17 beta-estradiol affects proliferation and apoptosis of canine bone marrow mesenchymal stem cells in vitro.

Emerging researches in humans, pigs and mice, highlighted that estrogen plays a pivotal role in self-renewal and differentiation of bone marrow mesenchymal stem cells (BMSCs). The present study aimed at evaluating effects of 17 beta-estradiol (E2) on proliferation and apop-tosis of canine-derived bone marrow mesenchymal stem cells (cBMSCs) in vitro. The results showed that E2 supplementation at the concentration of 10-11 M promoted the proliferation of cBMSCs by CCK-8 assay and RT-qPCR analysis for the proliferation-related genes, with proliferating cell nuclear antigen (PCNA), cyclin-D1 (CCND1) being up-regulated and cyclin--dependent kinase inhibitor 1B (CDKN1B) being down-regulated. Contrarily, analysis of fluores-cence-activated cell sorting (FACS) and RT-qPCR demonstrated that E2 supplementation above 10-11 M had inhibitory effects on the proliferation of cBMSCs and induced apoptosis. Intriguingly,cBMSCs still possessed the capability to differentiate into osteoblasts and adipocytes with 10-11 M E2 addition. Taken together, this study determined the optimal culture condition of cBMSCs in vitro, and has important implications for further understanding the regulatory effect of E2 on the self-renewal of cBMSCs, which are helpful for the clinical application of BMSCs.

Diagnostic potential of urinary monocyte chemoattractant protein-1 for Alzheimer's disease and amnestic mild cognitive impairment.

BACKGROUND AND PURPOSE: The chemokine monocyte chemoattractant protein-1 (MCP-1) is involved in the pathogenesis of Alzheimer's disease (AD). This study aimed to investigate whether urinary MCP-1 can distinguish patients with AD, patients with amnestic mild cognitive impairment (aMCI) and cognitively normal (CN) subjects. METHODS: A total of 754 participants, including 97 patients with AD, 50 patients with aMCI and 84 age- and sex-matched CN controls as well as a cohort of 523 CN subjects of different ages, were enrolled from five hospitals located in different areas of China. Urinary MCP-1 levels were determined using enzyme-linked immunosorbent assays. The correlations between urinary MCP-1 levels and cognition test scores or age were analysed. The optimal diagnostic sensitivity and specificity were determined using receiver operating characteristic curve analysis. RESULTS: In the cohort of CN subjects of different ages, urinary MCP-1 levels increased with ageing and were correlated with age. The urinary MCP-1 levels were higher in females than in males. In the cohort composed of patients with AD, aMCI and age- and sex-matched CN controls, urinary MCP-1 levels were significantly higher in patients with AD and aMCI than in CN controls. There were no differences in urine MCP-1 levels between the AD group and the aMCI group. The urinary MCP-1 levels were correlated with the Mini-Mental State Examination scores and age, and were able to differentiate patients with AD and aMCI from CN subjects. CONCLUSIONS: Urinary MCP-1 is a potential biomarker for the diagnosis of AD and aMCI.

Microvesicles containing microRNA-21 secreted by proximal tubular epithelial cells are involved in renal interstitial fibrosis by activating AKT pathway.

OBJECTIVE: To investigate the role of microvesicles containing microRNA-21 in renal interstitial fibrosis and its possible mechanism. MATERIALS AND METHODS: The renal interstitial fibrosis model was established by unilateral ureteral obstruction, and proximal tubule epithelial cell line (NRK52E) was used for cell model. The phenotype changes of the microvesicles containing microRNA-21 secreted by tubule cells during fibrosis were detected, and possible mechanisms responsible for the process were also analyzed. RESULTS: During the process of renal interstitial fibrosis, microRNA-21 level in the microvesicles secreted by tubule cells was increased. The microRNA-21 released from the damaged renal tubules inhibited the expression of PTEN and activated the AKT-mTOR signaling pathway, thereby exacerbating the renal interstitial fibrosis. CONCLUSIONS: MicroRNA-21 secreted by injured proximal tubule epithelial cells participated in renal interstitial fibrosis by activating the PTEN/AKT signaling pathway.

Naringenin regulates production of matrix metalloproteinases in the knee-joint and primary cultured articular chondrocytes and alleviates pain in rat osteoarthritis model

Inflammation of cartilage is a primary symptom for knee-joint osteoarthritis. Matrix metalloproteinases (MMPs) are known to play an important role in the articular cartilage destruction related to osteoarthritis. Naringenin is a plant-derived flavonoid known for its anti-inflammatory properties. We studied the effect of naringenin on the transcriptional expression, secretion and enzymatic activity of MMP-3 in vivo in the murine monosodium iodoacetate (MIA) osteoarthritis model. The assessment of pain behavior was also performed in the MIA rats. The destruction of knee-joint tissues was analyzed microscopically. Moreover, the effect of naringenin was also studied in vitro in IL-1β activated articular chondrocytes. The transcriptional expression of MMP-3, MMP-1, MMP-13, thrombospondin motifs (ADAMTS-4) and ADAMTS-5 was also studied in primary cultured chondrocytes of rats. Naringenin caused significant reduction in pain behavior and showed marked improvement in the tissue morphology of MIA rats. Moreover, a significant inhibition of MMP-3 expression in MIA rats was observed upon treatment with naringenin. In the in vitro tests, naringenin caused a significant reduction in the transcriptional expression, secretion and enzymatic activity of the studied degradative enzymes. The NF-κB pathway was also found to be inhibited upon treatment with naringenin in vitro. Overall, the study suggests that naringenin alleviated pain and regulated the production of matrix-metalloproteinases via regulation of NF-κB pathway. Thus, naringenin could be a potent therapeutic option for the treatment of osteoarthritis.

Bioinformatic analysis of RNA-seq data unveiled critical genes in rectal adenocarcinoma.

OBJECTIVE: RNA-seq data of rectal adenocarcinoma (READ) were analyzed with bioinformatics tools to unveil potential biomarkers in the disease. MATERIALS AND METHODS: RNA-seq data of READ were downloaded from The Cancer Genome Atlas (TCGA) database. Differential analysis was performed with package edgeR. False discovery rate (FDR) < 0.05 and |log2 (fold change)|>1 were set as cut-off values to screen out differentially expressed genes (DEGs). Gene coexpression network was constructed with package Ebcoexpress. Gene Ontology enrichment analysis was performed for the DEGs in the gene coexpression network with DAVID online tool. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was also performed for the genes with KOBASS 2.0. RESULTS: A total of 620 DEGs, 389 up-regulated genes, and 231 down-regulated genes, were identified from 163 READ samples and 9 normal controls. A gene coexpression network consisting of 71 DEGs and 253 edges were constructed. Genes were associated with ribosome and focal adhesion functions. Three modules were identified, in which genes were involved in muscle contraction, negative regulation of glial cell proliferation and extracellular matrix organization functions, respectively. Several critical hub genes were disclosed, such as RPS2, MMP1, MMP11 and FAM83H. Thirteen relevant small molecule drugs were identified, such as scriptaid and spaglumic acid. A total of 8 TFs and 5 miRNAs were acquired, such as MYC, NFY, STAT5A, miR-29, miR-200 and miR-19. CONCLUSIONS: Several critical genes and relevant drugs, TFs and miRNAs were revealed in READ. These findings could advance the understanding about the disease and benefit therapy development.

Evolution and functional significance of derived sternal ossification patterns in ornithothoracine birds.

The midline pattern of sternal ossification characteristic of the Cretaceous enantiornithine birds is unique among the Ornithodira, the group containing birds, nonavian dinosaurs and pterosaurs. This has been suggested to indicate that Enantiornithes is not the sister group of Ornithuromorpha, the clade that includes living birds and their close relatives, which would imply rampant convergence in many nonsternal features between enantiornithines and ornithuromorphs. However, detailed comparisons reveal greater similarity between neornithine (i.e. crown group bird) and enantiornithine modes of sternal ossification than previously recognized. Furthermore, a new subadult enantiornithine specimen demonstrates that sternal ossification followed a more typically ornithodiran pattern in basal members of the clade. This new specimen, referable to the Pengornithidae, indicates that the unique ossification pattern observed in other juvenile enantiornithines is derived within Enantiornithes. A similar but clearly distinct pattern appears to have evolved in parallel in the ornithuromorph lineage. The atypical mode of sternal ossification in some derived enantiornithines should be regarded as an autapomorphic condition rather than an indication that enantiornithines are not close relatives of ornithuromorphs. Based on what is known about molecular mechanisms for morphogenesis and the possible selective advantages, the parallel shifts to midline ossification that took place in derived enantiornithines and living neognathous birds appear to have been related to the development of a large ventral keel, which is only present in ornithuromorphs and enantiornithines. Midline ossification can serve to medially reinforce the sternum at a relatively early ontogenetic stage, which would have been especially beneficial during the protracted development of the superprecocial Cretaceous enantiornithines.

General practice education and training in southern China: recent development and ongoing challenges under the health care reform

China has launched a general practice (GP)-orientated primary care reform in 2009 to develop a more productive, coordinated, and cost-effective system to maintain and improve the health and wellbeing of one-fifth of the world population. The restructure of the health care system with a focus on primary care requires practitioners working on GP as gatekeepers for service delivery that is responsive to the needs of people. It is particularly prioritised to establish a sound education and training system to ensure that the competencies of practitioners are aligned with local health care needs. This article aims to provide a brief review of the development of GP, including exemplary model of education and training currently implemented in southern China, as well as the challenges to be addressed in the next step. There is a shortage of well-trained and qualified general practitioners in China where more than half of the licensed clinicians in primary care are educated below the undergraduate level. Although there is a stepwise increase in recognition that the capacity of GP is pivotal to the success of primary care development in China, challenges coming from resource restriction, rural and urban disparity, social attitude, and community involvement are highlighted as major bottlenecks that currently hinder the rapid development of GP in China. Supportive policy and guidelines are necessary to build up strong GP recognition and ensure adequate resources to underpin a robust primary care system to deliver affordable and effective health care services for the world’s largest population. It might share some similar experiences with other countries that are struggling to develop a GP-based primary care system.

Clinical study of transarterial chemoembolization combined with 3-dimensional conformal radiotherapy for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Monotherapy is not very effective for intermediate or advanced stage HCC. Efficacy of combined therapy using transarterial chemoembolization (TACE) with three-dimensional conformal radiotherapy (3-DCRT) for advanced HCC should be evaluated. METHODS: HCC patients were selected from our patient database. The sequence of treatments that patients underwent was several courses of TACE followed in 2-4 weeks by 3-DCRT. The median tumor irradiation dose was 44Gy. Toxicity, tumor response, and overall survival rate were analyzed. RESULTS: 140 HCC patients were followed up by the last follow-up time. Among these patients, hepatic toxicities due to treatment were notable in 15 cases. Gastrointestinal bleeding after the overall treatment occurred in 3 cases. Leukopenia of grade III was detected in 1 case. Radiation-induced liver disease (RILD) was observed in 3 patients. Among 140 patients, 27, 97, and 16 cases achieved partial response, stable disease, and progressive disease, respectively. The overall survival rates of 1-year, 3-years, and 5-years were 66%, 29%, and 13%, respectively, with a median survival time of 18 months. Both Child-Pugh grade and radiation dose were determined to be independent predictors for overall survival from multivariate analysis. CONCLUSION: The combined modality of TACE and 3-DCRT is a promising treatment for unresectable HCC. A large-scale, prospective randomized trial should be performed to confirm the utility of this combined therapy.

The synergistic effect of recombinant human endostatin (YH-16) combined with oxaliplatin on human colorectal carcinoma.

This study was designed to investigate the effect of recombinant human endostatin (YH-16) combined with oxaliplatin (L-OHP) on the growth of orthotopically-implanted human colorectal carcinoma in nude mice. Tumour volumes and weights were measured after therapy. Tumour cell morphology was observed by light and electron microscopy. Apoptosis was detected using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) assay, and vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) were determined by immunohistochemical staining. Tumour volumes in all treatment groups were significantly reduced compared with controls. YH-16 and L-OHP either alone or in combination caused tumour cell apoptosis, except in the YH-16 low-dose group. MVD was strongly inhibited in the treatment groups compared with the controls, although only YH-16 combined with L-OHP or L-OHP alone decreased VEGF expression. No obvious change in side-effects occurred. In conclusion, YH-16 combined with L-OHP inhibited growth and induced apoptosis in orthotopically-transplanted human colorectal carcinoma in nude mice without increasing side-effects.

Combined therapy of transcatheter arterial chemoembolisation and three-dimensional conformal radiotherapy for hepatocellular carcinoma.

To evaluate the toxicity and efficacy of combined therapy of three-dimensional conformal radiotherapy (3DCRT) and transcatheter arterial chemoembolisation (TACE) for hepatocellular carcinoma (HCC). 50 HCC patients treated by combined TACE and 3DCRT were selected from a patient database. Sequence of treatments was that TACE was performed first, followed by 3DCRT with an interval of about 4 weeks between. TACE was administered by 5-Fu 500-600 mg m(-2), cis-platinum 30-40 mg m(-2), epi-adriamycin 40-60 mg m(-2) mixed with iodized oil and Gelfoam embolisation. A median of two courses of TACE was given. 3DCRT was delivered by 4-6 coplanar or non-coplanar fields. The mean tumour dose was 43.0+/-6.3 Gy by conventional fractionation (2 Gy per fraction, five fractions a week), and mean dose to normal liver, 19.1+/-6.3 Gy. Acute hepatic toxicities were notable in five patients (10%) with Common Toxicity Criteria (CTC) grade 1 in two cases and grade 3 in three patients, but all recovered eventually. Two patients developed radiation-induced liver disease (RILD) and died soon after the onset of RILD. Four patients had Radiation Therapy Oncology Group (RTOG) grade 1 acute gastrointestinal complication and one patient had acute gastrointestinal bleeding. Five patients experienced RTOG Grade 1 leukopenia and Grade 2 in five cases. Nine patients achieved have partial response, and 37 patients were in stable disease. Four patients were observed to have progressive disease. The overall survival rates at 1 year, 2 years and 3 years were 60%, 38% and 28%, respectively, with a median survival period of 17 months. Irradiation dose, T-stage and hepatic cirrhosis were identified as independent predictors for overall survival by Cox proportional regression analysis. The 1 year, 2 years and 3 years local progression-free rates were 74%, 57% and 38%, and the 1 year, 2 years and 3 years distant metastasis rates were 15%, 21% and 40%, respectively. The combined modality of TACE and 3DCRT was tolerable for the majority of HCC patients, resulted in good outcome and warrants for further prospective trial.

The role of Schwann cell differentiation in perineural invasion of adenoid cystic and mucoepidermoid carcinoma of the salivary glands.

Twenty cases of adenoid cystic carcinoma (ACC) and 18 cases of mucoepidermoid carcinoma (MEC), were examined for expression of the Schwann cell markers S100 protein and glial fibrillary acidic protein (GFAP) by immunohistochemical staining. The relationship between expression of S100 and GFAP and the occurrence of perineural invasion was assessed. Ultrastructural localization of S100 and GFAP was examined by immunoelectron microscopy, and the co-expression of S100 and muscle actin by double fluorescence immunostain. Perineural invasion was found in 11 ACCs (55%) and 0 MECs (0%). S100 and GFAP were expressed in most of the ACCs but none of the MECs; the difference in the rate of perineural invasion and expression of S100 and GFAP was significant between ACC and MEC (P<0.001). There was a correlation between the expression of S100 and GFAP and perineural invasion in salivary malignancy (P<0.001). The ultrastructures of S100- and GFAP-positive cells were consistent with the characteristics of myoepithelial cells. Double fluorescence immunostain also showed that S100 and muscle actin were expressed in the same type of ACC cells. These results indicate that Schwann cell differentiation correlates with perineural invasion in salivary malignancy, and occurs in modified myoepithelial cells of ACC.

Polyreactive antigen-binding B (PAB-) cells are widely distributed and the PAB population consists of both B-1+ and B-1- phenotypes.

B cells that make polyreactive antibodies (PAB+ cells) express polyreactive Ig receptors on their surface and can bind a variety of different antigens. The present study shows that PAB+ cells are widely distributed, are present in varying numbers in different lymphoid organs and that their phenotype varies depending on the organs from which they are isolated. Up to 10 times more cells in PAB+ enriched populations bind antigens as compared to PAB- populations. Comparison of PAB+ with B-1+ cells showed that a high percentage of PAB+ cells are B-1+, but that many PAB+ cells do not express B-1 cell surface markers and, in fact, are B-1-. It is concluded that the B cell population consists of PAB+/B-1+, PAB+/B-1-, PAB-/B-1+, and PAB-/B-1- cells. The presence of PAB+ cells in the thymus points to the possibility that PAB+ cells may carry endogenous host antigens from peripheral tissues to the thymus where they may contribute to immunological tolerance.

Incomplete inactivation of voltage-dependent K+ channels in human B lymphoma cells.

The voltage-dependent K (KV) channel in Daudi human B lymphoma cells was characterized by using patch-clamp techniques. Whole-cell voltage-clamp experiments demonstrated that cell membrane depolarization induced a transient (time-dependent) outward current followed by a steady-state (time-independent) component. The time-dependent current resembled behavior of the type n channel, such as use dependence and a unique blockade by tetraethylammonium (TEA). Both time-dependent and time-independent currents were blocked by quinine with a similar IC50 (14.2 mM and 12.6 mM). Treatment with antisense oligonucleotide of human Kv1.3 gene significantly reduced both currents by 80%. Single-channel experiments showed that only one type of KV channel was recorded with a unitary conductance of approximately 19 pS. Consistent with whole-cell recordings, the channel activity in cell-attached patches remained in response to prolonged depolarization, and the remaining channel activity was blocked by quinine, but not TEA. Channel activity was scarcely seen in cell-attached patches after antisense treatment. Whole-cell current-clamp data showed that TEA, which blocks only the time-dependent current, caused a slight decrease in the membrane potential. In contrast, quinine and antisense, which block both time-dependent and -independent currents, strongly reduced the membrane potential. These data together suggest that the KV channel in Daudi cells does not completely inactivate and that the remaining channel activity due to this incomplete inactivation appears to be primarily responsible for maintaining the membrane potential.

Rabbit retinal neurons and glia express a variety of ENaC/DEG subunits.

Some members of the epithelial Na+ channel/degenerin (ENaC/DEG) family of ion channels have been detected in mammalian brain. Therefore, we examined the RNA and protein expression of these channels in another part of the central nervous system, the rabbit retina. We next sought to demonstrate physiological evidence for an amiloride-sensitive current in Müller glia, which, on the basis of a previous study, are thought to express alpha-ENaC (Golestaneh N, de Kozak Y, Klein C, and Mirshahi M. Glia 33: 160-168, 2001). RT-PCR of retinal RNA revealed the presence of alpha-, beta-, gamma-, and delta-ENaC as well as acid-sensing ion channel (ASIC)1, ASIC2, ASIC3, and ASIC4. Immunohistochemical localization with antibodies against alpha-ENaC and beta-ENaC showed labeling in Müller cells and neurons, respectively. The presence of alpha-ENaC, beta-ENaC, and ASIC1 was detected by Western blotting. Cultured Müller cells were whole cell patch clamped. These cells exhibited an inward Na+ current that was blocked by amiloride. These data demonstrate for the first time both the expression of a variety of ENaC and ASIC subunits in the rabbit retina as well as distinct cellular expression patterns of specific subunits in neurons and glia.