Metagenomic study of the gut microbiota associated with cow milk consumption in Chinese peri-/postmenopausal women.

Cow milk consumption (CMC) and alterations of gut bacterial composition are proposed to be closely related to human health and disease. Our research aims to investigate the changes in human gut microbial composition in Chinese peri-/postmenopausal women with different CMC habits. A total of 517 subjects were recruited and questionnaires about their CMC status were collected; 394 subjects were included in the final analyses. Fecal samples were used for studying gut bacterial composition. All the subjects were divided into a control group (n = 248) and a CMC group (n = 146) according to their CMC status. Non-parametric tests and LEfSe at different taxonomic levels were used to reveal differentially abundant taxa and functional categories across different CMC groups. Relative abundance (RA) of one phylum (p_Actinobacteria), three genera (g_Bifidobacterium, g_Anaerostipes, and g_Bacteroides), and 28 species diversified significantly across groups. Specifically, taxa g_Anaerostipes (p < 0.01), g_Bacteroides (p < 0.05), s_Anaerostipes_hadrus (p < 0.01), and s_Bifidobacterium_pseudocatenulatum (p < 0.01) were positively correlated with CMC levels, but p_Actinobacteria (p < 0.01) and g_Bifidobacterium (p < 0.01) were negatively associated with CMC levels. KEGG module analysis revealed 48 gut microbiome functional modules significantly (p < 0.05) associated with CMC, including Vibrio cholerae pathogenicity signature, cholera toxins (p = 9.52e-04), and cephamycin C biosynthesis module (p = 0.0057), among others. In conclusion, CMC was associated with changes in gut microbiome patterns including beta diversity and richness of some gut microbiota. The alterations of certain bacteria including g_Anaerostipes and s_Bifidobacterium_pseudocatenulatum in the CMC group should be important for human health. This study further supports the biological value of habitual cow milk consumption.

Pathogenic ubiquitination of GSDMB inhibits NK cell bactericidal functions.

Gasdermin B (GSDMB) belongs to a large family of pore-forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to human disease, its function in the immunological response to pathogens remains poorly understood. Here, we report a dynamic host-pathogen conflict between GSDMB and the IpaH7.8 effector protein secreted by enteroinvasive Shigella flexneri. We show that IpaH7.8 ubiquitinates and targets GSDMB for 26S proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of natural killer cells by suppressing granzyme-A-mediated activation of GSDMB. In contrast to the canonical function of most gasdermin family members, GSDMB does not inhibit Shigella by lysing host cells. Rather, it exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveal a mechanism employed by pathogens to counteract this host defense system.

Long-term dental intervention and laboratory examination in a patient with Vitamin D-dependent rickets type I: A case report.

RATIONALE: Vitamin D-dependent rickets type I (VDDR-I) is a rare form of rickets, which is an autosomal recessive disease caused by 1α-hydroxylase enzyme deficiency. However, long-term dental management and microscopic morphology of teeth remain largely unclear. PATIENT CONCERNS: We report the case of a 10-year-old Chinese boy complaining of yellowish-brown teeth with extensive caries. DIAGNOSES: Clinical and laboratory examinations were performed, and VDDR-I was confirmed. Scanning electron microscopy confirmed amelogenesis imperfecta. INTERVENTIONS: The patient had been taking drugs intervention for VDDR-I from the age of 3 years. The decayed teeth were treated, and metal-preformed crowns were placed to prevent further impairment. Sequence tooth extraction and remineralization therapy were also performed. OUTCOMES: After 3 years of follow-up, the patient exhibited normal tooth replacement and an acceptable oral hygiene status. However, the new erupted teeth had amelogenesis imperfecta. LESSONS: This case is the first to confirm amelogenesis imperfecta in a patient with VDDR-I that was not prevented by drug intervention. Importantly, it provides evidence that long-term dental intervention in patients with VDDR-I can result in an acceptable oral hygiene status. Therefore, early and long-term dental intervention is necessary in VDDR-I patients.

Gene Expression and RNA Splicing Imputation Identifies Novel Candidate Genes Associated with Osteoporosis.

CONTEXT: Though genome-wide association studies (GWASs) have identified hundreds of genetic variants associated with osteoporosis related traits, such as bone mineral density (BMD) and fracture, it remains a challenge to interpret their biological functions and underlying biological mechanisms. OBJECTIVE: Integrate diverse expression quantitative trait loci and splicing quantitative trait loci data with several powerful GWAS datasets to identify novel candidate genes associated with osteoporosis. DESIGN, SETTING, AND PARTICIPANTS: Here, we conducted a transcriptome-wide association study (TWAS) for total body BMD (TB-BMD) (n = 66 628 for discovery and 7697 for validation) and fracture (53 184 fracture cases and 373 611 controls for discovery and 37 857 cases and 227 116 controls for validation), respectively. We also conducted multi-SNP-based summarized mendelian randomization analysis to further validate our findings. RESULTS: In total, we detected 88 genes significantly associated with TB-BMD or fracture through expression or ribonucleic acid splicing. Summarized mendelian randomization analysis revealed that 78 of the significant genes may have potential causal effects on TB-BMD or fracture in at least 1 specific tissue. Among them, 64 genes have been reported in previous GWASs or TWASs for osteoporosis, such as ING3, CPED1, and WNT16, as well as 14 novel genes, such as DBF4B, GRN, TMUB2, and UNC93B1. CONCLUSIONS: Overall, our findings provide novel insights into the pathogenesis mechanisms of osteoporosis and highlight the power of a TWAS to identify and prioritize potential causal genes.

Mechanosensing by Gli1+ cells contributes to the orthodontic force-induced bone remodelling.

OBJECTIVES: Gli1+ cells have received extensive attention in tissue homeostasis and injury mobilization. The aim of this study was to investigate whether Gli1+ cells respond to force and contribute to bone remodelling. MATERIALS AND METHODS: We established orthodontic tooth movement (OTM) model to assess the bone response for mechanical force. The transgenic mice were utilized to label and inhibit Gli1+ cells, respectively. Additionally, mice that conditional ablate Yes-associated protein (Yap) in Gli1+ cells were applied in the present study. The tooth movement and bone remodelling were analysed. RESULTS: We first found Gli1+ cells expressed in periodontal ligament (PDL). They were proliferated and differentiated into osteoblastic cells under tensile force. Next, both pharmacological and genetic Gli1 inhibition models were utilized to confirm that inhibition of Gli1+ cells led to arrest of bone remodelling. Furthermore, immunofluorescence staining identified classical mechanotransduction factor Yap expressed in Gli1+ cells and decreased after suppression of Gli1+ cells. Additionally, conditional ablation of Yap gene in Gli1+ cells inhibited the bone remodelling as well, suggesting Gli1+ cells are force-responsive cells. CONCLUSIONS: Our findings highlighted that Gli1+ cells in PDL directly respond to orthodontic force and further mediate bone remodelling, thus providing novel functional evidence in the mechanism of bone remodelling and first uncovering the mechanical responsive property of Gli1+ cells.

Oxysterols provide innate immunity to bacterial infection by mobilizing cell surface accessible cholesterol.

Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene that converts cholesterol to the oxysterol 25-hydroxycholesterol (25HC). Circulating 25HC modulates essential immunological processes including antiviral immunity, inflammasome activation and antibody class switching; and dysregulation of CH25H may contribute to chronic inflammatory disease and cancer. Although 25HC is a potent regulator of cholesterol storage, uptake, efflux and biosynthesis, how these metabolic activities reprogram the immunological state of target cells remains poorly understood. Here, we used recently designed toxin-based biosensors that discriminate between distinct pools of plasma membrane cholesterol to elucidate how 25HC prevents Listeria monocytogenes from traversing the plasma membrane of infected host cells. The 25HC-mediated activation of acyl-CoA:cholesterol acyltransferase (ACAT) triggered rapid internalization of a biochemically defined fraction of cholesterol, termed 'accessible' cholesterol, from the plasma membrane while having little effect on cholesterol in complexes with sphingomyelin. We show that evolutionarily distinct bacterial species, L. monocytogenes and Shigella flexneri, exploit the accessible pool of cholesterol for infection and that acute mobilization of this pool by oxysterols confers immunity to these pathogens. The significance of this signal-mediated membrane remodelling pathway probably extends beyond host defence systems, as several other biologically active oxysterols also mobilize accessible cholesterol through an ACAT-dependent mechanism.

Sensory nerve-deficient microenvironment impairs tooth homeostasis by inducing apoptosis of dental pulp stem cells.

OBJECTIVES: The aim of this study is to investigate the role of sensory nerve in tooth homeostasis and its effect on mesenchymal stromal/stem cells (MSCs) in dental pulp. MATERIALS AND METHODS: We established the rat denervated incisor models to identify the morphological and histological changes of tooth. The groups were as follows: IANx (inferior alveolar nerve section), SCGx (superior cervical ganglion removal), IANx + SCGx and Sham group. The biological behaviour of dental pulp stromal/stem cells (DPSCs) was evaluated. Finally, we applied activin B to DPSCs from sensory nerve-deficient microenvironment to analyse the changes of proliferation and apoptosis. RESULTS: Incisor of IANx and IANx + SCGx groups exhibited obvious disorganized tooth structure, while SCGx group only showed slight decrease of dentin thickness, implying sensory nerve, not sympathetic nerve, contributes to the tooth homeostasis. Moreover, we found sensory nerve injury led to disfunction of DPSCs via activin B/SMAD2/3 signalling in vitro. Supplementing activin B promoted proliferation and reduced apoptosis of DPSCs in sensory nerve-deficient microenvironment. CONCLUSIONS: This research first demonstrates that sensory nerve-deficient microenvironment impairs tooth haemostasis by inducing apoptosis of DPSCs via activin B/SMAD2/3 signalling. Our study provides the evidence for the crucial role of sensory nerve in tooth homeostasis.

Analysis of Continuous Mutation and Evolution on Circulating SARS-CoV-2.

Monitoring the mutation and evolution of the virus is important for tracing its ongoing transmission and facilitating effective vaccine development. A total of 342 complete genomic sequences of SARS-CoV-2 were analyzed in this study. Compared to the reference genome reported in December 2019, 465 mutations were found, among which, 347 occurred in only 1 sequence, while 26 occurred in more than 5 sequences. For these 26 further identified as SNPs, 14 were closely linked and were grouped into 5 profiles. Phylogenetic analysis revealed the sequences formed 2 major groups. Most of the sequences in late period (March and April) constituted the Cluster II, while the sequences before March in this study and the reported S/L and A/B/C types in previous studies were all in Cluster I. The distributions of some mutations were specific geographically or temporally, the potential effect of which on the transmission and pathogenicity of SARS-CoV-2 deserves further evaluation and monitoring. Two mutations were found in the receptor-binding domain (RBD) but outside the receptor-binding motif (RBM), indicating that mutations may only have marginal biological effects but merit further attention. The observed novel sequence divergence is of great significance to the study of the transmission, pathogenicity, and development of an effective vaccine for SARS-CoV-2.

Installation of a cancer promoting WNT/SIX1 signaling axis by the oncofusion protein MLL-AF9.

BACKGROUND: Chromosomal translocation-induced expression of the chromatin modifying oncofusion protein MLL-AF9 promotes acute myelocytic leukemia (AML). Whereas WNT/ß-catenin signaling has previously been shown to support MLL-AF9-driven leukemogenesis, the mechanism underlying this relationship remains unclear. METHODS: We used two novel small molecules targeting WNT signaling as well as a genetically modified mouse model that allow targeted deletion of the WNT protein chaperone Wntless (WLS) to evaluate the role of WNT signaling in AML progression. ATAC-seq and transcriptome profiling were deployed to understand the cellular consequences of disrupting a WNT signaling in leukemic initiating cells (LICs). FINDINGS: We identified Six1 to be a WNT-controlled target gene in MLL-AF9-transformed leukemic initiating cells (LICs). MLL-AF9 alters the accessibility of Six1 DNA to the transcriptional effector TCF7L2, a transducer of WNT/ß-catenin gene expression changes. Disruption of WNT/SIX1 signaling using inhibitors of the Wnt signaling delays the development of AML. INTERPRETATION: By rendering TCF/LEF-binding elements controlling Six1 accessible to TCF7L2, MLL-AF9 promotes WNT/ß-catenin-dependent growth of LICs. Small molecules disrupting WNT/ß-catenin signaling block Six1 expression thereby disrupting leukemia driven by MLL fusion proteins.

Contributions of Bioactive Molecules in Stem Cell-Based Periodontal Regeneration.

Periodontal disease is a widespread disease, which without proper treatment, may lead to tooth loss in adults. Because stem cells from the inflammatory microenvironment created by periodontal disease exhibit impaired regeneration potential even under favorable conditions, it is difficult to obtain satisfactory therapeutic outcomes using traditional treatments, which only focus on the control of inflammation. Therefore, a new stem cell-based therapy known as cell aggregates/cell sheets technology has emerged. This approach provides sufficient numbers of stem cells with high viability for treating the defective site and offers new hope in the field of periodontal regeneration. However, it is not sufficient for regenerating periodontal tissues by delivering cell aggregates/cell sheets to the impaired microenvironment in order to suppress the function of resident cells. In the present review, we summarize some promising bioactive molecules that act as cellular signals, which recreate a favorable microenvironment for tissue regeneration, recruit endogenous cells into the defective site and enhance the viability of exogenous cells.

Chemical Modulation of WNT Signaling in Cancer.

Genetically based observations stemming from defects in development and in regeneration form the foundation of our understanding regarding how the secreted WNT proteins control coordinated cell fate decision-making in adult tissues. At the same time, our anticipation of potential benefits and unwanted toxicities associated with candidate anticancer agents targeting WNT signal transduction are also reliant upon this blueprint of WNT-associated physiology. Despite the long established role of WNT signaling in cancer, the emergence of WNT signaling as a suppressor of immunological attack in melanoma reveals an unanticipated anticancer potential in targeting WNT signaling. Here we review the literature associated with WNT signaling in cancer and discuss potential challenges that may be associated with the chemical attack of this important cellular process in achieving therapeutic goals. Although a number of small molecules targeting WNT signaling are introduced here, we center our discussion on antagonists of the WNT acyltransferase porcupine (PORCN) given the recent entry of two candidate molecules in clinical testing.

Extra-mitochondrial prosurvival BCL-2 proteins regulate gene transcription by inhibiting the SUFU tumour suppressor.

Direct interactions between pro- and anti-apoptotic BCL-2 family members form the basis of cell death decision-making at the outer mitochondrial membrane (OMM). Here we report that three anti-apoptotic BCL-2 proteins (MCL-1, BCL-2 and BCL-XL) found untethered from the OMM function as transcriptional regulators of a prosurvival and growth program. Anti-apoptotic BCL-2 proteins engage a BCL-2 homology (BH) domain sequence found in SUFU (suppressor of fused), a tumour suppressor and antagonist of the GLI DNA-binding proteins. BCL-2 proteins directly promote SUFU turnover, inhibit SUFU-GLI interaction, and induce the expression of the GLI target genes BCL-2, MCL-1 and BCL-XL. Anti-apoptotic BCL-2 protein/SUFU feedforward signalling promotes cancer cell survival and growth, and can be disabled with BH3 mimetics-small molecules that target anti-apoptotic BCL-2 proteins. Our findings delineate a chemical strategy for countering drug resistance in GLI-associated tumours and reveal unanticipated functions for BCL-2 proteins as transcriptional regulators.

Blockade to pathological remodeling of infarcted heart tissue using a porcupine antagonist.

The secreted Wnt signaling molecules are essential to the coordination of cell-fate decision making in multicellular organisms. In adult animals, the secreted Wnt proteins are critical for tissue regeneration and frequently contribute to cancer. Small molecules that disable the Wnt acyltransferase Porcupine (Porcn) are candidate anticancer agents in clinical testing. Here we have systematically assessed the effects of the Porcn inhibitor (WNT-974) on the regeneration of several tissue types to identify potentially unwanted chemical effects that could limit the therapeutic utility of such agents. An unanticipated observation from these studies is proregenerative responses in heart muscle induced by systemic chemical suppression of Wnt signaling. Using in vitro cultures of several cell types found in the heart, we delineate the Wnt signaling apparatus supporting an antiregenerative transcriptional program that includes a subunit of the nonfibrillar collagen VI. Similar to observations seen in animals exposed to WNT-974, deletion of the collagen VI subunit, COL6A1, has been shown to decrease aberrant remodeling and fibrosis in infarcted heart tissue. We demonstrate that WNT-974 can improve the recovery of heart function after left anterior descending coronary artery ligation by mitigating adverse remodeling of infarcted tissue. Injured heart tissue exposed to WNT-974 exhibits decreased scarring and reduced Col6 production. Our findings support the development of Porcn inhibitors as antifibrotic agents that could be exploited to promote heart repair following injury.

Mass spectrometry based proteomics profiling of human monocytes.

Human monocyte is an important cell type which is involved in various complex human diseases. To better understand the biology of human monocytes and facilitate further studies, we developed the first comprehensive proteome knowledge base specifically for human monocytes by integrating both in vivo and in vitro datasets. The top 2000 expressed genes from in vitro datasets and 779 genes from in vivo experiments were integrated into this study. Altogether, a total of 2237 unique monocyte-expressed genes were cataloged. Biological functions of these monocyte-expressed genes were annotated and classified via Gene Ontology (GO) analysis. Furthermore, by extracting the overlapped genes from in vivo and in vitro datasets, a core gene list including 541 unique genes was generated. Based on the core gene list, further gene-disease associations, pathway and network analyses were performed. Data analyses based on multiple bioinformatics tools produced a large body of biologically meaningful information, and revealed a number of genes such as SAMHD1, G6PD, GPD2 and ENO1, which have been reported to be related to immune response, blood biology, bone remodeling, and cancer respectively. As a unique resource, this study can serve as a reference map for future in-depth research on monocytes biology and monocyte-involved human diseases.

Delivery of the Porcupine Inhibitor WNT974 in Mice.

We describe here a technique for delivering the porcupine inhibitor WNT974 (formerly LGK974) in mice. The protocol entails once-a-day oral delivery of WNT974 for up to 3 months at a concentration sufficient to achieve systemic Wnt pathway inhibition with limited toxicity as measured by weight change. This route of delivery enables extended durations of Wnt signaling inhibition in a mammalian model organism.

Quantitative proteomics and integrative network analysis identified novel genes and pathways related to osteoporosis.

UNLABELLED: Osteoporosis is mainly characterized by low bone mineral density (BMD), and can be attributed to excessive bone resorption by osteoclasts. Migration of circulating monocytes from blood to bone is important for subsequent osteoclast differentiation and bone resorption. Identification of those genes and pathways related to osteoclastogenesis and BMD will contribute to a better understanding of the pathophysiological mechanisms of osteoporosis. In this study, we applied the LC-nano-ESI-MS(E) (Liquid Chromatograph-nano-Electrospray Ionization-Mass Spectrometry) for quantitative proteomic profiling in 33 female Caucasians with discordant BMD levels, with 16 high vs. 17 low BMD subjects. Protein quantitation was accomplished by label-free measurement of total ion currents collected from MS(E) data. Comparison of protein expression in high vs. low BMD subjects showed that ITGA2B (p=0.0063) and GSN (p=0.019) were up-regulated in the high BMD group. Additionally, our protein-RNA integrative analysis showed that RHOA (p=0.00062) differentially expressed between high vs. low BMD groups. Network analysis based on multiple tools revealed two pathways: "regulation of actin cytoskeleton" (p=1.13E-5, FDR=3.34E-4) and "leukocyte transendothelial migration" (p=2.76E-4, FDR=4.71E-3) that are functionally relevant to osteoporosis. Consistently, ITGA2B, GSN and RHOA played crucial roles in these two pathways respectively. All together, our study strongly supported the contribution of the genes ITGA2B, GSN and RHOA and the two pathways to osteoporosis risk. BIOLOGICAL SIGNIFICANCE: Mass spectrometry based quantitative proteomics study integrated with network analysis identified novel genes and pathways related to osteoporosis. The results were further verified in multiple level studies including protein-RNA integrative analysis and genome wide association studies.

Novel APP K724M mutation causes Chinese early-onset familial Alzheimer's disease and increases amyloid-ß42 to amyloid-ß40 ratio.

Alzheimer's disease (AD) is the most common neurodegenerative disorder among the elderly individuals. Although there are several million cases of AD estimated in China with the most population in the world, no Chinese early-onset familial AD caused by new APP gene mutation has ever been reported. Here, we first described a Chinese family with early-onset AD that was inherited in autosomal dominant manner, and the age of onset was 46.6 ± 7.7 years (n = 5; range, 40-58 years). By using genetic analysis of 3 collected patients' DNA samples, we identified a heterozygous APP gene mutation (g.275363A>T, K724M according to APP770). Finally, when APP695 with K724M mutation was ectopically expressed in HEK293 cell, the ratio of amyloid-ß42 to amyloid-ß40 was 2.23-fold higher than that of wild-type control. Together, our data suggest that APP K724M gene mutation may contribute to the cause of this Chinese early-onset familial AD.

Suggestion of GLYAT gene underlying variation of bone size and body lean mass as revealed by a bivariate genome-wide association study.

Bone and muscle, two major tissue types of musculoskeletal system, have strong genetic determination. Abnormality in bone and/or muscle may cause musculoskeletal diseases such as osteoporosis and sarcopenia. Bone size phenotypes (BSPs), such as hip bone size (HBS), appendicular bone size (ABS), are genetically correlated with body lean mass (mainly muscle mass). However, the specific genes shared by these phenotypes are largely unknown. In this study, we aimed to identify the specific genes with pleiotropic effects on BSPs and appendicular lean mass (ALM). We performed a bivariate genome-wide association study (GWAS) by analyzing ~690,000 SNPs in 1,627 unrelated Han Chinese adults (802 males and 825 females) followed by a replication study in 2,286 unrelated US Caucasians (558 males and 1,728 females). We identified 14 interesting single nucleotide polymorphisms (SNPs) that may contribute to variation of both BSPs and ALM, with p values <10(-6) in discovery stage. Among them, the association of three SNPs (rs2507838, rs7116722, and rs11826261) in/near GLYAT (glycine-N-acyltransferase) gene was replicated in US Caucasians, with p values ranging from 1.89 × 10(-3) to 3.71 × 10(-4) for ALM-ABS, from 5.14 × 10(-3) to 1.11 × 10(-2) for ALM-HBS, respectively. Meta-analyses yielded stronger association signals for rs2507838, rs7116722, and rs11826261, with pooled p values of 1.68 × 10(-8), 7.94 × 10(-8), 6.80 × 10(-8) for ALB-ABS and 1.22 × 10(-4), 9.85 × 10(-5), 3.96 × 10(-4) for ALM-HBS, respectively. Haplotype allele ATA based on these three SNPs was also associated with ALM-HBS and ALM-ABS in both discovery and replication samples. Interestingly, GLYAT was previously found to be essential to glucose metabolism and energy metabolism, suggesting the gene's dual role in both bone development and muscle growth. Our findings, together with the prior biological evidence, suggest the importance of GLYAT gene in co-regulation of bone phenotypes and body lean mass.