Effects of vegetation and soil changes on microbial biomass carbon and nitrogen in the Napahai meadow under N deposition.

To explore the responses of soil microorganisms to short-term nitrogen deposition in alpine meadow, we set three treatments of low nitrogen (5 g N·m-2·a-1), medium nitrogen (10 g N·m-2·a-1), and high nitrogen (15 g N·m-2·a-1) addition to investigate the effects of nitrogen-deposition induced alterations in plant diversity and soil physicochemical properties on microbial biomass carbon (MBC) and nitrogen (MBN) in a typical alpine meadow community of Carex nubigena in Napahai. The results showed that nitrogen addition significantly increased soil MBC, MBN, and their quotients, with the increases of MBC being as high as 139.3% under medium nitrogen treatment. Both MBC and MBN showed significant decreases along the soil layer, with a reduction of 24.1% to 75.1%. Nitrogen addition significantly increased aboveground biomass and reduced Shannon and Simpson indices by 6.6%-65.4%. Nitrogen addition significantly decreased soil pH, increased the contents of organic matter, total nitrogen, ammonium nitrogen and nitrate nitrogen, with the highest reduction (7.0%-511.1%) being observed in medium nitrogen treatment. Soil pH increased while other physical and chemical indicators significantly decreased with the increases of soil layer, with a variation range of 19.5%-91.2%. Results of structural equation model showed that microbial biomass was significantly positively correlated with ammonium nitrogen, nitrate nitrogen and organic matter, but negatively correlated with pH and Shannon index. The interaction of plant and soil physicochemical properties explained 55%-77% of the variations in MBC, MBN and their quotient. Soil physicochemical properties had the highest effect value (0.56-0.95) on MBC, MBN and their quotients, followed by plant diversity and aboveground biomass. Therefore, nitrogen deposition increased soil MBC and MBN and their quotient, primarily through improving soil nutrient availability and plant aboveground biomass, whereas MBC and MBN and their quotient were suppressed by high-level nitrogen deposition due to soil acidification and plant diversity losses.

S100 calcium binding protein A6 and associated long noncoding ribonucleic acids as biomarkers in the diagnosis and staging of primary biliary cholangitis.

BACKGROUND: Primary biliary cholangitis (PBC) is a chronic and slowly progressing cholestatic disease, which causes damage to the small intrahepatic bile duct by immuno-regulation, and may lead to cholestasis, liver fibrosis, cirrhosis and, eventually, liver failure. AIM: To explore the potential diagnosis and staging value of plasma S100 calcium binding protein A6 (S100A6) messenger ribonucleic acid (mRNA), LINC00312, LINC00472, and LINC01257 in primary biliary cholangitis. METHODS: A total of 145 PBC patients and 110 healthy controls (HCs) were enrolled. Among them, 80 PBC patients and 60 HCs were used as the training set, and 65 PBC patients and 50 HCs were used as the validation set. The relative expression levels of plasma S100A6 mRNA, long noncoding ribonucleic acids LINC00312, LINC00472 and LINC01257 were analyzed using quantitative reverse transcription-polymerase chain reaction. The bile duct ligation (BDL) mouse model was used to simulate PBC. Then double immunofluorescence was conducted to verify the overexpression of S100A6 protein in intrahepatic bile duct cells of BDL mice. Human intrahepatic biliary epithelial cells were treated with glycochenodeoxycholate to simulate the cholestatic environment of intrahepatic biliary epithelial cells in PBC. RESULTS: The expression of S100A6 protein in intrahepatic bile duct cells was up-regulated in the BDL mouse model compared with sham mice. The relative expression levels of plasma S100A6 mRNA, log10 LINC00472 and LINC01257 were up-regulated while LINC00312 was down-regulated in plasma of PBC patients compared with HCs (3.01 ± 1.04 vs 2.09 ± 0.87, P < 0.0001; 2.46 ± 1.03 vs 1.77 ± 0.84, P < 0.0001; 3.49 ± 1.64 vs 2.37 ± 0.96, P < 0.0001; 1.70 ± 0.33 vs 2.07 ± 0.53, P < 0.0001, respectively). The relative expression levels of S100A6 mRNA, LINC00472 and LINC01257 were up-regulated and LINC00312 was down-regulated in human intrahepatic biliary epithelial cells treated with glycochenodeoxycholate compared with control (2.97 ± 0.43 vs 1.09 ± 0.08, P = 0.0018; 2.70 ± 0.26 vs 1.10 ± 0.10, P = 0.0006; 2.23 ± 0.21 vs 1.10 ± 0.10, P = 0.0011; 1.20 ± 0.04 vs 3.03 ± 0.15, P < 0.0001, respectively). The mean expression of S100A6 in the advanced stage (III and IV) of PBC was up-regulated compared to that in HCs and the early stage (II) (3.38 ± 0.71 vs 2.09 ± 0.87, P < 0.0001; 3.38 ± 0.71 vs 2.57 ± 1.21, P = 0.0003, respectively); and in the early stage (II), it was higher than that in HCs (2.57 ± 1.21 vs 2.09 ± 0.87, P = 0.03). The mean expression of LINC00312 in the advanced stage was lower than that in the early stage and HCs (1.39 ± 0.29 vs 1.56 ± 0.33, P = 0.01; 1.39 ± 0.29 vs 2.07 ± 0.53, P < 0.0001, respectively); in addition, the mean expression of LINC00312 in the early stage was lower than that in HCs (1.56 ± 0.33 vs 2.07 ± 0.53, P < 0.0001). The mean expression of log10 LINC00472 in the advanced stage was higher than those in the early stage and HCs (2.99 ± 0.87 vs 1.81 ± 0.83, P < 0.0001; 2.99 ± 0.87 vs 1.77 ± 0.84, P < 0.0001, respectively). The mean expression of LINC01257 in both the early stage and advanced stage were up-regulated compared with HCs (3.88 ± 1.55 vs 2.37 ± 0.96, P < 0.0001; 3.57 ± 1.79 vs 2.37 ± 0.96, P < 0.0001, respectively). The areas under the curves (AUC) for S100A6, LINC00312, log10 LINC00472 and LINC01257 in PBC diagnosis were 0.759, 0.7292, 0.6942 and 0.7158, respectively. Furthermore, the AUC for these four genes in PBC staging were 0.666, 0.661, 0.839 and 0.5549, respectively. The expression levels of S100A6 mRNA, log10 LINC00472, and LINC01257 in plasma of PBC patients were decreased (2.35 ± 1.02 vs 3.06 ± 1.04, P = 0.0018; 1.99 ± 0.83 vs 2.33 ± 0.96, P = 0.036; 2.84 ± 0.92 vs 3.69 ± 1.54, P = 0.0006), and the expression level of LINC00312 was increased (1.95 ± 0.35 vs 1.73 ± 0.32, P = 0.0007) after treatment compared with before treatment using the paired t-test. Relative expression of S100A6 mRNA was positively correlated with log10 LINC00472 (r = 0.683, P < 0.0001); serum level of collagen type IV was positively correlated with the relative expression of log10 LINC00472 (r = 0.482, P < 0.0001); relative expression of S100A6 mRNA was positively correlated with the serum level of collagen type IV (r = 0.732, P < 0.0001). The AUC for the four biomarkers obtained in the validation set were close to the training set. CONCLUSION: These four genes may potentially act as novel biomarkers for the diagnosis of PBC. Moreover, LINC00472 acts as a potential biomarker for staging in PBC.

Exploring prognostic potential of long noncoding RNAs in colorectal cancer based on a competing endogenous RNA network.

BACKGROUND: Colorectal cancer (CRC) is one of the most prevalent tumors worldwide. Recently, long noncoding RNAs (lncRNAs) have been shown to influence tumorigenesis and tumor progression by acting as competing endogenous RNAs (ceRNAs). It is difficult to extract prognostic lncRNAs and useful bioinformation from most ceRNA networks constructed previously. AIM: To construct a prognostic related ceRNA regulatory network and lncRNA related signature based on risk score in CRC. METHODS: RNA transcriptome profile and clinical information of 506 CRC patients were downloaded from the Cancer Genome Atlas database. R packages and Perl program were used for data processing. Cox regression analysis was used for prognostic model construction. Quantitative real-time polymerase chain reaction was used to detect the expression of lncRNAs. RESULTS: A prognostic-related ceRNA network was constructed, including 9 lncRNAs, 44 mRNAs, and 30 miRNAs. In addition, a four-lncRNA model was constructed using multivariate Cox regression analysis, which could be an independent prognostic model in CRC. The risk score for each patient was calculated, and the 506 patients were divided into high and low-risk groups (253 for each group) based on the median risk score. The results of the survival analysis showed that patients with a high-risk score had a poor survival rate. Furthermore, the predictive value of the four-lncRNA model was evaluated in GSE38832. Patient survival probabilities could be better predicted when combing the risk score and clinical features. Gene Set Enrichment Analysis results verified that a number of cancer-related signaling pathways were enriched with a high-risk score in CRC. Finally, we validated a novel lncRNA (LINC00488) using quantitative real-time polymerase chain reaction in 22 paired CRC patient tumor tissues compared to adjacent non-tumor tissues. CONCLUSION: The four-lncRNA model could give better predictive value for CRC patients. Our understanding of the lncRNA-related ceRNA regulatory mechanism could provide a potential diagnostic indicator for CRC patients.

Let-7f-5p regulates TGFBR1 in glucocorticoid-inhibited osteoblast differentiation and ameliorates glucocorticoid-induced bone loss.

Previous studies indicated that let-7 enhances osteogenesis and bone formation of human adipose-derived mesenchymal stem cells (MSCs). We also have confirmed that let-7f-5p expression was upregulated during osteoblast differentiation in rat bone marrow-derived MSCs (BMSCs) and was downregulated in the vertebrae of patients with glucocorticoid (GC)-induced osteoporosis (GIOP). The study was performed to determine the role of let-7f-5p in GC-inhibited osteogenic differentiation of murine BMSCs in vitro and in GIOP in vivo. Here, we report that dexamethasone (Dex) inhibited osteogenic differentiation of BMSCs and let-7f-5p expression, while increasing the expression of transforming growth factor beta receptor 1 (TGFBR1), a direct target of let-7f-5p during osteoblast differentiation under Dex conditions. In addition, let-7f-5p promoted osteogenic differentiation of BMSCs, as indicated by the promotion of alkaline phosphatase (ALP) staining and activity, Von Kossa staining, and osteogenic marker expression (Runx2,Osx, Alp, and Ocn), but decreased TGFBR1 expression in the presence of Dex. However, overexpression of TGFBR1 reversed the upregulation of let-7f-5p during Dex-treated osteoblast differentiation. Knockdown of TGFBR1 reversed the effect of let-7f-5p downregulation during Dex-treated osteogenic differentiation of BMSCs. We also found that glucocorticoid receptor (GR) mediated transcriptional silencing of let-7f-5p and its knockdown enhanced Dex-inhibited osteogenic differentiation. Further, when injected in vivo, agomiR-let-7f-5p significantly reversed bone loss induced by Dex, as well as increased osteogenic marker expression (Runx2, Osx, Alp, and Ocn) and decreased TGFBR1 expression in bone extracts. These findings indicated that the regulatory axis of GR/let-7f-5p/TGFBR1 may be important for Dex-inhibited osteoblast differentiation and that let-7f-5p may be a useful therapeutic target for GIOP.

IGF-1R/ß-catenin signaling axis is involved in type 2 diabetic osteoporosis.

Insulin-like growth factor-1 receptor (IGF-1R) is involved in both glucose and bone metabolism. IGF-1R signaling regulates the canonical Wnt/ß-catenin signaling pathway. In this study, we investigated whether the IGF-1R/ ß-catenin signaling axis plays a role in the pathogenesis of diabetic osteoporosis (DOP). Serum from patients with or without DOP was collected to measure the IGF-1R level using enzyme-linked immunosorbent assay (ELISA). Rats were given streptozotocin following a four-week high-fat diet induction (DOP group), or received vehicle after the same period of a normal diet (control group). Dual energy X-ray absorption, a biomechanics test, and hematoxylin-eosin (HE) staining were performed to evaluate bone mass, bone strength, and histomorphology, respectively, in vertebrae. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were performed to measure the total and phosphorylation levels of IGF-1R, glycogen synthase kinase-3ß (GSK-3ß), and ß-catenin. The serum IGF-1R level was much higher in patients with DOP than in controls. DOP rats exhibited strikingly reduced bone mass and attenuated compression strength of the vertebrae compared with the control group. HE staining showed that the histomorphology of DOP vertebrae was seriously impaired, which manifested as decreased and thinned trabeculae and increased lipid droplets within trabeculae. PCR analysis demonstrated that IGF-1R mRNA expression was significantly up-regulated, and western blotting detection showed that phosphorylation levels of IGF-1R, GSK-3ß, and ß-catenin were enhanced in DOP rat vertebrae. Our results suggest that the IGF-1R/ß-catenin signaling axis plays a role in the pathogenesis of DOP. This may contribute to development of the underlying therapeutic target for DOP.

Plastrum Testudinis Extracts Promote BMSC Proliferation and Osteogenic Differentiation by Regulating Let-7f-5p and the TNFR2/PI3K/AKT Signaling Pathway.

BACKGROUND/AIMS: Plastrum testudinis extracts (PTE) show osteoprotective effects on glucocorticoid-induced osteoporosis in vivo and in vitro. However, the underlying molecular mechanism of PTE in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. METHODS: BMSC proliferation was investigated using the Cell Counting Kit-8 assay. BMSC differentiation and osteogenic mineralization were assayed using alkaline phosphatase and Alizarin red staining, respectively. The mRNA expression levels of Let-7f-5p, Tnfr2, Traf2, Pi3k, Akt, ß-catenin, Gsk3ß, Runx2, and Ocn were measured using real time quantitative polymerase chain reaction. Protein levels of TNFR2, TRAF2, p-PI3K, p-AKT, p-ß-CATENIN, and p-GSK3ß were analyzed by western blotting. The functional relationship of Let-7f-5p and Tnfr2 was determined by luciferase reporter assays. RESULTS: The optimum concentration for PTE was 30 µg/ml. PTE significantly promoted BMSC osteogenic differentiation and mineralization after 7 and 14 days in culture, respectively. The combination of PTE and osteogenic induction exhibited significant synergy. PTE upregulated Let-7f-5p, ß-catenin, Runx2, and Ocn mRNA expression, and downregulated Tnfr2, Traf2, Pi3k, Akt, and Gsk3ß mRNA expression. PTE inhibited TNFR2, TRAF2, and p-ß-CATENIN protein expression, and promoted p-PI3K, p-AKT, and p-GSK3ß protein expression. In addition, Tnfr2 was a functional target of Let-7f-5p in 293T cells. CONCLUSIONS: Our results suggested that PTE may promote BMSC proliferation and osteogenic differentiation via a mechanism associated with the regulation of Let-7f-5p and the TNFR2/PI3K/AKT signaling pathway.

Effect of osteoporosis induced by ovariectomy on vertebral bone defect/fracture in rat.

Osteoporotic vertebral fracture (OVF) is a worldwide health concern and lacks sufficient basic studies. Suitable animal models should be the foundation for basic study and treatment of OVF. There have been few studies on the development of animal models of osteoporotic vertebral bone defects. OVF models using various animal species should be developed to evaluate the therapeutic strategy in preclinical testing. We developed an OVF model in rats. Rat osteoporosis was induced by ovariectomy (OVX), and 3 months after OVX, a 3 mm diameter hemispheric vertebral bone defect was developed in lumbar vertebra 6 (L6). Sagittal plain X-rays of the rats, their bone quantity, bone microarchitecture, and histomorphology were analyzed: 3 months after OVX, rats showed significantly lower bone quantity, relative bone volume, and total volume bone mineral density. After the vertebral bone defect had developed for 16 weeks, no significant indication of self-healing could be observed from the sagittal plain X-rays, three-dimensional images, and histomorphology. These results indicate that the rat model of osteoporotic vertebral bone defect, induced by OVX and a 3 mm diameter hemispheric vertebral bone defect, can sufficiently mimic OVF patients in clinic and provide a sound basis for subsequent studies.

[Risk factors for multiple level osteoporosis vertebral compression fractures].

OBJECTIVE: To identify risk factors associated with patients suffered multiple level osteoporosis vertebral compression fractures(OVCFs). METHODS: From March 2011 to March 2015, 199 patients suffered osteoporotic were classified into multiple level OVCFs group and single level OVCF group. Multivariate logistic regression analysis were performed to identify risks factors associated with multiple level OVCFs. RESULTS: All the patients underwent OVCF, including 71 multiple level OVCFs and 128 single level OVCF. There were no differences in the age, gender, BMI, hypertension and diabetes between two groups. While multiple level OVCFs were associated with spinal deformity index SDI[(2<=SDI<4, OR=2.587, 95% CI(1.148, 5.828);SDI>=-4, OR=7.775, 95% CI(3.272, 18.478)], BMD[(T<-4.5SD, OR=2.608, 95% CI(1.038, 6.551)]. CONCLUSIONS: SDI and BMD might be the risk factors for multiple level OVCFs.

[Analysis of clinical characteristics of 20 patients with pyogenic spondylitis].

OBJECTIVE: To investigate the diagnostic value of clinical manifestation, laboratory examination and imaging changes for pyogenic spondylitis and to summarize the clinical characteristics of patients with pyogenic spondylitis. METHODS: The clinical data, of 20 patients with pyogenic spondylitis were diagnosed by histopathological examination from March 2012 to March 2015, were retrospectively analyzed. There were 9 males and 11 females, aged from 43 to 72 years old with an average of 58.9 years. Included 3 cases of cervical vertebrae, 7 cases of thoracic vertebrae, 10 cases of lumbar vertebrae. Patients of blood analysis, erythrocyte sedimentation rate(ESR), C reactive protein(CRP), X rays, CT and MRI were performed before treatment. Visual analogue scale (VAS) was used to evaluate the pain of patients suffering from vertebral pain. RESULTS: All the patients had suffered from vertebral pain before treatment. VAS was 9 points in 4 cases, 8 points in 6 cases, 7 points in 1 case, 3 points in 6 cases, and 2 points in 3 cases. Among them, 7 patients complicated with neurological symptoms, 11 with aggravating night pain, 10 with fever. WBC and Neutrophil count (NEU) of 5 cases were increased and other 15 cases were normal;CRP of 19 cases were increased and 1 case was normal;ESR of all 20 cases were increased. X rays showed the intervertebral space narrowing in all 20 cases, 13 cases complicated with destruction of vertebral body; CT showed the lesions of vertebral body in the 20 cases and complicated with destruction, sclerosis of sclerotin; MRI showed that the lesions of the vertebral body in the T1 image had uneven medium low signal, in the T2 image of the 16 cases had uneven high signal and 2 cases had uniform and high signal, 2 cases had main high signal compliated with mixed signal. Thirteen patients underwent surgical treatment and 7 patients received conservative treatment, and the patients left hospital while VAS had significantly improved after treatment. CONCLUSIONS: Pyogenic spondylitis is easy to be misdiagnosed or missed in clinic. It can be combined with the clinical manifestations, laboratory examination and imaging characteristics in order to make a definite diagnosis for purulent spondylitis in early.

Diagnostic value of enzyme-linked immunospot assay using CFP10/ESAT6 fusion protein as antigen in spinal tuberculosis.

OBJECTIVE: To establish a method of detecting spinal tuberculosis (TB) infection by enzyme-linked immunospot (ELlSPOT) assay and evaluate the value of CFP10/ESAT6 fusion protein for diagnosis of spinal TB. METHODS: Suspected spinal TB patients were prospectively recruited in two hospitals (First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine; Nanfang Hospital, Southern Medical University) from May 2012 to December 2013. Data on clinical characteristics of the patients and conventional laboratory results were collected. Compare and analyze the positive detection rate in spinal TB diagnosis by different methods including ELISPOT detection and conventional detection methods. RESULTS: 47 patients with spinal TB had available biopsy or surgical specimens for histopathological examination and 41 specimens had pathological features consistent with a diagnosis of TB infection. Among the spinal TB patients and non-TB disease patients,the overall sensitivity, specificity, positive predictive value, and negative predictive value of the ELISPOT assay in spinal TB diagnosis were 82.7%,87.2%,89.6%, and 79.1%,respectively; the 4 indexes of the PPD skin test were 61.5%, 46.2%, 60.4%, and 47.4%, respectively;those of the antibody detection were 55.8%, 61.5%, 65.9%, and 51.1%. The positive rate of ELISPOT was significantly higher than those of PPD skin test and antibody detection test (82.7% vs. 61.5%, Χ² =5.786, P=0.016; 82.7% vs. 55.8%, Χ² =8.847, P=0.003), but not significantly different from the positive rate of pathological examination (82.7% vs. 87.2%, Χ² =0.396, P=0.529). Moderate agreement was found between pathological examination and the ELISPOT assay (87.2%, Κ=0.498, P=0.001). CONCLUSION: With high sensitivity and specificity, the ELISPOT assay using CFP10/ESAT6 fusion protein as antigen is an effective technique for auxiliary diagnosis of spinal TB.

Biomechanical effects of cement distribution in the fractured area on osteoporotic vertebral compression fractures: a three-dimensional finite element analysis.

BACKGROUND: According to some clinical studies, insufficient cement distribution (ID) in the fractured area and asymmetrical cement distribution around the fractured area were thought to be the reasons for unrelieved pain and recollapse after percutaneous vertebral augmentation (PVA) in the treatment of symptomatic osteoporotic vertebral compression fractures. METHODS: Finite element methods were used to investigate the biomechanical variance among three patterns of cement distribution (ID and sufficient cement distribution in the fractured area and asymmetrical cement distribution around the fractured area including upward [BU] and downward [BD] cement distribution). RESULTS: Compared with fractured vertebra before PVA, distribution of von Mises stress in the cancellous bone was transferred to be concentrated at the cancellous bone surrounding cement after PVA, whereas it was not changed in the cortical bone. Compared with sufficient cement distribution group, maximum von Mises stress in the cancellous bone and cortical bone and maximum displacement of augmented vertebra increased significantly in the ID group, whereas asymmetrical cement distribution around the fractured area in BU and BD groups mainly increased maximum von Mises stress in the cancellous bone significantly. Similar results could be seen in all loading conditions. CONCLUSIONS: ID in the fractured area may lead to unrelieved pain after PVA in the treatment of symptomatic osteoporotic vertebral compression fractures as maximum displacement of augmented vertebral body increased significantly. Both ID in the fractured area and asymmetrical cement distribution around the fractured area are more likely to induce recollapse of augmented vertebra because they increased maximum von Mises stress in the cancellous bone and cortical bone of augmented vertebra significantly.

Fatal multi-organ Clonorchis sinensis infection in dog: a case report.

We describe here a fatal case of a dog with extensive Clonorchis sinensis (C. sinensis) infection. C. sinensis were detected in organs including the abdomen, bladder and heart. The infection was very heavy with a total number of 155,183 worms. These worms were in different developmental stages, but the majority of them were adult.