RBMS3 Expression and Its Correlation with Clinicopathological Features and Prognosis of Epithelial Ovarian Cancer / 中山大学学报(医学科学版)

ObjectiveTo investigate the expression of RNA binding motif single stranded interacting protein 3 （RBMS3） in epithelial ovarian cancer （EOC） tissues and its relationship with the clinicopathological features and prognosis of EOC. MethodsThe study enrolled the paraffin-embedded tissues from 110 EOC cases and 73 benign epithelial ovarian tumor cases pathologically diagnosed in the first affiliated Hospital of Bengbu Medical College from January 2015 to December 2019. By using anti-RBMS3 polyclonal antibody， the immunohistochemical staining was employed to detect RBMS3 expression in the tissues and then its correlation with the clinicopathological parameters and prognosis of EOC was analyzed. ResultsRBMS3 was expressed in both EOC and benign epithelial ovarian tumor tissues. RBMS3 expression in EOC tissues， significantly related with International Federation of Gynecology and Obstetrics （FIGO） stage， histological grade， CEA levels and survival status， was significantly lower than that in benign epithelial ovarian tumor tissues （P<0.05）. Kaplan–Meier survival curve showed that low RBMS3 expression in EOC patients was correlated with decreased progression-free survival （PFS） and overall survival （OS） （P<0.05）. Univariate analysis showed that RBMS3 expression， FIGO stage， residual lesion size， intestinal metastasis and intraperitoneal implantation were associated with OS of EOC patients （P<0.05）； multivariate analysis showed that low RBMS3 expression and intestinal metastasis were independent risk factors for poor prognosis in EOC patients （P<0.05）. ConclusionsRBMS3 is expressed at low levels in EOC tissues， which is closely related to poor prognosis of EOC patients. RBMS3 may function as a tumor suppressor gene in EOC tissues and can be used as an EOC-independent prognostic marker for targeted therapy against EOC.

Identification of Traditional Chinese Medicine Seahorses Using Graphene Oxide-based Fluorescent Sensing Technology / 中国实验方剂学杂志

ObjectiveTo establish a method for seahorse identification based on graphene oxide fluorescence sensing technology， and to provide a new research idea for identification of traditional Chinese medicine. MethodThe fluorophore FAM was labeled at the 5' end of the specificity upstream primer Ja-F of Hippocampus japonicus as the nucleic acid probe FAM-ssDNA （single strand DNA）. The recognition site of RNA polymerase Ⅱ was added to its specific downstream primer Ja-R as Ja-R1. The seahorse samples were amplified with Ja-F/Ja-R1 primers， and the ssDNA of H. japonicus was obtained by reverse transcription of the amplification products using vitro transcription method. The 20 μL nucleic acid probe FAM-ssDNA （500 nmol·L-1） was incubated at 90 ℃ for 5 min， and was gradually cooled to room temperature. Different volume of graphene oxide solution （100 mg·L-1） and Tris hydroxymethyl amino methane HCl （Tris-HCl） buffer （50 mmol·L-1） were added into each probe solution to make a final reaction volume of 1 mL. The fluorescence intensity of each sample was measured after mixing and placing different times at room temperature away from the light. So that the most appropriate graphene oxide concentration and reaction time were screened for constructing the best nucleic acid probe-graphene oxide biosensor. Adding probe complementary sequence FAM-ssDNA-match solution into the nucleic acid probe-graphene oxide solution， the fluorescence intensity of the reaction mixture was measured after being placed different times at room temperature. Therefore， the optimal reaction time of fluorescence recovery was screened and the feasibility of the sensor was tested. The sensitivity was detected via adding ddH2O as the blank control and different concentration H. japonicus ssDNA into each nucleic acid probe-graphene oxide solution， respectively. Finally， the commercial hippocampal were identified using the optimal experimental condition， and the feasibility of this method for the identification of Chinese medicinal materials was verified. ResultThe fluorescence of 1 mL reaction mixture including 10 nmol·L-1 nucleic acid probe FAM-ssDNA and 12 mg·L-1 go solution for 20 min at room temperature away from the light could be completely quenched. Feasibility test of the biosensor showed that when probe complementary sequence FAM-ssDNA-match solution （final concentration 90 nmol·L-1） was added to the biosensor solution and reacted 1 h reaction at room temperature， the fluorescence signal was significantly enhanced. Sensitivity test showed that the minimum concentration of ssDNA detected by this method was about 10 mg·L-1. This method was used to detect commercial seahorses， and only H. japonicus samples had obvious fluorescence signal. ConclusionThe graphene oxide-based fluorescent sensing technology could be used for zoological origin survey of commercial hippocampus.

Preliminary study of ssDNA aptamer cytotoxicity on RhD+ red blood cells / 中国输血杂志

【Objective】 To investigate the effect of ssDNA aptamer of RhD blood group antigen on erythrocyte toxicity. 【Methods】 Two full-length ssDNA aptamers(82 bp) of RhD blood group antigen were obtained by gene synthesis.Five samples of whole blood with EDTA anticoagulant were collected to prepare red blood cell suspensions (4×107/mL), which were split into 10 tubes(100 μL/tube), corresponding to 5 experimental groups and 5 controls.Two monospecific full-length ssDNA sequences (100 pmol/μL, 5μL each) were added into the experimental group, while the same amount of normal saline into the control.After treatment, the experimental group and the control were incubated for 60 min at 37℃.After washing, they were suspended in LISS solution and stored at 4℃.The experimental group and the control were set according to different time point during storage (1 h, 1 d, 3 d, 10 d and 17 d), with 5 tubes in each group.For erythrocytes in LISS suspension at different storage time, Annexin V labeled with FITC was used as a probe to label the phosphatidylserine (PS) content and Fluo-4 to label Ca2+ .The eversion of PS and the change of Ca2+ concentration in red blood cells in LISS suspensions were determined by flow cytometry. 【Results】 After incubation, all groups were examined under the light microscope.No agglutination occurred in the experimental group, while agglutination occurred in the control.Flow cytometry showed that the number of Annexin V-FITC staining cells of suspended erythrocytes at the same storage time-point was similar between the experimental group and the control, with no significant differences.In the experimental group, apoptosis rate of Annexin V cells at 10-day storage(6.06±1.38) was significantly higher than that at 1-hour storage(P<0.05), so as at 17-day storage(7.77±1.23) than 1-hour, 1-day and 3-day storage(P<0.05). The apoptosis rate of Fluo-4 AM cell in suspended RBCs at the same storage time-point was similar between the two groups(P>0.05). In the experimental group, the apoptosis rate of Fluo-4 AM cell at the 3-day, 10-day and 17-day storage was 20.84±4.16, 22.35±3.37 and 27.06±2.81, respectively(P<0.05). 【Conclusion】 ssDNA aptamer was not found to have any cytotoxic effects on red blood cells, and RhD ssDNA aptamer may be used as a material for the detection and preparation of universal blood.

Investigation of Single-stranded Deoxyribonucleic Acid Translocation Through α-Hemolysin Nanopore with Ionic Liquid Supporting Electrolytes / 分析化学

Due to the difference in spatial configuration and charge of the bases in a DNA molecule, characteristic translocation current pulses through a single nanopore could be obtained. This could become the basis of DNA sequencing method. However, due to the fast translocation speed (sub-micro seconds) and the small current change (about pA), it is still a challenge to obtain the accurate molecular substructure with present electronic techniques. In this work, in order to control the translocation behavior of ssDNA, two kinds of ionic liquids with high viscosity and conductivity were introduced to establish a viscosity gradient with the α-hemolysin single nanopore interface and the acidity of the solution was optimized. The trans chamber contained pure BmimPF6 and the cis chamber contained 1 mol/ L BmimCl and 10 mmol/ L Tris-HCl ( pH 5. 5 ). Preliminary experiment results under this electrolyte configuration showed that poly ( dC) 15 , poly ( dC) 15 , poly(dC) 30 and poly(dC) 50 exhibited obvious long duration pulses with high current suppression ratio. The blocking depth reached more than 95％ of long blocking events. The duration time of long blocking events prolonged to tens or hundreds of milliseconds. Meanwhile, the peak-peak of baseline noise was reduced by about 30％ .

Study on Interaction of Protamine and Single-stranded DNA by Current Pulses Through Single Nanopore / 分析化学

Single nanopore current pulse method is a new, rapid and simple detection method, which is promising for single-molecule DNA sequencing and bio-sensing. Due to the short duration and the low current amplitude of the pulses caused by molecular translocation under normal conditions, pulse detection system with fast response and high sensitivity is required. In this work, based on a lab-established pulse detection system, the effect of protamine in the regulation of single-stranded (ssDNA) current pulses with α-hemolysin (α-HL) single nanopore interface was investigated. Experimental results showed that the pulses positive charged protamine and negative ssDNA probes were both well observed with the established system, and both the pulse amplitude and duration of ssDNA were increased as a result of interaction with protamine. This study provides a way to improve the resolving power of current pulses based on molecular interactions.

Establishment and verification of a mouse model of gene H435Y mutation / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a mouse model of H435Y mutation of gene using CRISPR/Cas9- mediated gene targeting.</p><p><b>METHODS</b>The single-stranded guide RNA (sgRNA) specific to the H435Y loci of gene was designed based on the sequence of gene. After activity assessment, the active sgRNA and Cas9 were transcribed into RNA and microinjected along with the donor DNA fragment with point mutations into fertilized mouse eggs. The microinjected eggs were transferred into pseudopregnant mice to obtain the F0 generation mice with the target gene mutation confirmed by PCR and gene sequencing. gene mutations in the offsprings of the F0 generation mice were analyzed.</p><p><b>RESULTS</b>Gene sequencing confirmed the successful establishment of mouse models carrying H435Y mutation of gene in 4 of the F0 generation mice. The positive F0 generation mice were crossed with wild-type C57BL/6J mice to obtain the F1 generation mice, and PCR confirmed the presence of H435Y mutations of gene in 6 of the F1 mice. Then F2 generation mice were obtained by F1 generation matting with each other. PCR showed that H435Y mutation of gene in F2 mice was found, indicating the mousemodel of gene mutation in H435Y was established and propagated successfully.</p><p><b>CONCLUSIONS</b>We successfully established gene H435Y mutant mouse models using CRISPR/Cas9 technique.</p>

Possible Role of Single Stranded DNA Binding Protein 3 on Skin Hydration by Regulating Epidermal Differentiation

BACKGROUND: Skin hydration is a common problem both in elderly and young people as dry skin may cause irritation, dermatological disorders, and wrinkles. While both genetic and environmental factors seem to influence skin hydration, thorough genetic studies on skin hydration have not yet been conducted. OBJECTIVE: We used a genome-wide association study (GWAS) to explore the genetic elements underlying skin hydration by regulating epidermal differentiation and skin barrier function. METHODS: A GWAS was conducted to investigate the genetic factors influencing skin hydration in 100 Korean females along with molecular studies of genes in human epidermal keratinocytes for functional study in vitro. RESULTS: Among several single nucleotide polymorphisms identified in GWAS, we focused on Single Stranded DNA Binding Protein 3 (SSBP3) which is associated with DNA replication and DNA damage repair. To better understand the role of SSBP3 in skin cells, we introduced a calcium-induced differentiation keratinocyte culture system model and found that SSBP3 was upregulated in keratinocytes in a differentiation dependent manner. When SSBP3 was overexpressed using a recombinant adenovirus, the expression of differentiation-related genes such as loricrin and involucrin was markedly increased. CONCLUSION: Taken together, our results suggest that genetic variants in the intronic region of SSBP3 could be determinants in skin hydration of Korean females. SSBP3 represents a new candidate gene to evaluate the molecular basis of the hydration ability in individuals.

Identification of circo-like virus-Brazil genomic sequences in raw sewage from the metropolitan area of São Paulo: evidence of circulation two and three years after the first detection

BACKGROUND Two novel viruses named circo-like virus-Brazil (CLV-BR) hs1 and hs2 were previously discovered in a Brazilian human fecal sample through metagenomics. CLV-BR hs1 and hs2 possess a small circular DNA genome encoding a replication initiator protein (Rep), and the two genomes exhibit 92% nucleotide identity with each other. Phylogenetic analysis based on the Rep protein showed that CLV-BRs do not cluster with circoviruses, nanoviruses, geminiviruses or cycloviruses. OBJECTIVE The aim of this study was to search for CLV-BR genomes in sewage and reclaimed water samples from the metropolitan area of São Paulo, Brazil, to verify whether the first detection of these viruses was an isolated finding. METHODS Sewage and reclaimed water samples collected concomitantly during the years 2005-2006 were purified and concentrated using methodologies designed for the study of viruses. A total of 177 treated reclaimed water samples were grouped into five pools, as were 177 treated raw sewage samples. Nucleic acid extraction, polymerase chain reaction (PCR) amplification and Sanger sequencing were then performed.e FINDINGS CLV-BR genomes were detected in two pools of sewage samples, p6 and p9. Approximately 28% and 51% of the CLV-BR genome was amplified from p6 and p9, respectively, including 76% of the Rep gene. The detected genomes are most likely related to CLV-BR hs1. Comparative analysis showed several synonymous substitutions within Rep-encoding sequences, suggesting purifying selection for this gene, as has been observed for other eukaryotic circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses. MAIN CONCLUSION The results therefore indicated that CLV-BR has continued to circulate in Brazil two and three years after first being detected.

Effects of silencing single-stranded DNA-binding protein 1 gene on proliferation and DNA repair of submandibular gland cells after irradiation / 中华放射医学与防护杂志

Objective To investigate the effects of silencing the gene of single-stranded DNA-binding protein 1 (SSB1) on proliferation and DNA repair of rat submandibular gland (SMG) cells after irradiation, and explore the relationship between SSB1 and DNA damage repair. Methods Primary rat SMG cells were obtained by mechanical-enzyme digestion and identified by immunohistochemistry. The cells were divided into three groups, including blank control, negative control and shRNA transfection group. The shRNA was transfected into cells by recombinant adenovirus vector. Real-time quantitative PCR ( qRT-PCR) was used to detect the expression of SSB1 after silencing. The cell viability was detected by CCK-8 assay. Immunofluorescence analysis was performed to observe the dynamic formation of γ-H2AX foci. Results The SMG cells were positively stained for both Pan CK and α-Amylase. The efficiency of shRNA transfection was about 90%at 72 h post-transfection. Compared with the blank control group, the expression of SSB1 was significantly decreased in the cells transfected with shRNA (t=16. 24, P<0. 05). The cell viability of shRNA transfection group without irradiation was decreased indistinctively and became lower than the blank control group significantly until 120 h(t=3. 29, P<0. 05). After radiation with 5 Gy of γ-rays, the cell viability of shRNA transfection group was lower than that of the control groups significantly (F=10. 19-30. 13, P<0. 05). Silencing the expression of SSB1 could increase the number ofγ-H2AX foci in SMG cells at different time of radiation. Conclusions After silencing of the expression of SSB1, the SMG cells could be more radiosensitive, which indicats that SSB1 may play an important role in DNA damage repair after radiation.

Effects of silencing single-stranded DNA-binding protein 1 gene on proliferation and DNA repair of submandibular gland cells after irradiation / 中华放射医学与防护杂志

Objective To investigate the effects of silencing the gene of single-stranded DNA-binding protein 1 (SSB1) on proliferation and DNA repair of rat submandibular gland (SMG) cells after irradiation, and explore the relationship between SSB1 and DNA damage repair. Methods Primary rat SMG cells were obtained by mechanical-enzyme digestion and identified by immunohistochemistry. The cells were divided into three groups, including blank control, negative control and shRNA transfection group. The shRNA was transfected into cells by recombinant adenovirus vector. Real-time quantitative PCR ( qRT-PCR) was used to detect the expression of SSB1 after silencing. The cell viability was detected by CCK-8 assay. Immunofluorescence analysis was performed to observe the dynamic formation of γ-H2AX foci. Results The SMG cells were positively stained for both Pan CK and α-Amylase. The efficiency of shRNA transfection was about 90%at 72 h post-transfection. Compared with the blank control group, the expression of SSB1 was significantly decreased in the cells transfected with shRNA (t=16. 24, P<0. 05). The cell viability of shRNA transfection group without irradiation was decreased indistinctively and became lower than the blank control group significantly until 120 h(t=3. 29, P<0. 05). After radiation with 5 Gy of γ-rays, the cell viability of shRNA transfection group was lower than that of the control groups significantly (F=10. 19-30. 13, P<0. 05). Silencing the expression of SSB1 could increase the number ofγ-H2AX foci in SMG cells at different time of radiation. Conclusions After silencing of the expression of SSB1, the SMG cells could be more radiosensitive, which indicats that SSB1 may play an important role in DNA damage repair after radiation.

RBMS3 inhibits invasion and epithelial-mesenchymal transition of gastric cancer cells via regulating Wnt/β-catenin signal pathway / 肿瘤

Objective: To investigate the effect of RNA-binding motif, single-stranded-interacting protein 3 (RBMS3) on the invasion of gastric cancer cells, and to explore its possible action mechanism. Methods: The expression levels of RBMS3 mRNA and protein in human normal gastric mucosa epithelial cells (GES-1) and gastric cancer cells (MKN-28, MKN-45, NCI-N87 and SGC-7901) were detected by real-time fluorescent quantitative PCR (RFQ-PCR) and Western blotting, respectively. The RBMS3 overexpression vectors [RBMS3-pcDNA3.1(+)] were transfected into gastric cancer MKN-45 and SGC-7901 cells by using LipofectAMINE 2000, while the corresponding empty vector-transfection group and untransfection group were arranged as the negative control and blank control, respectively. The efficiency of RBMS3 overexpression was verified by RFQ-PCR and Western blotting. The cell invasion ability was detected by Transwell assay. The expression levels of epithelial-mesenchymal transition-related proteins and Wnt signal pathway protein β-catenin were detected by RFQ-PCR and Western blotting. After RBMS3 overexpressed MKN-45 and SGC-7901 cells were treated with Wnt pathway agonist lithium chloride (LiCl), the change of cell invasion ability was retested by Transwell assay. Results: The mRNA and protein levels of RBMS3 in 4 gastric cancer cell lines were obviously lower than those in normal gastric mucosa epithelial cells (all P < 0.05). After transfection of RBMS3 overexpression vectors, RBMS3 gene was overexpressed in gastric cancer MKN-45 and SGC-7901 cells (both P < 0.05), the invasion abilities of these two cells were significantly declined (both P < 0.05). In RBMS3 gene-overexpressed MKN-45 and SGC-7901 cells, the expressions of N-cadherin and β-catenin were significantly decreased (all P < 0.05), while the expression of E-cadherin was significantly increased (both P < 0.05). After LiCl treatment, the inhibitory effect of RBMS3 on invasion of gastric cancer MKN-45 and SGC-7901 cells was partially counteracted (both P < 0.05). Conclusion: RBMS3 may inhibit the invasion and epithelial-mesenchymal transition of human gastric cancer cells by suppressing Wnt/β-catenin signal pathway.

DNA damage to human genetic disorders with neurodevelopmental defects

Although some mutations are beneficial and are the driving force behind evolution, it is important to maintain DNA integrity and stability because it contains genetic information. However, in the oxygen-rich environment we live in, the DNA molecule is under constant threat from endogenous or exogenous insults. DNA damage could trigger the DNA damage response (DDR), which involves DNA repair, the regulation of cell cycle checkpoints, and the induction of programmed cell death or senescence. Dysregulation of these physiological responses to DNA damage causes developmental defects, neurological defects, premature aging, infertility, immune system defects, and tumors in humans. Some human syndromes are characterized by unique neurological phenotypes including microcephaly, mental retardation, ataxia, neurodegeneration, and neuropathy, suggesting a direct link between genomic instability resulting from defective DDR and neuropathology. In this review, rare human genetic disorders related to abnormal DDR and damage repair with neural defects will be discussed.

ASSOCIATION BETWEEN MURINE DOUBLE MINUTE 2 - T309G polymorphism and recurrence of hepatocellular carcinoma after surgical treatment

Background - Discovery and incorporation of biomarker panels to cancer studies enabled the understanding of genetic variation and its interference in carcinogenesis at molecular level. The potential association between single nucleotide polymorphism (SNP) 309 and increased development of tumors, such as hepatocellular carcinoma, has been subject to several studies. This is the first study on this association conducted in Brazil. Methods - 62 cases of cirrhotic patients with hepatocellular carcinoma surgically treated by partial hepatectomy (HPT) or by liver transplantation (LTX) from 2000 to 2009 at Santa Casa Hospital Complex, in the city of Porto Alegre, were retrospectively analyzed. Tumor samples from surgical specimen were collected and prepared for study in paraffin blocks. Results - Overall survival was 26.7 months in the HPT group and 62.4 months in the LTX group (P <0.01). Overall tumor recurrence was 66.7% in the HPT group (10/15) and 17% in the LTX group (8/47) (X²=13.602, P <0.01). Alpha-fetoprotein levels >200ng/mL, microvascular invasion and histological grade were associated with tumor recurrence (P <0.01). Recurrence rates in each surgical group and analysis of factors associated with tumor recurrence, when stratified for each genotypic pattern, were both not statistically significant. Conclusion - G/G genotype was not associated with tumor recurrence after surgical treatment and it did not show any correlation with other prognostic factors.

Contexto - A descoberta e incorporação de painéis de biomarcadores aos estudos do câncer permitiram o conhecimento de variações genéticas e sua interferência no processo de carcinogênese. A possibilidade de associação do polimorfismo de nucleotídeo simples T309G do gene MDM2 com o aumento da formação de tumores, dentre eles o hepatocarcinoma, tem sido alvo de diversos estudos. Objetivo - Analisar a influência do polimorfismo T309G do gene MDM2 na recidiva tumoral de pacientes cirróticos com hepatocarcinoma submetidos a tratamento cirúrgico. Métodos - Foram analisados retrospectivamente pacientes cirróticos com carcinoma hepatocelular submetidos a tratamento cirúrgico (hepatectomia parcial ou transplante hepático) no período de 2000 a 2009, na Santa Casa Hospital Complex in Porto Alegre, South Brazil. Foram coletadas amostras de fragmentos tumorais da peça operatória (fígado explantado ou segmento hepático), as quais foram preparadas para estudo em bloco parafinado. Resultados - A sobrevida global foi de 26,7 meses para o grupo hepatectomias e 62,4 meses para o grupo transplante hepático (P <0,01), havendo 66,7% de recidiva global no grupo hepatectomias (10/15), e 17% no grupo transplante hepático (8/47) (X²=13,602, P <0.01). Níveis de AFP>200ng/mL correlacionaram-se com a recidiva tumoral em ambos os subgrupos cirúrgicos. Observou-se que 83,3% dos pacientes com recidiva também apresentaram invasão microvascular ao exame anátomo-patológico (P <0,01). Não houve significância estatística quando a recidiva neoplásica foi avaliada para os diferentes genótipos e analisada para cada subgrupo cirúrgico. A análise dos fatores prognósticos relacionados à recidiva do hepatocarcinoma, quando estratificada para cada padrão genotípico, também não se mostrou significante. Conclusão - O nosso estudo revelou que o genótipo G/G não esteve associado à recidiva tumoral após o tratamento cirúrgico, seja nas hepatectomias parciais ou transplante hepático. Além disso, a presença desse genótipo não mostrou correlação com os fatores prognósticos estudados.

Assessing extent of single stranded DNA damage in oral mucosal cells of patients with oral squamous cell carcinoma and its correlation with TNM staging.

Context: This study was carried out on the assumption that oral mucosal cells might show DNA damage in oral squamous cell carcinoma (OSCC). Aims: To evaluate the extent of DNA damage in oral smears of patients with OSCC and determine correlation if any of the extent of DNA damage to TNM staging of oral cancer. Settings and design: A randomized controlled study at a regional cancer centre was designed for this project. Smears were taken from lesion proper of 30 patients with OSCC and from the buccal mucosa of 30 normal healthy volunteers. Materials and methods: Collected cells were centrifuged and single-cell gel electrophoresis (SCGE) assay was performed. DNA damage was visualized under a fluorescent microscope. Statistical analysis used : Mean DNA damage levels of both the groups were measured and statistically analyzed with students' test. The extent of DNA damage was correlated with the TNM stages by employing the one way ANOVA 'F' technique. Results: High statistical significance (P < 0.0001) was found in DNA damage levels between control and study groups. A stepwise increase in DNA damage levels with high statistical significance (P < 0.005) was also found between all the TNM stages. Conclusions: Statistically significant increased DNA damage levels in OSCC patients and their correlation to clinical staging suggest that comet assay may be used effectively to assess the prognosis of OSCC.

Detection of Specific DNA Sequence with a Non-labeled Molecular Beacon and a Nucleic Acid Dye Hoechst 33258 / 分析化学

A highly sensitive and selective method for specific DNA sequence detection is developed using a non-labeled molecular beacon (MB) and a nucleic acid dye Hoechst 33258. It is demonstrated by a specific DNA sequence of wild-type HBV as a model system. In this strategy, the stem of MB is completely designed as C/G base pairs. In the absence of target DNA, the interaction between Hoechst 33258 and the MBs is very weak,and the fluorescence signals of Hoechst 33258 is very low. In the presence of target DNA, the MBs hybridize with the target DNA and form double-stranded structure. Hoechst 33258 binds to dsDNA, and the fluorescence intensity is significantly enhanced. Under the optimum conditions, the fluorescence intensity of Hoechst 33258 exhibits good linear dependence on target DNA concentration in the range of 2 × 10-10-2 × 10-8 mol/L. The fitted regression equation is △I=3. 3439C(10-10 mol/L) ﹢18. 6949(R2=0. 9982) with a correlation coefficient of 0. 9982 (R2), and the detection limit is 9 × 10-11 mol/L (3σ). The proposed method has good precision, simple operation, fast detection speed, low detection limit, high accuracy and high sensitivity.

Molecular characterization of drug-resistant and drug-sensitive Aspergillus isolates causing infectious keratitis.

Purpose: To study the susceptibilities of Aspergillus species against amphotericin B in infectious keratitis and to find out if drug resistance had any association with the molecular characteristics of the fungi. Materials and Methods: One hundred and sixty Aspergillus isolates from the corneal scrapings of patients with keratitis were tested for susceptibilities to amphotericin B by broth microdilution method. These included Aspergillus flavus (64 isolates), A. fumigatus (43) and A. niger (53). Fungal DNA was extracted by glass bead vertexing technique. Polymerase chain reaction (PCR) assay was standardized and used to amplify the 28S rRNA gene. Single-stranded conformational polymorphism (SSCP) of the PCR product was performed by the standard protocol. Results: Of the 160 isolates, 84 (52.5%) showed low minimum inhibitory concentration (MIC) values (≤ 1.56 μg/ml) and were designated as amphotercin B-sensitive. Similarly, 76 (47.5%) had high MICs (≥ 3.12 μg/ml) and were categorized as amphotericin B-resistant. MIC50 and MIC90 values ranged between 3.12-6.25 μg/ml and 3.12-12.5 μg/ml respectively. A. flavus and A. niger showed higher MIC50 and MIC90 values than A. fumigatus. The SSCP pattern exhibited three extra bands (150 bp, 200 bp and 250 bp each) in addition to the 260 bp amplicon. Strains (lanes 1 and 7) lacking the 150 bp band showed low MIC values (≤ 1.56 μg/ml). Conclusion: A. niger and A. flavus isolates had higher MICs compared to A. fumigatus, suggesting a high index of suspicion for amphotericin B resistance. PCR-SSCP was a good molecular tool to characterize Aspergillus phenotypes in fungal keratitis.

Detection of miRNA gene sequence variations in multiple myeloma and its significance / 白血病·淋巴瘤

Objective To investigate sequence variations of 12 miRNA genes in multiple myeloma(MM) in order to find whether sequence variations in miRNA genes are associated with tumorigenesis and discuss the clinical significance of MM associated with miRNA genes mutations. Methods The miRNA gene mutations in 20 cases of MM, 4 MM-derived cell lines and 20 controls were detected by the methods of polymerase chain reaction single stranded conformation polymorphism (PCR-SSCP) and silver staining technique. Both clinical features and laboratory results were analyzed simultaneously. Results The electrophoretic patterns showed a total of three variations in miR-19a, miR-19b and miRNA-335,which were observed in 3 MM cells (15 %, 3/20). We also found variations of miRNA-335 in MM-derived cell lines KM-3and RPMI8226. However, no sequence alteration in the miRNA genes was observed in our set of controls. One of the three MM patients died, and two of them were detected mutations at the terminal stage of the disease.Conclusion A relative high frequency of miRNA gene mutation was found in MM and MM derived cell lines, which suggests possibility of a main mechanism underlying tumorigenesis. And, detecting miRNA gene mutations in MM might be benefit to evaluate the progression and prognosis of disease.

In vitro functional characterization of human anti-bullous pemphigoid 180 (BP180) single-chain Fv antibody / 中华皮肤科杂志

Objective To characterize the function of human anti-BP180 single-chain Fv antibody (scFv) in vitro. Methods The IgG autoantibodies against BP180 were purified by affinity chromatography from the sera of patients with BP. The inhibitive effect of previously constructed anti-BP180 scFv on the binding of anti-BP180 IgG autoantibodies to the recombinant NC16A domain of human BP180 antigen was observed by competitive ELISA, competitive immunofluorescence assay and competitive inhibition test for complement activation. Results As ELISA revealed, the scFv significantly inhibited the binding of anti-BP180 IgG autoantibodies to the corresponding antigen (P ＜ 0.01 ), and the inhibitive effect was dose-dependent within the concentration range from 0 to 60 μg/ml. The inhibitive rate peaked at 69.50%. The deposition of anti-BP180-IgG autoantibodies in basement membrane zone and the IgG autoantibody-mediated complement C3 activation were both suppressed by the scFv of 40 μg/ml. Conclusion The genetically engineered anti-BP180 scFv has a certain inhibitive effect on the binding of BP-IgG autoantibodies to BP180 antigen and on the subsequent complement activation in vitro.

Detection of alphaB-crystallin mRNA using Single-stranded DNA Probe in Oligodendrocytes of the Developing Chick Retina / 체질인류학회지

In situ hybridization (ISH) using single-stranded DNA probe (ssDNA probe) is a useful method for observing the specific transcripts in cells, since it is convenient to prepare probe which is specific and sensitive. In this study, ssDNA probe for detection of alphaB-crystallin (aBC) mRNA, transcript of a heat shock protein, was prepared and aBC mRNA-expressed cells were spatiotemporally observed in the retina of the developing chick embryos. Single-stranded antisense probe produced by reverse transcription and polymerase chain reaction was identified as a specific probe for aBC mRNA in comparison to negative control using sense probe and immunohistochemistry for aBC protein. In the ISH experiment, aBC mRNA was expressed only in the peripapillary glial cells which are a specific cell type located in the avian retina adjacent to the optic nerve at E12 and E14 retinas. At E16, a small number of aBC mRNA-expressed cells were identified in the nerve fiber layer (NFL) of the retina. At E18, aBC mRNA-expressed cells were observed in the ganglion cell layer (GCL) as well as the NFL. At E20, the number of aBC mRNA-expressed cells was increased in the GCL and the NFL. Based on the same localization of nkx2.2 immunoreactive cells and aBC mRNA-expressed cells, aBC mRNA-expressed cells were identified as oligodendrocytes. These results indicate that ssDNA probe for aBC mRNA detection is very useful tool for oligodendrocyte research such as distribution, migration and differentiation of the cells.

Gene diagnosis of X-linked spondyloepiphyseal dysplasia tarda / 中华检验医学杂志

ObjectiveTo investigate the molecular pathogenesis of a pedigree of X-linked spondyloepiphyseal dysplasia atarda (SEDL) and to establish methods of gene diagnosis. Methods Clinical diagnosis was made based on height measurement, radiological examination and pedigree analysis. Peripheral blood samples of relevant family members were collected. After genomic DNA extraction, single strand conformation polymorphism (SSCP) followed with DNA sequencing was used to detect SEDL gene exons 36. Microsatellite marker DXS16 was selected for linkage analysis. Results The abnormal electrophoretic bands were detected in exon 4 of probands by PCR-SSCP. A c. 218C ＞ T mutation in exon 4 of SEDL gene was found in three probands, which resulted in a change in amino acid sequence S37L. The heterozygous exon 4 mutation was identified in three carriers, but not in healthy individuals, and no mutations were detect in exon 3, 5 and 6 of probands. Three unmarried young females (Ⅲ10, Ⅳ6 and Ⅳ7) were found to harbor the mutation by DNA sequencing analysis. ConclusionsA c. 218C ＞ T missense mutation in exon 4 of SEDL gene is the cause of molecular pathogenesis of the pedigree. SSCP and DNA sequencing can be used for prenatal gene diagnosis.