Transcriptional response of Streptococcus pneumoniae to varying concentrations of carbohydrates and metal ions

S. pneumoniae is one of the most common human pathogen that resides in nasopharynx. It is responsible for millions of death every year all over the world, especially in young children. S. pneumoniae can spread from nasopharynx to different parts of the human body where it may encounter different nutrients including various concentration of metal ions and carbohydrates. It has an ability to survive on these nutrients by tuning it’s genome according. This thesis contributes to the understanding of sugar- and metal-ions dependent regulation of virulence genes/operons including fcs operon, PsaR regulon and AdcR regulon. Chapter 2 deals with the optimization of conditions for bacterial transcriptome analysis from cell harvest to DNA microarray analysis. Taking into account the time, costs and accuracy of the experiments, this technology platform proves to be very useful and universally applicable for studying bacterial transcriptomes. Here, we perform DNA microarray analysis with Streptococcus pneumoniae as a case-study by comparing the transcriptional responses of S. pneumoniae grown in the presence of varying L-serine concentrations in the medium. Total RNA was isolated by using a Macaloid method using an RNA isolation kit and the quality of RNA was checked by using an RNA quality check kit. cDNA was prepared using reverse transcriptase and the cDNA samples were labelled using one of two amine-reactive fluorescent dyes, . Homemade DNA microarray slides were used for hybridization of the labelled cDNA samples and microarray data were analyzed by using a cDNA microarray data pre-processing framework (Microprep). Finally, Cyber-T was used to analyze the data generated using Microprep for the identification of statistically significant differentially expressed genes. Furthermore, in-house built software packages (PePPER, FIVA, DISCLOSE, PROSECUTOR, Genome2D) were used to analyze data. In Chapter 3 explores the impact of fucose on the transcriptome of S. pneumoniae D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (fcs operon), was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the fcs operon, as a transcriptional activator of the fcs operon. We also predict a 19-bp putative FcsR regulatory site (5’-ATTTGAACATTATTCAAGT-3’) in the promoter region of the fcs operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the fcs operon. Manganese (Mn2+)-, zinc (Zn2+)- and copper (Cu2+) play significant roles in transcriptional gene regulation, physiology and virulence of Streptococcus pneumoniae. So far, the effect of the important transition metal ion cobalt (Co2+) on gene expression of S. pneumoniae has not yet been explored. Chapter 4 reveals the impact of Co2+ stress on the transcriptome of S. pneumoniae strain D39. BLAST searches revealed that the genome of S. pneumoniae encodes a putative Co2+-transport operon (cbi operon), the expression of which we show here to be induced by a high Co2+ concentration. Furthermore, we found that Co2+, as has been shown previously for Zn2+, can cause derepression of the genes of the PsaR virulence regulon, encoding the Mn2+-uptake system PsaBCA, the choline binding protein PcpA and the cell-wall associated serine protease PrtA. Interestingly, although Mn2+ represses expression of the PsaR regulon and Co2+ leads to derepression, both metal ions stimulate interaction of PsaR with its target promoters. These data will be discussed in the light of previous studies on similar metal-responsive transcriptional regulators. Chapter 5 examines the Ni2+-dependent regulation of PsaR regulon (pcpA, psaBCA and prtA). Here, we combine qRT-PCR analysis, metal accumulation assays, β-galactosidase assays and electrophoretic mobility shift assays to show that the elevated concentration of Ni2+ leads to the higher expression of PsaR regulon. Our ICP-MS data demonstrated that the induced expression of PsaR regulon is directly linked to high concentration of Ni2+, which reduces the cell-associated concentration of Mn2+. In vitro studies with purified PsaR protein showed that Ni2+ diminishes the Mn2+-dependent interaction of PsaR to the promoter regions of its target genes. Additionally, Ni2+-dependent role of transcriptional regulator PsaR in the regulation of PsaR regulon is studied by the DNA microarray analysis. Chapter 6 explains the transcriptome analysis of S. pneumoniae D39 in presence of Ni2+. The regulation of virulence genes in the human pathogen Streptococcus pneumoniae is an important characteristic of transition metal ions. Metal ions such as Mn2+, Zn2+, Cu2+and Co2+ have been shown to regulate the expression of various virulence genes. The effect of Ni2+ on global gene expression of S. pneumoniae has not yet been investigated. Here, we identify a number of differentially expressed genes/ operons in the presence of a high extracellular concentration of Ni2+. The virulence genes belonging to the AdcR regulon (adcRCBA, phtA, phtB, phtD and phtE), the PsaR regulon (prtA, pcpA and psaBCA) and Zn2+/ Co2+ -efflux system czcD were highly upregulated in the presence of Ni2+. By means of DNA microarray analysis, electrophoretic mobility shift assays and transcriptional lacZ-reporter studies, we show that Ni2+ is directly involved in the derepression of adcRCBA, adcAII-phD, phtA, phtB and phtE via the Zn2+-dependent repressor AdcR. The opposite effect of Zn2+ and Ni2+ on the AdcR regulon is also explored in detail. In conclusion, the role of Ni2+ in the regulation of virulence genes belonging to the AdcR regulon has been further elucidated

Transcriptome analysis of Streptococcus pneumoniae D39 in the presence of cobalt

Cobalt (Co2+) is an important transition metal ion that plays a vital role in cellular physiology of bacteria. The role of Co2+ in the regulation of several genes/operons in Streptococcus pneumoniae has recently been reported [1]. The data described in this article relate to the genome-wide transcriptional profiling of Streptococcus pneumoniae D39, either in the presence or absence of 0.5 mM Co2+ in chemically defined medium (CDM) using DNA microarray analysis. Genes belonging to a broad range of cellular processes such as virulence, transport and efflux systems, stress response and surface attachment were differentially expressed in the presence of Co2+. We used transcriptional lacZ assays and electrophoretic mobility shift assays (EMSAs) to confirm our results [1]. The dataset is publicly available at the Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/) with accession number GSE57696

Ni2+-Dependent and PsaR-Mediated Regulation of the Virulence Genes pcpA, psaBCA, and prtA in Streptococcus pneumoniae

Previous studies have shown that the transcriptional regulator PsaR regulates the expression of the PsaR regulon consisting of genes encoding choline binding protein (PcpA), the extracellular serine protease (PrtA), and the Mn2+-uptake system (PsaBCA), in the presence of manganese (Mn2+), zinc (Zn2+), and cobalt (Co2+). In this study, we explore the Ni2+-dependent regulation of the PsaR regulon. We have demonstrated by qRT-PCR analysis, metal accumulation assays, β-galactosidase assays, and electrophoretic mobility shift assays that an elevated concentration of Ni2+ leads to strong induction of the PsaR regulon. Our ICP-MS data show that the Ni2+-dependent expression of the PsaR regulon is directly linked to high, cell-associated, concentration of Ni2+, which reduces the cell-associated concentration of Mn2+. In vitro studies with the purified PsaR protein showed that Ni2+ diminishes the Mn2+-dependent interaction of PsaR to the promoter regions of its target genes, confirming an opposite effect of Mn2+ and Ni2+ in the regulation of the PsaR regulon. Additionally, the Ni2+-dependent role of PsaR in the regulation of the PsaR regulon was studied by transcriptome analysis

Characterizing Circular Supply Chain Practices in Industry 5.0 With Respect to Sustainable Manufacturing Operations

The current research investigated the significance of circular supply chain practices in Industry 5.0 with respect to their sustainable manufacturing operations. Through a comprehensive review of the literature, the current study identified key circular supply chain practices and their potential benefits for Industry 5.0. The findings indicated that closed-loop supply chains, sustainable sourcing, product design for circularity, and waste reduction may assist Industry 5.0 firms to achieve their sustainability objectives while enhancing the operational efficiency. Moreover, the study also highlighted the challenges associated with the implementation of circular supply chain practices including the necessity for collaboration among supply chain partners, investment in new technologies and infrastructure, and the development of new skills and capabilities. From a practical and managerial perspective, the implications suggest that firms aiming to adopt circular supply chain practices in Industry 5.0 should prioritize collaboration and coordination, make investments in new technologies and infrastructure, and foster the acquisition of new skills and capabilities. To complement this research, future studies could employ empirical research methods in order to validate the findings and recommendations as well as explore potential barriers to the implementation of circular supply chain practices in Industry 5.0

Course and outcomes of complicated gallstone disease in pregnancy: a single centre experience

Background: The incidence of acute abdomen during pregnancy is approximately 1 in 500 pregnancies. The incidence of symptomatic gallstone disease in pregnancy is reported in approximately 0.2-0.5 per 1,000 pregnancies. Symptoms are similar to those in the nonpregnant state. A delay in diagnosis may increase the risk of perforation. Treatment in most cases is conservative. However, recent trends, newer instrumentation and skilled personnel encourage arranging laparoscopic cholecystectomy at the time of diagnosis.Methods: This study was a retrospective study, included 117 pregnant patients with acute gallstone disease, who were treated and followed-up at Government Medical College, Srinagar, Department of General Surgery and Department of Gynae And Obstetrics, between January 2015 and April 2017.Results: The mean age of patients in our study was 28.6 years. Majority of patients 56 (47.86%) were in is trimester of pregnancy. Parity of the patients varied from 1 to 6, with a mean parity of 2.67. The presentation of majority of patients was colicky pain right upper abdomen,108 (92.30%). All patients had gallstones on USG scan.101(86.32%) patients had acute cholecystitis, while 8 (6.83%) patients had predominant features of acute pancreatitis,8(6.83%) patients had accompanying choledocholithiasis The average wall thickness of gallbladder in our patients was 4.62 mm. Majority 106 (90.59%) patients were managed conservatively. 8 (6.83%) patients underwent cholecystectomy in same admission, after failure of conservative management, 7 patients underwent laparoscopic cholecystectomy and one underwent open cholecystectomy. 3 patients (2.56%), who had features of cholangitis were managed by ERCP. The average length of hospital stay in our patient group was 8.61 days. There was one maternal death reported in our study, there were a total of 8 (6.83%) preterm deliveries.Conclusions: Symptomatic gallstone disease in pregnancy is a common surgical problem. Diagnosis during pregnancy can be difficult, majority of cases can be managed conservatively, intervention whenever indicated must be undertaken

The Regulation of the AdcR Regulon in Streptococcus pneumoniae Depends Both on Zn(2+)- and Ni(2+)-Availability

By using a transcriptomic approach, we have elucidated the effect of Ni(2+) on the global gene expression of S. pneumoniae D39 by identifying several differentially expressed genes/operons in the presence of a high extracellular concentration of Ni(2+). The genes belonging to the AdcR regulon (adcRCBA, adcAII-phtD, phtA, phtB, and phtE) and the PsaR regulon (pcpA, prtA, and psaBCA) were highly upregulated in the presence of Ni(2+). We have further studied the role of Ni(2+) in the regulation of the AdcR regulon by using ICP-MS analysis, electrophoretic mobility shift assays and transcriptional lacZ-reporter studies, and demonstrate that Ni(2+) is directly involved in the derepression of the AdcR regulon via the Zn(2+)-dependent repressor AdcR, and has an opposite effect on the expression of the AdcR regulon compared to Zn(2+)

Cysteine-Mediated Gene Expression and Characterization of the CmbR Regulon in Streptococcus pneumoniae

In this study, we investigated the transcriptomic response of Streptococcus pneumoniae D39 to cysteine. Transcriptome comparison of the D39 wild-type grown at a restricted concentration of cysteine (0.03 mM) to one grown at a high concentration of cysteine (50 mM) in chemically-defined medium (CDM) revealed elevated expression of various genes/operons, i.e., spd-0150, metQ, spd-0431, metEF, gshT, spd-0618, fhs, tcyB, metB-csd, metA, spd-1898, yvdE, and cysK, likely to be involved in the transport and utilization of cysteine and/or methionine. Microarray-based data were further confirmed by quantitative RT-PCR. Promoter lacZ-fusion studies and quantitative RT-PCR data showed that the transcriptional regulator CmbR acts as a transcriptional repressor of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE, putatively involved in cysteine uptake and utilization. The operator site of CmbR in the promoter regions of CmbR-regulated genes is predicted and confirmed by mutating or deleting CmbR operator sites from the promoter regions of these genes

Canto & Its Evolution in Urdu Literature

Poetry of any literature is the mirror of that language in which it is being produced. Urdu literature, under the influence of globalization, has changed so much and adopted many forms and molds of poetry from modern literature of the western world. Urdu poetry especially in the nearer past has adopted many new styles, techniques, and Genres from the languages of the world. In this article, it is discussed to what extent Urdu literature has adopted the genre of Canto and what are the different ways of exploration and innovation of Urdu poets in this regard. It presents the contemporary situation of Canto along with its tradition in Urdu literature