Endometrial transcriptome profiling of patients with recurrent implantation failure during hormone replacement therapy cycles.

Background: The molecular mechanisms underlying window of implantation (WOI) displacement in patients with recurrent implantation failure (RIF) remain unclear. This study aims to explore the transcriptomic signatures of endometrium with normal and displaced WOIs and to identify the causes of endometrial receptivity (ER) abnormalities and WOI displacement in RIF patients. Methods: In this study, 40 RIF patients were recruited and underwent personalized embryo transfer (pET) guided by the predicted results of endometrial receptivity diagnosis (ERD) model. Transcriptome analysis of endometrium from patients with clinical pregnancies after pET was performed to identify differentially expressed genes (DEGs) associated with WOI displacement. Gene expression data from HRT and natural cycle endometrium were compared to identify specific gene expression patterns of ER-related genes during WOI. Results: The ERD results indicated that 67.5% of RIF patients (27/40) were non-receptive in the conventional WOI (P+5) of the HRT cycle. The clinical pregnancy rate in RIF patients improved to 65% (26/40) after ERD-guided pET, indicating the effectiveness of transcriptome-based WOI prediction. Among the 26 patients with clinical pregnancy, the gene expression profiles of P+5 endometrium from advanced (n=6), normal (n=10) and delayed (n=10) WOI groups were significantly different from each other. Furthermore, 10 DEGs identified among P+5 endometrium of 3 groups were involved in immunomodulation, transmembrane transport and tissue regeneration, which could accurately classify the endometrium with different WOIs. Additionally, a large number of ER-related genes showed significant correlation and similar gene expression patterns in P+3, P+5, and P+7 endometrium from HRT cycles and LH+5, LH+7, and LH+9 endometrium from natural cycles. Conclusion: Our study shows that ER-related genes share similar gene expression patterns during WOI in both natural and HRT cycles, and their aberrant expression is associated with WOI displacements. The improvement of pregnancy outcomes in RIF patients by adjusting ET timing according to ERD results demonstrates the importance of transcriptome-based endometrial receptivity assessment and the clinical efficiency of ERD model.

[Concentrations and Community Structures of Culturable Bacteria in Aerosols of Household Garbage Stations in Shanghai].

The airborne microorganism has attracted increasing attention, and household garbage carries various pathogenic bacteria that affect the surrounding environment and public health. In this study, the culturable bacteria in the air were collected by using a six-level Anderson sampler, and the temperature, relative humidity, PM2.5, and PM10 in the garbage stations and their surrounding environment were recorded. The relationships between environmental factors and culturable bacterial pollution in the air were also analyzed. The results showed that the culturable bacterial concentrations in five sampling sites (the garbage station of a villa and the area downwind, the garbage station of a campus, the roof of an office building, and the garbage station of a residential area) were (1254±92), (280±123), (172±47), (84±18), and (175±174) CFU·m-3, respectively. The concentrations of the culturable bacteria in the garbage station of the villa were significantly higher than those of other sampling sites, mainly because there were biochemical treatment facilities for the on-site treatment of wet garbage in the garbage house. The sizes of the culturable bacteria in the garbage station of the villa mainly ranged from 1.1-4.7 µm, and the bacterial sizes at the other four sampling sites were primarily larger than 7 µm, with a few bacteria ranging from 1.1-2.1 µm. In this study, Proteobacteria and Firmicutes were the dominant phyla, and Corynebacterium and Bacillus were the dominant genera. More importantly, some opportunistic pathogens such as Corynebacterium, Staphylococcus, and Acinetobacter were also detected. The concentrations of the culturable bacteria in the garbage station of the villa were highly correlated with temperature, relative humidity, PM2.5, and PM10. Exiguobacterium in the air was highly correlated with PM10, temperature, and relative humidity. The health hazard quotient (HQ) values of the five sampling sites were all less than 1; however, the results of microbial quantitative risk assessment showed that the health risks of the male and female staff in the three garbage houses were all higher than the corresponding reference values. This study revealed the influence of garbage stations on the bioaerosol in the surrounding environment and provided references for the evaluation of air quality in and around garbage stations.

An Allele-Specific Functional SNP Associated with Two Systemic Autoimmune Diseases Modulates IRF5 Expression by Long-Range Chromatin Loop Formation.

Both systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) are autoimmune diseases sharing similar genetic backgrounds. Genome-wide association studies have constantly disclosed numerous genetic variants conferring to both disease risks at 7q32.1, but the functional mechanisms underlying them are still largely unknown. Through a series of bioinformatics and functional analyses, we prioritized a potential independent functional single-nucleotide polymorphism (rs13239597) within TNPO3 promoter region, residing in a putative enhancer element and validated that IRF5 is the distal target gene (∼118 kb) of rs13239597, which is a key regulator involved in pathogenic autoantibody dysregulation, increasing risk of both SLE and SSc. We experimentally validated the long-range chromatin interactions between rs13239597 and IRF5 using chromosome conformation capture assay. We further demonstrated that rs13239597-A acted as an allele-specific enhancer regulating IRF5 expression, independently of TNPO3 by using dual-luciferase reporter assays and CRISPR-Cas9. Particularly, the transcription factor EVI1 could preferentially bind to rs13239597-A allele and increase the enhancer activity to regulate IRF5 expression. Taken together, our results uncovered a mechanistic insight of a noncoding functional variant acting as an allele-specific distal enhancer to directly modulate IRF5 expression, which might obligate in understanding of complex genetic architectures of SLE and SSc pathogenesis.

BabaoDan cures hepatic encephalopathy by decreasing ammonia levels and alleviating inflammation in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: BabaoDan (BBD) is a famous traditional Chinese formula frequently used in TCM clinics to eliminate jaundice and treat infectious viral hepatitis. This paper assesses BBD's preventive and therapeutic effects on hepatic encephalopathy after liver cirrhosis (CHE) and acute liver failure (AHE) in rats and explains its possible mechanism of action. METHODS: CHE rat model was established by injection of carbon tetrachloride (CCl4) twice a week for a total of 9 weeks and then by injection of thioacetamide (TAA) to induce hepatic encephalopathy. AHE rat model was established by injection of TAA once a day for a total of 3 days. In CHE rat model, BBD was gavaged once a day at the end of the 6th week until the experiment ended. In AHE rat model,BBD was gavaged once a day 3 days before TAA injection until the experiment ended. The preventive and therapeutic effects of BBD on brain dysfunction, as well as liver injury, pathology and fibrosis were evaluated in vivo. The role of BBD in the regulation of inflammatory factors and myeloid differentiation factor 88/Toll-like receptor 4/nuclear factor kappa-B (TLR4/MyD88/NK-κ B) pathway was detected in both liver and brain in vivo. The rat bone marrow derived macrophages (BMDMs) were activated by Lipopolysaccharide (LPS), and the role of BBD in the regulation of inflammatory factors and NK-κ B pathway were detected in vitro. RESULTS: In CHE rat model: BBD significantly improved the total distance as well as the activity rate of rats. BBD also improved the learning and memory abilities of rats compared with the control group. In addition, BBD effectively decreased ammonia levels and significantly decreased the levels of alanine aminotransferase (ALT), aspartate transaminase (AST), total bilirubin (TBil) and total bile acid (TBA), as well as improved the levels of total protein (TP) and albumin (Alb). In the liver, BBD not only inhibited the gene expressions of tumor necrosis factor alpha (TNF-α), interleukini-6 (IL-6), TLR4, MyD88, and NF-κ B but also inhibited the protein expressions of TLR4, MyD88, NK-κ B and TNF-α. In the brain, BBD inhibited the gene expressions of iNOS, IL-6, TNF-α, TLR-4, MyD88, and NF-κ B, as well as inhibited the protein expressions of TLR4, MyD88, P65 TNF-α and ionized calcium binding adapter molecule 1 (Iba-1). BBD also decreased NO and TNF-α in the blood. IN AHE RAT MODEL: BBD improved neurological scores, blood ammonia levels and the brain inflammatory gene expressions of iNOS, TNF-α and IL-1ß. BBD also improved liver function biomarkers such as ALT, TBil, TBA, TP, ALB and inflammatory and apoptotic gene expressions of TNF-α, IL-1ß, IL-6, Bax, Bcl-2, caspase-9, caspase-3 and NF-κ B. In LPS-activated rat BMDMs, BBD decreased NO and TNF-α production in BMDM culture supernatant. In addition, BBD inhibited the gene expressions of TNF-α, IL-1 ß and IL-6 as well as the phosphorylation of P65. CONCLUSION: BBD can prevent and cure hepatic encephalopathy (HE) derived from both chronic and acute liver diseases. BBD can reduce hyperammonemia as well as the systematic and neurological inflammation. Inflammation is likely an important target of BBD to treat HE. The anti-inflammatory role of BBD may lie in its regulation of the TLR4/MyD88/NF-κ B pathways.

Concomitant adenosquamous carcinoma and cystadenocarcinoma of the extrahepatic bile duct: A case report.

BACKGROUND: Infiltrative adenosquamous carcinoma (ASC) of the extrahepatic bile duct is reported infrequently, which is an unusual variant of the ordinary adenocarcinoma. The simultaneous development of ASC and cystadenocarcinoma in the extrahepatic biliary tree is rare. In addition, the accurate preoperative diagnosis of concomitant carcinoma in the multiple biliary trees at an early stage is often difficult. Thus, awareness of the risk of the multiplicity of biliary tumors is perhaps the most important factor in identifying these cases. CASE SUMMARY: Here, we report a case of a 63-year-old female with jaundice, who was referred to Shuguang Hospital because of abdominal pain for 1 mo. An abdominal contrast-enhanced computed tomography revealed a type I choledochal cyst and intraluminal masses suggestive of adenoma of the common bile duct. In addition, a preoperative diagnosis of a concomitant Klatskin tumor and type I choledochal cyst was made. The patient underwent anti-inflammatory therapy, followed by radical surgery due to hilar cholangiocarcinoma and resection of the choledochal cyst. Examination of the surgical specimen revealed a papillary tumor of the common bile duct, which arose from the malignant transformation of a pre-existing cystadenoma. Histologic examination confirmed a special type of cholangiocarcinoma; the tumor in the hilar bile duct was an ASC, whereas the tumor in the common bile duct was a moderately differentiated cystadenocarcinoma. The patient showed rapid deterioration 8 mo after surgery. CONCLUSION: Although concomitant ASC and cystadenocarcinoma of the extrahepatic bile duct is difficult to diagnose before surgery, and the prognosis is poor after surgery, surgical resection is still the preferred treatment.

An Osteoporosis Risk SNP at 1p36.12 Acts as an Allele-Specific Enhancer to Modulate LINC00339 Expression via Long-Range Loop Formation.

Genome-wide association studies (GWASs) have reproducibly associated variants within intergenic regions of 1p36.12 locus with osteoporosis, but the functional roles underlying these noncoding variants are unknown. Through an integrative functional genomic and epigenomic analyses, we prioritized rs6426749 as a potential causal SNP for osteoporosis at 1p36.12. Dual-luciferase assay and CRISPR/Cas9 experiments demonstrate that rs6426749 acts as a distal allele-specific enhancer regulating expression of a lncRNA (LINC00339) (∼360 kb) via long-range chromatin loop formation and that this loop is mediated by CTCF occupied near rs6426749 and LINC00339 promoter region. Specifically, rs6426749-G allele can bind transcription factor TFAP2A, which efficiently elevates the enhancer activity and increases LINC00339 expression. Downregulation of LINC00339 significantly increases the expression of CDC42 in osteoblast cells, which is a pivotal regulator involved in bone metabolism. Our study provides mechanistic insight into how a noncoding SNP affects osteoporosis by long-range interaction, a finding that could indicate promising therapeutic targets for osteoporosis.

Multiple Functional Variants at 13q14 Risk Locus for Osteoporosis Regulate RANKL Expression Through Long-Range Super-Enhancer.

RANKL is a key regulator involved in bone metabolism, and a drug target for osteoporosis. The clinical diagnosis and assessment of osteoporosis are mainly based on bone mineral density (BMD). Previous powerful genomewide association studies (GWASs) have identified multiple intergenic single-nucleotide polymorphisms (SNPs) located over 100 kb upstream of RANKL and 65 kb downstream of AKAP11 at 13q14.11 for osteoporosis. Whether these SNPs exert their roles on osteoporosis through RANKL is unknown. In this study, we conducted integrative analyses combining expression quantitative trait locus (eQTL), genomic chromatin interaction (high-throughput chromosome conformation capture [Hi-C]), epigenetic annotation, and a series of functional assays. The eQTL analysis identified six potential functional SNPs (rs9533090, rs9594738, r8001611, rs9533094, rs9533095, and rs9594759) exclusively correlated with RANKL gene expression (p < 0.001) at 13q14.11. Co-localization analyses suggested that eQTL signal for RANKL and BMD-GWAS signal shared the same causal variants. Hi-C analysis and functional annotation further validated that the first five osteoporosis SNPs are located in a super-enhancer region to regulate the expression of RANKL via long-range chromosomal interaction. Particularly, dual-luciferase assay showed that the region harboring rs9533090 in the super-enhancer has the strongest enhancer activity, and rs9533090 is an allele-specific regulatory SNP. Furthermore, deletion of the region harboring rs9533090 using CRISPR/Cas9 genome editing significantly reduced RANKL expression in both mRNA level and protein level. Finally, we found that the rs9533090-C robustly recruits transcription factor NFIC, which efficiently elevates the enhancer activity and increases the RANKL expression. In summary, we provided a feasible method to identify regulatory noncoding SNPs to distally regulate their target gene underlying the pathogenesis of osteoporosis by using bioinformatics data analyses and experimental validation. Our findings would be a potential and promising therapeutic target for precision medicine in osteoporosis. © 2018 American Society for Bone and Mineral Research.

Genetics association study and functional analysis on osteoporosis susceptibility gene BDNF.

To explore the relationship between brain-derived neurotrophic factor (BDNF) gene and bone mineral density (BMD) in Chinese Han population, we performed association analysis of 14 tag SNPs on BDNF gene with hip/spine BMD in 1300 Han Chinese samples from Shaanxi Province. We found that 8 of the 14 SNPs were significantly associated with hip or spine BMD (P < 0.05). Moreover, the SNP rs16917237 was significantly associated with both hip and spine BMD, with significant Bonferroni correlation (P value 0.05/14 = 0.0036) in hip BMD. To further explore the regulatory mechanism of BDNF gene in osteoporosis, we further performed a set of data analyses, including linkage disequilibrium and haplotype analysis, epigenetic annotation, expression quantitative trait locus (eQTL) analysis and metabolic pathway analysis. Further, we have established a mouse pre-osteoblasts differentiation cell model (MC3T3-E1) by recombination human bone morphogenetic protein (rh-BMP2) induction. siRNA- mediated knock down of BDNF in this cell model showed that all 14 SNPs are in the same haplotype block. Strong signals of active histone H3K4me1, H3K4me3, H3K27ac modifications and P300 binding were observed in osteoblasts, in the region surrounding the most significant SNP rs16917237, suggesting that this SNP might have a regulatory function in osteoblasts. Furthermore, analysis of genotype data of rs16917237 and BDNF expression in multiple tissues from GTEx showed that rs16917237 SNP could significantly affect the expression of BDNF in 11 tissues. Through analysis of the various BDNF pathways, we showed that BDNF participates in the MAPK pathway, which is a vital and well-established pathway affecting osteoblasts proliferation and differentiation. siRNA knock down of BDNF significantly decreased the mRNA and protein levels of CREB, which is important in the MAPK pathway in osteoblast differentiation. These findings suggest that BDNF might affect osteoblast differentiation via regulation of CREB expression. In conclusion, our results from combined genetic association and functional analyses show that BDNF is a vital osteoporosis susceptibility gene, which can affect BMD not only in Chinese Han but also likely in other populations.

[Expression of long isoform leptin receptor and shortest membrane bound variant in peripheral blood mononuclear cells from the obese and normal individuals].

OBJECTIVE: To investigate the expression of the long isoform leptin receptor (OB-R(L)) and the shortest membrane bound variant (OB-R(S)) in peripheral blood mononuclear cells from the obese and normal individuals (6.08 ng/ml or 31.21 ng/ml). METHODS: Mononuclear cells were obtained from 30 obese individuals (BMI > 25 kg/m2) and 20 normal individuals (BMI < 23 kg/m2). RT-PCR was used to detect the expression of OB-R(S) and OB-R(L). The level of serum leptin was measured with immunoradiometric assay. RESULTS: OB-R(S) was expressed in all individuals, and OB-R(L) was expressed in 38 individuals. OB-R(L) was not expressed in the 12 obese individuals with the BMI > 39 kg/m2. The expression level of OB-R(S) was 4 times higher than that of OB-R(L). There was no significant difference in the expression of either isoforms between men and women. The relative expression of both OB-R(S) and OB-R(L) isoforms was significantly lower and the serum leptin level was significantly higher in the obese individuals (6.08 ng/ml or 31.21 ng/ml). The leptin receptor expression levels were significantly negatively correlated with BMI and serum leptin level. CONCLUSION: Both OB-R(S) and OB-R(L) are expressed in the peripheral blood mononuclear cells from normal and the medium obese subjects with a consistent predominance of OB-R(S). There is no significant difference in the expression of OB-R(S) and OB-R(L) between men and women. Compared with normal individuals, the expression of OB-R(S) and OB-R(L) in human mononuclear cells is lower.

Optimising gene therapy of hypoparathyroidism with hematopoietic stem cells.

BACKGROUND: The treatment of hypoparathyroidism (HPT) is still a difficult clinical problem, which necessitates a new therapy. Gene therapy of HPT has been valuable, but how to improve the gene transfer efficiency and expression stability is a problem. This study was designed to optimize the gene therapy of HPT with hematopoietic stem cells (HSCs) recombined with the parathyroid hormone (PTH) gene. METHODS: The human PTH gene was amplified by polymerase chain reaction (PCR) from pcDNA3.1-PTH vectors and inserted into murine stem cell virus (MSCV) vectors with double enzyme digestion (EcoRI and XhoI). The recombinant vectors were transfected into PA317 packaging cell lines by the lipofectin method and screened by G418 selective medium. The condensed recombinant retroviruses were extracted and used to infect HSCs, which were injected into mice suffering from HPT. The change of symptoms and serum levels of PTH and calcium in each group of mice were investigated. RESULTS: The human PTH gene was inserted into MSCV vectors successfully and the titres were up to 2 x 10(7) colony forming unit (CFU)/ml in condensed retroviral solution. The secretion of PTH reached 15 ng.10(-6).cell(-1) per 48 hours. The wild type viruses were not detected via PCR amplification, so they were safe for use. The mice suffering from HPT recovered quickly and the serum levels of calcium and PTH remained normal for about three months after the HSCs recombined with PTH were injected into them. The therapeutic effect of this method was better than simple recombinant retroviruses injection. CONCLUSIONS: The recombinant retroviral vectors MSCV-PTH and the high-titre condensed retroviral solution recombined with the PTH gene are obtained. The recombinant retroviral solution could infect HSCs at a high rate of efficiency. The infected HSCs could cure HPT in mice. This method has provided theoretical evidence for the clinical gene therapy of HPT.

[The effects of mutant site G81R of RAB5A on RAB5A function].

To study the effects of mutant site G81R of RAB5A, two antisense RNA of RAB5A G81R and RAB5A were inserted into pcDNA3. 1/V5-His TOPO expression vector, and transfected into Anip973, respectively, then detected the protein expressional level of RAB5A by Western blot. Finally, we synchronized Anip973 and the two transfected cells at G0/G1, released cells at G0/G1 by culture medium containing FBS, and sequentially analyzed the percentage of cells at G0/G1, S and G2/M by FCM. The expression of RAB5A may be completely blocked by the antisense RNA of RAB5A G81R, while the antisense RNA of RAB5A partially blocked expression of RAB5A. Furthermore, cell cycle of Anip973 was reciprocity to the protein expressional level of RAB5A. The antisense RNA of RAB5A G81R effectively blocked the expression of RAB5A in Anip973. Cell cycle was lengthened by blocking or reducing expression of RAB5A G81R.

[Sequence analysis and expression study of BRI gene in two lung adenocarcinoma cell lines with different metastatic potential].

OBJECTIVE: To investigate the sequence of amyloid fibrils (BRI) gene and its expression in two lung adenocarcinoma cell lines AGZY83-a and Anip973 with the same tumor origin but different metastatic potential. METHODS: DNA sequencing, sequential G banding fluorescence in situ hybridization (FISH) and Northern blot were used to analyze the sequence and expression of BRI gene was in two lung adenocarcinoma cell lines with different metastatic potential. RESULTS: The expression of BRI gene was up-regulated in the highly metastatic cell line Anip973 and was down-regulated in the low metastatic cell line AGZY83-a from which the Anip973 was derived. FISH results disclosed that in the two cell lines, the same rearrangements existed in the chromosome region where BRI gene was located, but in Anip973, amplification took place in the chromosome region where BRI gene was located. DNA sequencing results showed different mutations in the 5' untranslated region of BRI gene in the two cell lines. CONCLUSION: The above results revealed that there was no relation between BRI gene differential expression and rearrangements of chromosome. The amplification of the chromosome region where BRI gene was located and the different mutations in the 5' untranslated region of BRI gene probably contributed to the differential expression.

[Mapping and expression analysis of a different expression cDNA fragment from lung adenocarcinoma cell line].

Lung cancer is one of the most common malignant tumors in humans. Metastasis is the basic biological feature of malignant tumors, which is the main cause of death. Molecular mechanism of metastasis is still unclear, although lots of studies have been done in tumor metastasis. To study and explore the molecular basis of metastasis in lung cancer, and isolate tumor metastasis-related genes, two human lung adenocarcinoma cell lines AGZY 83-a and Anip 973 were chosen as research materials. The Anip973 was derived from AGZY83-a, but manifested much higher metastasis potential than the parent line. Using mRNA differential display technique, an unknown cDNA fragment, OPB7-1, which is over-expressive in Anip973 cell line, was obtained. It was used as a template to isolate its corresponding cDNA through dbEST searching and PCR. To search and clone lung adenocarcinoma metastasis-related candidate gene, and to explore the molecular basis of development of lung carcinoma, differential expression of OPB7-1 cDNA fragment among 9 human lung adenocarcinoma cell lines and 12 normal human tissues were detected using cell culture, cDNA clone, Northern blot analysis and bioinformation technology. Results showed that there were significant differences in OPB7-1 expression among 9 human lung adenocarcinoma cell lines. High expression tendency was observed in Anip973 cell line with high metastasis potential, TKB-18 cell line with high invasion potential and GLC-82 cell line with low differentiation potential. Besides, a bigger fragment can be found in Anip973 cell line on the Northern blot hybridization. The 3.0 kb transcriptions were found in various tissues. Over-expression in heart and skeletal muscle could be observed, whereas expression in spleen, liver, kidney, placental and lung could be found except colon, thyroid gland and small intestine. These manifests indicate that OPB7-1 gene has a wide-rage expression in human multiple tissues. A 1.0 kb cDNA fragment was acquired by linking up EST fragments homologous match 5' end and PCR. BLAST analysis revealed that OPB7-1 gene has extremely low sequence identity with any known genes from GenBank and any sequences from EST database. The chromosomal localization of it was determined by RH location method. The OPB7-1 fragment was localized to chromosome 1p31-34. That OPB7-1 gene has an extensive expression pattern, may be a novel tumor gene related to lung carcinoma. Further research needs to be done to obtain the full-length cDNA of OPB7-1 gene. It will be helpful to investigate the expression in lung cancer cases and other tumor tissues for further determining the function of OPB7-1 gene in development of tumor.