[Functional characterization of SLC12A1 gene variants in 3 patients with Bartter syndrome type â ].

Objective: To clarify the molecular basis of patients with Bartter syndrome type I and explore the therapeutic effect of trafficking-defective variations by chemical chaperone 4-Phenylbutyric acid(4-PBA). Methods: The clinical characteristics, laboratory findings and genetic data of 3 patients diagnosed with Bartter syndrome type I who were admitted to Department of Nephrology, Children's Hospital of Nanjing Medical University from 2017 to 2018 were retrospectively analyzed. Wild type and variant SLC12A1 gene constructs were transiently overexpressed in HEK293 cells. Western blotting was used to detect the expression levels of Na+-K+-2Cl-cotransporter(NKCC2) protein. Immunofluorescent staining was applied to investigate the subcellular localization of NKCC2 protein. In addition, the effect of the chemical chaperone 4-PBA on the expression and localization of the SLC12A1 gene variants was investigated. Unpaired t test was used for statistical analysis of 4-PBA treatment. Results: All the 3 patients (2 males and 1 female), aged 3.0, 4.0 and 1.2 years, respectively. All patients had antenatal onset with polyhydramnios and were born prematurely. After birth, all patients presented with hypochlorine alkalosis accompanied by hypokalemia and hyponatremia. Sequencing analysis revealed that the 3 patients were homozygotes or compound heterozygotes for variants in the SLC12A1 gene. In HEK293 cells, the surface expression of NKCC2 in 3 variants (p.L463S, p.L479V, p.507-510del) are all lower than in wild type (0.718±0.039, 0.287±0.081, 0.025±0.156 vs. 1.001±0.028, t=5.92, 8.35, 30.49, all P<0.01). Moreover, the total protein expression of p.L479V and p.507-510del group were all lower than that in wild type group (0.630±0.032, 0.043±0.003 vs. 1.000±0.111, t=3.21, 8.65, all P<0.05). 4-PBA treatment increased the mature protein expression level of the p.L463S and p. L479V group in 4-PBA treatment group are all higher than the untreated group (0.459±0.018 vs. 1.123±0.024, 0.053±0.012 vs. 1.256±0.037, t=2.75, 18.35, all P<0.05). Cytoplasmic retention of the L479V and 507-510del variants were observed by immunofluorescent staining. 4-PBA treatment could rescue a number of NKCC2 L479V variants to the membrane. Conclusions: The 3 SLC12A1 variants cause expression or subcellular localization defects of the protein. The findings that plasma membrane expression and activity can be rescued by 4PBA might help to develop novel therapeutic strategy for Bartter syndrome type Ⅰ.

Novel lncRNA-ZNF281 regulates cell growth, stemness and invasion of glioma stem-like U251s cells.

Glioma is the most common sub-type of brain tumor. Due to the presence of stem-like cells, it is characterized by poor prognosis, aggressive ability and high post-surgical recurrence rates. Hence, there is critical need to identify molecular mechanisms of glioma stem-like cells. We found a novel lncRNA in the ZNF281 gene and named it lncRNA-ZNF281. We detected the expression of lncRNA-ZNF281 in glioma stem-like cells (U251s), the glioma cell line (U251) and also in normal brain tissue. The expression of lncRNA-ZNF281 was lower in glioma stem-like cells (U251s) and this indicates that lncRNA-ZNF281 can regulate the self-renewal capacity of glioma stem-like cells and stem cell marker expression. Most significantly, lncRNA-ZNF281 inhibits the invasion of glioma stem-like cells by regulating the expression of the NF-κB1 signaling pathway. Our data demonstrates that lncRNA-ZNF281 inhibits the self-renewing ability and invasion of GSCs in vitro and in vivo and can reduce tumorigenicity in the glioma stem-like cell (U251s). The underlying mechanisms may involve the regulation of stem cell markers (CD133, Nestin, OCT4 and Nanog) to reduce the self-renewal ability and regulate the NF-κB1 signaling pathway and inhibit U251s glioma stem-like cell invasion. These finding suggest that lncRNA-ZNF281 could be a successful new therapeutic target in glioma.

Management of clinically negative nodes (N0) in supraglottic laryngeal carcinoma: A systematic review.

The purpose of this study was to evaluate the treatment of clinically negative cervical lymph nodes in supraglottic carcinoma by a meta-analysis. The search words were "supraglottic carcinoma", "cervical lymph nodes negative/cN0", "radical neck dissection", and "radiotherapy". The databases included the Chinese biomedical literature database, Medline, Cochrane library, EMBASE database, journals, and theses, etc. from 1989 onwards. Using the 5-year overall survival, disease-free survival, and disease-specific survival rates, and the recurrence and distant metastasis rates as observation indexes, the proper model and method were selected after a heterogeneity test to allow combined statistic tests, sensitivity analysis, and publication bias analysis to be conducted. Four studies (807 cases) were included in the analysis. Comparisons of the 5-year overall survival, disease-free survival, and disease-specific survival rates as well as lymph node metastasis and the recurrence rate for radical neck dissection and radiotherapy showed no significant differences. There was no advantage of radical neck dissection in supraglottic carcinoma with clinically negative cervical lymph nodes compared to radiotherapy. However, owing to the lack of a prospective study and large number of cases, selection bias and measurement bias may still exist.

Evaluation of the stability of reference genes in bone mesenchymal stem cells from patients with avascular necrosis of the femoral head.

This study aimed to evaluate 12 genes (18S, GAPDH, B2M, ACTB, ALAS1, GUSB, HPRT1, PBGD, PPIA, PUM1, RPL29, and TBP) for their reliability and stability as reference sequences for real-time quantitative PCR (RT-qPCR) in bone marrow-derived mesenchymal stem cells (BMSCs) isolated from patients with avascular necrosis of the femoral head (ANFH). BMSCs were isolated from 20 ANFH patients divided into four groups according to etiology, and four donors with femoral neck fractures. Total RNA was isolated from BMSCs and reverse transcribed into complementary DNA, which served as a template for RT-qPCR. Three commonly used programs were then used to analyze the results. Reference gene expression varied within each group, between specific groups, and among all five groups. Based on comparisons of all five groups, two of the programs used suggested that HPRT1 was the most stable reference gene, while 18S and ACTB were the most variable. Among the 12 candidate reference genes, HPRT1 exhibited the greatest reliability, followed by PPIA. Thus, these sequences could be used as references for the normalization of RT-qPCR results.

Improving energy accumulation of microbial fuel cells by metabolism regulation using Rhodoferax ferrireducens as biocatalyst.

AIMS: To study the physiology and metabolism of microbial cells in the performance of microbial fuel cells (MFCs). METHODS AND RESULTS: A dual-chamber MFCs was constructed, and Rhodoferax ferrireducens was used as biocatalyst. To examine the physiology of microbial cells in the performance of MFCs, the anode media containing planktonic cells was replaced with fresh media in which KH(2)PO(4) and/or NH(4)Cl were excluded. The replacing of anode media containing planktonic cells with fresh media excluded of KH(2)PO(4) and NH(4)Cl made the coulombic yield remarkably increased by a factor of 68% (from 29.1 to 46.8C). The results showed that the electricity could be generated with cells in biofilms as biocatalyst, and coulombic yield was improved by limiting cell growth via removal of ingredients in anode media. By supplementation of glucose to the anode media when current declined to baseline, MFCs achieved about same platform current values immediately. MFCs could continue to produce electricity for about 30 h even after glucose was below detection. CONCLUSIONS: Biofilms and metabolism of glucose play important roles in the performance of MFCs. Coulombic yield of MFCs could be improved by regulating the media ingredients using the stable biofilms-electrode system. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first attempt to study the effect of ingredient compositions of anode media on the performance of MFCs. The observed results that MFCs continued to produce electricity after glucose was below detection was helpful to better understand the mechanism of microbial electricity production.

Identification and characterization of a novel zinc finger protein (HZF1) gene and its function in erythroid and megakaryocytic differentiation of K562 cells.

A novel zinc finger protein (HZF1) gene was identified and characterized by screening a human bone marrow cDNA library, using a new expression sequence tag probe that contains sequences encoding zinc finger motifs. There are at least three transcripts that may result from different splicing of the pre-mRNA, but the differences among them are only involved in 5' non-translation region of HZF1 mRNA. HZF1 gene contains four exons and three introns. The putative protein consists of 670 amino-acid residues including 15 typical C2H2 and 2 C2RH zinc finger motifs. This structure characterization of HZF1 and the nuclear location of the protein suggest that HZF1 may function as a transcription factor. HZF1 mRNA expression was detected in ubiquitous tissues and various hematopoietic cell lines. Increased HZF1 mRNA expression was observed following erythroid differentiation of K562 cells induced by hemin or megakaryocytic differentiation of K562 cells induced by phorbol myristate acetate (PMA). Both of the antisense method and RNA interference assay revealed that repression of the intrinsic expression of HZF1 blocked the hemin-induced erythroid differentiation and reduced the PMA-induced megakaryocytic differentiation of K562 cells, which suggested that HZF1 play important roles in erythroid and megakaryocytic differentiation.

[Expression of retinoic acid receptor gamma gene in ES cells and its effect on their differentiation and apoptosis].

We have constructed pSG5-RAR gamma-neo plasmid containing mouse retinoic acid receptor gamma (RAR gamma) gene and neo gene, and introduced it into embryonic stem ES-5 cells, by calcium phosphate mediated transfection. Some G418-resistant clones were isolated and from RNA dot blot analysis of these clones, a clone overexpressing RAR gamma gene was established, designated as ES-gamma cell line. Northern blot hydridization and Southern blot hydridization analysis of ES-gamma cells (Fig 3, 4) demonstrated that ES-gamma cells overexpressed exogenous RAR gamma mRNA and the exogenous RAR gamma cDNA integrated into the genome of ES cells. ES-gamma cells retained undifferentiated morphology and positive alkaline phosphatase activity (Plate I, Fig. 1, 2), so it resembled ES-5 cells in terms of stem cell characteristics. When ES-gamma cells were subcutaneously inoculated into nude mouse and differentiated in vivo, tumorous nodules containing various tissue structures were obtained, demonstrating their pluripotent properties just like parent ES-5 cells. Contrasting with ES-5 cells, the histological features of tumors showed no cartilage tissues, but abundant muscle tissues and keratinized cyst like structures constituted by stratified squamous epithelia (Plate I, Fig. 3). Differentiating in vitro by hanging drop culture methods, ES-gamma cells differentiated mostly into fibroblast-like cells, (Plate II, Fig. 1-5). The above results indicated that overexpression of RAR gamma gene changed the cell type of ES cells differentiating in vivo and in vitro. During the differentiation of ES-5 cells induced by RA, a large number of cells rounded up, detached from the dish and tended to die. We suspected that this phenomenon may be apoptosis. The ultrastructure appearance of the dying cells displayed typical apoptotic changes including chromatin condensation and nuclear fragmentation (Plate I, Fig. 4, 5). Detection of DNA fragments using agarose gel electrophoresis showed characteristic laddered patterns of apoptotic DNA fragments (Fig. 5). The above results indicated that RA induced apoptosis of ES-5 cells in the course of differentiation. The percentage of apoptosis of ES-5 cells increased accordingly, with the increase of RA concentration (Fig. 6). With the same concentration of RA 10(-7) mol/L, the percentage of apoptotic of ES-gamma death was roughly one times more than that of ES-5 cells (Fig. 7), a fact indicating that RAR gamma may mediate the apoptotic signal transduction of ES cells by RA.

Growth-inhibitory and differentiation-inducing activity of dimethylformamide in cultured human malignant glioma cells.

OBJECTIVE: To determine the growth-inhibitory and differentiation-inducing activity of dimethylformamide (DMF) on a human glioma cell line (SHG-44). DMF is a type of polar solvent and a potent differentiation-inducing agent in many kinds of human solid tumors, yet its effect on human glioma remains unclear. METHODS: The effects of DMF on cell proliferation using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, cell cycle distribution (with flow cytometry), colony-forming efficiency in double-layer soft agar, tumorigenicity in athymic nude mice, morphological changes, and glial fibrillary acidic protein expression were studied. RESULTS: At dose ranges of 0.25, 0.5, 0.75, and 1.0%, DMF caused a dose-dependent proliferation inhibitory effect in monolayers and a marked dose-dependent suppression of colony-forming efficiency in double-layer soft agar with a complete loss of colony-forming ability in cells exposed to 0.75 and 1.0% DMF. Accumulation of cells in G0/G1 phases was observed in DMF-treated (0.5 and 1.0%) cells, also in a dose-dependent manner. SHG-44 cells exposed to DMF (0.5 and 1.0%) for 15 days changed morphologically from small spindle-shaped to large polygonal and flattened stellate cells with multiple slender processes. These cells were still tumorigenic in athymic nude mice, but the growth of xenografts was remarkably reduced, especially in the 1.0% DMF-treated group. The expression of glial fibrillary acidic protein was notably increased by DMF (0.5 and 1.0%). Washout experiments revealed that the effects of DMF on cell proliferation and cell cycle distribution were reversible. CONCLUSION: Our results suggest that DMF drove the SHG-44 cells to a more mature phenotype with inhibited growth.

Modulation effects of hexamethylene bisacetamide on growth and differentiation of cultured human malignant glioma cells.

The modulation effects of hexamethylene bisacetamide (HMBA), a differentiation-inducing agent, on growth and differentiation of cells from human malignant glioma cell line SHG-44 were studied. At cytostatic doses (2.5 mM, 5 mM, 7.5 mM, and 10 mM for 15 days), HMBA exerted a marked inhibitory effect on cell proliferation. Exposure to HMBA (5 mM and 10 mM for 12 days) also resulted in an accumulation of cells in G0/G1 phase and a decrease of cells in S phase as analyzed by flow cytometry. The reversible effects of 7.5 mM HMBA and 10 mM HMBA on cell proliferation and 10 mM HMBA on disruption of cell cycle distribution were observed when HMBA was removed from culture media on Day 6 and replaced with HMBA-free media. Colony-forming efficiency (CFE) in soft agar was remarkably decreased by HMBA (2.5 mM, 5 mM, 7.5 mM, and 10 mM for 14 days), and in 7.5 mM HMBA- and 10 nM HMBA treated cells, the CFEs were reduced to 25% and 12.5%, respectively, of that in untreated cells. Cells treated with HMBA (5 mM and 10 mM for 15 days) remained tumorigenic in athymic nude mice, but the growth rates of the xenografts were much slower than those in the control group. The effects of HMBA on cell proliferation, cell cycle distribution, CFE, and growth of xenografts were dose dependent. A more mature phenotype was confirmed by the morphological changes from spindle shape to large polygonal stellate shape and remarkably elevated expression of glial fibrillary acidic protein in cells exposed to HMBA (5 mM, 10 mM for 15 days). Our results showed that a more differentiated phenotype with marked growth arrest was induced in SHG-44 cells by HMBA.

Methodologic study of direct preparation for chromosome analysis of human solid brain tumors.

Human malignant glioma grown in athymic nude mice (NHG-1) and three freshly resected human solid gliomas were used in the study of factors influencing the direct preparation (DP) for chromosome analysis of human solid tumors. The results showed that: 1) the length of time after the blood supply was obstructed was a major factor in reducing the success rate of DP, i.e., a 2-hour delay resulted in a significantly lowered metaphase number and after 4 hours almost no metaphases could be seen; 2) preserving tumor cells at 4 degrees C may prolong the time limit to about 4 hours; 3) culture medium (RPMI 1640 and Eagle MEM) and bovine calf serum concentration (0%, 10%, 20%, and 30%) did not influence the success rate significantly; 4) colchicine concentration (0.025 micrograms/mL, 0.05 micrograms/mL, 0.1 micrograms/mL) and time of treatment (30 min, 90 min, or 180 min) mainly affected the quality of chromosomes observed but had little effect on the quantity of metaphases that might be obtained. Based on these results, we had a success rate of more than 80% in 72 xenografts and 22 human brain tumors.

[Dibutyryl cyclic adenosine monophosphate (DBc-AMP) in the treatment of recurrent malignant gliomas--report of 7 patients].

It has been demonstrated, by experiments, that DBc-AMP can induce biochemical and morphological changes in cultured glioma cells. In this paper, seven recurrent malignant glioma patients, who had received operation, radiotherapy and chemotherapy, were treated with DBc-AMP by injecting into the tumor feeding artery in 1 patient and directly into the tumor cavities in 6 patients. The results showed that although no improvement of symptoms was observed except the case by intraarterial injection, the IgG and IgA levels were elevated and tumor volume was reduced as shown by enhancement CT scan in 4 patients. It suggests that this method be a better adjuvant therapy for recurrent malignant gliomas.

[Establishment of human glioma cell line--nude mice solid tumor model NHG-1 and its characteristics].

Establishment and its characteristics of a nude mice solid tumor model NHG-1 from human glioma cell line are reported. 5-8 week old NC nude mice of both sexes and SHG-44 cell line used in this experiment were from our laboratory. The initial successful transplantation rate was 7/11 (64%) and that of 30 passages in the subsequent 4 years was 100%. After subcutaneous inoculation, growth curve showed a latent period in week 1-2, slow growing period in week 3-4, rapid growing period in week 5-6 and a final plateau period in week 7. The doubling time was 7 days and cell cycle time was 2.5 days. The cells in G1, S and G2M phases comprised 56%, 27% and 17%, respectively. The survival time of the host was 54 +/- 15 days. The tumor tissues showed a tendency towards invading the surrounding soft tissues. By morphological observation with light and electron microscopes, LDH isozyme assay, PAP immuno-histochemistry labelling GFAP and chromosome analysis, it is confirmed that the transplantable tumor possesses the characteristics of human malignant glioma. The estrogen receptor in the transplantable tumors demonstrated by cytochemical assay indicates that the glioma carcinogenesis is related to endocrine factor of the host. The therapeutic effects of anticancer drugs, such as ACNU, BCNU and 10-hydroxy-2-decenoic acid from the royal jelly on NHG-1 model are evaluated.