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1.  Human Mucin MUC1 RNA Undergoes Different Types of Alternative Splicing Resulting in Multiple Isoforms 
MUC1 is a transmembrane mucin with important functions in normal and transformed cells carried out by the extracellular domain or the cytoplasmic tail. A characteristic feature of the MUC1 extracellular domain is the variable number of tandem repeats (VNTR) region. Alternative splicing may regulate MUC1 expression and possibly function. We developed an RT-PCR method for efficient isolation of MUC1 mRNA isoforms that allowed us to evaluate the extent of alternative splicing of MUC1 and elucidate some of the rules that govern this process. We cloned and analyzed 21, 24, and 36 isoforms from human tumor cell lines HeLa, MCF7, and Jurkat respectively, and 16 from normal activated human T cells. Among the 78 MUC1 isoforms we isolated, 76 are new and different cells showed varied MUC1 expression patterns. The VNTR region of exon 2 was recognized as an intron with a fixed 5′ splice site but variable 3′ splice sites. We also report that the 3506 A/G SNP in exon 2 can regulate 3′ splice sites selection in intron 1 and produce different MUC1 short isoform proteins. Furthermore, the SNP A to G mutation was also observed in vivo, during de novo tumor formation in MUC1+/−KrasG12D/+PtenloxP/loxP mice. No specific functions have been associated with previously reported short isoforms. We now report that one new G SNP-associated isoform MUC1/Y-LSP, but not the A SNP-associated isoform MUC1/Y, inhibits tumor growth in immunocompetent but not immunocompromised mice.
PMCID: PMC3538090  PMID: 22941036
MUC1; alternative splicing; isoforms; tumor
2.  Molecular Networking as a Dereplication Strategy 
Journal of natural products  2013;76(9):1686-1699.
A major goal in natural product discovery programs is to rapidly dereplicate known entities from complex biological extracts. We demonstrate here that molecular networking, an approach that organizes MS/MS data based on chemical similarity, is a powerful complement to traditional dereplication strategies. Successful dereplication with molecular networks requires MS/MS spectra of the natural product mixture along with MS/MS spectra of known standards, synthetic compounds, or well-characterized organisms, preferably organized into robust databases. This approach can accommodate different ionization platforms, enabling cross correlations of MS/MS data from ambient ionization, direct infusion, and LC-based methods. Molecular networking not only dereplicates known molecules from complex mixtures, it also captures related analogs, a challenge for many other dereplication strategies. To illustrate its utility as a dereplication tool, we apply mass spectrometry-based molecular networking to a diverse array of marine and terrestrial microbial samples, illustrating the dereplication of 58 molecules including analogs.
PMCID: PMC3936340  PMID: 24025162
3.  Regulatory factors for the assembly of thylakoid membrane protein complexes 
Major multi-protein photosynthetic complexes, located in thylakoid membranes, are responsible for the capture of light and its conversion into chemical energy in oxygenic photosynthetic organisms. Although the structures and functions of these photosynthetic complexes have been explored, the molecular mechanisms underlying their assembly remain elusive. In this review, we summarize current knowledge of the regulatory components involved in the assembly of thylakoid membrane protein complexes in photosynthetic organisms. Many of the known regulatory factors are conserved between prokaryotes and eukaryotes, whereas others appear to be newly evolved or to have expanded predominantly in eukaryotes. Their specific features and fundamental differences in cyanobacteria, green algae and land plants are discussed.
PMCID: PMC3497070  PMID: 23148269
regulatory factors; biogenesis; assembly; thylakoid membrane protein complex; chloroplast
4.  The identification and characterization of breast cancer CTCs competent for brain metastasis 
Science translational medicine  2013;5(180):10.1126/scitranslmed.3005109.
Brain metastatic breast cancer (BMBC) is uniformly fatal and increasing in frequency. Despite its devastating outcome, mechanisms causing BMBC remain largely unknown. The mechanisms that implicate circulating tumor cells (CTCs) in metastatic disease, notably in BMBC, remain elusive. Here we characterize CTCs isolated from peripheral blood mononuclear cells (PBMCs) of patients with breast cancer, and also develop CTC lines from three of these patients. In epithelial cell adhesion molecule (EpCAM)–negative CTCs, we identified a potential signature of brain metastasis comprising “brain metastasis selected markers (BMSM)” HER2+/EGFR+/HPSE+/Notch1+. These CTCs—which are not captured by the CellSearch platform because of their EpCAM negativity—were analyzed for cell invasiveness and metastatic competency in vivo. CTC lines expressing the BMSM signature were highly invasive and capable of generating brain and lung metastases when xenografted in nude mice. Notably, increased brain metastatic capabilities, frequency, and quantitation were detected in EpCAM− CTCs overexpressing the BMSM signature. The presence of proteins of the BMSM CTC signature was also detected in the metastatic lesions of animals. Collectively, we provide evidence of isolation, characterization, and long-term culture of human breast cancer CTCs, leading to the description of a BMSM protein signature that is suggestive of CTC metastatic competency to the brain.
PMCID: PMC3863909  PMID: 23576814
5.  3DScapeCS: application of three dimensional, parallel, dynamic network visualization in Cytoscape 
BMC Bioinformatics  2013;14:322.
The exponential growth of gigantic biological data from various sources, such as protein-protein interaction (PPI), genome sequences scaffolding, Mass spectrometry (MS) molecular networking and metabolic flux, demands an efficient way for better visualization and interpretation beyond the conventional, two-dimensional visualization tools.
We developed a 3D Cytoscape Client/Server (3DScapeCS) plugin, which adopted Cytoscape in interpreting different types of data, and UbiGraph for three-dimensional visualization. The extra dimension is useful in accommodating, visualizing, and distinguishing large-scale networks with multiple crossed connections in five case studies.
Evaluation on several experimental data using 3DScapeCS and its special features, including multilevel graph layout, time-course data animation, and parallel visualization has proven its usefulness in visualizing complex data and help to make insightful conclusions.
PMCID: PMC3835703  PMID: 24225050
6.  EEG feature comparison and classification of simple and compound limb motor imagery 
Motor imagery can elicit brain oscillations in Rolandic mu rhythm and central beta rhythm, both originating in the sensorimotor cortex. In contrast with simple limb motor imagery, less work was reported about compound limb motor imagery which involves several parts of limbs. The goal of this study was to investigate the differences of the EEG patterns between simple limb motor imagery and compound limb motor imagery, and discuss the separability of multiple types of mental tasks.
Ten subjects participated in the experiment involving three tasks of simple limb motor imagery (left hand, right hand, feet), three tasks of compound limb motor imagery (both hands, left hand combined with right foot, right hand combined with left foot) and rest state. Event-related spectral perturbation (ERSP), power spectral entropy (PSE) and spatial distribution coefficient were adopted to analyze these seven EEG patterns. Then three algorithms of modified multi-class common spatial patterns (CSP) were used for feature extraction and classification was implemented by support vector machine (SVM).
The induced event-related desynchronization (ERD) affects more components within both alpha and beta bands resulting in more broad ERD bands at electrode positions C3, Cz and C4 during left/right hand combined with contralateral foot imagery, whose PSE values are significant higher than that of simple limb motor imagery. From the topographical distribution, simultaneous imagination of upper limb and contralateral lower limb certainly contributes to the activation of more areas on cerebral cortex. Classification result shows that multi-class stationary Tikhonov regularized CSP (Multi-sTRCSP) outperforms other two multi-class CSP methods, with the highest accuracy of 84% and mean accuracy of 70%.
The work implies that there exist the separable differences between simple limb motor imagery and compound limb motor imagery, which can be utilized to build a multimodal classification paradigm in motor imagery based brain-computer interface (BCI) systems.
PMCID: PMC3853015  PMID: 24119261
Compound limb motor imagery; Event-related desynchronization; Event-related spectral perturbation; Power spectral entropy; Spatial distribution coefficient; Common spatial patterns; Support vector machine
7.  Identification of novel inhibitors of M. tuberculosis growth using whole cell based high-throughput screening 
ACS chemical biology  2012;7(8):1377-1384.
Despite the urgent need for new antitubercular drugs, few are on the horizon. To combat the problem of emerging drug resistance, structurally unique chemical entities that inhibit new targets will be required. Here we describe our investigations using whole cell screening of a diverse collection of small molecules as a methodology for identifying novel inhibitors that target new pathways for Mycobacterium tuberculosis drug discovery. We find that conducting primary screens using model mycobacterial species may limit the potential for identifying new inhibitors with efficacy against M. tuberculosis. In addition, we confirm the importance of developing in vitro assay conditions that are reflective of in vivo biology for maximizing the proportion of hits from whole cell screening that are likely to have activity in vivo. Finally, we describe the identification and characterization of two novel inhibitors that target steps in M. tuberculosis cell wall biosynthesis. The first is a novel benzimidazole that targets mycobacterial membrane protein large 3 (MmpL3), a proposed transporter for cell wall mycolic acids. The second is a nitro-triazole that inhibits decaprenylphosphoryl-β-d-ribose 2′-epimerase (DprE1), an epimerase required for cell wall biosynthesis. These proteins are both among the small number of new targets that have been identified by forward chemical genetics using resistance generation coupled with genome sequencing. This suggests that methodologies currently employed for screening and target identification may lead to a bias in target discovery, and that alternative methods should be explored.
PMCID: PMC3560293  PMID: 22577943
8.  Urease Operon and Urease Activity in Commensal and Disease-Causing Nontypeable Haemophilus influenzae 
Journal of Clinical Microbiology  2013;51(2):653-655.
The ure operon was significantly more prevalent in Haemophilus influenzae isolates causing otitis media and chronic obstructive pulmonary disease (COPD)-associated bronchitis than in those from throats of healthy individuals (97% versus 78.1%, P < 0.001). Strains lacking the ure operon are over 8 times more likely to be from the throat than either otitis media or COPD isolates.
PMCID: PMC3553914  PMID: 23224103
9.  Design and validation of a supragenome array for determination of the genomic content of Haemophilus influenzae isolates 
BMC Genomics  2013;14:484.
Haemophilus influenzae colonizes the human nasopharynx as a commensal, and is etiologically associated with numerous opportunistic infections of the airway; it is also less commonly associated with invasive disease. Clinical isolates of H. influenzae display extensive genomic diversity and plasticity. The development of strategies to successfully prevent, diagnose and treat H. influenzae infections depends on tools to ascertain the gene content of individual isolates.
We describe and validate a Haemophilus influenzae supragenome hybridization (SGH) array that can be used to characterize the full genic complement of any strain within the species, as well as strains from several highly related species. The array contains 31,307 probes that collectively cover essentially all alleles of the 2890 gene clusters identified from the whole genome sequencing of 24 clinical H. influenzae strains. The finite supragenome model predicts that these data include greater than 85% of all non-rare genes (where rare genes are defined as those present in less than 10% of sequenced strains). The veracity of the array was tested by comparing the whole genome sequences of eight strains with their hybridization data obtained using the supragenome array. The array predictions were correct and reproducible for ~ 98% of the gene content of all of the sequenced strains. This technology was then applied to an investigation of the gene content of 193 geographically and clinically diverse H. influenzae clinical strains. These strains came from multiple locations from five different continents and Papua New Guinea and include isolates from: the middle ears of persons with otitis media and otorrhea; lung aspirates and sputum samples from pneumonia and COPD patients, blood specimens from patients with sepsis; cerebrospinal fluid from patients with meningitis, as well as from pharyngeal specimens from healthy persons.
These analyses provided the most comprehensive and detailed genomic/phylogenetic look at this species to date, and identified a subset of highly divergent strains that form a separate lineage within the species. This array provides a cost-effective and high-throughput tool to determine the gene content of any H. influenzae isolate or lineage. Furthermore, the method for probe selection can be applied to any species, given a group of available whole genome sequences.
PMCID: PMC3723446  PMID: 23865594
10.  Azole Susceptibility and Transcriptome Profiling in Candida albicans Mitochondrial Electron Transport Chain Complex I Mutants 
Mitochondrial dysfunction in pathogenic fungi or model yeast causes altered susceptibilities to antifungal drugs. Here we have characterized the role of mitochondrial complex I (CI) of Candida albicans in antifungal susceptibility. Inhibitors of CI to CV, except for CII, increased the susceptibility of both patient and lab isolates, even those with a resistance phenotype. In addition, in a C. albicans library of 12 CI null mutants, 10 displayed hypersusceptibility to fluconazole and were severely growth inhibited on glycerol, implying a role for each gene in cell respiration. We chose two other hypersusceptible null mutants of C. albicans, the goa1Δ and ndh51Δ mutants, for transcriptional profiling by RNA-Seq. Goa1p is required for CI activity, while Ndh51p is a CI subunit. RNA-Seq revealed that both the ndh51Δ mutant and especially the goa1Δ mutant had significant downregulation of transporter genes, including CDR1 and CDR2, which encode efflux proteins. In the goa1Δ mutant, we noted the downregulation of genes required for the biogenesis and replication of peroxisomes, as well as metabolic pathways assigned to peroxisomes such as β-oxidation of fatty acids, glyoxylate bypass, and acetyl coenzyme A (acetyl-CoA) transferases that are known to shuttle acetyl-CoA between peroxisomes and mitochondria. The transcriptome profile of the ndh51Δ mutant did not include downregulation of peroxisome genes but had, instead, extensive downregulation of the ergosterol synthesis gene family. Our data establish that cell energy is required for azole susceptibility and that downregulation of efflux genes may be an outcome of that dysfunction. However, there are mutant-specific changes that may also increase the susceptibility of both of these C. albicans mutants to azoles.
PMCID: PMC3535965  PMID: 23147730
11.  Both Host and Pathogen Factors Predispose to Escherichia coli Urinary-Source Bacteremia in Hospitalized Patients 
We determined host and pathogen risk factors for urinary-source bacteremia in a prospective study of patients with Escherichia coli bacteriuria. Both host (urinary retention; history of urogenital surgery) and pathogen factors (a capsule characteristic) were independent predictors of bacteremia.
Background. The urinary tract is the most common source for Escherichia coli bacteremia. Mortality from E. coli urinary-source bacteremia is higher than that from urinary tract infection. Predisposing factors for urinary-source E. coli bacteremia are poorly characterized.
Methods. In order to identify urinary-source bacteremia risk factors, we conducted a 12-month prospective cohort study of adult inpatients with E. coli bacteriuria that were tested for bacteremia within ±1 day of the bacteriuria. Patients with bacteremia were compared with those without bacteremia. Bacterial isolates from urine were screened for 16 putative virulence genes using high-throughput dot-blot hybridization.
Results. Twenty-four of 156 subjects (15%) had E. coli bacteremia. Bacteremic patients were more likely to have benign prostatic hyperplasia (56% vs 19%; P = .04), a history of urogenital surgery (63% vs 28%; P = .001), and presentation with hesitancy/retention (21% vs 4%; P = .002), fever (63% vs 38%; P = .02), and pyelonephritis (67% vs 41%; P = .02). The genes kpsMT (group II capsule) (17 [71%] vs 62 [47%]; P = .03) and prf (P-fimbriae family) (13 [54%] vs 40 [30%]; P = .02) were more frequent in the urinary strains from bacteremic patients. Symptoms of hesitancy/retention (odds ratio [OR], 7.8; 95% confidence interval [CI], 1.6–37), history of a urogenital procedure (OR, 5.4; 95% CI, 2–14.7), and presence of kpsMT (OR, 2.9; 95% CI, 1–8.2) independently predicted bacteremia.
Conclusions. Bacteremia secondary to E. coli bacteriuria was frequent (15%) in those tested for it. Urinary stasis, surgical disruption of urogenital tissues, and a bacterial capsule characteristic contribute to systemic invasion by uropathogenic E. coli.
PMCID: PMC3357479  PMID: 22431806
12.  Effects of 14-Alpha-Lipoyl Andrographolide on Quorum Sensing in Pseudomonas aeruginosa 
Antimicrobial Agents and Chemotherapy  2012;56(12):6088-6094.
In Pseudomonas aeruginosa, the quorum-sensing (QS) system is closely related to biofilm formation. We previously demonstrated that 14-alpha-lipoyl andrographolide (AL-1) has synergistic effects on antibiofilm and antivirulence factors (pyocyanin and exopolysaccharide) of P. aeruginosa when combined with conventional antibiotics, while it has little inhibitory effect on its growth. However, its molecular mechanism remains elusive. Here we investigated the effect of AL-1 on QS systems, especially the Las and Rhl systems. This investigation showed that AL-1 can inhibit LasR–3-oxo-C12-homoserine lactone (HSL) interactions and repress the transcriptional level of QS-regulated genes. Reverse transcription (RT)-PCR data showed that AL-1 significantly reduced the expression levels of lasR, lasI, rhlR, and rhlI in a dose-dependent manner. AL-1 not only decreased the expression level of Psl, which is positively regulated by the Las system, but also increased the level of secretion of ExoS, which is negatively regulated by the Rhl system, indicating that AL-1 has multiple effects on both the Las and Rhl systems. It is no wonder that AL-1 showed synergistic effects with other antimicrobial agents in the treatment of P. aeruginosa infections.
PMCID: PMC3497202  PMID: 22802260
13.  Human Papillomavirus Genital Infections among Men, China, 2007–2009 
Emerging Infectious Diseases  2013;19(6):992-995.
To determine prevalence of genital human papillomavirus (HPV) infection among men in rural China, we analyzed genital swab specimens. Among 2,236 male residents of rural Henan Province, HPV infection prevalence was 17.5%. The most common oncogenic and nononcogenic types were HPV-16 and HPV-3, respectively. Infection was associated with younger age and multiple sex partners.
PMCID: PMC3713808  PMID: 23735236
HPV; men; China; viruses; genital infection; human papillomavirus
14.  Expression and clinical significance of the Trop-2 gene in advanced non-small cell lung carcinoma 
Oncology Letters  2013;6(2):375-380.
The Trop-2 gene has been examined in various carcinomas and is reported to be significantly associated with prognosis. Little is known with regard to Trop-2 gene expression in advanced non-small cell lung carcinoma (NSCLC). The present study investigated the expression of Trop-2 and its association with the prognosis of advanced NSCLC. The clinical records of 87 patients with advanced NSCLC, consisting of 37 cases of squamous cell carcinoma (SCC) and 50 cases of adenocarcinoma (AdC), together with 17 tumor-adjacent normal tissues, were retrospectively evaluated. Trop-2 expression was measured using an immunohistochemical method and its association with clinicopathological data and prognosis was also evaluated. The expression of Trop-2 was significantly higher in the cancer tissues compared with the tumor-adjacent normal tissues, and significantly higher in SCC compared with AdC (P=0.018). In SCC, the overexpression of Trop-2 was only correlated with the histological grade of the tumor (P= 0.035) and no correlation was observed with gender, age, lymph node metastasis, TNM stage or Eastern Cooperative Oncology Group (ECOG) performance status (PS). In AdC, the over-expression of Trop-2 was correlated with the histological grade, lymph node metastasis and TNM stage (P= 0.01, 0.024 and 0.015, respectively), while no correlation with gender, age or ECOG-PS was observed. The survival frequency was significantly higher in the Trop-2-negative patients compared with the Trop-2-positive patients [17.25 months (95% CI, 14.922–19.577) vs. 13.274 months (95% CI, 11.507–15.041); P= 0.008]. The survival time was significantly longer in the Trop-2-negative AdC patients [17.275 months (95% CI, 14.575–19.975) vs. 11.469 months (95% CI, 11.507–15.041); P= 0.002], but not in the SCC patients [17.167 months (95% CI, 12.428–21.906) vs. 14.647 months (95% CI, 12.062–17.232); P= 0.276]. The multivariate analysis revealed that Trop-2 expression [hazard ratio (HR) 2.381; P= 0.038], TNM stage (HR, 2.193; P= 0.03) and ECOG-PS (HR, 2.696; P= 0.007) were independent predictors for the survival outcome of patients with AdC. These results suggest that Trop-2 overexpression is closely correlated with an unfavorable prognosis in advanced NSCLC. Trop-2 is an independent prognostic marker and a potential new therapeutic target in advanced AdC.
PMCID: PMC3789024  PMID: 24137332
non-small cell lung carcinoma; trop-2; prognosis; survival
15.  Patient characteristics but not virulence factors discriminate between asymptomatic and symptomatic E. coli bacteriuria in the hospital 
BMC Infectious Diseases  2013;13:213.
Escherichia coli is a common cause of asymptomatic and symptomatic bacteriuria in hospitalized patients. Asymptomatic bacteriuria (ASB) is frequently treated with antibiotics without a clear indication. Our goal was to determine patient and pathogen factors suggestive of ASB.
We conducted a 12-month prospective cohort study of adult inpatients with E. coli bacteriuria seen at a tertiary care hospital in St. Louis, Missouri, USA. Urine cultures were taken at the discretion of treating physicians. Bacterial isolates were tested for 14 putative virulence genes using high-throughput dot-blot hybridization.
The median age of the 287 study patients was 65 (19–101) years; 78% were female. Seventy percent had community-acquired bacteriuria. One-hundred ten (38.3%) patients had ASB and 177 (61.7%) had symptomatic urinary tract infection (sUTI). Asymptomatic patients were more likely than symptomatic patients to have congestive heart failure (p = 0.03), a history of myocardial infarction (p = 0.01), chronic pulmonary disease (p = 0.045), peripheral vascular disease (p = 0.04), and dementia (p = 0.03). Patients with sUTI were more likely to be neutropenic at the time of bacteriuria (p = 0.046). Chronic pulmonary disease [OR 2.1 (95% CI 1.04, 4.1)] and dementia [OR 2.4 (95% CI 1.02, 5.8)] were independent predictors for asymptomatic bacteriuria. Absence of pyuria was not predictive of ASB. None of the individual virulence genes tested were associated with ASB nor was the total number of genes.
Asymptomatic E. coli bacteriuria in hospitalized patients was frequent and more common in patients with dementia and chronic pulmonary disease. Bacterial virulence factors could not discriminate symptomatic from asymptomatic bacteriurias. Asymptomatic E. coli bacteriuria cannot be predicted by virulence screening.
PMCID: PMC3658957  PMID: 23663267
Escherichia coli; Bacteriuria; Urinary tract infection; Asymptomatic; Virulence factors
16.  ContigScape: a Cytoscape plugin facilitating microbial genome gap closing 
BMC Genomics  2013;14:289.
With the emergence of next-generation sequencing, the availability of prokaryotic genome sequences is expanding rapidly. A total of 5,276 genomes have been released since 2008, yet only 1,692 genomes were complete. The final phase of microbial genome sequencing, particularly gap closing, is frequently the rate-limiting step either because of complex genomic structures that cause sequence bias even with high genomic coverage, or the presence of repeat sequences that may cause gaps in assembly.
We have developed a Cytoscape plugin to facilitate gap closing for high-throughput sequencing data from microbial genomes. This plugin is capable of interactively displaying the relationships among genomic contigs derived from various sequencing formats. The sequence contigs of plasmids and special repeats (IS elements, ribosomal RNAs, terminal repeats, etc.) can be displayed as well.
Displaying relationships between contigs using graphs in Cytoscape rather than tables provides a more straightforward visual representation. This will facilitate a faster and more precise determination of the linkages among contigs and greatly improve the efficiency of gap closing.
PMCID: PMC3651407  PMID: 23627759
ContigScape; Repeat contig; Microbial; Visualization; Linkage; Gap closing
17.  Channel Selection Based on Phase Measurement in P300-Based Brain-Computer Interface 
PLoS ONE  2013;8(4):e60608.
Most EEG-based brain-computer interface (BCI) paradigms include specific electrode positions. As the structures and activities of the brain vary with each individual, contributing channels should be chosen based on original records of BCIs. Phase measurement is an important approach in EEG analyses, but seldom used for channel selections. In this paper, the phase locking and concentrating value-based recursive feature elimination approach (PLCV-RFE) is proposed to produce robust-EEG channel selections in a P300 speller. The PLCV-RFE, deriving from the phase resetting mechanism, measures the phase relation between EEGs and ranks channels by the recursive strategy. Data recorded from 32 electrodes on 9 subjects are used to evaluate the proposed method. The results show that the PLCV-RFE substantially reduces channel sets and improves recognition accuracies significantly. Moreover, compared with other state-of-the-art feature selection methods (SSNRSF and SVM-RFE), the PLCV-RFE achieves better performance. Thus the phase measurement is available in the channel selection of BCI and it may be an evidence to indirectly support that phase resetting is at least one reason for ERP generations.
PMCID: PMC3623913  PMID: 23593261
18.  Complete Genome Sequence of Amycolatopsis mediterranei S699 Based on De Novo Assembly via a Combinatorial Sequencing Strategy 
Journal of Bacteriology  2012;194(20):5699-5700.
The genome of Amycolatopsis mediterranei S699 was resequenced and assembled de novo. By comparing the sequences of S699 previously released and that of A. mediterranei U32, about 10 kb of major indels was found to differ between the two S699 genomes, and the differences are likely attributable to their different assembly strategies.
PMCID: PMC3458657  PMID: 23012281
19.  White-Opaque Switching in Natural MTLa/α Isolates of Candida albicans: Evolutionary Implications for Roles in Host Adaptation, Pathogenesis, and Sex 
PLoS Biology  2013;11(3):e1001525.
All Mating Type Locus strain types of Candida albicans show white-opaque switching competency, not just MTL homozygotes, which allows them to adapt better to environmental changes.
Phenotypic transitions play critical roles in host adaptation, virulence, and sexual reproduction in pathogenic fungi. A minority of natural isolates of Candida albicans, which are homozygous at the mating type locus (MTL, a/a or α/α), are known to be able to switch between two distinct cell types: white and opaque. It is puzzling that white-opaque switching has never been observed in the majority of natural C. albicans strains that have heterozygous MTL genotypes (a/α), given that they contain all of the opaque-specific genes essential for switching. Here we report the discovery of white-opaque switching in a number of natural a/α strains of C. albicans under a condition mimicking aspects of the host environment. The optimal condition for white-to-opaque switching in a/α strains of C. albicans is to use N-acetylglucosamine (GlcNAc) as the sole carbon source and to incubate the cells in 5% CO2. Although the induction of white-to-opaque switching in a/α strains of C. albicans is not as robust as in MTL homozygotes in response to GlcNAc and CO2, opaque cells of a/α strains exhibit similar features of cellular and colony morphology to their MTL homozygous counterparts. Like MTL homozygotes, white and opaque cells of a/α strains differ in their behavior in different mouse infection models. We have further demonstrated that the transcriptional regulators Rfg1, Brg1, and Efg1 are involved in the regulation of white-to-opaque switching in a/α strains. We propose that the integration of multiple environmental cues and the activation and inactivation of a set of transcriptional regulators controls the expression of the master switching regulator WOR1, which determines the final fate of the cell type in C. albicans. Our discovery of white-opaque switching in the majority of natural a/α strains of C. albicans emphasizes its widespread nature and importance in host adaptation, pathogenesis, and parasexual reproduction.
Author Summary
Phenotypic transitions enable fungal pathogens to better adapt to their ever-changing environments. Approximately 10% of natural Candida albicans strains, which are homozygous at the mating type locus (MTL, a/a and α/α), can switch between two distinguishable morphological forms: white and opaque. The two cell types differ in a number of biological aspects including virulence, susceptibility to host immune attacks, and mating competency. Here, we demonstrate that white-opaque switching competency is not restricted to the MTL homozygous strains, but is a general characteristic of all MTL strain types of C. albicans (a/a, α/α, and a/α). Two host environmental cues, N-acetylglucosamine and CO2, promote white-to-opaque switching and stabilize the opaque phenotype. Thus, although switching is normally blocked in a/α cells, this block can be overcome through specific environmental changes. We further show that three transcriptional regulators (Rfg1, Brg1, and Efg1) help to regulate white-opaque switching in MTL heterozygotes of C. albicans. This study generalizes white-opaque switching to strains with all mating-type configurations and emphasizes its importance in host adaptation, pathogenesis, and parasexual reproduction.
PMCID: PMC3608550  PMID: 23555196
20.  Revised Genome Sequence of Burkholderia thailandensis MSMB43 with Improved Annotation 
Journal of Bacteriology  2012;194(17):4749-4750.
There is growing interest in discovery of novel bioactive natural products from Burkholderia thailandensis. Here we report a significantly improved genome sequence and reannotation of Burkholderia thailandensis MSMB43, which will facilitate the discovery of new natural products through genome mining and studies of the metabolic versatility of this bacterium.
PMCID: PMC3415525  PMID: 22887659
21.  N-Acetylglucosamine Induces White-to-Opaque Switching and Mating in Candida tropicalis, Providing New Insights into Adaptation and Fungal Sexual Evolution 
Eukaryotic Cell  2012;11(6):773-782.
Pathogenic fungi are capable of switching between different phenotypes, each of which has a different biological advantage. In the most prevalent human fungal pathogen, Candida albicans, phenotypic transitions not only improve its adaptation to a continuously changing host microenvironment but also regulate sexual mating. In this report, we show that Candida tropicalis, another important human opportunistic pathogen, undergoes reversible and heritable phenotypic switching, referred to as the “white-opaque” transition. Here we show that N-acetylglucosamine (GlcNAc), an inducer of white-to-opaque switching in C. albicans, promotes opaque-cell formation and mating and also inhibits filamentation in a number of natural C. tropicalis strains. Our results suggest that host chemical signals may facilitate this phenotypic switching and mating of C. tropicalis, which had been previously thought to reproduce asexually. Overexpression of the C. tropicalis WOR1 gene in C. albicans induces opaque-cell formation. Additionally, an intermediate phase between white and opaque was observed in C. tropicalis, indicating that the switching could be tristable.
PMCID: PMC3370467  PMID: 22544905
22.  Crystal Structure of Arabidopsis Deg2 Protein Reveals an Internal PDZ Ligand Locking the Hexameric Resting State* 
The Journal of Biological Chemistry  2012;287(44):37564-37569.
Background: The PDZ protease Deg2 is involved in chloroplast protein quality control through a yet unknown molecular mechanism.
Results: A novel PDZ domain with an internal ligand mediates hexamer formation and locks Deg2 into the resting state.
Conclusion: Formation of the resting hexamer may be a common strategy in a Deg protease subfamily.
Significance: We provide structural insights into the PDZ domain-mediated regulation of Deg proteases.
Eukaryotic organelles have developed elaborate protein quality control systems to ensure their normal activity, among which Deg/HtrA proteases play an essential role. Plant Deg2 protease is a homologue of prokaryotic DegQ/DegP proteases and is located in the chloroplast stroma, where its proteolytic activity is required to maintain the efficiency of photosynthetic machinery during stress. Here, we demonstrate that Deg2 exhibits dual protease-chaperone activities, and we present the hexameric structure of Deg2 complexed with co-purified peptides. The structure shows that Deg2 contains a unique second PDZ domain (PDZ2) following a conventional PDZ domain (PDZ1), with PDZ2 orchestrating the cage assembly of Deg2. We discovered a conserved internal ligand for PDZ2 that mediates hexamer formation and thus locks the protease in the resting state. These findings provide insight into the diverse modes of PDZ domain-mediated regulation of Deg proteases.
PMCID: PMC3481350  PMID: 22961982
Chaperone Chaperonin; Protease; Protein Structure; Structural Biology; X-ray Crystallography; Deg/HtrA Protease; PDZ Domain; Oligomerization; Protease-Chaperone; Protein Quality Control
23.  Nontypeable Haemophilus influenzae Genetic Islands Associated with Chronic Pulmonary Infection 
PLoS ONE  2012;7(9):e44730.
Haemophilus influenzae (Hi) colonizes the human respiratory tract and is an important pathogen associated with chronic obstructive pulmonary disease (COPD). Bacterial factors that interact with the human host may be important in the pathogenesis of COPD. These factors, however, have not been well defined. The overall goal of this study was to identify bacterial genetic elements with increased prevalence among H. influenzae strains isolated from patients with COPD compared to those isolated from the pharynges of healthy individuals.
Methodology/Principal Findings
Four nontypeable H. influenzae (NTHi) strains, two isolated from the airways of patients with COPD and two from a healthy individual, were subjected to whole genome sequencing using 454 FLX Titanium technology. COPD strain-specific genetic islands greater than 500 bp in size were identified by in silico subtraction. Open reading frames residing within these islands include known Hi virulence genes such as lic2b, hgbA, iga, hmw1 and hmw2, as well as genes encoding urease and other enzymes involving metabolic pathways. The distributions of seven selected genetic islands were assessed among a panel of 421 NTHi strains of both disease and commensal origins using a Library-on-a-Slide high throughput dot blot DNA hybridization procedure. Four of the seven islands screened, containing genes that encode a methyltransferase, a dehydrogenase, a urease synthesis enzyme, and a set of unknown short ORFs, respectively, were more prevalent in COPD strains than in colonizing strains with prevalence ratios ranging from 1.21 to 2.85 (p≤0.0002). Surprisingly, none of these sequences show increased prevalence among NTHi isolated from the airways of patients with cystic fibrosis.
Our data suggest that specific bacterial genes, many involved in metabolic functions, are associated with the ability of NTHi strains to survive in the lower airways of patients with COPD.
PMCID: PMC3435294  PMID: 22970300
24.  Cloning and characterization of a novel 2-ketoisovalerate reductase from the beauvericin producer Fusarium proliferatum LF061 
BMC Biotechnology  2012;12:55.
The ketoisovalerate reductase (EC ) is required for the formation of beauvericin via the nonribosomal peptide synthetase biosynthetic pathway. It catalyzes the NADPH-specific reduction of ketoisovaleric acid to hydroxyisovalerate. However, little is known about the bioinformatics’ data about the 2-Kiv reductase in Fusarium. To date, heterologous production of the gene KivRFp from Fusarium has not been achieved.
The KivRFp gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene KivRFp contained a 1,359 bp open reading frame (ORF) encoding a polypeptide of 452 amino acids with a molecular mass of 52 kDa. Sequence analysis indicated that it showed 61% and 52% amino acid identities to ketoisovalerate reductase from Beauveria bassiana ATCC 7159 (ACI30654) and Metarhizium acridum CQMa 102 (EFY89891), respectively; and several conserved regions were identified, including the putative nucleotide-binding signature site, GXGXXG, a catalytic triad (Glu405, Asn184, and Lys285). The KivRFp exhibited the highest activity at 35°C and pH 7.5 respectively, by reduction of ketoisovalerate. It also exhibited the high level of stability over wide temperature and pH spectra and in the presence of metal ions or detergents.
A new ketoisovalerate reductase KivRFp was identified and characterized from the depsipeptide-producing fungus F. proliferatum. KivRFp has been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering and directed evolution, towards the goal of developing efficient enzyme for downstream biotechnological applications.
PMCID: PMC3478992  PMID: 22916830
2-Kiv Reductase; Beauvericin; Fusarium proliferatum LF061
25.  Effect of Sex, Age, and Race on the Clinical Presentation of Tuberculosis: A 15-Year Population-Based Study 
Extrapulmonary tuberculosis (EPTB) is an important health problem that may cause serious morbidity and diagnostic challenges. We conducted a case–control study involving 5,684, approximately 99% of bacteriologically confirmed TB patients (including 1,925 EPTB cases) diagnosed in Denmark and Greenland during 1992–2007 to gain insight to the role of host factors in EPTB pathogenesis. Among patients from Somalia and Asia, persons 25–44 and 45–64 years of age were more likely to have EPTB than persons 15–24 years of age. In contrast, among persons from Greenland, the two oldest age groups were significantly less likely to have EPTB than the youngest age group. For all the age groups, the odds for having EPTB was significantly higher among patients from Somalia and Asia and significantly lower among the patients from Greenland than among patients from Denmark. Furthermore, the occurrence of specific types of EPTB significantly varied among different age groups or origins.
PMCID: PMC3144827  PMID: 21813849

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