardiac fibrosis.

Schlafen 5 suppresses human immunodeficiency virus type 1 transcription by commandeering cellular epigenetic machinery.

Schlafen-5 (SLFN5) is an interferon-induced protein of the Schlafen family, which are involved in immune responses and oncogenesis. To date, little is known regarding its anti-HIV-1 function. Here, the authors report that overexpression of SLFN5 inhibits HIV-1 replication and reduces viral mRNA levels, whereas depletion of endogenous SLFN5 promotes HIV-1 replication. Moreover, they show that SLFN5 markedly decreases the transcriptional activity of HIV-1 long terminal repeat (LTR) via binding to two sequences in the U5-R region, which consequently represses the recruitment of RNA polymerase II to the transcription initiation site. Mutagenesis studies show the importance of nuclear localization and the N-terminal 1-570 amino acids fragment in the inhibition of HIV-1. Further mechanistic studies demonstrate that SLFN5 interacts with components of the PRC2 complex, G9a and Histone H3, thereby promoting H3K27me2 and H3K27me3 modification leading to silencing HIV-1 transcription. In concert with this, they find that SLFN5 blocks the activation of latent HIV-1. Altogether, their findings demonstrate that SLFN5 is a transcriptional repressor of HIV-1 through epigenetic modulation and a potential determinant of HIV-1 latency.

Design of novel broad-spectrum antiviral nucleoside analogues using natural bases ring-opening strategy.

The global prevalence of RNA virus infections has presented significant challenges to public health in recent years, necessitating the expansion of its alternative therapeutic library. Due to its evolutional conservation, RNA-dependent RNA polymerase (RdRp) has emerged as a potential target for broad-spectrum antiviral nucleoside analogues. However, after over half a century of structural modification, exploring unclaimed chemical space using frequently-used structural substitution methods to design new nucleoside analogues is challenging. In this study, we explore the use of the "ring-opening" strategy to design new base mimics, thereby using these base mimics to design new nucleoside analogues with broad-spectrum antiviral activities. A total of 29 compounds were synthesized. Their activity against viral RdRp was initially screened using an influenza A virus RdRp high-throughput screening model. Then, the antiviral activity of 38a was verified against influenza virus strain A/PR/8/34 (H1N1), demonstrating a 50% inhibitory concentration (IC50) value of 9.95 µM, which was superior to that of ribavirin (the positive control, IC50 = 11.43 µM). Moreover, 38a also has inhibitory activity against coronavirus 229E with an IC50 of 30.82 µM. In addition, compounds 42 and 46f exhibit an 82% inhibition rate against vesicular stomatitis virus at a concentration of 20 µM and hardly induce cytotoxicity in host cells. This work demonstrates the feasibility of designing nucleoside analogues with "ring-opening" bases and suggests the "ring-opening" nucleosides may have greater polarity, and designing prodrugs is an important aspect of optimizing their antiviral activity. Future research should focus on enhancing the conformational restriction of open-loop bases to mimic Watson-Crick base pairing better and improve antiviral activity.

METTL3 boosts mitochondrial fission and induces cardiac fibrosis by enhancing LncRNA GAS5 methylation.

Dysregulated mitochondrial metabolism occurs in several pathological processes characterized by cell proliferation and migration. Nonetheless, the role of mitochondrial fission is not well appreciated in cardiac fibrosis, which is accompanied by enhanced fibroblast proliferation and migration. We investigated the causes and consequences of mitochondrial fission in cardiac fibrosis using cultured cells, animal models, and clinical samples. Increased METTL3 expression caused excessive mitochondrial fission, resulting in the proliferation and migration of cardiac fibroblasts that lead to cardiac fibrosis. Knockdown of METTL3 suppressed mitochondrial fission, inhibiting fibroblast proliferation and migration for ameliorating cardiac fibrosis. Elevated METTL3 and N6-methyladenosine (m6A) levels were associated with low expression of long non-coding RNA GAS5. Mechanistically, METTL3-mediated m6A methylation of GAS5 induced its degradation, dependent of YTHDF2. GAS5 could interact with mitochondrial fission marker Drp1 directly; overexpression of GAS5 suppressed Drp1-mediated mitochondrial fission, inhibiting cardiac fibroblast proliferation and migration. Knockdown of GAS5 produced the opposite effect. Clinically, increased METTL3 and YTHDF2 levels corresponded with decreased GAS5 expression, increased m6A mRNA content and mitochondrial fission, and increased cardiac fibrosis in human heart tissue with atrial fibrillation. We describe a novel mechanism wherein METTL3 boosts mitochondrial fission, cardiac fibroblast proliferation, and fibroblast migration: METTL3 catalyzes m6A methylation of GAS5 methylation in a YTHDF2-dependent manner. Our findings provide insight into the development of preventative measures for cardiac fibrosis.

NADP(+)-IDH Mutations Promote Hypersuccinylation that Impairs Mitochondria Respiration and Induces Apoptosis Resistance.

Elucidating the tumorigenic mechanism of R-2-hydroxyglutarate (R-2HG) is critical for determining how NADP(+)-IDH mutations cause cancer. Here we report that R-2HG induces cancerous metabolism and apoptosis resistance through promoting hypersuccinylation. By competitive inhibition of the mitochondrial tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH), R-2HG preferentially induced succinyl-CoA accumulation and hypersuccinylation in the mitochondria. IDH1 mutation-bearing glioma samples and cells were hypersuccinylated in the mitochondria. IDH1 mutation or SDH inactivation resulted in hypersuccinylation, causing respiration inhibition and inducing cancerous metabolism and mitochondrial depolarization. These mitochondrial dysfunctions induced BCL-2 accumulation at the mitochondrial membrane, leading to apoptosis resistance of hypersuccinylated cells. Relief of hypersuccinylation by overexpressing the desuccinylase SIRT5 or supplementing glycine rescued mitochondrial dysfunctions, reversed BCL-2 accumulation, and slowed the oncogenic growth of hypersuccinylated IDH1(R132C)-harboring HT1080 cells. Thus, R-2HG-induced hypersuccinylation contributes to the tumorigenicity of NADP(+)-IDH mutations, suggesting the potential of hypersuccinylation inhibition as an intervention for hypersuccinylation-related tumors.

Identification of prognostic and therapeutic biomarkers in type 2 papillary renal cell carcinoma.

BACKGROUND: Papillary renal cell carcinoma (PRCC) can be divided into type 1 (PRCC1) and type 2 (PRCC2) and PRCC2 share a more invasive phenotype and worse prognosis. This study aims to identify potential prognostic and therapeutic biomarkers in PRCC2. METHODS: A cohort from The Cancer Genome Atlas and two datasets from Gene Expression Omnibus were examined. Common differentially expressed genes (DEGs) were screened and potential biomarkers were explored by using Kaplan-Meier method and cox regression analysis. Functional enrichment analysis was utilized to evaluate the potential biological functions. Tumor infiltrating immune cells were estimated by CIBERSORT algorithm. Ninety-two PRCC2 samples from Fudan University Shanghai Cancer Center were obtained, and immunostaining was performed to validate prognostic and therapeutic significance of the potential biomarker. RESULTS: PRCC2 has worse overall survival and shares distinct molecular characteristics from PRCC1. There was significant higher expression level of Targeting protein for Xklp2 (TPX2) in PRCC2 compared with normal tissues. Higher expression level of TPX2 was significantly associated with worse overall survival in PRCC2 and kinesin family genes expression were found significantly elevated in high risk PRCC2. Abundance of tumor infiltrating M1 macrophage was significantly higher in PRCC2 and it was also associated with worse overall survival. In the FUSCC cohort, higher TPX2 expression was significantly correlated with worse overall and progression-free survival. Retrospective analysis indicated that mTOR inhibitor (everolimus) had greater efficacy in the high-risk group than in the low-risk group (overall response rate: 28.6% vs. 16.7%) and that everolimus had greater efficacy than sunitinib in the high-risk group (overall response rate: 28.6% vs. 20%). CONCLUSIONS: TPX2 was a prognostic and therapeutic biomarker in PRCC2. Higher abundance of tumor infiltrating M1 macrophage was significantly associated with worse overall survival in PRCC2. mTOR inhibitors may have good efficacy in patients with high-risk PRCC2.

Spinal Sirtuin 3 Contributes to Electroacupuncture Analgesia in Mice With Chronic Constriction Injury-Induced Neuropathic Pain.

BACKGROUND: Electroacupuncture (EA) is a traditional Chinese therapeutic technique that has a beneficial effect on neuropathic pain; however, the specific mechanism remains unclear. In this study, we investigated whether EA inhibits spinal Ca/calmodulin-dependent protein kinase II (CaMKIIα) phosphorylation through Sirtuin 3 (SIRT3) protein, thus relieving neuropathic pain. MATERIALS AND METHODS: We used wild-type and SIRT3 knockout (SIRT3-/-) mice and used chronic constriction injury (CCI) as a pain model. We performed Western blotting, immunostaining, von Frey, and Hargreaves tests to gather biochemical and behavioral data. Downregulation and overexpression and spinal SIRT3 protein were achieved by intraspinal injection of SIRT3 small interfering RNA and intraspinal injection of lentivirus-SIRT3. To test the hypothesis that CaMKIIα signaling was involved in the analgesic effects of EA, we expressed CaMKIIα-specific designer receptors exclusively activated by designer drugs (DREADDs) in the spinal dorsal horn (SDH) of mice. RESULTS: These results showed that the mechanical and thermal hyperalgesia induced by CCI was related to the decreased spinal SIRT3 expression in the SDH of mice. A significant reduction of mechanical and thermal thresholds was found in the SIRT3-/- mice. SIRT3 overexpression or EA treatment alleviated CCI-induced neuropathic pain and prevented the spinal CaMKIIα phosphorylation. Most importantly, EA increased the expression of spinal SIRT3 protein in the SDH. Downregulation of spinal SIRT3 or CaMKIIα Gq-DREADD activation inhibited the regulatory effect of EA on neuropathic pain. CONCLUSION: Our results showed that CaMKIIα phosphorylation was inhibited by spinal SIRT3 in neuropathic pain and that EA attenuated CCI-induced neuropathic pain mainly by upregulating spinal SIRT3 expression in the SDH of mice.

IMB-BZ as an Inhibitor Targeting ESX-1 Secretion System to Control Mycobacterial Infection.

BACKGROUND: Resistance to anti-tuberculosis (TB) drugs is a major issue in TB control, and demands the discovery of new drugs targeting the virulence factor ESX-1. METHODS: We first established a high-throughput screen (HTS) assay for the discovery of ESX-1 secretion inhibitors. The positive hits were then evaluated for the potency of diminishing the survival of virulent mycobacteria and reducing bacterial virulence. We further investigated the probability of inducing drug resistance and the underlying mechanism using mycobacterial protein fragment complementation. RESULTS: A robust HTS assay was developed to identify small molecules that inhibit ESX-1 secretion without impairing bacterial growth in vitro. A hit named IMB-BZ specifically inhibits the secretion of CFP-10 and reduces virulence in an ESX-1-dependent manner, therefore resulting in significant reduction in intracellular and in vivo survival of mycobacteria. Blocking the CFP-10-EccCb1 interaction directly or indirectly underlies the inhibitory effect of IMB-BZ on the secretion of CFP-10. Importantly, our finding shows that the ESX-1 inhibitors pose low risk of drug resistance development by mycobacteria in vitro as compared with traditional anti-TB drugs, and exhibit high potency against chronic mycobacterial infection. CONCLUSIONS: Targeting ESX-1 may lead to the development of novel therapeutics for tuberculosis. IMB-BZ holds the potential for future development into a new anti-TB drug.

Biallelic DNAH9 mutations are identified in Chinese patients with defective left-right patterning and cilia-related complex congenital heart disease.

Defective left-right (LR) pattering results in a spectrum of laterality disorders including situs inversus totalis (SIT) and heterotaxy syndrome (Htx). Approximately, 50% of patients with primary ciliary dyskinesia (PCD) displayed SIT. Recessive variants in DNAH9 have recently been implicated in patients with situs inversus. Here, we describe six unrelated family trios and 2 sporadic patients with laterality defects and complex congenital heart disease (CHD). Through whole exome sequencing (WES), we identified compound heterozygous mutations in DNAH9 in the affected individuals of these family trios. Ex vivo cDNA amplification revealed that DNAH9 mRNA expression was significantly downregulated in these patients carrying biallelic DNAH9 mutations, which cause a premature stop codon or exon skipping. Transmission electron microscopy (TEM) analysis identified ultrastructural defects of the outer dynein arms in these affected individuals. dnah9 knockdown in zebrafish lead to the disturbance of cardiac left-right patterning without affecting ciliogenesis in Kupffer's vesicle (KV). By generating a Dnah9 knockout (KO) C57BL/6n mouse model, we found that Dnah9 loss leads to compromised cardiac function. In this study, we identified recessive DNAH9 mutations in Chinese patients with cardiac abnormalities and defective LR pattering.

Bi-allelic Mutations in TTC21A Induce Asthenoteratospermia in Humans and Mice.

Male infertility is a major concern affecting human reproductive health. Asthenoteratospermia can cause male infertility through reduced motility and abnormal morphology of spermatozoa. Several genes, including DNAH1 and some CFAP family members, are involved in multiple morphological abnormalities of the sperm flagella (MMAF). However, these known genes only account for approximately 60% of human MMAF cases. Here, we conducted further genetic analyses by using whole-exome sequencing in a cohort of 65 Han Chinese men with MMAF. Intriguingly, bi-allelic mutations of TTC21A (tetratricopeptide repeat domain 21A) were identified in three (5%) unrelated, MMAF-affected men, including two with homozygous stop-gain mutations and one with compound heterozygous mutations of TTC21A. Notably, these men consistently presented with MMAF and additional abnormalities of sperm head-tail conjunction. Furthermore, a homozygous TTC21A splicing mutation was identified in two Tunisian cases from an independent MMAF cohort. TTC21A is preferentially expressed in the testis and encodes an intraflagellar transport (IFT)-associated protein that possesses several tetratricopeptide repeat domains that perform functions crucial for ciliary function. To further investigate the potential roles of TTC21A in spermatogenesis, we generated Ttc21a mutant mice by using CRISPR-Cas9 technology and revealed sperm structural defects of the flagella and the connecting piece. Our consistent observations across human populations and in the mouse model strongly support the notion that bi-allelic mutations in TTC21A can induce asthenoteratospermia with defects of the sperm flagella and head-tail conjunction.

Design, synthesis, and anti-influenza A virus activity evaluation of novel indole containing derivatives of triazole.

We designed and synthesized 18 substituted indole derivatives containing a triazole scaffold as novel anti-influenza A virus candidates using a bio-isosteric and scaffold-hopping strategy from the lead compound 4-32-2. Most of the target compounds (eg: 6, 7a, 7d, 7f-j, 7l, 7m, 7o, 7q) exhibited potent anti-influenza A virus activity and low cytotoxicity in vitro. In particular, 7a exhibited the most potent anti-IAV activity (IC50: 1.34 ± 0.13 µM) with low cytotoxicity (CC50: > 100 µM), and high selectivity index (SI: > 74.63), which provides a new chemical scaffold for the development of novel anti-IAV drug.

Identification of Darunavir Derivatives for Inhibition of SARS-CoV-2 3CLpro.

The effective antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed around the world. The 3C-like protease (3CLpro) of SARS-CoV-2 plays a pivotal role in virus replication; it also has become an important therapeutic target for the infection of SARS-CoV-2. In this work, we have identified Darunavir derivatives that inhibit the 3CLpro through a high-throughput screening method based on a fluorescence resonance energy transfer (FRET) assay in vitro. We found that the compounds 29# and 50# containing polyphenol and caffeine derivatives as the P2 ligand, respectively, exhibited favorable anti-3CLpro potency with EC50 values of 6.3 µM and 3.5 µM and were shown to bind to SARS-CoV-2 3CLpro in vitro. Moreover, we analyzed the binding mode of the DRV in the 3CLpro through molecular docking. Importantly, 29# and 50# exhibited a similar activity against the protease in Omicron variants. The inhibitory effect of compounds 29# and 50# on the SARS-CoV-2 3CLpro warrants that they are worth being the template to design functionally improved inhibitors for the treatment of COVID-19.

High maternal blood lipid levels during early pregnancy are associated with increased risk of congenital heart disease in offspring.

INTRODUCTION: This study aimed to investigate whether maternal blood lipid levels during early pregnancy are associated with the occurrence of congenital heart disease (CHD) in their offspring. MATERIAL AND METHODS: In this single-center case-control study, mothers of offspring with CHD (n = 230) and without CHD (n = 381) were included. Maternal lipid levels were determined on fasting blood samples taken in the first trimester. Relevant demographic and clinical data were extracted from the medical records. Maternal lipid profile was compared between the two groups, and regression analysis was performed to evaluate the association between lipid profile and CHD risk in offspring. RESULTS: Compared with the control group, levels of triglyceride, apolipoprotein-A1, and apolipoprotein-B in early pregnancy were significantly higher in the CHD group. Multivariate analyses showed that triglyceride (odds ratio [OR] 2.46, 95% CI 1.62-3.73, p < 0.01), total/high-density lipoprotein cholesterol (OR 2.10, 95% CI 1.07-4.13, p = 0.03), and apolipoprotein-A1 (OR 2.73, 95% CI 1.16-6.40, p = 0.02) were positively associated with CHD risk in offspring. CONCLUSIONS: Elevated maternal lipid profile was associated with increased risk of CHD in offspring.

Anthraquinone analogues with inhibitory activities against influenza a virus from Polygonatum odoratum.

Three anthraquinone analogues (1-3) were isolated by phytochemical work on EtOAc-soluble ingredients extracted from the roots of Polygonatum odoratum. The structures of all isolates were elucidated by NMR, MS and CD experiments, of which 1 (polygodoquinone A) was identified as a new anthraquinone derivative. Specifically, 1 represents an unusual structure composed of a naphthoquinone derivative linked to an anthraquinone via a C-C bond. 1-3 exhibited remarkable influenza A virus inhibitory activity with IC50 values of 11.4, 11.0, and 2.3 µM, respectively, which were better than ribavirin as the positive control.

Cell-Wide Survey of Amide-Bonded Lysine Modifications by Using Deacetylase CobB.

BACKGROUND: Lysine post-translational modifications are important regulators of protein function. Proteomic and biochemical approaches have resulted in identification of several lysine modifications, including acetylation, crotonylation, and succinylation. Here, we developed an approach for surveying amide-bonded lysine modifications in the proteome of human tissues/cells based on the observation that many lysine modifications are amide-bonded and that the Salmonella enterica deacetylase, CobB, is an amidase. RESULTS: After the proteome of human tissues/cells was denatured and the non-covalently bonded metabolites were removed by acetone washes, and the amide-bonded modifiers were released by CobB and analyzed using liquid- and/or gas chromatography/mass spectrometry metabolomic analysis. This protocol, which required 3-4 days for completion, was used to qualitatively identify more than 40 documented and unreported lysine modifications from the human proteome and to quantitatively analyze dynamic changes in targeted amide-bonded lysine modifications. CONCLUSIONS: We developed a method that was capable of monitoring and quantifying amide-bonded lysine modifications in cells of different origins.

An integrative genomic analysis of transcriptional profiles identifies characteristic genes and patterns in HIV-infected long-term non-progressors and elite controllers.

BACKGROUND: Despite that most HIV-infected individuals experience progressive CD4+ T cell loss and develop AIDS, a minority of HIV-infected individuals remain asymptomatic and maintain high level CD4+ T cell counts several years after seroconversion. Efforts have been made to understand the determinants of the nonprogressive status, exemplified by the clinical course of elite controllers (ECs) who maintain an undetectable viremia and viremic nonprogressors (VNPs) who have a normal CD4+ count in spite of circulating viral load. However, the intrinsic mechanism underlying nonprogression remained elusive. In this study, we performed an integrative analysis of transcriptional profiles to pinpoint the underlying mechanism for a naturally occurring viral control. METHODS: Three microarray datasets, reporting mRNA expression of the LTNPs or ECs in HIV-infected patients, were retrieved from Gene Expression Ominbus (GEO) or Arrayexpress databases. These datasets, profiled on the same type of microarray chip, were selected and merged by a bioinformatic approach to build a meta-analysis derived transcriptome (MADNT). In addition, we investigated the different transcriptional pathways and potential biomarkers in CD4+ and CD8+ cells in ECs and whole blood in VNPs compared to HIV progressors. The combined transcriptome and each subgroup was subject to gene set enrichment analysis and weighted co-expression network analysis to search potential transcription patterns related to the non-progressive status. RESULTS: 30 up-regulated genes and 83 down-regulated genes were identified in lymphocytes from integrative meta-analysis of expression data. The interferon response and innate immune activation was reduced in both CD4+ and CD8+ T cells from ECs. Several characteristic genes including CMPK1, CBX7, EIF3L, EIF4A and ZNF395 were indicated to be highly correlated with viremic control. Besides that, we indicated that the reduction of ribosome components and blockade of translation facilitated AIDS disease progression. Most interestingly, among VNPs who have a relatively high viral load, we detected a two gene-interaction networks which showed a strong correlation to immune control even with a rigorous statistical threshold (p value = 2-e4 and p value = 0.004, respectively) by WGCNA. CONCLUSIONS: We have identified differentially expressed genes and transcriptional patterns in ECs and VNPs compared to normal chronic HIV-infected individuals. Our study provides new insights into the pathogenesis of HIV and AIDS and clues for the therapeutic strategies for anti-retroviral administration.

Susceptibility to congenital heart defects associated with a polymorphism in TBX2 3' untranslated region in the Han Chinese population.

BACKGROUND: Tbx2 plays a critical role in determining fates of cardiomyocytes. Little is known about the contribution of TBX2 3' untranslated region (UTR) variants to the risk of congenital heart defect (CHD). Thus, we aimed to determine the association of single-nucleotide polymorphisms (SNPs) in TBX2 3' UTR with CHD susceptibility. METHODS: We recruited 1285 controls and 1241 CHD children from China. SNPs identification and genotyping were detected using Sanger Sequencing and SNaPshot. Stratified analysis was conducted to explore the association between rs59382073 polymorphism and CHD subtypes. Functional analyses were performed by luciferase assays in HEK-293T and H9c2 cells. RESULTS: Among five TBX2 3'UTR variants identified, rs59382073 minor allele T carriers had a 1.89-fold increased CHD risk compared to GG genotype (95% CI = 1.48-2.46, P = 4.48 × 10-7). The most probable subtypes were right ventricular outflow tract obstruction, conotruncal, and septal defect. G to T variation decreased luciferase activity in cells. This discrepancy was exaggerated by miR-3940 and miR-708, while their corresponding inhibitors eliminated it. CONCLUSION: T allele of rs59382073 in TBX2 3'UTR contributed to greater CHD risk in the Han Chinese population. G to T variation created binding sites for miR-3940 and miR-708 to inhibit gene expression.