Shiau-Ting Hu

National Yang Ming University, Taipei, Taipei, Taiwan

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Publications (15)51.45 Total impact

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    ABSTRACT: The occurrence of diseases caused by rapidly growing mycobacteria (RGM) is increasing in Taiwan. In this study, the in vitro antimicrobial activities of tigecycline, minocycline, tetracycline and doxycycline were evaluated against 160 clinical RGM isolates, including 34 Mycobacterium abscessus sensu stricto (s.s.), 44 Mycobacterium massiliense, 1 Mycobacterium bolletii, 58 Mycobacterium fortuitum and 23 Mycobacterium chelonae. Clarithromycin and amikacin were tested alone as well as for synergistic effect with tigecycline. Both amikacin and tigecycline showed excellent activities against the RGM. More than 85% of each of the five RGM species isolates showed susceptibility to the two drugs. The MIC(50) and MIC(90) values (drug concentrations at which 50% and 90%, respectively, of the tested isolates did not show any visible growth) of amikacin were 1-4mg/L and 2-8mg/L, respectively, whilst those of tigecycline were 0.125-1mg/L and 0.5-2.0mg/L. Clarithromycin had only moderate activity, with ≥42.9% but ≤87.5% of each RGM species isolates showing susceptibility. The other three drugs had limited or no antimicrobial activity, with <40% of each RGM species isolates showing susceptibility. Combined with clarithromycin, tigecycline had synergistic activity against 92.9%, 68.8%, 100%, 35.7% and 46.2% of M. abscessus s.s., M. massiliense, M. bolletii, M. fortuitum and M. chelonae isolates, respectively. However, tigecycline combined with amikacin had synergistic activity against <25% but antagonistic activity against >18% of each RGM species. Thus, tigecycline alone may be an alternative for treating RGM diseases in patients who are intolerant to cefoxitin, imipenem or amikacin. However, it should be used with caution or not used in combination with amikacin for RGM diseases.
    International journal of antimicrobial agents 01/2013; · 3.03 Impact Factor
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    ABSTRACT: Mycobacterium abscessus is a non-tuberculous mycobacterium. It can cause diseases in both immunosuppressed and immunocompetent patients and is highly resistant to multiple antimicrobial agents. M. abscessus displays two different colony morphology types: smooth and rough morphotypes. Cells with a rough morphotype are more virulent. The purpose of this study was to identify genes responsible for M. abscessus morphotype switching. With transposon mutagenesis, a mutant with a Tn5 inserted into the promoter region of the mab_3168c gene was found to switch its colonies from a rough to a smooth morphotype. This mutant had a higher sliding motility but a lower ability to form biofilms, aggregate in culture, and survive inside macrophages. Results of bioinformatic analyses suggest that the putative Mab_3168c protein is a member of the GCN5-related N-acetyltransferase superfamily. This prediction was supported by the demonstration that the mab_3168c gene conferred M. abscessus and M. smegmatis cells resistance to amikacin. The multiple roles of mab_3168c suggest that it could be a potential target for development of therapeutic regimens to treat diseases caused by M. abscessus.
    PLoS ONE 01/2013; 8(6):e67563. · 3.53 Impact Factor
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    ABSTRACT: BACKGROUND: Mycobacteria can be quickly and simply identified by PCR restriction-enzyme analysis (PRA), but misidentification can occur because of similarities in band sizes that are critical for discriminating among species. Capillary electrophoresis can provide computer-aided band discrimination. The aim of this research was to develop an algorithm for identifying mycobacteria by combined rpoB duplex PRA (DPRA) and hsp65 PRA with capillary electrophoresis. RESULTS: Three hundred and seventy-six acid-fast bacillus smear-positive BACTEC cultures, including 200 Mycobacterium tuberculosis complexes (MTC) and 176 non-tuberculous mycobacteria (NTM) were analyzed. With combined hsp65 and rpoB DPRA, the accuracy rate was 100 % (200 isolates) for the MTC and 91.4 % (161 isolates) for the NTM. Among the discordant results (8.6 %) for the NTM, one isolate of Mycobacterial species and the an isolate of M. flavescens were found as new sub-types in hsp65 PRA. CONCLUSIONS: This effective and novel identification algorithm using combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can rapidly identify mycobacteria and find new sub-types in hsp65 PRA. In addition, it is complementary to 16S rDNA sequencing.
    BMC Microbiology 07/2012; 12(1):137. · 2.98 Impact Factor
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    ABSTRACT: Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium pathogen, whose incidence and prevalence have been increasing in the last decade. M. kansasii can cause pulmonary tuberculosis clinically and radiographically indistinguishable from that caused by Mycobacterium tuberculosis infection. Unlike the widely-studied M. tuberculosis, little is known about the innate immune response against M. kansasii infection. Although inflammasome activation plays an important role in host defense against bacterial infection, its role against atypical mycobacteria remains poorly understood. In this report, the role of inflammasome activity in THP-1 macrophages against M. kansasii infection was studied. Results indicated that viable, but not heat-killed, M. kansasii induced caspase-1-dependent IL-1β secretion in macrophages. The underlying mechanism was found to be through activation of an inflammasome containing the NLR (Nod-like receptor) family member NLRP3 and the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Further, potassium efflux, lysosomal acidification, ROS production and cathepsin B release played a role in M. kansasii-induced inflammasome activation. Finally, the secreted IL-1β derived from caspase-1 activation was shown to restrict intracellular M. kansasii. These findings demonstrate a biological role for the NLRP3 inflammasome in host defense against M. kansasii.
    PLoS ONE 01/2012; 7(4):e36292. · 3.53 Impact Factor
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    ABSTRACT: Early secretory antigen 6 (ESAT-6) and cell filtrate protein 10 (CFP-10) are two antigens secreted as a complex by the replicating Mycobacterium tuberculosis complex (MTC). Recently, an immunochromatographic assay (ICA) using a monoclonal antibody against the ESAT-6/CFP-10 complex was developed for the purpose of MTC detection. In this study, the efficacy of the assay was tested with 603 BACTEC cultures that were incubated for 3 additional days after positive signals appeared in the BACTEC MGIT 960 system. Bacterial isolates were recovered from these 603 BACTEC cultures, and 332 MTC isolates, 270 nontuberculosis mycobacterial isolates, and 1 Nocardia isolate were identified by using standard biochemical assays. The ESAT-6/CFP-10 assay detected 322 MTC cultures, resulting in a sensitivity of 97% and a specificity of 97.4%. To reduce the false-negative rate and improve the sensitivity, either serpentine cording in an acid-fast bacillus stain of the cultural smear, the ESAT-6/CFP-10 assay, or a combination of both was used for MTC detection. The sensitivity was then increased to 99.1%, and the negative predictive value increased to 98.9%, but the specificity decreased to 94.8% and the positive predictive value decreased to 95.9%. However, a combination of serpentine cording in cultural smears and the positivity of the ICA resulted in the specificity and positive predictive values of 100%. Therefore, BACTEC cultures with both serpentine cording and positivity of the ESAT-6/CFP-10 assay could be reported to contain MTC directly. The ESAT-6/CFP-10 assay may be an alternative of the Capilia assay (MPB64-ICA) as a convenient and cost-effective method for identification of MTC in culture.
    Journal of clinical microbiology 03/2011; 49(3):902-7. · 4.16 Impact Factor
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    ABSTRACT: BtuB (B twelve uptake) is an outer membrane protein of Escherichia coli. It serves as a receptor for cobalamines uptake or bactericidal toxin entry. A decrease in the production of the BtuB protein would cause E. coli to become resistant to colicins. The production of BtuB has been shown to be regulated at the post-transcriptional level. The secondary structure of 5' untranslated region of btuB mRNA and the intracellular concentration of adenosylcobalamin (Ado-Cbl) would affect the translational efficiency and RNA stability of btuB gene. The transcriptional regulation of btuB expression is still unclear. To determine whether the btuB gene is also transcriptionally controlled by trans-acting factors, a genomic library was screened for clones that enable E. coli to grow in the presence of colicin E7, and a plasmid carrying gadX and gadY genes was isolated. The lacZ reporter gene assay revealed that these two genes decreased the btuB promoter activity by approximately 50%, and the production of the BtuB protein was reduced by approximately 90% in the presence of a plasmid carrying both gadX and gadY genes in E. coli as determined by Western blotting. Results of electrophoretic mobility assay and DNase I footprinting indicated that the GadX protein binds to the 5' untranslated region of the btuB gene. Since gadX and gadY genes are more highly expressed under acidic conditions, the transcriptional level of btuB in cells cultured in pH 7.4 or pH 5.5 medium was examined by quantitative real-time PCR to investigate the effect of GadX. The results showed the transcription of gadX with 1.4-fold increase but the level of btuB was reduced to 57%. Through biological and biochemical analysis, we have demonstrated the GadX can directly interact with btuB promoter and affect the expression of btuB. In conclusion, this study provides the first evidence that the expression of btuB gene is transcriptionally repressed by the acid responsive genes gadX and gadY.
    BMC Microbiology 02/2011; 11:33. · 2.98 Impact Factor
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    ABSTRACT: Dengue virus (DENV) is one of the most common infectious pathogens worldwide. One major clinical and pathogenic feature of DENV infection is the elevation of interleukin-8 (IL-8) expression; however, little is known about the molecular mechanism of DENV-induced chemokine production. The positive transcription elongation factor b (P-TEFb) composed of CDK9 and cyclin T1 stimulates gene expression by enhancing RNA polymerase II (RNA pol II) processivity. This study examined the possibility that P-TEFb mediates DENV-induced IL-8 expression. The treatment of either a pharmacological inhibitor of P-TEFb, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) or cyclin T1 siRNA prior to DENV infection abolished the elevation of IL-8, indicating that P-TEFb is essential for IL-8 induction. Moreover, DENV core protein participated in the activation of IL-8 promoter in a P-TEFb-dependent manner. Immunostaining and co-immunoprecipitation assays demonstrated the association between P-TEFb and DENV core protein. Finally, chromatin immunoprecipitation (ChIP) results indicated that P-TEFb and DENV core protein were recruited to the transcriptionally active IL-8 gene promoter. Taken together, this study showed that P-TEFb and DENV core protein work in concert to enhance IL-8 gene expression by DENV infection. This is the first demonstration of P-TEFb being directly involved in virus-induced host gene expression by interacting with a viral structural protein.
    Cellular Microbiology 11/2010; 12(11):1589-603. · 4.81 Impact Factor
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    ABSTRACT: The aim of this study was to determine whether clofazimine, dapsone and cycloserine may be suitable antimicrobial agents for the treatment of infections due to rapidly growing mycobacteria (RGM). The antimicrobial activity of the three drugs against 117 Mycobacterium abscessus isolates, 48 Mycobacterium fortuitum isolates and 20 Mycobacterium chelonae isolates was evaluated based on their broth microdilution minimal inhibitory concentrations (MICs) against the isolates. Clofazimine was highly efficacious against these RGM. The vast majority of M. abscessus, M. fortuitum and M. chelonae isolates (99.1%, 91.7% and 100%, respectively) had clofazimine MICs of <or=1mg/L. MIC(50) values (MIC for 50% of the organisms) of clofazimine against the isolates ranged from 0.25mg/L to 0.5mg/L and MIC(90) values (MIC for 90% of the organisms) ranged from 0.5mg/L to 1.0mg/L. Cycloserine and dapsone had little or no activity against the isolates. The effects of combined application of clofazimine and amikacin on 40 M. abscessus isolates, 48 M. fortuitum isolates and 20 M. chelonae isolates were evaluated. Addition of 0.25x MIC of amikacin for the isolates to clofazimine reduced clofazimine MICs in all of the M. abscessus and M. chelonae isolates and in 48% of the M. fortuitum isolates tested. Clofazimine, either alone or combined with amikacin, may serve as a promising drug for the treatment of RGM infections.
    International journal of antimicrobial agents 02/2010; 35(4):400-4. · 3.03 Impact Factor
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    ABSTRACT: The effect of factor for inversion stimulation (Fis) protein on IS2 transposition was investigated. A full-length IS2 was found to transpose at a frequency 64 times lower in a normal Escherichia coli than in a fis- mutant. To investigate whether Fis affects IS2 transposition by DNA binding, gel retardation and DNase I footprinting experiments were performed. Analysis of Fis binding to the left terminus of IS2 revealed that Fis binds to nucleotide number 44-60 located between the -35 and -10 regions of the major IS2 promoter. To further determine whether Fis binding affects IS2 transcription, the major IS2 promoter was fused to a luciferase gene and assayed for its transcription efficiency in the presence or absence of Fis. The results showed that Fis reduced transcription from the major IS2 promoter by approximately sixfold. Analysis of Fis binding to the right terminal repeat of IS2 revealed that Fis binds to the inner end of the repeat, which is the same region as the place where the IS2 transposase binds. These results suggest that Fis inhibits IS2 transposition by blocking the binding sites of IS2 transposase and by repressing the transcription of IS2 genes.
    FEMS Microbiology Letters 11/2007; 275(1):98-105. · 2.05 Impact Factor
  • Chang-Chieh Chen, Shiau-Ting Hu
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    ABSTRACT: IS629 is 1,310 bp in length with a pair of 25-bp imperfect inverted repeats at its termini. Two partially overlapping open reading frames, orfA and orfB, are present in IS629, and two putative translational frameshift signals, TTTTG (T4G) and AAAAT (A4T), are located near the 3'-end of orfA. With the lacZ gene as the reporter, both T4G and A4T motifs are determined to be a -1 frameshift signal. Two peptides representing the two transframe products designated OrfAB' and OrfAB, are identified by a liquid chromatography-tandem mass spectrometric approach. Results of transposition assays show that OrfAB' is the transposase and that OrfAB aids in the transposition of IS629. Pulse-chase experiments and Escherichia coli two-hybrid assays demonstrate that OrfAB binds to and stabilizes OrfAB', thus increasing the transposition activity of IS629. This is the first transposable element in the IS3 family shown to have two functional frameshifted products involved in transposition and to use a transframe product to regulate transposition.
    Journal of Biological Chemistry 09/2006; 281(31):21617-28. · 4.65 Impact Factor
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    ABSTRACT: The ColE7 operon is an SOS response regulon, which encodes bacteriocin ColE7 to kill susceptible Escherichia coli and its related enterobacteria under conditions of stress. We have observed for the first time that polyamines confer limited resistance against ColE7 on E. coli cells. Thus, this study aims to investigate the role of polyamines in modulating the protective effect of the E. coli cells against colicin. In the experiments, we surprisingly found that endogenous polyamines are also essential for ColE7 production, and the rate of polyamine synthesis is directly related to the SOS response. Our experimental results further indicated that exogenous polyamines suppress the expression of TolA, BtuB, OmpF, and OmpC proteins that are responsible for ColE7 uptake. Moreover, two-dimensional gel electrophoresis revealed that the production of two periplasmic proteins, PotD and OppA, is increased in E. coli cells under ColE7 exposure. Based on these observations, we propose that endogenous polyamines may play a dual role in the ColE7 system. Polyamines may participate in initiating the expression of the SOS response of the ColE7 operon and simultaneously down-regulate proteins that are essential for colicin uptake, thus conferring a survival advantage on colicin-producing E. coli under stress conditions in the natural environment.
    Journal of Biological Chemistry 06/2006; 281(19):13083-91. · 4.65 Impact Factor
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    ABSTRACT: Translational frameshifting, a ubiquitous mechanism used to produce alternative proteins for different biological purposes, appears in a variety of genes in probably all organisms. In the past, the combinational use of sophisticated expression vectors, specific endopeptidases, and Edman degradation has been the main approach for identification of the translational frameshift sites. Although Edman degradation is highly reliable, it is also time-consuming and costly. In this article, we report a new liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach for identifying the -1 translational frameshift sites. The approach consists of three steps: (i) LC-MS/MS analysis of the protein digests, (ii) primary data analysis using the known mRNA sequence, and (iii) advanced data analysis using a new database containing distinct mRNA sequences with single insertion at particular positions. We first validated our approach by analyzing the previously documented slippery sequence, A4G, from IS3. With this approach, we further determined whether the TTTTTTG (T6G) sequence of IS1372 from Streptomyces lividans had the -1 translational frameshifting potential. The identified amino acid sequence of the transframe peptide indicated that the -1 frameshifting occurred at the T6G motif, as predicted previously. The results on IS3 (A4G) and IS1372 (T6G) suggested that this approach is effective for the translational frameshifting studies.
    Analytical Biochemistry 05/2005; 339(1):83-93. · 2.58 Impact Factor
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    Shao-Hung Wang, Wan-Jr Syu, Shiau-Ting Hu
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    ABSTRACT: Dengue virus causes dengue haemorrhagic fever or dengue shock syndrome with a high mortality rate. The genome of dengue virus is a positive-sense, single-stranded RNA encoding three structural and seven non-structural proteins. The core protein is one of the three structural proteins and is the building block of the nucleocapsid of dengue virus. The core protein of dengue virus type 2 (DEN2) is composed of 100 aa with four alpha-helix domains. An internal hydrophobic domain located at aa 44-60 was identified. The DEN2 core protein was shown to form homodimers. Deletion of aa 1-36 or 73-100 decreased but did not completely abolish the core-to-core homotypic interaction, whereas deletion of a portion (aa 44-60) within aa 37-72 completely abolished the ability of the DEN2 core proteins to interact with each other. A recombinant DEN2 core protein corresponding to aa 37-72 was able to undergo homotypic interaction and bound to a native DEN2 core protein. The results of this study indicated that the homotypic interaction domain of the DEN2 core protein is located at aa 37-72 and that the internal hydrophobic domain located at aa 44-60 plays a pivotal role in core-to-core homotypic interaction.
    Journal of General Virology 09/2004; 85(Pt 8):2307-14. · 3.13 Impact Factor
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    ABSTRACT: In dengue virus (DEN) particles, the core protein is a structural protein of the nucleocapsid. The core protein is known to be present in the nucleus of DEN-infected cells but there have been conflicting reports as to whether it is also present in the nucleolus. To clarify this, the intracellular location of the core protein was examined using a monoclonal antibody, 15B11, which was produced in this study. Immunofluorescence staining with this antibody demonstrated that the core protein first appeared in the cytoplasm and then in the nuclei and nucleoli of infected cells. Nuclear localization of the core protein was determined to be independent of other DEN proteins, since recombinant core proteins still entered the nuclei and nucleoli of cells transfected with only the core protein gene. Three putative nuclear localization signal motifs have been predicted to be present on the core protein. Deletion of the first one (KKAR), located at aa 6-9, and mutation of the second one (KKSK), located at aa 73-76, did not eliminate the nuclear localization property of the core protein. The third motif with a bipartite structure, RKeigrmlnilnRRRR, located at aa 85-100, was determined to be responsible for the nuclear localization of the core protein, since the core protein without this motif was located exclusively in the cytoplasm of DEN-infected cells and that this motif mediated nuclear localization of a normally cytoplasmic protein.
    Journal of General Virology 01/2003; 83(Pt 12):3093-102. · 3.13 Impact Factor
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    ABSTRACT: Three sequences similar to that of the consensus binding sequence of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex were found in the major IS2 promoter region. Experiments were performed to determine whether the cAMP-CRP complex plays a role in the regulation of IS2 transposition. In the gel retardation assay, the cAMP-CRP complex was found to be able to bind the major IS2 promoter. A DNA footprinting assay confirmed that the cAMP-CRP complex binds to the sequences mentioned above. With an IS2 promoter-luciferase gene fusion construct, the cAMP-CRP complex was shown to inhibit transcription from the major IS2 promoter. IS2 was found to transpose at a frequency approximately 200-fold higher in an Escherichia coli host defective for CRP or adenyl cyclase than in a wild-type host. These results suggest that the cAMP-CRP complex is a negative regulator of IS2 transposition.
    Journal of Bacteriology 05/1998; · 3.19 Impact Factor