High-efficient stabilization and solidification of municipal solid waste incineration fly ash by synergy of alkali treatment and supersulfated cement.

Municipal solid waste incineration fly ash (IFA) designated as hazardous waste poses risks to environment and human health. This study introduces a novel approach for the stabilization and solidification (S/S) of IFA: a combined approach involving alkali treatment and immobilization in low-carbon supersulfated cement (SSC). The impact of varying temperatures of alkali solution on the chemical and mineralogical compositions, as well as the pozzolanic reactivity of IFA, and the removal efficiency of heavy metals and metallic aluminum (Al) were examined. The physical characteristics, hydration kinetics and effectiveness of SSC in immobilizing IFA were also analyzed. Results showed that alkali treatment at 25 °C effectively eliminated heavy metals like manganese (Mn), barium (Ba), nickel (Ni), and chromium (Cr) to safe levels and totally removed the metallic Al, while enhancing the pozzolanic reactivity of IFA. By incorporating the alkali-treated IFA and filtrate, the density, compressive strength and hydration reaction of SSC were improved, resulting in higher hydration degree, finer pore structure, and denser microstructure compared to untreated IFA. The rich presence of calcium-aluminosilicate-hydrate (C-(A)-S-H) and ettringite (AFt) in SSC facilitated the efficient stabilization and solidification of heavy metals, leading to a significant decrease in their leaching potential. The use of SSC for treating Ca(OH)2- and 25°C-treated IFA could achieve high strength and high-efficient immobilization.

Recycling of waste glass and incinerated sewage sludge ash in glass-ceramics.

Disposal of waste glass and incinerated sewage sludge ash (ISSA) in landfills is a waste of resources and poses significant environmental risks. This work aims to recycle waste glass and ISSA together to form value-added glass-ceramics. The physical and mechanical properties, leaching behaviour, and microstructure of the glass-ceramics produced with different proportions of waste glass powder (WGP) and ISSA were investigated. Thermodynamic calculations were performed to predict the formation of crystalline phases and the phase transformation involved. The results showed the potential of WGP and ISSA as raw materials in glass-ceramics production. WGP effectively densified the microstructure of the glass-ceramics by forming a viscous phase. As WGP content increased, the total porosity of glass-ceramics decreased whereas the density increased, accompanied by the formed anorthite transforming into wollastonite. The incorporation of WGP densified and refined the pore structure of the glass-ceramics, thereby improving the mechanical properties and reducing the water absorption. The glass-ceramics produced with a 50:50 blend of WGP and ISSA exhibited the highest compressive strength of 43.7 MPa and the lowest water absorption of 0.3 %. All fabricated glass-ceramics exhibited innocuous heavy metal leaching. The co-sintering of ISSA and WGP can produce additive-free glass-ceramics, characterized by reduced energy consumption and notable heavy metal immobilization capacity. These materials hold promise for utilization in construction as building materials.

Effect of air pollution-controlled residue of a sewage sludge incinerator on the drying shrinkage and the pore structure of alkali-activated materials.

Recycling air pollution-controlled residues (APCR) generated from sewage sludge incinerators can be used for waste management, but the leaching of potentially toxic heavy metals from APCR poses environmental and human health issues. The present paper describes a procedure using APCR to produce alkali-activated materials and thereby realize their disposal. The effect of APCR on the compressive strength and drying shrinkage of the alkali-activated slag/glass powder was investigated. The pore structure characteristics were analyzed for clarifying its relationship with drying shrinkage. The results indicated that the drying shrinkage of the alkali-activated material was related to the mesopore volume. The drying shrinkage was slightly increased after the incorporation of the 10 % APCR, which was likely attributed to the high volume of mesopores compared to the 20 % APCR that lowered the drying shrinkage and compressive strength. This decrease in drying shrinkage was due to the recrystallization of sodium sulfate in the pore solution that can act as expansive agents and aggregates. The growth stress of the crystalline sodium sulfate within the matrix can offset the tension stress caused by the water loss. In addition, leaching studies using the SW-846 Method 1311 showed that recycling APCR into the alkali-activated system did not present a toxicity leaching risk or release unacceptable concentrations of heavy metals. The incorporation of waste APCR and waste glass can make AAMs a very promising and safe environmental technology.

Physicochemical and pozzolanic properties of municipal solid waste incineration fly ash with different pretreatments.

Swelling caused by gas generated from municipal solid waste incineration fly ash (MSWIFA) when it is mixed with alkali limits its uses. Besides, the leaching of anion salts and heavy metals contained in MSWIFA poses a high risk to environment. This study presents the feasibility of a one-step alkaline washing, one-step thermal quenching and two-step combination of alkaline washing and thermal quenching pretreatment methods in altering the key properties of MSWIFA for promoting its reusability. It was found that apart from H2(gas), NH3(gas) was also generated during the alkaline washing of the MSWIFA. Besides, pretreatments led to the reduction in particle size, the increase in pore volume and specific surface area of the MSWIFA, as well as the removal of chloride and sulfate anions. All the pretreatment methods were effective in reducing leaching of heavy metals to below levels of nonhazardous waste except Cd and Pb with alkaline washing. Furthermore, both the chemical Frattini test and the mechanical activity index test showed improvement in pozzolanic activities of the MSWIFA after the pretreatments. Overall, the combined pretreatment method was most effective in eliminating gas emission, and reducing leaching of metal ions and anions from the ash, while enhancing the pozzolanic activity of the ash.

Induction of adhesion molecule expression in co-culture of human bronchial epithelial cells and neutrophils suppressed by puerarin via down-regulating p38 mitogen-activated protein kinase and nuclear factor κB pathways.

OBJECTIVE: In this study, we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system, assess the effects of puerarin on suppressing these adhesion molecules expressions, and explore the roles of two crucial signal-transduction elements p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa B (NF-κB) in modulating adhesion molecules expressions. METHODS: Neutrophils and BEAS-2B cells (one human bronchial epithelial cell line) were co-cultured, and adhesion molecules expressions on cell surface were detected using flow cytometry. The mRNA levels of adhesion molecules were assessed by real-time quantitative polymerase chain reaction (real-time qPCR). Phosphorylated p38 MAPK and inhibitor κB were analyzed by Western blot. RESULTS: In co-culture system, adhesion molecules expressions on BEAS-2B cells and neutrophils were enhanced significantly (P<0.05). Correspondingly, the mRNA levels of adhesion molecules were also increased greatly. Moreover, the pretreatment of peurarin obviously suppressed adhesion molecules expressions on cell surface. Furthermore, phosphorylated p38 MAPK and inhibitor κB in BEAS-2B cells and neutrophils were elevated in co-culture system, but decreased significantly after upon the treatment of peurarin (P<0.05). CONCLUSIONS: Coculture boosted the interactions between human bronchial epithelial cells and neutrophils mimicking airway inflflammation, whereas peurarin decreased the expression of adhesion molecules on cell surface by suppressing the activities of p38 MAPK and NF-κB pathways, and exhibiting its anti-inflflammation activity.

[Study on MSO/GO-based determination method for trace amount of aqueous Hg2+].

OBJECTIVE: To establish a highly sensitive fluorometric nanobiosensor for determination of aqueous mercury ions (Hg(2+)) using optimized mercury-specific oligonucleotide (MSO) probes and graphene oxide (GO). METHODS: The nanobiosensor was assembled by attaching the self-designed MSO(1) (5' end labeled with fluorophore carboxyfluorescein (FAM), denoted as FAM-MSO(1)) and MSO(2) to the surface of GO through strong non-covalent bonding forces. Upon the addition of Hg(2+), the formation of the T-Hg(2+)-T configuration desorbed the FAM-MSO(1) and MSO(2) from the surface of GO, resulting in a restoration of the fluorescence of FAM-MSO(1). Using the specific mispairing of T-Hg(2+)-T and the changes in fluorescent signals in solutions, quantitative analysis of Hg(2+) could be performed. RESULTS: The average thickness of the prepared GO sheets was only 1.4 nm. For the Hg(2+) nanobiosensor, the optimum concentrations of FAM-MSO(1) and MSO(2) were both 1 µmol/L, the optimum volume of 0.5 g/L GO was 5 µL, and the limit of detection was 10 pmol/L; it had low cross-reactivity with 10 other kinds of non-specific metal ions; the fluorescence recovery efficiency was up to 65% in the re-determination of Hg(2+) after addition of Na(2)S(2)O(3). CONCLUSION: The MSO/GO-based nanobiosensor is convenient to operate, highly sensitive, highly specific, highly accurate, and reusable. It can be applied to determine trace amount of Hg(2+) in aqueous solutions.

[A primary investigation on the isoniazid-induced alterations in efflux gene expression among the isoniazid resistant Mycobacterium tuberculosis clinical isolates].

OBJECTIVE: To detect the changes on the expression of putative drug efflux genes caused by isoniazid-inducement in single resistance to the isoniazid Mycobacterium tuberculosis (M. tuberculosis) clinical isolates, for exploring the putative efflux genes which causing M. tuberculosis isoniazid resistance as well as the mechanism related to high expression of the putative efflux genes. METHODS: We selected 35 M. tuberculosis clinical isolates which were only resistant to isoniazid as well as 10 sensitive M. tuberculosis clinical isolates and using H37Rv as control. Each strain was cultured in 7H9 liquid medium without isoniazid and with subinhibitory isoniazid concentration (1/4 MIC) induction. After RNA extraction and reverse transcription, real-time PCR was conducted to assess the expression changes of 27 putative drug efflux pump genes with formula 2(-ΔΔCT) to calculate the expression of each putative drug efflux pump genes. RESULTS: Of the 27 putative genes, 13 of them were expressed at high level. High expression of Rv1258c gene had the maximum number of 6 strains, followed by high expression of Rv0849 and Rv2265 which both had 5 strains. Fourteen strains (40.00%) out of the 35 strains had high expression pump genes. Six strains (17.14%) had only one highly expressed putative efflux pump gene. Eight strains (22.86%) had two or more highly expressed putative efflux pump genes, including two, four, five, seven genes that highly expressed in 4, 2, 1, 1 strains respectively. For the 27 putative genes, ten sensitive strains and H37Rv did not show highly expressed genes. CONCLUSION: Rv1258c, Rv2265, Rv0849, etc. genes might be the putative efflux pumps genes of M. tuberculosis resistant to isoniazid. Isoniazid might serve as the inducer of M. tuberculosis part putative efflux pump genes, inducing activation and causing high expression of these putative efflux pump genes.

[Mitochondrial tRNAIle A4317G mutation may influence the phenotypic manifestation of deafness-associated 12S rRNA A1555G mutation].

Mitochondrial 12S rRNA A1555G mutation has been associated with both aminoglycoside-induced and nonsyndromic hearing loss. In this report, we performed a clinical and genetic evaluation, and mitochondrial genome analysis of one hearing-impaired Chinese family carrying the A1555G mutation. Strikingly, the penetrances of hearing loss in this family, which were 81% and 66.7%, respectively, when aminoglycoside-induced hearing loss was included or excluded. The penetrances of hearing loss in this family were significantly higher than those in other Chinese families carrying the A1555G mutation. Sequence analysis of their mitochondrial genomes revealed the presence of homoplasmic tRNAIle A4317G mutations and 38 mtDNA polymorphisms belonging to East-Asian haplogroup B4c1b2. Further analysis revealed that other mitochondrial DNA variants were not functional significantly, while the A4317G mutation is localized to a highly conserved nucleotide (conventional site 59) at tRNAIle TΨC loop of tRNAIle. The mutation may alter secondary structure and function of this tRNA, thereby leading to mitochondrial dysfunction. Allelic analysis showed that this mutation was absent in 961 hearing normal Chinese controls. Thus, the altered tRNAIle metabolism by the A4317G mutation may aggravate mitochondrial dysfunction associated with the A1555G mutation, and contribute to the higher penetrance of hearing loss. Therefore, the tRNAIle A4317G mutation may act as a mitochondrial modifier to influence the phenotypic manifestation of the A1555G mutation.

[Mitochondrial 12S rRNA A1555G mutation associated with nonsyndromic hearing loss in twenty-five Han Chinese pedigrees].

Mitochondrial 12S rRNA A1555AG mutation is one of the important causes of aminoglycoside-induced and nonsyndromic deafness. We report here the clinical, genetic and molecular characterization of 25 Chinese families carrying the A1555G mutation.Clinical and genetic characterizations of these Chinese families exhibited a wide range of penetrance, severity and age-at-onset of hearing impairment. The average penetrances of deafness were 28.1% and 21.5%, respectively, when aminoglycoside-induced hearing loss was included or excluded. Furthermore, the average age-of-onset for deafness without aminoglycoside exposure ranged from 1 and 15 years old. Their mitochondrial genomes exhibited distinct sets of polymorphisms including 16 novel variants, belonging to ten Eastern Asian haplogroups A, B, D, F, G, M, N and R, respectively. Strikingly, these Chinese families carrying mitochondrial haplogroup B exhibited higher penetrance and expressivity of hearing loss. In addition, 7 known secondary mutations and 21 variants resided at the highly conservative residues may enhance the penetrace of hearing loss in these Chinese families. Moreover, the absence of mutation in GJB2 gene suggested that GJB2 may not be a modifier for the phenotypic expression of the A1555G mutation in these Chinese families. These observations suggested that mitochondrial haplotypes and other modifiers may modulate the variable penetrance and expressivity of deafness among these Chinese families.

Inhibition of sperm capacitation and fertilizing capacity by adjudin is mediated by chloride and its channels in humans.

STUDY QUESTION: Does adjudin disrupt chloride ion (Cl⁻) ion transport function in human sperm and impede sperm capacitation and fertilizing ability in vitro? SUMMARY ANSWER: In this study the results indicate that adjudin is a potent blocker of Cl⁻ channels: disrupting Cl⁻ ion transport function results in a decline in sperm capacitation and fertilizing ability in humans in vitro. WHAT IS KNOWN ALREADY: Although our previous studies have demonstrated that adjudin exerts its effect by disrupting sertoli-germ cell adhesion junctions, most notably apical ectoplasmic specialization by targeting testin and actin filament bundles that disrupts the actin-based cytoskeleton in sertoli cells, it remains unclear whether adjudin impedes Cl⁻ ion transport function in the human sperm. STUDY DESIGN, SIZE AND DURATION: Semen samples were obtained from 45 fertile men (aged 25-32). Spermatozoa were isolated from the semen in the human tube fluid (HTF) medium by centrifugation through a discontinuous Percoll gradient, and incubated with adjudin at 10 nM-10 µM and/or other reagents under capacitating conditions for 0-5 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated the effect of adjudin and different reagents on sperm functions with which they were incubated at 37 °C. Sperm motility and hyperactivation were analyzed by a computer-assisted sperm analysis (CASA) system. Sperm capacitation and the acrosome reaction were assessed by chlortetracycline fluorescence staining. Sperm fertilizing ability was evaluated by sperm penetration of zona-free hamster egg assay, and cellular cAMP levels in spermatozoa were quantified by the EIA kit. The proteins tyrosine, serine and threonine phosphorylation in the presence or absence of adjudin were analyzed by means of a immunodetection of spermatozoa, especially, compared the effect of adjudin on sperm hyperactivation and capacitation in the complete HTF medium with the Cl⁻-deficient HTF medium as well as the various Cl⁻ channel blockers. MAIN RESULTS AND THE ROLE OF CHANCE: Adjudin significantly inhibited sperm hyperactivation but not sperm motility. Adjudin-induced inhibition of sperm capacitation was reversible, and it was found to block the rhuZP3ß- and progesterone-induced acrosome reaction in a dose-dependent manner. Adjudin also blocked sperm penetration of zona-free hamster eggs, and significantly inhibited both forskolin-activated transmembrane adenylyl cyclase and soluble adenylyl cyclase activities leading to a significant decline in the cellular cAMP levels in human spermatozoa. Adjudin failed to reduce sperm protein tyrosine phosphorylation but it did prevent sperm serine and threonine protein phosphorylation. Interestingly, adjudin was found to exert its inhibitory effects on sperm capacitation and capacitation-associated events only in the complete Cl⁻-HTF medium but not Cl⁻-deficient medium, illustrating the likely involvement of Cl⁻. Adjudin inhibits the fertility capacity of human sperm is mediated by disrupting chloride ion and its transport function. LIMITATIONS, REASONS FOR CAUTION: This study has examined the effect of adjudin only on human sperm capacitation and fertilizing ability in vitro and thus has some limitations. Further investigations in vivo are needed to confirm adjudin is a potent male contraceptive. WIDER IMPLICATIONS OF THE FINDINGS: Our studies demonstrated that adjudin inhibition of capacitation is reversible and its toxicity is low, opening the door for the examination of adjudin as a mediator of male fertility control. Adjudin may be a safe, efficient and reversible male antifertility agent and applicable to initial clinical trials of adjudin as a male antifertility agent in humans. STUDING FUNDING/COMPETING INTEREST(S): This work was supported by the National Basic Research Program of China (2006CB504002), the Nature Science Foundation of China (Nos. 81000244 and 81170554), Zhejiang Project of Science and Technology (2011C23046), the Nature Science Fund of Zhejiang province (Nos.Y2100058 and Y2090236), the key Science and Technology Innovation Team of Zhejiang Province (No.2012R10048-07) and the National Institutes of Health (NICHD U54 HD029990 project 5), USA. The authors declare no conflict of interest.

Brief Report: Single-nucleotide polymorphisms in VKORC1 are risk factors for systemic lupus erythematosus in Asians.

OBJECTIVE: The increased risk of thrombosis in systemic lupus erythematosus (SLE) may be partially explained by interrelated genetic pathways for thrombosis and SLE. The present study was undertaken to investigate whether 33 established and novel single-nucleotide polymorphisms (SNPs) in 20 genes involved in hemostasis pathways that have been associated with deep venous thrombosis (DVT) in the general population are risk factors for SLE among Asian subjects. METHODS: Patients in the discovery cohort were enrolled in 1 of 2 North American SLE cohorts. Patients in the replication cohort were enrolled in 1 of 4 Asian or 2 North American cohorts. We first genotyped 263 Asian patients with SLE and 357 healthy Asian control subjects for 33 SNPs in the discovery phase, and then genotyped 5 SNPs in up to an additional 1,496 patients and 993 controls in the replication phase. Patients were compared to controls for bivariate association with minor alleles. Principal components analysis was used to control for intra-Asian ancestry in the replication cohort. RESULTS: Two genetic variants in the gene VKORC1 were highly significant in both the discovery and replication cohorts: rs9934438 (in the discovery cohort, odds ratio [OR] 2.45, P=2×10(-9); in the replication cohort, OR 1.54, P=4×10(-6)) and rs9923231 (in the discovery cohort, OR 2.40, P=6×10(-9); in the replication cohort, OR 1.53, P=5×10(-6)). These associations were significant in the replication cohort after adjustment for intra-Asian ancestry: for rs9934438, OR 1.34, P=0.0029; for rs9923231, OR 1.34, P=0.0032. CONCLUSION: Genetic variants in VKORC1, which are involved in vitamin K reduction and associated with DVT, correlate with SLE development in Asian subjects. These results suggest that there may be intersecting genetic pathways for the development of SLE and thrombosis.

Aptasensors for rapid detection of Escherichia coli O157:H7 and Salmonella typhimurium.

Herein we reported the development of aptamer-based biosensors (aptasensors) based on label-free aptamers and gold nanoparticles (AuNPs) for detection of Escherichia coli (E. coli) O157:H7 and Salmonella typhimurium. Target bacteria binding aptamers are adsorbed on the surface of unmodified AuNPs to capture target bacteria, and the detection was accomplished by target bacteria-induced aggregation of the aptasensor which is associated as red-to-purple color change upon high-salt conditions. By employing anti-E. coli O157:H7 aptamer and anti-S. typhimurium aptamer, we developed a convenient and rapid approach that could selectively detect bacteria without specialized instrumentation and pretreatment steps such as cell lysis. The aptasensor could detect as low as 105colony-forming units (CFU)/ml target bacteria within 20 min or less and its specificity was 100%. This novel method has a great potential application in rapid detection of bacteria in the near future.

[Detection of mercury (II) ions (Hg(2+)) in water by a nanobiosensor].

OBJECTIVE: To develop a nanobiosensor for rapid colorimetric detecting Mercury (II) Ions (Hg(2+)) in water by mercury-specific oligonucleotides (MSOs) probe and gold nanoparticles. METHODS: The nanobiosensor was assembled by adsorbing the optimized MSOs on the surface of gold nanoparticles. A direct colorimetric probe of Hg(2+) which relied on the T-T mismatches in DNA duplexes was used to selectively and strongly capture Hg(2+). Hg(2+) induces the aggregation of gold nanoparticles with appropriate amount of salts, resulting the color change (red to blue). RESULTS: The diameter and concentration of the gold nanoparticle preparation were 15 nm and 2.97 nmol/L, respectively. Truncated MSOs (9 bp) showed the similar Hg(2+)-binding activity. The optimum concentration of the NaNO3 solution was 0.5 mol/L. The nanobiosensor could detect Hg(2+)in a range of 10 ∼ 1000 µmol/L within few minutes and the specificity was 100%. CONCLUSION: A new nanobiosensor is developed successfully for rapid colorimetric detecting Hg(2+) in water, avoiding either MSOs labeling or gold nanoparticles modification. This technique is simple, convenient and rapid detecting method with high sensitivity and specificity.

[Spectrum and frequency of mitochondrial 12S rRNA variants in the Chinese subjects with nonsynrdomic hearing loss in Zhejiang Province].

Mitochondrial DNA (mtDNA) mutations are one of the important causes of deafness. In particular, the 12S rRNA gene is the hot spots for mutations associated with both aminoglycoside ototoxicity and nonsyndromic deafness. In this report, a total of 318 Chinese pediatric hearing-impaired subjects were recruited from otology clinics in the Zhejiang Province, China. These subjects underwent clinical, genetic evaluation and molecular analysis of 12S rRNA gene. Mutational analysis identified 34 variants in the 12S rRNA gene in this cohort. The incidences of the known deafness-associated 1555A>G, 1494C>T and 1095T>C mutations were 9.1%, 0.6% and 1.25% in this cohort, respectively. Other mtDNA variants were evaluated by structural and phylogenetic analysis. Of these, the 839A>G and 1452T>C variants could confer increased sensitivity to aminoglycosides or nonsyndromic deafness as they were not present in 449 Chinese controls and localized at highly conserved nucleotides of the 12S rRNA. However, other variants appeared to be polymorphisms. These data further support the idea that mitochondrial 12S rRNA is one of major targets for aminoglycoside ototoxicity. These data have been providing valuable information to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside antibiotic therapy, and eventually to decrease the incidence of deafness.

[Mutations in mitochondrial DNA associated with hypertension].

Mutations in mitochondrial DNA (mtDNA) are one of the molecular bases of hypertension. Among these, the tRNAMet A4435G, tRNAMet/tRNAGln A4401G, tRNAIle A4263G, T4291C and A4295G mutations have been reported to be associated with essential hypertension. These mutations alter the structure of the corresponding mitochondrial tRNAs and cause failures in tRNA metabolism. These shortages of these tRNAs lead to an impairment of mitochondrial protein synthesis and a failure in the oxidative phosphorylation function. These result in a deficit in ATP synthesis and an increase of generation of reactive oxygen species. As a result, these mitochondrial dysfunctions may contribute to the development of hypertension. Furthermore, the tissue specificity of these pathogenic mtDNA mutations might be associated with tRNA metabolism and nuclear modifier genes. These mtDNA mutations should be considered as inherited risk factors for future molecular diagnosis. Thus, these findings provide new insights into the molecular mechanism, management and treatment of maternally inherited hypertension. This review summarized the association between mtDNA mutations and hypertension.

Gene expression using the PALM system.

The PALM Robot MicroBeam laser microdissection system can isolate specified cells from complex tissues section, in a rapid and precise manner. Combined with other methods, PALM may be used for gene expression elucidating the role of specialized cell type in physiological and pathological activity. This chapter describes the application of the PALM MicroBeam system to isolate RNA from cells in a complex tissue for subsequent gene expression analysis. Protocols show the steps from preparation of tissue samples to the final quantitative results. The process is articulated in several steps, each of which requires optimal choices in order to obtain reliable data from a limited number of cells (500-10,000 cells). Furthermore, the notes regarding tissue preparation, microdissection of the interested cells, are also emphasized.

[HIV-1 infection affects the expression of host cell factor TSG101 and Alix].

To investigate the effects of HIV-1 infection on the expression of host factors TSG101 (Tumor Susceptibility Gene 101) and Alix (ALG-2-interacting protein X). HIV-1 infectious clone pNL4-3 was used to infect TZM-bl, PM1, Jurkat cell lines and human peripheral blood mononuclear cells (PBMC). Twenty-four hours post-infection, the infected or uninfected cells were harvested respectively for extraction of total RNAs and total cellular proteins, which were subsequently used in RT-PCR and Western-blotting respectively to quantify TSG101 and Alix, respectively. Our data showed that HIV-1 infection resulted in various influences on the expression of TSG101 and Alix in the cell lines and the primary PBMC. A down-regulation was mainly observed in the cell lines, whereas an up-regulation of TSG101 was identified in primary PBMC. Three patterns were observed for down-regulation, including dual down-regulation of TSG101 and Alix for Jurkat cells, single down-regulation of Alix for TZM-bl cells and marginal or no influence on PM1 cells. The dual down-regulation of Alix and TSG101 in Jurkat cells coincided with less expression of HIV-1 p24 protein. This is the first-line evidence that HIV-1 infection affects the expression of host factors TSG101 and Alix, the down-regulation of these molecules may influence the HIV-1 replication. The underlying mechanism remains to be addressed.

[Effect of hepatocyte growth factor gene transfection on biological features of lymphoma cells].

OBJECTIVE: To investigate the biological effect of hepatocyte growth factor (HGF) on HGF gene-transfected Raji cells. METHODS: Total RNA was extracted from human hepatic tissue, HGF gene cDNA was amplified by RT-PCR, and then cloned into vector pVITRO2-mcs to construct recombinant eukaryotic expression vector pVITRO2-mcs-HGF. The recombinant vector was transfected to Raji cells, and the stably transfected cells were selected by homomycin B in serial passages, and confirmed by real-time fluorescent quantitative PCR, ELISA, immunocytohistochemistry. The biological features of transfected Raji cells were evaluated by semisolid culture. RESULTS: RT-PCR results showed that Raji cells were transfected successfully with recombinant eukaryotic expression vector pVITRO2-mcs-HGF. HGF mRNA and protein were expressed successfully in Raji cells. Expression of HGF gene enhanced proliferation, metastasis and invasion of Raji cells. CONCLUSION: HGF gene has been cloned and recombined to construct recombinant eukaryotic expression vector pVITRO2-mcs-HGF successfully. Transfected HGF may change the biological features of Raji cells.

CFTR is essential for sperm fertilizing capacity and is correlated with sperm quality in humans.

BACKGROUND: Our previous studies have demonstrated the cystic fibrosis transmembrane conductance regulator (CFTR) is important for capacitation and male fertility in mouse and guinea pig spermatozoa. However, the exact function of CFTR on human sperm fertilizing capacity, and correlation with sperm quality has not been established. The present study may shed light on some unexplained male infertility, and on a possible new method for diagnosis of male infertility and strategy for male contraception. METHODS: To assess the effect of CFTR on human sperm fertilizing capacity, we examined sperm capacitation and the acrosome reaction using chlortetracycline staining, analyzed sperm hyperactivation by computer-assisted semen analysis (CASA), measured intracellular cAMP levels using ElA and evaluated sperm penetration of zona-free hamster eggs assay in fertile men. The percentage of spermatozoa expressing CFTR from fertile, healthy and infertile men (mainly teratospermic, asthenoteratospermic, asthenospermic and oligospermic) was conducted by indirect immunofluorescence staining. RESULTS: Progesterone significantly facilitated human sperm capacitation and ZP3 triggered the acrosome reaction, both were significantly inhibited by CFTR inhibitor-172 (CFTRinh-172; 10 nM-1 microM) in a dose-dependent manner. The presence of 100 nM CFTRinh-172 markedly depressed intracellular cAMP levels, sperm hyperactivation and sperm penetration of zona-free hamster eggs. In addition, the percentage of spermatozoa expressing CFTR in the fertile men was significantly higher than healthy and infertile men categories (P < 0.01). CONCLUSIONS: CFTR is essential for human sperm fertilizing capacity and the impairment of CFTR expression in spermatozoa is correlated with a reduction of sperm quality. These results suggest that defective expression of CFTR in human sperm may lead to the reduction of sperm fertilizing capacity.

[Mitochondrial D-Loop gene polymorphisms in the patients with type 2 diabetes mellitus].

Polymerase chain reaction (PCR) and direct nucleotide sequencing were used to analyze the mitochondrial D-loop gene of 199 Zhejiang patients with T2DM and 102 controls and the relationship between D-Loop gene variations and the main clinical symptoms. The mitochondrial D-Loop gene was a hypervariable area and np73A-G, np263A-G, np16223C-T, and np16519T-C were four high variations, and 29 unreported new variations were found. np193A-G, np234A-G, and np16108C-T were related to diabetes mellitus with family history. These results showed that there are various forms of polymorphism in mitochondrial DNA D-Loop gene in a Zhejiang population, some of which are related to diabetes mellitus.