Relationship Between Drug Resistance Characteristics and Biofilm Formation in Klebsiella Pneumoniae Strains.

Objective: To conduct epidemiological analysis of Klebsiella pneumoniae (K. pneumoniae) with hypervirulence, and to investigate its drug resistance phenotype, Extended-spectrum ß-lactamase (ESBLs) gene, virulence factor, capsular serotype and biofilm formation, so as to provide theoretical basis for further understanding of the drug resistance mechanism of K. pneumoniae with hypervirulence. Methods: K. Pneumoniae were isolated from clinical samples collected from inpatients. All strains were identified by VITEK2 Compact using fully automatic microbial analyzer, the minimal inhibitory concentration (MIC) of antibiotics was determined by microbroth dilution test. The double disk diffusion method was used to detect the production of ESBLs, modified carbapenem inactivation method (mCIM) was used to detect the production of carbapenemase, and hypermucoviscosity phenotype was detected by wire drawing test. PCR was used to detect ESBLs gene, virulence factor and capsular serotype. Crystal violet staining was used to detect the ability of biofilm formation. Results: The ESBLs genes detected in this study included strains blaTEM 35 (36.5%), blaSHV 51 (53.1%), and blaCTX-M 49 (51.0%). Most strains carried multiple ESBLs genes, but not all of them produce ESBLs. K1 and K2 accounted for 14.6% and 11.5% respectively. Most (91.7%) strains carried the fimH gene, and the other virulence genes were ybtS (53.1%), entB (46.9%), rmpA (41.7%), aerobactin (32.3%), allS (15.6%), kfu (15.6%). Of all the Klebsiella pneumoniae strains, 33 (34.4%) exhibited ESBLs phenotype, 16 (16.7%) were carbapenemase-producing, and 20 (20.8%) with ESBLs phenotype tested were resistant to all four drugs. The correlation between ESBLs-producing strains and biofilm formation was significantly increased compared to strains without ESBLs phenotype (P=0.035). Conclusion: Compared to hypervirulent Klebsiella pneumoniae (hvKP), classical Klebsiella pneumoniae (cKP) has a tendency to acquire antibiotic resistance. Our study showed that genes encoding rmpA, K1 or K2, and kfu were highly associated with hvKP.

Clinical application of serum tumor abnormal protein combined with tumor markers in lung cancer patients.

Aim: To explore the clinical application of tumor abnormal protein (TAP) combined with tumor markers in the diagnosis of lung cancer. Methods: Samples from 248 lung cancer patients and 59 patients with benign lung diseases were tested for TAP and tumor markers pro-gastrin-releasing peptide, carcinoembryonic antigen, NSE, CYFRA 21-1 and CA72-4. Results: The sensitivity of TAP and CYFRA 21-1 in the lung cancer group was significantly higher than that of the other indexes. TAP combined with NSE and CYFRA 21-1 or combined with NSE, CYFRA 21-1 and squamous cell carcinoma antigen detection could reduce detection indicators under the premise it does not reduce the sensitivity and accuracy of lung cancer diagnosis, and at the same time could improve the specificity, positive predictive value and positive likelihood ratio of detection. Conclusion: TAP detection represents a promising diagnostic tool. It is also suggested that combination with established tumor markers and comprehensive judgment could improve the accuracy of lung cancer auxiliary diagnosis.

The presence of tumor abnormal protein (TAP) is closely related to the development and progression of many cancers. During metabolism, cancer cells can release complicated abnormal glycoproteins as well as calcium histone proteins which constitute TAP. In essence, TAP results from the glycosylation changes related to cancer cells. In this study, TAP and tumor markers were assessed for their diagnostic value in lung cancer. The serum levels of TAP, pro-gastrin-releasing peptide, carcinoembryonic antigen, CYFRA 21-1 and CA72-4 in the patients with lung cancer were significantly higher than in those with benign lung diseases. TAP combined with CYFRA 21-1 and CA72-4 or with CYFRA 21-1, CA72-4 and squamous cell carcinoma antigen could further improve the sensitivity of lung cancer diagnosis and is suitable for lung cancer screening in both normal and high-risk populations.

Transcriptomic profiling of chemical exposure reveals roles of Yap1 in protecting yeast cells from oxidative and other types of stresses.

Transcriptomic profiles are generated by comparing wild-type and the yeast yap1 mutant to various chemicals in an attempt to establish a correlation between this gene mutation and chemical exposure. Test chemicals include ClonNAT as a non-genotoxic agent, methyl methanesulphonate (MMS) as an alkylating agent, tert-butyl hydroperoxide (t-BHP) as an oxidative agent and the mixture of t-BHP and MMS to reflect complex natural exposure. Differentially expressed genes (DEGs) were identified and specific DEGs were obtained by excluding overlapping DEGs with the control group. In the MMS exposure group, deoxyribonucleotide biosynthetic processes were upregulated, while oxidation-reduction processes were downregulated. In the t-BHP exposure group, metabolic processes were upregulated while peroxisome and ion transport pathways were downregulated. In the mixture exposure group, the proteasome pathway was upregulated, while the aerobic respiration was downregulated. Homologue analysis of DEGs related to human diseases showed that many of DEGs were linked to cancer, ageing and neuronal degeneration. These observations confirm that the yap1 mutant is more sensitive to chemicals than wild-type cells and that the susceptible individuals carrying the YAP1-like gene defect may enhance risk to chemical exposure. Hence, this study offers a novel approach to environmental risk assessment, based on the genetic backgrounds of susceptible individuals.

RNA sequencing provides insights into the toxicogenomic response of ZF4 cells to methyl methanesulfonate.

Whole genome transcriptomic studies are powerful for characterizing the molecular mechanisms underlying the physiological effects of chemicals, and are informative for environmental health risk assessment. Alkylating agents are an abundant class of chemicals that can damage DNA in the environment, and are used for anticancer treatments. Currently, little is known regarding the molecular mechanisms of toxic alkylating agents in zebrafish cell lines. In this study, RNA-sequencing was used to investigate the transcriptomic responses of zebrafish ZF4 cells following exposure to the model genotoxicant methyl methanesulfonate (MMS). The half-maximal inhibitory concentration (IC50 ) of MMS was 639.16 ± 61.8 µm, and apoptosis was induced within 24 h of exposure. RNA sequencing identified 3601 differentially expressed genes (DEGs) that were upregulated and 3037 that were downregulated. Gene ontology enrichment analysis revealed that most DEGs belonged to synthesis and metabolism categories. RNA-associated processes were the most upregulated, while cell cycle and adhesion were the most repressed processes, and neuron-related processes were the most downregulated developmental process. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis identified DNA damage repair, cell cycle, apoptosis and spliceosome as overrepresented terms. Six types of alternative splicing were detected. In total, 1156 alternative splicing DEGs were specifically expressed following MMS treatment, many of which belonged to metabolism and catabolic process categories. Cluster analysis of orthologs was able to extrapolate toxicotranscriptomic data between zebrafish and yeast. These results provide insight into the genome-wide response of ZF4 cells following exposure to MMS, and this knowledge will inform future toxicogenomic data analysis and environmental health risk assessment.

Assessment of the toxicity of CuO nanoparticles by using Saccharomyces cerevisiae mutants with multiple genes deleted.

To develop applicable and susceptible models to evaluate the toxicity of nanoparticles, the antimicrobial effects of CuO nanoparticles (CuO-NPs) on various Saccharomyces cerevisiae (S. cerevisiae) strains (wild type, single-gene-deleted mutants, and multiple-gene-deleted mutants) were determined and compared. Further experiments were also conducted to analyze the mechanisms associated with toxicity using copper salt, bulk CuO (bCuO), carbon-shelled copper nanoparticles (C/Cu-NPs), and carbon nanoparticles (C-NPs) for comparisons. The results indicated that the growth inhibition rates of CuO-NPs for the wild-type and the single-gene-deleted strains were comparable, while for the multiple-gene deletion mutant, significantly higher toxicity was observed (P < 0.05). When the toxicity of the CuO-NPs to yeast cells was compared with the toxicities of copper salt and bCuO, we concluded that the toxicity of CuO-NPs should be attributed to soluble copper rather than to the nanoparticles. The striking difference in adverse effects of C-NPs and C/Cu-NPs with equivalent surface areas also proved this. A toxicity assay revealed that the multiple-gene-deleted mutant was significantly more sensitive to CuO-NPs than the wild type. Specifically, compared with the wild-type strain, copper was readily taken up by mutant strains when cell permeability genes were knocked out, and the mutants with deletions of genes regulated under oxidative stress (OS) were likely producing more reactive oxygen species (ROS). Hence, as mechanism-based gene inactivation could increase the susceptibility of yeast, the multiple-gene-deleted mutants should be improved model organisms to investigate the toxicity of nanoparticles.

Yeast Surface Display of Capsid Protein VP7 of Grass Carp Reovirus: Fundamental Investigation for the Development of Vaccine Against Hemorrhagic Disease.

VP7, an outer capsid protein of grass carp reovirus (GCRV), was expressed and displayed on the surface of Saccharomyces cerevisiae for developing an efficient vaccine against hemorrhagic disease of grass carp. The result of flow cytometry analysis indicated that protein VP7 could be displayed on the surface of yeast cells after inducing with galactose. The expression of VP7 was confirmed by western blot analysis and further visualized with confocal microscopy. The specific antibodies against VP7 generated from mice were detectable from all immune groups except the control group, which was immunized with untransformed yeast cells. The displaying VP7 on glycosylation-deficient strain EBYΔMnn9 was detected to induce a relatively low level of specific antibody amongst the three strains. However, the antiserum of EBYΔM9-VP7 showed relative high capacity to neutralize GCRV. Further neutralization testing assays indicated that the neutralizing ability of antiserum of the EBYΔM9-VP7 group appeared concentration dependent, and could be up to 66.7% when the antiserum was diluted to 1:50. This result indicates that appropriate gene modification of glycosylation in a yeast strain has essential effect on the immunogenicity of a yeast-based vaccine.

[Subcellular localization of ataxin-3 and its effect on the morphology of cytoplasmic organoids].

OBJECTIVE: To explore the subcellular localization of ataxin-3 and the effect of polyglutamine (polyQ) expansion mutation on the morphology of mitochondrion, golgi apparatus and endoplasmic reticulum. METHODS: Transient transfection was employed to build cell models expressing wild-type or mutant ataxin-3 proteins. Indirect immunofluorescence was applied to identify markers of organelle membrane. The results were observed under a laser scanning confocal microscope. RESULTS: No co-localization was observed for ataxin-3 protein and mitochondrial marker TOM20, but the percentage of cells with mitochondrial fragmentation has increased in cells expressing mutant ataxin-3 (P<0.05). No co-localization was observed for ataxin-3 protein and golgi marker GM130, and mutant ataxin-3 did not cause golgi fragmentation. Wide type and polyQ-expanded ataxin-3 both showed partial co-localization with ER marker calnexin. The latter showed more overlap with calnexin, and the overlapping signals were mostly located in the places where aggregates were situated. CONCLUSION: PolyQ-expanded ataxin-3 protein may indirectly affect the integrity of mitochondria, but may cause no effect on the structure and functions of golgi apparatus. Endoplasmic reticulum may be another place where extended ataxin-3 protein can induce cytotoxicity in addition to the nucleus.

Investigation of effect of 17α-ethinylestradiol on vigilin expression using an isolated recombinant antibody.

Vigilin, a highly conserved protein from yeast to mammals, is a multifunctional protein in eukaryotic organisms. One biological function of vigilin is to stabilize the expression level of vitellogenin (VTG). This study aimed to develop vigilin as a new estrogen-inducible biomarker that correlates with the widely applied estrogen-inducible biomarker VTG and expand the ability to detect it in various species. Here, a recombinant monoclonal antibody with high specificity against the conserved C-terminal region of vigilin from zebrafish (Danio rerio) was successfully isolated from a phage display antibody library and found to recognize vigilin proteins from multiple vertebrate species. The effect of 17α-ethinylestradiol (EE2) on vigilin expression in the liver of zebrafish and juvenile crucian carp (Carassius auratus) was investigated. Although vigilin mRNA was expressed in all tissues analyzed from male zebrafish, vigilin protein was detected exclusively in the testis of male zebrafish, as well as the liver of female zebrafish and juvenile crucian carp at a lower level without exposure to EE2. Significant induction of vigilin mRNA by exposure to EE2 was observed in the liver and testis of male zebrafish, even at a low dose of 6.25 ng/L (21.09 pmol/L). In Hela cells, the expression of vigilin coincided with high protein synthesis activity but not dose-dependently by EE2 exposure. Therefore, the recombinant antibody may be used as a detection tool to screen for mammalian cell lines or organs with estrogen-inducible expression of vigilin.

Quantification of GFP signals by fluorescent microscopy and flow cytometry.

Green fluorescent protein (GFP) is an ideal reporter in in vivo studies. Flow cytometry and fluorescent microscopy are two conventional tools to detect the GFP signal; flow cytometry is an effective and sensitive technique to quantitatively analyze fluorescent intensity, while fluorescent microscopy can visualize the subcellular location and expression of GFP. In this chapter, we describe a method using GFP as a reporter under the control of a target gene promoter. The system allows measurement of the levels of target gene expression by both fluorescent microscopy and flow cytometry. This method can be applied not only to dissect the target gene promoter but also as a sensor to detect environmental pollutants.

Distribution of vitellogenin in zebrafish (Danio rerio) tissues for biomarker analysis.

Vitellogenin (VTG), the precursor of yolk proteins, is a sensitive biomarker of estrogenic contamination in aquatic environments. Traditionally, VTG was believed to be synthesized under the control of estrogen in the livers of mature females and then secreted into the blood, before being taken up by the ovaries and cleaved into lipovitellin and phosvitin, which provide nutrition for developing embryos. However, recent studies have reported that the liver is not the only tissue to express VTG and this has led to questions over the precise tissue distribution of VTG in zebrafish. Moreover, studies in zebrafish on the expression of the VTG protein are rare. Using Western blotting and reverse-transcriptase polymerase chain reaction, this present study reports that the VTG protein and VTG1 mRNA were detected not only in the liver, but also in various extrahepatic tissues, including the heart, spleen, kidney, skin, muscle, gill, eye and brain tissues, of female and 17α-ethinylestradiol (EE2)-treated male zebrafish. Due to the high expression levels of VTG and the ease of taking samples, skin and eye tissues were chosen to evaluate the effects of varying doses and exposure times of EE2 on male zebrafish. The VTG gene and protein were induced by EE2 exposure in liver, skin and eye tissues of male zebrafish in dose- and time-dependent patterns. Therefore, we suggest that zebrafish skin and eye tissues may be alternatives to plasma and liver tissues for VTG biomarker analysis.

Selection and characterization of single-chain recombinant antibodies against spring viraemia of carp virus from mouse phage display library.

Antibody-displaying phage library was selected after three rounds of panning against spring viraemia of carp virus (SVCV) by phage display technology. Eight positive clones which could produce soluble single-chain fragment variable (scFv) antibody induced by isopropyl-beta-d-thiogalactopyranoside (IPTG) were obtained. Dot blot results showed that the eight scFv antibodies could recognize SVCV. The soluble scFv antibodies showed a molecular weight 29 kD by Western blot. All scFv antibodies could recognize SVCV proteins specifically without cross-reaction with other virus proteins by ELISA. Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the SVCV-infected cells. These scFv antibodies will be useful tools to establish immunological detection methods for SVCV.

Risk assessment of xenoestrogens in a typical domestic sewage-holding lake in China.

Release of domestic sewage leads to accumulation of xenoestrogens in holding waters, especially in closed or semi-enclosed waters such as lakes. In the study, the occurrence, distribution, estrogenic activity and risk of eight xenoestreogens were evaluated in Lake Donghu, China. Nonylphenol (NP), octylphenol (OP), and bisphenol A (BPA) were identified as the main xenoestrogens ranging from tens of ngL(-1) (in the surface water) or ng g(-1)dw (in the suspended particles and sediment) to tens of µgL(-1) or µg g(-1)dw. The sum of 17ß-estradiol equivalents (∑EEQs) ranged from 0.32 to 45.02 ngL(-1) in the surface water, 0.53 to 71.86 ng g(-1)dw in the suspended particles, and 0.09 to 24.73 ng g(-1)dw in the sediment. Diethylstilbestrol (DES) was determined as the main contributor to ∑EEQs followed by NP. The risk assessment showed a higher risk in the surface water than in the suspended particles and sediment in such domestic sewage-holding lake.

Sulforaphane inhibits CYP1A1 activity and promotes genotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro.

Increasing environmental pollution by carcinogens such as some of persistent organic pollutants (POPs) has prompted growing interest in searching for chemopreventive compounds which are readily obtainable. Sulforaphane (SFN) is isolated from cruciferous vegetables and has the potentials to reduce carcinogenesis through various pathways. In this study, we studied the effects of SFN on CYP1A1 activity and genotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results showed that SFN inhibited TCDD-induced CYP1A1 activity in H4IIE cells by directly inhibiting CYP1A1 activity, probably through binding to aryl hydrocarbon receptor and/or CYP1A1 revealed by molecular docking. However, SFN promoted TCDD-induced DNA damage in yeast cells and reduced the viability of initiated yeast cells. Besides, it is surprising that SFN also failed to reduce genotoxicity induced by other genotoxic reagents which possess different mechanisms to lead to DNA damage. Currently, it is difficult to predict whether SFN has the potentials to reduce the risk of TCDD based on the conflicting observations in the study. Therefore, further studies should be urgent to reveal the function and mechanism of SFN in the stress of such POPs on human health.

Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation.

In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos. A T7 phage display cDNA library was constructed from zebrafish tailbud embryos and used to identify the antigens potentially recognized by scFv1, which showed the highest frequency and strongest binding against the tailbud embryos. We acquired 4 candidate epitopes using scFv1 and the corresponding genes showed significantly higher expression levels at tailbud stage than at 30%-epiboly. The most potent epitope of scFv1 was the clone scFv1-2, which showed strong homology to zebrafish myristoylated alanine-rich C-kinase substrate b (Marcksb). Western blot analysis confirmed the high expression of marcksb in the post-gastrula embryos, and the endogenous expression of Marcksb was interfered by injection of scFv1. Zebrafish marcksb showed dynamic expression patterns during embryonic development. Knockdown of marcksb strongly affected gastrulation movements. Moreover, we revealed that zebrafish marcksb is required for cell membrane protrusion and F-actin alignment. Thus, our study uncovered 4 types of scFvs binding to zebrafish post-gastrula embryos, and the epitope of scFv1 was found to be required for normal gastrulation of zebrafish. To our knowledge, this was the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates, and we were able to identify zebrafish marcksb that was required for gastrulation.

Construction and evaluation of two biosensors based on yeast transcriptional response to genotoxic chemicals.

It has been well established that essentially all microbial mutagens are rodent carcinogens, yet current mutagen detection systems are limited by their detection sensitivity. Here we report the construction of a pair of hypersensitive biosensors by optimizing both reporters and the host strain. The resulting RNR3-yEGFP and HUG1-yEGFP reporters and the septuple yeast mutant in combination with the automated protocol not only remarkably enhance the detection sensitivity, but also allow a high throughput screen of environmental genotoxins. This system is deemed much more sensitive than similar yeast and bacterium-based tests for all selected chemicals examined in this study.

Antigenic analysis of grass carp reovirus using single-chain variable fragment antibody against IgM from Ctenopharyngodon idella.

Grass carp (Ctenopharyngodon idella) is an important species of freshwater aquaculture fish in China. However, grass carp reovirus (GCRV) can cause fatal hemorrhagic disease in yearling populations. Until now, a strategy to define the antigenic capacity of the virus's structural proteins for preparing an effective vaccine has not been available. In this study, some single-chain variable fragment antibodies (scFv), which could specifically recognize grass carp IgM, were selected from a constructed mouse naïve antibody phage display cDNA library. The identified scFv C1B3 clone was shown to possess relatively higher specific binding activity to grass carp IgM. Furthermore, ELISA analysis indicated that the IgM level in serum from virus-infected grass carp was more than two times higher than that of the control group at 5-7 days post infection. Moreover, Western blot analysis demonstrated that the outer capsid protein VP7 has a specific immuno-binding-reaction with the serum IgM from virus-infected grass carp. Our results suggest that VP7 can induce a stronger immune response in grass carp than the other GCRV structural proteins, which implies that VP7 protein could be used as a preferred immunogen for vaccine design.

Molecular cloning of grass carp (Ctenopharyngodon idellus) T-bet and GATA-3, and their expression profiles with IFN-γ in response to grass carp reovirus (GCRV) infection.

Both T-bet and GATA-3, Th1/Th2 lineage-specific transcription factors, play important roles in the development of T cells and Th1/Th2 differentiation. In this study, T-bet and GATA-3 genes were cloned from grass carp (Ctenopharyngodon idellus). The putative primary structure of the polypeptide deduced from the cDNA sequence of grass carp T-bet contained 608 aa, which possessed a T-box DNA binding domain. The putative primary structure of the polypeptide deduced from the cDNA sequence of grass carp GATA-3 contained 396 aa, which possessed two consensus zinc finger domains (C-X(2)-C-X(17)-C-X(2)-C). The YxKxHxxxRP motif, KRRLSA and LMEKs/n sequences were also conserved in this GATA-3. Phylogenetic analysis indicated that grass carp T-bet and GATA-3 group with their known counterparts with zebrafish T-bet and GATA-3 as the closest neighbor, respectively. RT-qPCR results showed that grass carp T-bet gene was highly expressed in head kidney, followed by spleen, and low expressed in gill, liver, kidney, and intestine, while GATA-3 gene was highly expressed in intestine, followed by spleen, and low expressed in gill, liver, kidney, and head kidney. Grass carp is one of the "four important domestic fish" in China and often infected by grass carp reovirus (GCRV). As yet, there is no evidence that T-bet and GATA-3 (Th1/Th2 subsets) are involved in anti-virus immune of teleost fish. In this study, by RT-qPCR, we analyzed the expression dynamics of grass carp T-bet and GATA-3 genes with IFN-γ gene in response to GCRV infection for the first time. The expression dynamics showed that three genes might be crucially modulated by in vivo GCRV infection: (1) GCRV mainly induced a T-bet expression profile comparing to the GATA-3 expression, while the higher expression profiles of IFN-γ correlated with the up-regulation of T-bet; (2) T-bet/IFN-γ and GATA-3 expression changes suggest that in GCRV-infected grass carp, the common immune state of head kidney further heightens, whereas the common physiological state of intestine transforms to an anti-virus immune state. From this finding, we realize that GCRV mainly induces a Th1 response, and Th1 cell-mediated recognition mechanisms play very important roles in anti-virus cellular immune of grass carp.

[Genesis, development and application prospect of antibody library: a review].

Antibodies are immunoglobulins specifically introduced by immunity response of high animals, with the responsibility for recognising and cleaning out specific antigens. Antibody is not only a powerful weapon against pathogen invasion in the organism, but also a tool for specific molecular recognition used in basic scientific research. The diversity of antibody molecules resulted in the concept of antibody library; each individual animal is a natural antibody library. In the post-genome era, in order to fit various "omics", especially for proteomics requirement of high throughput technology, some gene engineering antibody libraries and antibody alternative libraries have been constructed based on phage display technology. Yet, more and more in vitro display systems such as ribosome display, mRNA display have been used for antibody library study, and that present more advantages than phage display. This mini review outlines the genesis, development and application prospect of antibody libraries according to the published reviews and research articles, and offers up to date development and application prospect of antibody library technology.

[Linkage analysis of susceptibility loci in 2 target chromosomes in pedigrees with paranoid schizophrenia and undifferentiated schizophrenia].

OBJECTIVE: To investigate the relationship of susceptibility loci in chromosomes 1q21-25 and 6p21-25 and schizophrenia subtypes in Chinese population. METHODS: A genomic scan and parametric and non-parametric analyses were performed on 242 individuals from 36 schizophrenia pedigrees, including 19 paranoid schizophrenia and 17 undifferentiated schizophrenia pedigrees, from Henan province of China using 5 microsatellite markers in the chromosome region 1q21-25 and 8 microsatellite markers in the chromosome region 6p21-25, which were the candidates of previous studies. All affected subjects were diagnosed and typed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revised (DSM-IV-TR; American Psychiatric Association, 2000). All subjects signed informed consent. RESULTS: In chromosome 1, parametric analysis under the dominant inheritance mode of all 36 pedigrees showed that the maximum multi-point heterogeneity Log of odds score method (HLOD) score was 1.33 (α = 0.38). The non-parametric analysis and the single point and multi-point nonparametric linkage (NPL) scores suggested linkage at D1S484, D1S2878, and D1S196. In the 19 paranoid schizophrenias pedigrees, linkage was not observed for any of the 5 markers. In the 17 undifferentiated schizophrenia pedigrees, the multi-point NPL score was 1.60 (P= 0.0367) at D1S484. The single point NPL score was 1.95(P= 0.0145) and the multi-point NPL score was 2.39 (P= 0.0041) at D1S2878. Additionally, the multi-point NPL score was 1.74 (P= 0.0255) at D1S196. These same three loci showed suggestive linkage during the integrative analysis of all 36 pedigrees. In chromosome 6, parametric linkage analysis under the dominant and recessive inheritance and the non-parametric linkage analysis of all 36 pedigrees and the 17 undifferentiated schizophrenia pedigrees, linkage was not observed for any of the 8 markers. In the 19 paranoid schizophrenias pedigrees, parametric analysis showed that under recessive inheritance mode the maximum single-point HLOD score was 1.26 (α = 0.40) and the multi-point HLOD was 1.12 (α = 0.38) at D6S289 in the chromosome 6p23. In nonparametric analysis, the single-point NPL score was 1.52 (P= 0.0402) and the multi-point NPL score was 1.92 (P= 0.0206) at D6S289. CONCLUSION: Susceptibility genes correlated with undifferentiated schizophrenia pedigrees from D1S484, D1S2878, D1S196 loci, and those correlated with paranoid schizophrenia pedigrees from D6S289 locus are likely present in chromosome regions 1q23.3 and 1q24.2, and chromosome region 6p23, respectively.

Inactivation of YAP1 enhances sensitivity of the yeast RNR3-lacZ genotoxicity testing system to a broad range of DNA-damaging agents.

Despite the great advances by using microorganism-based genotoxicity testing systems to assess environmental genotoxic compounds, most of them respond poorly, particularly to oxidative agents. In this study, we systematically examined the RNR3-lacZ reporter gene expression in Saccharomyces cerevisiae mutant strains defective in the protection against reactive oxygen species and found that only YAP1 deletion resulted in a significant enhancement in the detection of oxidative damage. To our surprise, YAP1 deletion also caused an increased cellular sensitivity to a variety of DNA damage. This altered sensitivity appears to be independent of oxidative damage because under conditions in which vitamin C treatment rescued oxidative damage, it failed to reverse the phenotypes caused by other types of DNA damage. Furthermore, although inactivation of cell permeability genes enhanced the RNR3-lacZ detection sensitivity particularly to large molecular weight compounds, their effects on small molecular oxidative agents are minimal. Taken together, this study helps to create a hypersensitive genotoxicity testing system to a broad range of DNA-damaging agents by deleting a single yeast gene.