Elevated MIC Values of Imidazole Drugs against Aspergillus fumigatus Isolates with TR34/L98H/S297T/F495I Mutation.

The use of azole fungicides in agriculture is believed to be one of the main reasons for the emergence of azole resistance in Aspergillus fumigatus Though widely used in agriculture, imidazole fungicides have not been linked to resistance in A. fumigatus This study showed that elevated MIC values of imidazole drugs were observed against A. fumigatus isolates with TR34/L98H/S297T/F495I mutation, but not among isolates with TR34/L98H mutation. Short-tandem-repeat (STR) typing analysis of 580 A. fumigatus isolates from 20 countries suggested that the majority of TR34/L98H/S297T/F495I strains from China were genetically different from the predominant major clade comprising most of the azole-resistant strains and the strains with the same mutation from the Netherlands and Denmark. Alignments of sterol 14α-demethylase sequences suggested that F495I in A. fumigatus was orthologous to F506I in Penicillium digitatum and F489L in Pyrenophora teres, which have been reported to be associated with imidazole resistance. In vitro antifungal susceptibility testing of different recombinants with cyp51A mutations further confirmed the association of the F495I mutation with imidazole resistance. In conclusion, this study suggested that environmental use of imidazole fungicides might confer selection pressure for the emergence of azole resistance in A. fumigatus.

High prevalence and clonal dissemination of OXA-72-producing Acinetobacter baumannii in a Chinese hospital: a cross sectional study.

BACKGROUND: Carbapenem resistance in Acinetobacter baumannii in China was mainly mediated by OXA-23-like carbapenemases, while OXA-24/40-like carbapenemases were rarely identified. OXA-72 is one variant of OXA-24/40-like carbapenemases. This study aimed to demonstrate the epidemiology and characterizations of OXA-72-producing A. baumannii in a Chinese hospital. METHODS: A total of 107 clinical A. calcoaceticus-A. baumannii (Acb) complex isolates were collected in a Chinese hospital during between 2014 and 2016. These isolates were identified using Vitek 2 system and gyrB multiplex PCR. Vitek 2 system was used for antibiotic susceptibility testing. Genes encoding for major classes of carbapenemases were investigated by PCR. Rep-PCR was used for genotyping of all the A. baumannii isolates. The risk factors for carriage of OXA-72-producing or OXA-23-producing A. baumannii were analyzed through univariate and multivariate logistic regression. RESULTS: Of the 107 Acb isolates collected, 101 isolates (94.4%) and 6 isolates (5.6%) were identified as A. baumannii and A. pittii, respectively. 78 A. baumannii isolates (77.2%) were carbapenem resistant and mainly cultured from intensive care unit (ICU). blaOXA-72 and blaOXA-23 genes were identified in 45(57.7%) and 33(42.3%) carbapenem-resistant A. baumannii (CRAB), respectively. Multivariate risk factor analyses showed that prior carbapenem usage and nasogastric intubation were significantly associated with carriage of OXA-72-producing A. baumannii or OXA-23-producing A. baumannii. Rep-PCR analysis showed that 9 and 22 Rep-PCR types were assigned to 78 CRAB isolates and 23 carbapenem-susceptible A. baumannii (CSAB) isolates, respectively. A higher diverstiy of Rep-PCR patterns was observed among OXA-72-producing A. baumannii isolates than OXA-23-producing A. baumannii isolates, but all of them belonged to the same clone complex. MLST analysis suggested that the OXA-72 isolates from this study correspond to CC92/CC2 clone complex. CONCLUSIONS: This study demonstrates high prevalence and potential clonal spread of closely related genotypes of OXA-72-producing A. baumannii within a Chinese hospital. Continuous surveillance is necessary to monitor the dissemination of these strains in other healthcare settings to guide infection control policies in order to curb the spread of this bacterium.

Epidemiology and Molecular Characterizations of Azole Resistance in Clinical and Environmental Aspergillus fumigatus Isolates from China.

Azole resistance in Aspergillus fumigatus has emerged as a worldwide public health problem. We sought here to demonstrate the occurrence and characteristics of azole resistance in A. fumigatus from different parts of China. A total of 317 clinical and 144 environmental A. fumigatus isolates from 12 provinces were collected and subjected to screening for azole resistance. Antifungal susceptibility, cyp51A gene sequencing, and genotyping were carried out for all suspected azole-resistant isolates and a subset of azole-susceptible isolates. As a result, 8 (2.5%) clinical and 2 (1.4%) environmental A. fumigatus isolates were identified as azole resistant. Five azole-resistant strains exhibit the TR34/L98H mutation, whereas four carry the TR34/L98H/S297T/F495I mutation in the cyp51A gene. Genetic typing and phylogenetic analysis showed that there was a worldwide clonal expansion of the TR34/L98H isolates, while the TR34/L98H/S297T/F495I isolates from China harbored a distinct genetic background with resistant isolates from other countries. High polymorphisms existed in the cyp51A gene that produced amino acid changes among azole-susceptible A. fumigatus isolates, with N248K being the most common mutation. These data suggest that the wide distribution of azole-resistant A. fumigatus might be attributed to the environmental resistance mechanisms in China.

Oncogenic miR-23a in Pancreatic Ductal Adenocarcinogenesis Via Inhibiting APAF1.

BACKGROUND: miR-23a, which participates in invasion of pancreatic ductal adenocarcinoma cells into the mesothelial barrier, is a critical regulator in many cancers. It, however, is still unknown whether miR-23a regulates pancreatic cell proliferation and apoptosis or not. AIMS: We sought to investigate the role of miR-23a in regulation of pancreatic cell proliferation and apoptosis. METHODS: miRNA, mRNA, and protein expressions were determined by qRT-PCR and Western blot, respectively. Dual-luciferase reporter assay was used in detection for binding ability of miR-23a to APAF1. Ectopic miR-23a and APAF 1 were introduced to pancreatic cells, and their roles in proliferation and apoptosis were detected by MTT, colony formation, and apoptosis assays, respectively. RESULTS: Up-regulation of miR-23a and down-regulation of APAF 1 were found in pancreatic ductal cancer, respectively. miR-23a significantly inhibited the luciferase activity by targeting APAF 1 3'UTR. Ectopic miR-23a significantly suppressed the APAF 1 gene expression in pancreatic cancer cells. Similar to siAPAF1, miR-23a significantly promoted pancreatic cancer cell proliferation and repressed apoptosis. Furthermore, miR-23a inhibitor and exogenous APAF 1 could recover the effects. CONCLUSIONS: It is suggested that miR-23a, acting as an oncogenic regulator by directly targeting APAF 1 in pancreatic cancer, is a useful potential biomarker in diagnosis and treatment of pancreatic cancer.

MicroRNA-27a promotes proliferation and suppresses apoptosis by targeting PLK2 in laryngeal carcinoma.

BACKGROUND: miRNA-27a has been confirmed as an important regulator in carcinogenesis and other pathological processes. Whether and how it plays a role in the laryngeal carcinoma is unknown. METHODS: Mature miRNA-27a expression in laryngeal cancer was detected by qRT-PCR. Gain-of-function studies using mature miR-27a were performed to investigate cell proliferation and apoptosis in the Hep2 cells. In silico database analysis and luciferase reporter assay were applied to predict and validate the direct target, respectively. Loss-of-function assays were performed to investigate the functional significance of the miR-27a target gene. qRT-PCR and Western blot were used to evaluate mRNA and protein levels of the target, respectively. RESULTS: miR-27a was significantly up-regulated in the laryngeal tumor tissues compared to the adjacent non-tumor tissues. In silico database analysis result revealed that PLK2 is a potential target of miR-27a. luciferase reporter assay result showed the direct inhibition of miR-27a on PLK2-3'UTR. In the cases with miR-27a up-regulation, PLK2 protein expression level was significantly lower in cancer tissues than that in the adjacent non-tumor tissues, which showed a negative correlation with miR-27a expression level. Both miR-27a and knockdown of PLK2 caused the increase of the cell viability and colony formation and inhibition of the late apoptosis in the Hep2 cell lines. Moreover, miR-27a but not PLK2 also repressed the early apoptosis in the Hep2 cells. Additionally, no alteration of the Hep2 cell cycle induced by miR-27a was detected. CONCLUSIONS: miR-27a acts as an oncogene in laryngeal squamous cell carcinoma through down-regulation of PLK2 and may provide a novel clue into the potential mechanism of LSCC oncogenesis or serve as a useful biomarker in diagnosis and therapy in laryngeal cancer.

[Expression of WNK4 gene regulated by aldosterone].

OBJECTIVE: To study the expression and mechanism of WNK4 gene regulated by aldosterone. METHODS: Wistar rats were treated with aldosterone and potassium water. Serum aldosterone and ion as well as urine ion were measured. The expression of WNK4 gene in kidney tissues was detected by real-time PCR. Kidney-derived HEK293 cells were cultured, transfected with pGL3-WNK4, and then stimulated by aldosterone. After 24 h of transfection, luciferase activities of the plasmid were detected. RESULTS: Compared with those of the controls, serum aldosterone and urine K(+) of experimental rats were significantly elevated, whilst urine Na(+) was significantly decreased. And urine Cl(-) was significantly increased only in the group of high K(+). Serum K(+), Na(+) and Cl(-) showed no significant difference. Expression of WNK4 gene in kidney tissues was significantly decreased. The luciferase activity of pGL3-WNK4-484 plasmid has decreased after stimulated with aldosterone, while the activity of pGL3-WNK4-275 showed no change. CONCLUSION: Aldosterone can down-regulate the expression of WNK4 through binding with regulatory element in the upstream of the gene.

Promoter hypermethylation-induced transcriptional down-regulation of the gene MYCT1 in laryngeal squamous cell carcinoma.

BACKGROUND: MYCT1, previously named MTLC, is a novel candidate tumor suppressor gene. MYCT1 was cloned from laryngeal squamous cell cancer (LSCC) and has been found to be down-regulated in LSCC; however, the regulatory details have not been fully elucidated. METHODS: Here, we sought to investigate the methylation status of the CpG islands of MYCT1 and mRNA levels by bisulfite-specific PCR (BSP) based on sequencing restriction enzyme digestion, reverse transcription and real-time quantitative polymerase chain reaction (RQ-PCR). The function of specific sites in the proximal promoter of MYCT1 in LSCC was measured by transient transfection, luciferase assays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). RESULTS: The results suggested hypermethylation of 12 CpG sites of the promoter in both laryngeal cancer tissues and the laryngeal cancer line Hep-2 cell. The hypermethylation of the site CGCG (-695 to -692), which has been identified as the c-Myc binding site, was identified in laryngeal cancer tissues (59/73) compared to paired mucosa (13/73); in addition, statistical analysis revealed that the methylation status of this site significantly correlated with cancer cell differentiation(p < 0.01). The mRNA level of MYCT1 increased in Hep-2 cells treated with 5-aza-C (p < 0.01). The luciferase activity from mutant transfectants pGL3-MYCT1m (-852/+12, mut-695-C > A, mut-693-C > G) was significantly reduced compared with the wild type pGL3-MYCT1 (-852/+12), while the luciferase activity from wild transfectants pGL3-MYCT1 (-852/+12) rose after 5-aza treatment in Hep-2 cells. Finally, EMSA and ChIP confirmed that the methylation of the CGCG (-695 to -692) site prevented c-Myc from binding of the site and demethylation treatment of the 5' flanking region of MYCT1 by 5-aza induced the increased occupation of the core promoter by c-Myc (p < 0.01). CONCLUSION: In summary, this study concluded that hypermethylation contributed to the transcriptional down-regulation of MYCT1 and could inhibit cancer cell differentiation in LSCC. DNA methylation of the CGCG site (-695 to -692) of MYCT1 altered the promoter activity by interfering with its binding to c-Myc in LSCC. Epigenetic therapy of reactivating MYCT1 by 5-aza should be further evaluated in clinical trails of LSCC.

[Molecular and prenatal diagnosis of a pedigree with spinocerebellar ataxia].

OBJECTIVE: To identify the type of a pedigree with spinocerebellar ataxia, and carry out asymptomatic carrier detection and prenatal diagnosis. METHODS: The blood samples of two patients in the spinocerebellar ataxia pedigree were collected. Based on the clinical characteristics of the pedigree and the disease incidence in China, the regions containing the CAG repeat of the SCA1, SCA2 and SCA3/MJD genes were amplified by polymerase chain reaction (PCR). The numbers of CAG repeats in the normal and abnormal allele fragments were identified by using agarose gel electrophoresis and DNA sequencing. We further carried out tests on the children of the patients and fetus to identify the presence of the abnormal allele. RESULTS: The numbers of CAG repeat in the SCA1 and SCA2 genes were in the normal range. The CAG repeat number in one allele of SCA3/MJD gene was in the normal range, while that in the other allele was in the abnormal range. One of the children of the patients and the fetus carried the abnormal allele. CONCLUSION: It was confirmed that the pedigree was SCA3/MJD by gene diagnosis. One of the children of the patients was asymptomatic carrier and the fetus also carried the abnormal allele.

Silencing of TBX20 gene expression in rat myocardial and human embryonic kidney cells leads to cell cycle arrest in G2 phase.

Congenital heart diseases (CHDs) are the most common birth defects due to abnormal cardiac development. The T-box 20 (TBX20) gene is a member of the T­box family of transcription factors and encodes TBX20, which is essential for early heart development. In the present study, reduced TBX20 expression was observed in CHD tissue samples compared with normal tissues, and the function of TBX20 in Rattus norvegicus myocardial cells [H9c2(2-1)] and human embryonic kidney cells (HEK293) was investigated. TBX20 was silenced in H9c2 and HEK293 cells via transfection of small interfering RNA and short hairpin RNA duplexes, respectively, and TBX20 mRNA and protein levels were subsequently examined using reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analysis. Cell proliferation was assessed using a cell counting kit and proliferating cell nuclear antigen expression was determined by western blotting. Analysis of cell apoptosis was achieved by annexin V­fluorescein isothiocyanate/propidium iodide staining and a fluorometric terminal deoxynucleotidyl transferase dUTP nick­end labeling system. Cell cycle analysis was achieved using fluorescence­activated cell sorting, and, an RT­qPCR array was used to profile the expression of TBX20­related genes. Silencing of TBX20 in H9c2 and HEK293 cells significantly inhibited cell proliferation, induced cell apoptosis and led to G2/M cell cycle arrest. A reduction in cyclin B1 mRNA levels and an increase in cyclin­dependent kinase inhibitor 1B mRNA levels was observed, which indicated that cells were arrested in G2 phase. Concurrently, the mRNA levels of GATA binding protein 4 were increased in both cell lines, which may provide an explanation for the abnormal cardiac hypertrophy observed in patients with congenital heart disease. These results suggest that TBX20 is required for heart morphogenesis, and inhibition of TBX20 expression may lead to the suppression of cell proliferation and cell cycle arrest.

Methylation Status of SP1 Sites within miR-23a-27a-24-2 Promoter Region Influences Laryngeal Cancer Cell Proliferation and Apoptosis.

DNA methylation plays critical roles in regulation of microRNA expression and function. miR-23a-27a-24-2 cluster has various functions and aberrant expression of the cluster is a common event in many cancers. However, whether DNA methylation influences the cluster expression and function is not reported. Here we found a CG-rich region spanning two SP1 sites in the cluster promoter region. The SP1 sites in the cluster were demethylated and methylated in Hep2 cells and HEK293 cells, respectively. Meanwhile, the cluster was significantly upregulated and downregulated in Hep2 cells and HEK293 cells, respectively. The SP1 sites were remethylated and the cluster was significantly downregulated in Hep2 cells into which methyl donor, S-adenosyl-L-methionine, was introduced. Moreover, S-adenosyl-L-methionine significantly increased Hep2 cell viability and repressed Hep2 cell early apoptosis. We also found that construct with two SP1 sites had highest luciferase activity and SP1 specifically bound the gene cluster promoter in vitro. We conclude that demethylated SP1 sites in miR-23a-27a-24-2 cluster upregulate the cluster expression, leading to proliferation promotion and early apoptosis inhibition in laryngeal cancer cells.

High microRNA-23a expression in laryngeal squamous cell carcinoma is associated with poor patient prognosis.

BACKGROUND: MicroRNA-23a (miR-23a) has been demonstrated to play an important role in the development of several types of cancer, but its role in tumorigenesis of laryngeal carcinoma is still unclear. The aim of this study was to investigate the expression patterns and clinical implications of miR-23a in laryngeal cancer. METHODS: Quantitative RT-PCR was performed to evaluate the expression level of miR-23a in 52 pairs of laryngeal cancer. Analysis between miR-23a expression and clinical features of laryngeal carcinomas was performed by appropriate statistical methods. Role of miR-23a in laryngeal cancer cell migration and invasion was detected via transwell and matrigel assays, respectively. RESULTS: miR-23a was significantly up-regulated in laryngeal cancer tissues compared to normal adjacent laryngeal tissues (P < 0.01). Tumors with high miR-23a expression had significantly greater extent of lymph node metastasis (P < 0.01), worse clinical stage (P < 0.05) and shorter overall five-year survival (P < 0.01) than those with low miR-23a expression. Both univariate and multivariate Cox hazard regression analysis results showed that clinical stage and miR-23a expression were significantly correlated with patient five-year survival (P < 0.01). miR-23a overexpression also significantly promoted laryngeal cancer cell migration and invasion in vitro. CONCLUSIONS: miR-23a, an independent prognostic factor for laryngeal cancer, participates in the onset and progression of laryngeal cancer. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2021488014982305.

A very rare case of trisomy 4q32.3-4q35.2 and trisomy 21q11.2-21q22.11 in a patient with recombinant chromosomes 4 and 21.

We report the case of a patient with a clinical phenotype consistent with Down Syndrome (DS) who has a novel karyotypic abnormality. Karyotypic analyses were performed to investigate the cause of two spontaneous abortions. A balanced translocation between chromosomes 4 and 21 was identified, along with an additional abnormal chromosome 21. We performed high-resolution banding, comparative genomic hybridization (CGH), and FISH studies in both the patient and her mother to define the abnormality and determine its origin. CGH revealed a gain in copy number on the long arm of chromosome 4, spanning at least 24.4 Mb, and a gain in copy number on the long arm of chromosome 21, spanning at least 16.2 Mb. FISH analysis using a chromosome 21 centromere probe and chromosome 4 long arm telomere (4pter) probe confirmed the origin of the marker chromosome. It has been confirmed by the State Key Laboratory of Medical Genetics of China that this is the first reported instance of the karyotype 47,XX,t(4;21)(q31.3;q11.2),+der(21)t(4;21)mat reported in the world.

Expression of MTLC gene in gastric carcinoma.

AIM: To investigate the expression of c-myc target from laryngeal cancer cells (MTLC) gene in gastric carcinoma (GC) tissues and the effect of MTLC over-expression on gastric carcinoma cell line BGC823. METHODS: RT-PCR was performed to determine the expression of MTLC mRNA in GC and matched control tissues. BGC823 cells were transfected with an expression vector pcDNA3.1-MTLC by liposome and screened by G418. Growth of cells expressing MTLC was observed daily by manual counting. Apoptotic cells were determined by TdT-mediated dUTP nick-end labeling (TUNEL) assay. RESULTS: The expression of MTLC mRNAs was down-regulated in 9(60%) of 15 cases of GC tissues. The growth rates of the BGC823 cells expressing MTLC were indistinguishable from that of control cells. A marked acceleration of apoptosis was observed in MTLC-expressing cells. CONCLUSION: MTLC was down-regulated in the majority of GC tissues and could promote apoptosis of GC cell lines, which suggests that MTLC may play an important role in the carcinogenesis of gastric carcinoma.

[Studies of the deletion and expression of cytokeratin 13 gene in laryngeal squamous cell carcinoma].

In order to investigate the role of Cytokeratin 13(CK13) gene in laryngeal carcinogenesis, we detected the deletion of CK13 gene through LOH analysis indirectly at DNA level using 5 STR primers within and near CK13 gene in 72 cases of laryngeal squamous cell carcinoma, then detected the differential expression between 16 cases of paired normal and cancerous tissue by Northern blot, and performed immunohistochemistry using well characterized monoclonal antibody against CK13 in squamous cell carcinoma of different stages. We found that all of the microsatellite loci exist LOH, and the LOH frequencies were 18.03%, 28.13%, 27.42%, 39.68% and 34.85% at D17S1964E, D17S2092, D17S791, D17S1665 and D17S808 respectively. The LOH+ cases accounted for 77.78% (56/72), and the frequencies of LOH were not related to the type of laryngeal carcinoma and the lymphoid metastasis; but significantly related to the differentiation, P < 0.05. CK13 gene is expressed significantly higher in 16 cases of normal tissues than in paired cancerous tissues, and the immunostain revealed that CK13 was expressed in normal laryngeal squamous cell or high differentiation stage, and its expression decreased or disappeared in poor ones, P < 0.01. CK13 gene might play an important role in the laryngeal carcinogenesis, acting as a novel tumor suppressor gene, and may be relevant to laryngeal squamous cell carcinoma diagnosis and prognosis. Further research will contribute to conform it.

Cloning of hTERT cDNA fragment and application of anti-hTERT monoclonal antibody in mechanism of laryngeal carcinogenesis.

The mechanism of telomerase activation is still not clear till now. In order to understand the expressional mode of telomerase and the mechanism of telomerase activation in laryngeal carcinogenesis, we cloned a fragment of hTERT cDNA and prepared a monoclonal antibody against hTERT. We performed immuno-histochemical staining in laryngeal cancer tissues using this antibody. We found that the frequencies of hTERT positive cells were positively correlated with undifferentiation of cancer tissues and that the expression of hTERT was positively correlated with levels of c-Myc, which suggested that c-Myc might play an important role in activation of telomerase. These results revealed that overexpression of c-Myc upregulated telomerase, which in turn resulted in immortalization of laryngeal squamous cells, and this mechanism existed not only in the initiation of laryngeal carcinogenesis but also in the whole process of cancer development.

[Cloning and characterization of MTLC, a novel gene in 6q25].

OBJECTIVE: To identify and characterize laryngeal cancer related novel genes located on chromosome 6q25. METHODS: Electric hybridization was performed in human genome database using EST (expression sequence tag) as probe. Novel genes were deduced by software from positive DNA clones and their cDNAs were amplified by RT-PCR using primers designed according to the sequence of the putative genes. RESULTS: A novel gene was cloned successfully. The full length of this gene was about 21 kb. It contained two exons and produced a 1006 bp transcript coding a protein with 235 amino acid residues. It's 5'flanking sequence contained two binding sites of oncoprotein c-Myc, thus it was named MTLC (c-Myc target from laryngeal cancer cells). Homologous assay showed that MTLC exhibited little overall homology to known human proteins but it exhibited good overall homology to mouse MT-MC1 protein with an identity of 78%. The primary structure of MTLC protein contained a nuclear location signal motif, but it did not have other conserved domains. The results of subcellular location experiment showed that MTLC expressed in nuclei of human hepatocellular carcinoma cell line Bel7402 cells, while a wide distribution of MTLC in various tissues was demonstrated by Northern blotting. CONCLUSION: MTLC may play an important role as a target gene of c-Myc and as a transcription factor in keeping the normal physiological process of cells.

[Analysis on genetic polymorphism of 14 short tandem repeat loci on chromosome 7p14-15 and 12q13 in Chinese north Hans].

OBJECTIVE: To analyze the genetic polymorphism of 6 short tandem repeat (STR) loci on chromosome 7p14-15 and 8 STR loci on chromosome 12q13 in Chinese north Hans. METHODS: Fluorescence-labeling polymerase chain reaction and capillary electrophoresis were used to analyze the genetic polymorphism of 100 randomly selected individuals from Chinese north Han nationality at 6 STR loci (D7S1808, D7S2250, D7S2251, D7S683, D7S656 and D7S528) on chromosome 7p14-15 and 8 STR loci(D12S1056, D12S1293, D12S83, D12S1655, D12S1662, D12S334, D12S137 and D12S102) on chromosome 12q13. RESULTS: In the Chinese north Han population, 7 alleles and 24 genotypes, 8 alleles and 27 genotypes, 7 alleles and 22 genotypes, 4 alleles and 10 genotypes, 6 alleles and 17 genotypes, 5 alleles and 13 genotypes were observed at D7S1808, D7S2250, D7S2251, D7S683, D7S656 and D7S528. The heterozygosities at the above 6 STR loci were 86%, 88%, 83%, 79%, 85% and 80%, respectively. Five alleles and 15 genotypes, 5 alleles and 15 genotypes, 8 alleles and 29 genotypes, 6 alleles and 17 genotypes, 6 alleles and 17 genotypes, 6 alleles and 19 genotypes, 5 alleles and 13 genotypes, 7 alleles and 24 genotypes were observed at D12S1056, D12S1293, D12S83, D12S1655, D12S1662, D12S334, D12S137 and D12S102. The heterozygosities at the above 8 STR loci were 86%, 84%, 87%, 82%, 84%, 85%, 81% and 89%, respectively. CONCLUSION: The distributions of allele frequencies of 6 STR loci on chromosome 7p14-15 and of 8 STR loci on chromosome 12q13 were consistent with the Hardy-Weinberg equilibrium. The highly genetic polymorphism was observed in Chinese north Han population.

[Studies on the mutation and expression of TBX5 gene in human simple congenital heart disease].

This work is to investigate the mutation and expression of TBX5 gene in human simple congenital heart disease. The mutations of eight exons of TBX5 gene in 61 CHD family members (a total of 216 individuals including 65 patients and 151 normal relatives) were examined by PCR-DGGE. Using beta-actin as internal control, the differential expression between 34 myocardium samples from simple congenital heart disease patients and three normal controls was conducted by RT-PCR. There is no mutation detected in all samples; The mRNA expression levels of TBX5 gene show descent tendency in samples of simple congenital heart disease compared with normal controls. The mutations in coding region of TBX5 gene do not cause human simple congenital heart disease, but the abnormality in transcription level of TBX5 gene maybe a kind of mechanism causing human simple congenital heart disease.

Clinical value of serum HMGB1 in diagnosis and prognosis of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignancies in the head and neck region. Recently, aberrantly expressed high-mobility group box 1 (HMGB1) has received a great deal of attention as a potential biomarker for cancer diagnosis and prognosis. Therefore, the aim of this study was to estimate HMGB1 levels in serum in LSCC patients and healthy controls and evaluated the potential of serum HMGB1 as a noninvasive biomarker for diagnosis and prognosis in LSCC. Serum HMGB1 levels were analyzed in 71 LSCC patients and 50 healthy controls. The serum HMGB1 level was significantly higher in LSCC patients compared with the healthy controls (4.81 ± 2.33 vs. 3.21 ± 1.08 ng/mL, P < 0.001). High serum HMGB1 was significantly associated with T classification (P = 0.005), N classification (P = 0.002), and clinical stage (P = 0.001). The area under ROC curve was 0.716, and the sensitivity and specificity were 42.3 and 92.0%, respectively. The Kaplan-Meier plots showed that patients with high serum HMGB1 had a poorer overall survival than those with low serum HMGB1 (P = 0.036). Serum HMGB1 levels are significantly associated with the progression of LSCC. In this population, HMGB1 has a poor sensitivity, but a high specificity for the diagnosis of LSCC. Serum HMGB1 level has potential as a biomarker for the prognosis in LSCC patients.

S100A4 is upregulated via the binding of c-Myb in methylation-free laryngeal cancer cells.

DNA hypomethylation is correlated with the overexpression of the S100A4 gene in several types of cancers including laryngeal cancer, but the molecular mechanism is unknown. We speculated that the methylation status of the promoter affects its binding to the corresponding transcription factors. In the present study, luciferase reporter assay results indicated that the sequences -485 - +73 and -486 - -530 of the S100A4 promoter may harbor the positive and negative cis-acting elements, respectively; and moreover, the luciferase activity promoted by the sequence -485 - +73 increased and the S100A4 gene was significantly upregulated in 5-Aza-induced HEp2 cells. This implies that the methylation status of the sequence is important in regulating the expression of S100A4. Four transcription factor binding motifs including c-Myb, C/EBpα, Ap2 and Msx-1 in the region were predicted by P-Match software. c-Myb and C/EBpα but not Ap2 and Msx-1 were confirmed by EMSA and ChIP as transcription factors of S100A4. The decreased luciferase activity in methylation-free HEp2 cells transfected by the mutant c-Myb motif related to the methylated cytosine suggests that the hypomethylation of the c-Myb motif upregulates the S100A4 expression in laryngeal cancer.