Phylogenetic characterization of bacteria in the gut of house flies (Musca domestica L.)

House flies (Musca domestica L.) are cosmopolitan, ubiquitous, synanthropic insects that serve as mechanical or biological vectors for various microorganisms. To fully assess the role of house flies in the epidemiology of human diseases, it is essential to understand the diversity of microbiota harbored by natural fly populations. This study aimed to identify the diversity of house fly gut bacteria by both culture-dependent and culture-independent approaches. A total of 102 bacterial strains were isolated from the gut of 65 house flies collected from various public places including a garden, public park, garbage/dump area, public toilet, hospital, restaurant/canteen, mutton shop/market, and house/human habitation. Molecular phylogenetic analyses placed these isolates into 22 different genera. The majority of bacteria identified were known potential pathogens of the genera Klebsiella, Aeromonas, Shigella, Morganella, Providencia, and Staphylococcus. Culture-independent methods involved the construction of a 16S rRNA gene clone library, and sequence analyses supported culture recovery results. However, additional bacterial taxa not determined via culture recovery were revealed using this methodology and included members of the classes Alphaproteobacteria, Deltaproteobacteria, and the phylum Bacteroidetes. Here, we show that the house fly gut is an environmental reservoir for a vast number of bacterial species, which may have impacts on vector potential and pathogen transmission.     

Erratum: Interactions Between Cadmium and Zinc in the Biological Samples of Pakistani Smokers and Nonsmokers Cardiovascular Disease Patients

The pathogenesis of some cardiovascular diseases (CVDs) has been altered with changes in the balance of certain trace and toxic elements. The aim of the present study was to assess the role of zinc (Zn) and cadmium (Cd) in smoker and nonsmoker male CVD patients (n = 457) of two age groups (31–45) and (46–60). The both elements were determined in biological samples (scalp hair, blood, and urine) of CVD patients and healthy referents for comparison purpose. The concentrations of Zn and Cd were measured by atomic absorption spectrophotometer prior to microwave-assisted acid digestion. It was observed that the mean values of Cd were significantly higher in the biological samples of smokers CVD as compared to nonsmoker CVD patients, while the level of Zn was lower in both smoker and nonsmoker patients. The concentrations of Zn in whole blood and scalp hair samples were lower in CVD patients as compared to referents (p > 0.001). Results showed significant changes of levels of Cd and Zn in blood and scalp hair samples of CVD patients when compared with healthy referents, while reverse in the case of urine samples. It was observed that low Zn levels were associated with both smoker and nonsmoker CVD patients, while increased cadmium accumulation was observed in smoker patients as compared to nonsmoker patients (p > 0.025).     

Synthesis and in-vitro anticancer activity of 3,5-bis(indolyl)-1,2,4-thiadiazoles

A series of 3,5-bis(indolyl)-1,2,4-thiadiazoles were synthesized and evaluated for their cytotoxicity against selected human cancer cell lines. The reaction of indole-3-thiocarboxamide 3 with iodobenzene diacetate underwent oxidative dimerization to give 3,5-bis(indolyl)-1,2,4-thiadiazoles 4a-n. Among the synthesized bis(indoly)-1,2,4-thiadiazoles, the compound 4h with 4-chlorobenzyl and methoxy substituents showed the most potent activity.     

Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42. 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.     

Xylanase production by Burkholderia sp. DMAX strain under solid state fermentation using distillery spent wash

Xylanase production by a newly isolated strain of Burkholderia sp. was studied under solid state fermentation using anaerobically treated distillery spent wash. Response surface methodology (RSM) involving Box-Behnken design was employed for optimizing xylanase production. The interactions between distillery effluent concentration, initial pH, moisture ratio and inoculum size were investigated and modeled. Under optimized conditions, xylanase production was found to be in the range of 5200-5600 U/g. The partially purified enzyme recovered after ammonium sulphate fractionation showed maximum activity at 50 degrees C and pH 8.6. Kinetic parameters like Km and Vmax for xylan were found to be 12.75 mg/ml and 165 micromol/mg/min. In the presence of metal ions such as Ca2+, Co2+, Mn2+, Ba2+, Mg2+ and protein disulphide reducing agents such as beta-mercaptoethanol and dithiotheritol (DTT) the activity of enzyme increased, where as strong inhibition of enzyme activity was observed in the presence of Cu2+, Ag+, Fe2+ and SDS. The crude enzyme hydrolysed lignocellulosic substrate, wheat bran as well as industrial pulp.     

A general framework for analyzing data from two short time-series microarray experiments

We propose a general theoretical framework for analyzing differentially expressed genes and behavior patterns from two homogenous short time-course data. The framework generalizes the recently proposed Hilbert-Schmidt Independence Criterion (HSIC)-based framework adapting it to the time-series scenario by utilizing tensor analysis for data transformation. The proposed framework is effective in yielding criteria that can identify both the differentially expressed genes and time-course patterns of interest between two time-series experiments without requiring to explicitly cluster the data. The results, obtained by applying the proposed framework with a linear kernel formulation, on various data sets are found to be both biologically meaningful and consistent with published studies.     

Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation*

Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in fucosylation could be caused by the detected changes in enzymes belonging to the glycan biosynthesis pathways of protein fucosylation observed in our proteomic analysis. The altered protein fucosylation forms have great potential in aiding our understanding of castration resistance and may lead to the development of novel therapeutic approaches and specific detection strategies for prostate cancer.Androgen is important for the development, function, and proliferation of both normal and cancerous prostate cells (1). At the earliest stage of prostate cancer, prostate cancer cells are dependent on the presence of androgen, and androgen-deprivation therapy (ADT)¹ is used to treat prostate cancer (2). However, cells become androgen-independent as a result of androgen deprivation therapy, and they become more aggressive. This results in androgen-independent remission of prostate cancer (3). LNCaP and PC3 cell lines have been widely used as models of prostate cancer. LNCaP is an androgen-dependent cancer cell line, whereas PC3 is an androgen-independent cell line. The LNCaP cell line is less aggressive as compared with PC3 cells that have a high metastatic potential. LNCaP and PC3 cells have been previously studied by genomics and proteomics approaches to understand the mechanism(s) responsible for the aggressive and metastatic nature of prostate cancer (4–8).Post-translational modifications (PTMs) such as phosphorylation are important in the function of the androgen-dependent pathway. Androgen receptors bind to androgen and are then phosphorylated before translocating into the nucleus (3). However, protein PTMs cannot be directly inferred from gene expression. Glycosylation is an abundant PTM and most cell surface or secreted proteins are expected to be glycosylated (9). Glycosylation is one of the more complex PTMs because of the fact that different glycosylation machineries are present in different cells, multiple glycosylation sites exist on many glycoproteins and each glycosylation site can be modified by several different glycans (10, 11). Such microheterogeneity of glycan structures at each glycosylation site with different site occupancy significantly increases the structural diversity of each glycoprotein that is specific to the microenvironment of the cells where each glycoprotein is produced. Although these characteristics of protein glycosylation pose considerable challenges to the structural and functional analyses of glycoproteins, we expect that cell and cell microenvironment-specific glycoproteins differ according to the physiological and pathological states of the cells. Aberrant glycosylation is the result of alterations in glycosylation genes that may lead to the development of cancer. A systematic approach to analyze proteins, glycoproteins, and glycosylation is expected to permit the identification of the glycoprotein alterations that are specific to each cell state and aid the understanding of the functions of glycosylation because alterations in glycosylation can affect glycoprotein abundance or function (12, 13). A detailed analysis of glycoproteins in cancer cells with different functions is needed to understand tumor biology and how glycoproteins can function as therapeutic targets or diagnostic biomarkers (14, 15).In this study, a comprehensive proteomic and glycoproteomic platform was designed to investigate the differences in proteins, glycoproteins, and site-specific glycosylation forms of glycoproteins between LNCaP and PC3 cells (Fig. 1). To our knowledge, this is the first report to characterize glycoproteins with respect to protein abundance, glycosylation occupancy, and glycosylation heterogeneity at specific glycosites. These altered glycosylation patterns among proteins between LNCaP and PC3 cell lines have a significant potential to aid our understanding of the altered glycoprotein expression in prostate cancer cells, thus leading to novel specific methods to detect aggressive prostate cancer.Open in a separate windowFig. 1.Schematic representation of the workflow for the integrated analysis of glycosite-containing peptides, global protein expression, and intact glycopeptides. Proteins were obtained from LNCaP and PC3 cell lines followed by tryptic digestion and iTRAQ labeling. Labeled peptide samples were then combined and separated into two aliquots. One aliquot was enriched for glycosite-containing peptides using Solid Phase Extraction of Glycopeptides (SPEG) and the other aliquot was used for bRPLC separation followed by the analysis of global proteins and intact glycopeptides. Finally, peptides were analyzed using LC-MS/MS.