DNA Binding in High Salt: Analysing the Salt Dependence of Replication Protein A3 from the Halophile Haloferax volcanii

Halophilic archaea maintain intracellular salt concentrations close to saturation to survive in high-salt environments and their cellular processes have adapted to function under these conditions. Little is known regarding halophilic adaptation of the DNA processing machinery, particularly intriguing since protein-DNA interactions are classically salt sensitive. To investigate such adaptation, we characterised the DNA-binding capabilities of recombinant RPA3 from Haloferax volcanii (HvRPA3). Under physiological salt conditions (3M KCl), HvRPA3 is monomeric, binding 18 nucleotide ssDNA with nanomolar affinity, demonstrating that RPAs containing the single OB-fold/zinc finger architecture bind with broadly comparable affinity to two OB-fold/zinc finger RPAs. Reducing the salt concentration to 1M KCl induces dimerisation of the protein, which retains its ability to bind DNA. On circular ssDNA, two concentration-dependent binding modes are observed. Conventionally, increased salt concentration adversely affects DNA binding but HvRPA3 does not bind DNA in 0.2M KCl, although multimerisation may occlude the binding site. The single N-terminal OB-fold is competent to bind DNA in the absence of the C-terminal zinc finger, albeit with reduced affinity. This study represents the first quantitative characterisation of DNA binding in a halophilic protein in extreme salt concentrations

In vivo investigation of interactions between replisome components in Escherichia coli: An expanded model for the processivity switch

Background: Protein interactions within the replisome (a highly coordinated protein complex) are crucial to maintain temporal and spatial regulation for high fidelity DNA synthesis in Escherichia coli (E. coli). A key component of these interactions is the processivity switch, ensuring smooth transition of the replicative DNA polymerase III (Pol III) between Okazaki fragments on the lagging strand. Multiple interaction studies between replisome components have been performed to indicate the essential roles of Pol III (DnaE), β-clamp, DnaB helicase, DNA and the t (DnaX) subunit for this switch.Methods: Known interacting regions of both DnaE and various truncated versions of t were chosen for co-expression in E. coli. Differences in the growth pattern of cells co-expressing various truncated versions of DnaX and DnaE, on liquid and solid media were subsequently analyzed. Based on in vivo analyses to explore the interactions between these components, an expanded model for the processivity switch is presented here.Results: The analyses suggest that residues 481-643 of t are sufficient to establish a functional interaction with the DnaB helicase and DnaE during replication, while residues 461-480 of t interact with the C-terminal tail of DnaE to disengage Pol III from the β-clamp during processivity switching. We also propose that residues 430-460 of t are involved in sensing the DNA structure required for the processivity switch.Conclusion: These observations expand the current understanding of processivity switching and help dissect the regions of t utilized for binding to different replisome components such as DnaB helicase, polymerase and DNA.Keywords: Processivity Switch; Clamp Loader; DnaE; DnaX; DnaB Helicas

Simultaneous detection of phylogroups and ESBL genes in E. coli using Multiplex PCR

Background: Escherichia coli (E. coli) strains harbor various allelic versions of beta lactamase genes and their identification using conventional phenotypic tests is a tedious and time consuming task. In the present study, multiplex PCR is performed for the simulatanoues detection of E. coli phylogroups and extended-spectrum β-lactamase- (ESBL) genes.Methods: A total of 128 E. coli isolates from urine samples were screened for antibiotic resistance and expression of ESBL activity using phenotypic and genotypic methods. Uniplex and multiplex PCRs were used to detect E. coli phylogroup detrminants and blaCTX-M-15, blaOXA-1 and TEM1 genes. Chi Square test of independence was employed for evaluating significant levels at P value < 0.05. Results: Phylogroup B2 was detected as the predominant group (36%) followed by group D (30%), A (25%) and B1 (9%). The highest resistance was seen against nalidixic acid (100%) and the lowest against amoxicillin-clavulanic acid (55%). Significant P values were observed for resistance against cefotaxime and cefepime in the phylogroup B2 while resistance against cefoxitin, sulfamethoxazole and fosfomycin was significantly associated with group D. Combination disc diffusion test (CDDT) showed ESBL activity in 42% E. coli isolates. A significant association of blaCTX-M-15 gene was observed for phylogroup B2 (P = 0.007). Moreover, a combination genotype of blaCTX-M-15 and TEM1 was also found statistically prevalent in phylogroup B2 (P = 0.006).Conclusion: The study highlights the alarming rise in antibiotic resistance and delineates B2 a predominant phylogoup with a high prevalence of blaCTX-M-15 and TEM1 genes in urinary E. coli isolates.Keywords: UTI; E. Coli; Phylogenetic groups; ESBL genes; Multiplex PCR 

Molecular surveillance of HCV mono-infection and HCV-HBV co-infection in symptomatic population at Hyderabad, Pakistan

Background: Pakistan is endemic to hepatitis B and C infections. Alarming rise in hepatitis C virus (HCV) infection has been noticed in some areas of Sindh with an increasing risk for co-infection frequency in this region.Objective: To estimate the burden of HBV/HCV infection in Hyderabad Pakistan. Methods: ELISA and Nucleic acid Amplification test were performed to detect viruses. SPSS and online calculator were used for statistical analysis.Results: From a total of 108 seropositive hepatitis patients, 36.1% (n=39) were found HCV RNA-positive. Non-significant differences were observed in the frequencies of HCV infection for both genders [OR=0.735, CI (95%) 0.307-1.761, p&lt;0.05]. The percentage of HBV DNA detection among 108 HCV-seropositive cases was 17.9% (n=19). However, HCV-HBV co-infection in HCV-RNA positive cases was determined in 48.7% (n=19) cases with non-significant difference in both genders [OR=1.51, CI (95%) = 0.38 - 5.96, p&lt; 0.05]. Analysis suggested weakly positive correlation between HCV mono-infection and HCV-HBV co-infection and age (r =0.184, and r =0.1231), respectively.Conclusion: The study demonstrates a high prevalence of HBV co-infection among active hepatitis C patients of Hyderabad.Keywords: HCV mono-infection, HCV-HBV co-infection, molecular surveillance, Nucleic acid Amplification Test, active hepatitis C, Hyderabad, Sindh

Co-expression and purification of the RadA recombinase with the RadB paralog from Haloferax volcanii yields heteromeric ring-like structures

The study of archaeal proteins and the processes to which they contribute poses particular challenges due to the often extreme environments in which they function. DNA recombination, replication and repair proteins of the halophilic euryarchaeon, Haloferax volcanii (Hvo) are of particular interest as they tend to resemble eukaryotic counterparts in both structure and activity, and genetic tools are available to facilitate their analysis. In the present study, we show using bioinformatics approaches that the Hvo RecA-like protein RadA is structurally similar to other recombinases although is distinguished by a unique acidic insertion loop. To facilitate expression of Hvo RadA a co-expression approach was used, providing its lone paralog, RadB, as a binding partner. At present, structural and biochemical characterization of Hvo RadA is lacking. Here, we describe for the first time co-expression of Hvo RadA with RadB and purification of these proteins as a complex under in vitro conditions. Purification procedures were performed under high salt concentration (>1 M sodium chloride) to maintain the solubility of the proteins. Quantitative densitometry analysis of the co-expressed and co-purified RadAB complex estimated the ratio of RadA to RadB to be 4 : 1, which suggests that the proteins interact with a specific stoichiometry. Based on a combination of analyses, including size exclusion chromatography, Western blot and electron microscopy observations, we suggest that RadA multimerizes into a ring-like structure in the absence of DNA and nucleoside co-factor

Molecular surveillance of HCV mono-infection and HCV-HBV co-infection in symptomatic population at Hyderabad, Pakistan

Background: Pakistan is endemic to hepatitis B and C infections. Alarming rise in hepatitis C virus (HCV) infection has been noticed in some areas of Sindh with an increasing risk for co-infection frequency in this region. Objective: To estimate the burden of HBV/HCV infection in Hyderabad Pakistan. Methods: ELISA and Nucleic acid Amplification test were performed to detect viruses. SPSS and online calculator were used for statistical analysis. Results: From a total of 108 seropositive hepatitis patients, 36.1% (n=39) were found HCV RNA-positive. Non-significant differences were observed in the frequencies of HCV infection for both genders [OR=0.735, CI (95%) 0.307-1.761, p&lt;0.05]. The percentage of HBV DNA detection among 108 HCV-seropositive cases was 17.9% (n=19). However, HCV-HBV co-infection in HCV-RNA positive cases was determined in 48.7% (n=19) cases with non-significant difference in both genders [OR=1.51, CI (95%) = 0.38 - 5.96, p&lt; 0.05]. Analysis suggested weakly positive correlation between HCV mono-infection and HCV-HBV co-infection and age (r =0.184, and r =0.1231), respectively. Conclusion: The study demonstrates a high prevalence of HBV co-infection among active hepatitis C patients of Hyderabad

Taxonomy for internet of things tools for monitoring personal effects

Human capacities to track and identify peers, products and activities have benefited from the wide proliferation of networked electronic devices; developments in smart-materials, near-field communication and computervision technology further advance these capabilities. During this investigation into The Internet of Things (IoT) a new taxonomy was created together with a set of prototype applications and accompanying architecture. In addition to presenting these taxonomies the creation of a peer-to-peer network with a distributed database is discussed. The development of a system that enables users to track and exchange objects or services using a secure and robust data repository model is also proposed

Forecasting tax revenues using time series techniques – a case of Pakistan

The objective of this research was to forecast the tax revenue of Pakistan for the fiscal year 2016–17 using three different time series techniques and also to analyse the impact of indirect taxes on the working class. The study further analysed the efficiency of three different time series models such as the Autoregressive model (A.R. with seasonal dummies), Autoregressive Integrated Moving Average model (A.R.I.M.A.), and the Vector Autoregression (V.A.R.) model. In any economy, tax analysis and forecasting of revenues is of paramount importance to ensure the economic and fiscal policies. This study is important to identify significant variables affecting tax revenue specifically in Pakistan. The data used for this paper was from July 1985 to December 2016 (monthly) and focused on forecasting for 2017. For the forecasting of total tax revenue, we used components of tax revenues such as direct tax, sales tax, federal excise duty and customs duties. The results of this study revealed that among these models the A.R.I.M.A. model gives better-forecasted values for the total tax revenues of Pakistan. The results further demonstrated that major tax revenue is generated by indirect taxes, which cause more inflation that directly hits the working class of Pakistan