A new rapid and efficient system with dominant selection developed to inactivate and conditionally express genes in Candida albicans.

Candida albicans is an important human fungal pathogen but its study has been hampered for being a natural diploid that lacks a complete sexual cycle. Gene knock-out and essential gene repression are used to study gene function in C. albicans. To effectively study essential genes in wild-type C. albicans, we took advantage of the compatible effects of the antibiotics hygromycin B and nourseothricin, the recyclable CaSAT1-flipper and the tetracycline-repressible (Tet-off) system. To allow deleting two alleles simultaneously, we created a cassette with a C. albicans HygB resistance gene (CaHygB) flanked with the FLP recombinase target sites that can be operated alongside the CaSAT1-flipper. Additionally, to enable conditionally switching off essential genes, we created a CaHygB-based Tet-off cassette that consisted of the CaTDH3 promoter, which is used for the constitutive expression of the tetracycline-regulated transactivator and a tetracycline response operator. To validate the new systems, all strains were constructed based on the wild-type strain and selected by the two dominant selectable markers, CaHygB and CaSAT1. The C. albicans general transcriptional activator CaGCN4 and its negative regulator CaPCL5 genes were targeted for gene deletion, and the essential cyclin-dependent kinase CaPHO85 gene was placed under the Tet-off system. Cagcn4, Capcl5, the conditional Tet-off CaPHO85 mutants, and mutants bearing two out of the three mutations were generated. By subjecting the mutants to various stress conditions, the functional relationship of the genes was revealed. This new system can efficiently delete genes and conditionally switch off essential genes in wild-type C. albicans to assess functional interaction between genes.

Viral Respiratory Tract Infections in Adult Patients Attending Outpatient and Emergency Departments, Taiwan, 2012-2013: A PCR/Electrospray Ionization Mass Spectrometry Study.

Viral etiologies of respiratory tract infections (RTIs) have been less studied in adult than in pediatric populations. Furthermore, the ability of PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) to detect enteroviruses and rhinoviruses in respiratory samples has not been well evaluated. We sought to use PCR/ESI-MS to comprehensively investigate the viral epidemiology of adult RTIs, including testing for rhinoviruses and enteroviruses. Nasopharyngeal or throat swabs from 267 adults with acute RTIs (212 upper RTIs and 55 lower RTIs) who visited a local clinic or the outpatient or emergency departments of a medical center in Taiwan between October 2012 and June 2013 were tested for respiratory viruses by both virus isolation and PCR/ESI-MS. Throat swabs from 15 patients with bacterial infections and 27 individuals without active infections were included as control samples. Respiratory viruses were found in 23.6%, 47.2%, and 47.9% of the 267 cases by virus isolation, PCR/ESI-MS, and both methods, respectively. When both methods were used, the influenza A virus (24.3%) and rhinoviruses (9.4%) were the most frequently identified viruses, whereas human coronaviruses, human metapneumovirus (hMPV), enteroviruses, adenoviruses, respiratory syncytial virus, and parainfluenza viruses were identified in small proportions of cases (<5% of cases for each type of virus). Coinfection was observed in 4.1% of cases. In the control group, only 1 (2.4%) sample tested positive for a respiratory virus by PCR/ESI-MS. Patients who were undergoing steroid treatment, had an active malignancy, or suffered from chronic obstructive pulmonary disease (COPD) were at risk for rhinovirus, hMPV, or parainfluenza infections, respectively. Overall, immunocompromised patients, patients with COPD, and patients receiving dialysis were at risk for noninfluenza respiratory virus infection. Rhinoviruses (12.7%), influenza A virus (10.9%), and parainfluenza viruses (7.3%) were the most common viruses involved in the 55 cases of lower RTIs. The factors of parainfluenza infection, old age, and immunosuppression were independently associated with lower RTIs. In conclusion, PCR/ESI-MS improved the diagnostic yield for viral RTIs. Non-influenza respiratory virus infections were associated with patients with comorbidities and with lower RTIs. Additional studies that delineate the clinical need for including non-influenza respiratory viruses in the diagnostic work-up in these populations are warranted.

Affinity purification of Candida albicans CaCdc4-associated proteins reveals the presence of novel proteins involved in morphogenesis.

Candida albicans CDC4 is nonessential and plays a role in suppressing filamentous growth, in contrast to its evolutionary counterparts involved in the G1-S transition of the cell cycle. Genetic epistasis analysis has indicated that proteins besides Sol1 are targets of C. albicans Cdc4. Moreover, no formal evidence suggests that C. albicans Cdc4 functions through the ubiquitin E3 ligase of the Skp1-Cul1/Cdc53-F-box complex. To elucidate the role of C. albicans CDC4, C. albicans Cdc4-associated proteins were sought by affinity purification. A 6xHis epitope-tagged C. albicans Cdc4 expressed from Escherichia coli was used in affinity purifications with the cell lysate of C. albicans cdc4 homozygous null mutant. Candida albicans Cdc4 and its associated proteins were resolved by SDS-PAGE and visualized by silver staining. The candidate proteins were recovered and trypsin-digested to generate MALDI-TOF spectra profiles, which were used to search against those of known proteins in the database to reveal their identities. Two out of four proteins encoded by GPH1 and THR1 genes were further verified to interact with C. albicans Cdc4 using a yeast two-hybrid assay. We conclude that in vitro affinity purification using C. albicans Cdc4 generated from E. coli as the bait and proteins from cell lysate of C. albicans cdc4 homozygous null mutant as a source of prey permit the identification of novel proteins that physically interact and functionally associate with C. albicans Cdc4.

Association of DAZL haplotypes with spermatogenic failure in infertile men.

OBJECTIVE: To identify novel DAZL single nucleotide polymorphisms (SNPs) and to compare allele/genotype frequencies, linkage disequilibrium (LD) characteristics, and DAZL haplotypes between fertile and infertile men. DESIGN: Prospective case study. SETTING: University genetics laboratory and reproductive clinics. PATIENT(S): Two hundred thirty-one infertile men and 191 men with proven fertility. INTERVENTION(S): Single strand conformation polymorphism and sequence analysis for DAZL gene polymorphism screening were done for all subjects enrolled. MAIN OUTCOME MEASURE(S): Novel SNPs, allele/genotype frequencies, LD characteristics, and DAZL haplotypes between fertile and infertile men. RESULT(S): Five SNPs were identified: 260A>G, 386A>G, 520+34c>a, 584+28c>t and 796+36g>a. SNP 386A>G was significantly associated with spermatogenic failure and was mainly heterozygous in infertile patients. The major haplotypes in infertile men were AACTA (45.8%), followed by AACCG, AAATA, AAACG, and GGACG for 260A>G/386A>G/520+34c>a/584+28c>t/796+36g>a. The major haplotypes for the control subjects were AACCG (41.7 %), AAATA, and AACTA. Of all haplotypes, five showed significant differences in frequency between infertile men and control subjects. Haplotypes AACTA, AAACG, and GGACG were overtransmitted in patients with spermatogenic failure, whereas haplotypes AACCG and AAATA were undertransmitted in these patients. CONCLUSION(S): Our study suggests the association of autosomal DAZL haplotypes with human spermatogenic failure.

TPMD: a database and resources of microsatellite marker genotyped in Taiwanese populations.

Taiwan Polymorphic Marker Database (TPMD) (http://tpmd.nhri.org.tw/) is a marker database designed to provide experimental details and useful marker information allelotyped in Taiwanese populations accompanied by resources and technical supports. The current version deposited more than 372,000 allelotyping data from 1425 frequently used and fluorescent-labeled microsatellite markers with variation types of dinucleotide, trinucleotide and tetranucleotide. TPMD contains text and map displays with searchable and retrievable options for marker names, chromosomal location in various human genome maps and marker heterozygosity in populations of Taiwanese, Japanese and Caucasian. The integration of marker information in map display is useful for the selection of high heterozygosity and commonly used microsatellite markers to refine mapping of diseases locus followed by identification of disease gene by positional candidate cloning. In addition, our results indicated that the number of markers with heterozygosity over 0.7 in Asian populations is lower than that in Caucasian. To increase accuracy and facilitate genetic studies using microsatellite markers, we also list markers with genotyping difficulty due to ambiguity of allele calling and recommend an optimal set of microsatellite markers for genotyping in Taiwanese, and possible extension of genotyping in other Mongoloid populations.

Successful matched-unrelated bone marrow transplantation in a patient with beta-thalassemia major.

The authors describe a 5-year-old boy with beta-thalassemia major who received bone marrow transplantation (BMT) from a human leukocyte antigen (HLA)-matched unrelated donor. The conditioning regimen consisted of 16 mg/kg busulfan and 200 mg/kg cyclophosphamide. The transplantation was complicated with grade II graft-versus-host disease, although prophylaxis with cyclosporine and short-term methotrexate was carried out. Cytomegalovirus disease occurred at 2 months after transplantation but was controlled successfully. The child remains disease-free and in good clinical condition 53 months after BMT. The authors suggest that BMT from an HLA-matched unrelated donor could be considered as an alternative treatment in patients with beta-thalassemia major when no HLA-matched donor is available.

Association study of novel human serotonin 5-HT(1B) polymorphisms with alcohol dependence in Taiwanese Han.

BACKGROUND: Abnormal serotonergic pathways are implicated in numerous neuropsychiatric disorders, such as depression, anxiety, migraine, substance abuse, and alcoholism. The human serotonin receptor 1B, encoded by the HTR1B gene, is a presynaptic serotonin autoreceptor that plays a role in regulating serotonin synthesis and release. Because the linkage of antisocial alcoholism to the HTR1B gene was recently reported in two populations, it was of interest to identify genetic variants at the HTR1B locus and study their association with alcoholism in the Taiwanese Han population. METHODS: We sequenced DNA from Taiwanese Han to screen for genetic variation in the coding, promoter, and partial 3' untranslated regions of the HTR1B locus of 158 alcohol-dependent cases with withdrawal symptoms and 149 control subjects, who either never drank or drank only occasionally and in low quantities. RESULTS: Seven variants were identified. Positive associations were found between variant A-161T and alcohol dependence at both the allelic and genotypic level. In addition, an expression study showed that the A-161T variant affected reporter gene activity. CONCLUSIONS: Our results support an association between HTR1B and alcohol dependence. The HTR1B A-161T polymorphism may be valuable both as a functional and as an anonymous genetic marker for HTR1B.