Congenital heart disease (CHD) is a worldwide problem with high morbidity and mortality. Early diagnosis of congenital heart disease is still a challenge in clinical work. In recent years, few studies indicated that placental methylation may be predictors of CHD. More studies are needed to confirm the association between placental methylation and CHD. The aim of this study was to investigate the association between prenatal placental DNA methylation and CHD. Placental tissues were obtained from four fetuses during the second trimester with isolated, non-syndromic congenital heart disease, including three cases with double outlet right ventricle (DORV) and one case with tetralogy of Fallot (TOF), and four unaffected fetuses as controls. The Illumina Infinium Human Methylation 850K BeadChip assay was employed to identify differential methylation sites (DMSs) and differential methylation regions (DMRs). Differential methylation was evaluated by comparing the β-values for individual CpG loci in cases vs. controls. In addition, the function of genes was assessed through KEGG enrichment analysis, Gene Ontology (GO) analysis and KEGG pathway analysis. Compared with the control group, we identified 9625 differential methylation genes on 26,202 DMSs (p < 0.05), of which 6997 were hyper-methylation and 2628 were hypo-methylation. The top 30 terms of GO biological process and KEGG enrichment analysis of DMSs were connected with multiple important pathways of heart development and disease. Ten differentially methylated regions and the genes related to DMRs, such as TLL1, CRABP1, FDFT1, and PCK2, were identified. The deformity caused by the loss of function of these genes is remarkably consistent with the clinical phenotype of our cases. The DNA methylation level of placental tissue is closely associated with fetal congenital heart disease.

Klebsiella pneumoniae is a gram-negative bacterium that can cause many diseases in hospitals and communities. Intestinal K. pneumoniae infections are relatively rare. Most K. pneumoniae infections begin with the colonization of the gastrointestinal system. In this study, clinically isolated K. pneumoniae strains were used to infect intestinal epithelial Caco-2 cells to study the possible intestinal translocation mechanism of K. pneumoniae. We found that of the three K. pneumoniae strains tested, KP1821 exhibited the strongest adhesive and invasive abilities and that the adhesion to Caco-2 intestinal epithelial cells was affected by the acidic environment of the stomach. Transcriptome sequencing revealed the involvement of molecules associated with the extracellular matrix and cell adhesion, inflammatory response, calcium ion and transforming growth factor β (TGF-β) signaling pathways, and other abnormalities in biological processes and cell signaling pathways. Additionally, tolloid-like protein 1 (TLL1) was significantly upregulated. Knocking down TLL1 with shRNA significantly reduced KP1821\'s ability to invade and adhere to intestinal epithelial cells. TLL1 is involved in the activation of the TGF-β signaling pathway. Inhibition of this pathway using the inhibitor SB431542 induced significantly reduced adhesion and invasion capabilities of KP1821. Our findings demonstrate that TLL1 participates in K. pneumoniae adhesion and invasion of intestinal epithelial cells by activating the TGF-β signaling pathway.

Periodontitis is caused by multiple factors involving a bacterial challenge and a susceptible host, although there is no report on gene mutation directly linked to this common disease. Mutations in the proteinase bone morphogenetic protein 1 (BMP1) were identified in patients with osteogenesis imperfecta, who display some dentin defects and alveolar bone loss. We previously reported essential roles of BMP1 and tolloid-like 1 (TLL1), two closely related extracellular proteinases with overlapping functions, in mouse periodontium growth by simultaneous knockout (KO) of both genes, although the separate roles of BMP1 and TLL1 have remained unclear. Here, we have investigated whether and how BMP1 and TLL1 separately maintain periodontal homeostasis by comparing single Bmp1 KO and Tll1 KO with double KO (dKO) phenotypes.
Floxed Bmp1 and/or Tll1 alleles were deleted in transgenic mice via ubiquitously expressed CreERT2 induced by tamoxifen treatment starting at 4-weeks of age (harvested at 18-weeks of age). Multiple approaches, including X-ray, micro-CT, calcein and alizarin red double-labeling, scanning electron microscopy, and histological and immunostaining assays, were used to analyze periodontal phenotypes and molecular mechanisms.
Both Bmp1 KO and double KO mice exhibited severe periodontal defects, characterized by periodontal ligament (PDL) fiber loss and ectopic ossification in the expanded PDL area, and drastic reductions in alveolar bone and cementum volumes, whereas Tll1 KO mice displayed very mild phenotypes. Mechanistic studies revealed a sharp increase in the uncleaved precursor of type I collagen (procollagen I), leading to defective extracellular matrices.
BMP1, but not TLL1, is essential for maintaining periodontal homeostasis. This occurs at least partly via biosynthetic processing of procollagen I, thereby maintaining appropriate levels of procollagen I and its activated products such as mature collagen I.

The bone morphogenetic protein (BMP) 1/tolloid (TLD) proteinase family is a group of important metalloproteinases, which play key roles in the growth and development of tissues and organs via regulating the biosynthetic processing of the extracellular matrix. Clinical reports have revealed that mutations in the genes encoding BMP1/TLD proteinases lead to dentinogenesis imperfecta type Ⅰ, accompanied with osteogenesis imperfecta. Therefore, this proteinase family is essential for the development of hard tissues. In this study, we review the research progress in the function and mechanism of the BMP1/TLD proteinase family in the development of teeth and bone.
骨形态发生蛋白（BMP）1/tolloid（TLD）蛋白酶家族是一类重要的基质金属蛋白酶，可通过调控细胞外基质的生物合成而在组织、器官生长发育中发挥重要作用。临床报道发现BMP1/TLD蛋白酶家族的编码基因发生突变可导致伴有成骨发育不全的Ⅰ型牙本质发育不全，提示该蛋白酶家族在牙及骨等硬组织发育中具有重要作用。本文将BMP1/TLD蛋白酶家族在牙和骨组织发育中的作用及其机制所取得的研究进展作一综述。.

Spinal muscular atrophy (SMA) is a rare neuromuscular disorder threating hundreds of thousands of lives worldwide. And the severity of SMA differs among different clinical types, which has been demonstrated to be modified by factors like SMN2, SERF1, NAIP, GTF2H2 and PLS3. However, the severities of many SMA cases, especially the cases within a family, often failed to be explained by these modifiers. Therefore, other modifiers are still waiting to be explored.
In this study, we presented a rare case of SMA discordant family with a mild SMA male patient and a severe SMA female patient. The two SMA cases fulfilled the diagnostic criteria defined by the International SMA Consortium. With whole exome sequencing, we confirmed the heterozygous deletion of exon7 at SMN1 on the parents\' genomes and the homozygous deletions on the two patients\' genomes. The MLPA results confirmed the deletions and indicated that all the family members carry two copies of SMN2, SERF1, NAIP and GTF2H2. Further genomic analysis identified compound heterozygous mutations at TLL2 on the male patient\'s genome, and compound heterozygous mutations at VPS13A and the de novo mutation at AGAP5 on female patient\'s genome. TLL2 is an activator of myostatin, which negatively regulates the growth of skeletal muscle tissue. Mutation in TLL2 has been proved to increase muscular function in mice model. VPS13A encodes proteins that control the cycling of proteins through the trans-Golgi network to endosomes, lysosomes and the plasma membrane. And AGAP5 was reported to have GTPase activator activity.
We reported a case of SMA discordant family and identified mutations at TLL2, VPS13A and AGAP5 on the patients\' genomes. The mutations at TLL2 were predicted to be pathogenic and are likely to alleviate the severity of the male SMA patient. Our finding broadens the spectrum of genetic modifiers of SMA and will contribute to accurate counseling of SMA affected patients and families.

Metzincins are key molecules in the degradation of the extracellular matrix and play an important role in cellular processes such as cell migration, adhesion, and cell fusion of malignant tumors, including cutaneous melanoma (CM). We hypothesized that genetic variants of the metzincin metallopeptidase family genes would be associated with CM-specific survival (CMSS). To test this hypothesis, we first performed Cox proportional hazards regression analysis to evaluate the associations between genetic variants of 75 metzincin metallopeptidase family genes and CMSS using the dataset from the genome-wide association study (GWAS) from The University of Texas MD Anderson Cancer Center (MDACC) which included 858 non-Hispanic white patients with CM, and then validated using the dataset from the Harvard GWAS study which had 409 non-Hispanic white patients with invasive CM. Four independent SNPs (MMP16 rs10090371 C>A, ADAMTS3 rs788935 T>C, TLL2 rs10882807 T>C and MMP9 rs3918251 A>G) were identified as predictors of CMSS, with a variant-allele attributed hazards ratio (HR) of 1.73 (1.32-2.29, 9.68E-05), 1.46 (1.15-1.85, 0.002), 1.68 (1.31-2.14, 3.32E-05) and 0.67 (0.51-0.87, 0.003), respectively, in the meta-analysis of these two GWAS studies. Combined analysis of risk genotypes of these four SNPs revealed a decreased CMSS in a dose-response manner as the number of risk genotypes increased (Ptrend  < 0.001). An improvement was observed in the prediction model (area under the curve [AUC] = 81.4% vs. 78.6%), when these risk genotypes were added to the model containing non-genotyping variables. Our findings suggest that these genetic variants may be promising prognostic biomarkers for CMSS.

Mutations in bone morphogenetic protein 1 (BMP1) in humans or deletion of BMP1 and related protease tolloid like 1 (TLL1) in mice lead to osteogenesis imperfecta (OI). Here, we show progressive periodontal defects in mice in which both BMP1 and TLL1 have been conditionally ablated, including malformed periodontal ligament (PDL) (recently shown to play key roles in normal alveolar bone formation), significant loss in alveolar bone mass ( P < 0.01), and a sharp reduction in cellular cementum. Molecular mechanism studies revealed a dramatic increase in the uncleaved precursor of type I collagen (procollagen I) and a reduction in dentin matrix protein 1 (DMP1), which is partially responsible for defects in extracellular matrix (ECM) formation and mineralization. We also showed a marked increase in the expression of matrix metallopeptidase 13 (MMP13) and tartrate-resistant acid phosphatase (TRAP), leading to an acceleration in periodontal breakdown. Finally, we demonstrated that systemic application of antibiotics significantly improved the alveolar bone and PDL damage of the knockdown phenotype, which are thus shown to be partially secondary to pathogen-induced inflammation. Together, identification of the novel roles of BMP1 and TLL1 in maintaining homeostasis of periodontal formation, partly via biosynthetic processing of procollagen I and DMP1, provides novel insights into key contributions of the extracellular matrix environment to periodontal homeostasis and contributes toward understanding of the pathology of periodontitis.

BACKGROUND: Mutations in the proteinase bone morphogenetic protein-1 (BMP1) were recently identified in patients with osteogenesis imperfecta, which can be associated with type 1 dentinogenesis imperfecta. BMP1 is co-expressed in various tissues and has overlapping activities with the closely related proteinase mammalian tolloid-like 1 (TLL1). In this study we investigated whether removing the overlapping activities of BMP1 and TLL1 affects the mineralization of tooth root dentin.
METHODS: Floxed alleles of the BMP1 and TLL1 genes were excised via ubiquitously expressed Cre induced by tamoxifen treatment beginning at 3 days of age (harvested at 3 weeks of age) or beginning at 4 weeks of age (harvested at 8 weeks of age). Multiple techniques, including x-ray analysis, double-labeling with calcein and alizarin red stains for measurement of dentin formation rate, and histologic and immunostaining assays, were used to analyze the dentin phenotype.
RESULTS: BMP1/TLL1 double knockout mice displayed short and thin root dentin, defects in dentin mineralization, and delayed tooth eruption. Molecular mechanism studies revealed accumulation of collagens in dentin and a sharp reduction in non-collagenous proteins such as dentin matrix protein 1 and dentin sialophosphoprotein. Furthermore, we found a strong reduction in tartrate-resistant acid phosphatase, which is likely caused by defects in bone cells.
CONCLUSIONS: BMP1/TLL1 appear to play crucial roles in maintaining extracellular matrix homeostasis essential to root formation and dentin mineralization.

The Toll pathway is one of the most important signaling pathways regulating insect innate immunity. Spatzle is a key protein that functions as a Toll receptor ligand to trigger Toll-dependent expression of immunity-related genes. In this study, a novel spatzle gene (ApSPZ) from the Chinese oak silkworm Antheraea pernyi was identified. The ApSPZ cDNA is 1065 nucleotides with an open reading frame (ORF) of 777 bp encoding a protein of 258 amino acids. The protein has an estimated molecular weight of 29.71 kDa and an isoelectric point (PI) of 8.53. ApSPZ is a nuclear and secretory protein with no conserved domains or membrane helices and shares 40% amino acid identity with SPZ from Manduca sexta. Phylogenetic analysis indicated that ApSPZ might be a new member of the Spatzle type 1 family, which belongs to the Spatzle superfamily. The expression patterns of several genes involved in the Toll pathway were examined at different developmental stages and various tissues in 5th instar larvae. The examined targets included A. pernyi spatzle, GNBP, MyD88, Tolloid, cactus and dorsalA. The RT-PCR results showed that these genes were predominantly expressed in immune-responsive fat body tissue, indicating that the genes play a crucial role in A. pernyi innate immunity. Moreover, A. pernyi infection with the fungus Nosema pernyi and the gram-positive bacterium Enterococcus pernyi, but not the gram-negative bacterium Escherichia coli, activated the Toll signaling pathway. These results represent the first study of the Toll pathway in A. pernyi, which provides insight into the A. pernyi innate immune system.

Maximal metabolic outputs for exercise and thermogenesis in birds presumably influence fitness through effects on flight and shivering performance. Because both summit (Msum, maximum thermoregulatory metabolic rate) and maximum (MMR, maximum exercise metabolic rate) metabolic rates are functions of skeletal muscle activity, correlations between these measurements and their mechanistic underpinnings might occur. To examine whether such correlations occur, we measured the effects of experimental cold and exercise training protocols for 3 weeks on body (Mb) and muscle (Mpec) masses, basal metabolic rate (BMR), Msum, MMR, pectoralis mRNA and protein expression for myostatin, and mRNA expression of TLL-1 and TLL-2 (metalloproteinase activators of myostatin) in house sparrows (Passer domesticus). Both training protocols increased Msum, MMR, Mb and Mpec, but BMR increased with cold training and decreased with exercise training. No significant differences occurred for pectoralis myostatin mRNA expression, but cold and exercise increased the expression of TLL-1 and TLL-2. Pectoralis myostatin protein levels were generally reduced for both training groups. These data clearly demonstrate cross-training effects of cold and exercise in birds, and are consistent with a role for myostatin in increasing pectoralis muscle mass and driving organismal increases in metabolic capacities.