Cosmid based mutagenesis causes genetic instability in Streptomyces coelicolor, as shown by targeting of the lipoprotein signal peptidase gene

Bacterial lipoproteins are extracellular proteins tethered to cell membranes by covalently attached lipids. Deleting the lipoprotein signal peptidase (lsp) gene in Streptomyces coelicolor results in growth and developmental defects that cannot be restored by reintroducing lsp. This led us to hypothesise that lsp is essential and that the lsp mutant we isolated previously had acquired compensatory secondary mutations. Here we report resequencing of the genomes of wild-type M145 and the cis-complemented ∆lsp mutant (BJT1004) to map and identify these secondary mutations but we show that they do not increase the efficiency of disrupting lsp and are not lsp suppressors. We provide evidence that they are induced by introducing the cosmid St4A10∆lsp, as part of ReDirect PCR mutagenesis protocol, which transiently duplicates a number of important cell division genes. Disruption of lsp using a suicide vector (which does not result in gene duplication) still results in growth and developmental delays and we conclude that loss of Lsp function results in developmental defects due to the loss of all lipoproteins from the cell membrane. Significantly, our results also indicate the use of cosmid libraries for the genetic manipulation of bacteria can lead to phenotypes not necessarily linked to the gene(s) of interest

Ultrasensitive, Label Free, Chemiresistive Nanobiosensor Using Multiwalled Carbon Nanotubes Embedded Electrospun SU-8 Nanofibers

This paper reports the synthesis and fabrication of aligned electrospun nanofibers derived out of multiwalled carbon nanotubes (MWCNTs) embedded SU-8 photoresist, which are targeted towards ultrasensitive biosensor applications. The ultrasensitivity (detection in the range of fg/mL) and the specificity of these biosensors were achieved by complementing the inherent advantages of MWCNTs such as high surface to volume ratio and excellent electrical and transduction properties with the ease of surface functionalization of SU-8. The electrospinning process was optimized to precisely align nanofibers in between two electrodes of a copper microelectrode array. MWCNTs not only enhance the conductivity of SU-8 nanofibers but also act as transduction elements. In this paper, MWCNTs were embedded way beyond the percolation threshold and the optimum percentage loading of MWCNTs for maximizing the conductivity of nanofibers was figured out experimentally. As a proof of concept, the detection of myoglobin, an important biomarker for on-set of Acute Myocardial Infection (AMI) has been demonstrated by functionalizing the nanofibers with anti-myoglobin antibodies and carrying out detection using a chemiresistive method. This simple and robust device yielded a detection limit of 6 fg/mL

The comparative study of applanation and optical coherence biometry methods for the intra ocular lens power calculation

Purpose: To compare applanation biometry (A-Scan) and optical coherence biometry (AL-Scan) methods for IOL power calculation based on Axial Length and post operative refractive outcome. Methodology: Prospective and Interventional Randomized Comparative Study, Sample size of 400, studied under two sub groups, for Axial Length readings and IOL power calculation by A-Scan (Biomedix) and AL-Scan (Nidek). Keratometry readings are taken only by AL-Scan.Results: Mean ± St. dev. of A.L. measured by App. Biometry was low (22.79 ± 0.9 mm) than Opt. Coh. Biometry (23.16 ± 0.78 mm) to be significant (P= .0001). Mean ± St. dev. IOL power was higher (21.75 ± 2.1D) than App. Biometry (20.88 ± 1.59 D) to be significant (P= 0.0001). Mean ± St. dev. of refractive status for Myopia is higher -0.97 ± 0.53 by App. Biometry than Opt. Coh. Biometry -0.5 ± 0.19, to be significant (P= 0.0001) and Mean ± St.dev. for Hyperopia is higher 0.98 ± 0.59 by App. Biometry than Opt. Coh. Biometry 0.46 ± 0.18, to be significant (P= 0.0001). Bland–Altman plots showed perfect agreement between both methods regarding A.L. and calculated IOL power. Further subgroup analysis revealed a statistically significant difference in different age groups and types of cataract for Posterior Sub capsular cataract alone and Nuclear Sclerosis with Posterior Sub capsular cataract (P= 0.001). Conclusion: There is significant difference between App. and Opt. Coh. Biometry; however, certain situations of Cataract is demanding mandatory role of App. Biometry

Analyzing the Complex Regulatory Landscape of Hfq – an Integrative, Multi-Omics Approach

The ability of bacteria to respond to environmental change is based on the ability to coordinate, redirect and fine-tune their genetic repertoire as and when required. While we can learn a great deal from reductive analysis of individual pathways and global approaches to gene regulation, a deeper understanding of these complex signaling networks requires the simultaneous consideration of several regulatory layers at the genome scale. To highlight the power of this approach we analyzed the Hfq transcriptional/translational regulatory network in the model bacterium Pseudomonas fluorescens. We first used extensive ‘omics’ analyses to assess how hfq deletion affects mRNA abundance, mRNA translation and protein abundance. The subsequent, multi-level integration of these datasets allows us to highlight the discrete contributions by Hfq to gene regulation at different levels. The integrative approach to regulatory analysis we describe here has significant potential, for both dissecting individual signaling pathways and understanding the strategies bacteria use to cope with external challenges

Translational control of the SigR-directed oxidative stress response in streptomyces via IF3-mediated repression of a noncanonical GTC start codon

The major oxidative stress response in Streptomyces is controlled by the sigma factor SigR and its cognate antisigma factor RsrA, and SigR activity is tightly controlled through multiple mechanisms at both the transcriptional and posttranslational levels. Here we show that sigR has a highly unusual GTC start codon and that this leads to another level of SigR regulation, in which SigR translation is repressed by translation initiation factor 3 (IF3). Changing the GTC to a canonical start codon causes SigR to be overproduced relative to RsrA, resulting in unregulated and constitutive expression of the SigR regulon. Similarly, introducing IF3* mutations that impair its ability to repress SigR translation has the same effect. Thus, the noncanonical GTC sigR start codon and its repression by IF3 are critical for the correct and proper functioning of the oxidative stress regulatory system. sigR and rsrA are cotranscribed and translationally coupled, and it had therefore been assumed that SigR and RsrA are produced in stoichiometric amounts. Here we show that RsrA can be transcribed and translated independently of SigR, present evidence that RsrA is normally produced in excess of SigR, and describe the factors that determine SigR-RsrA stoichiometry.IMPORTANCE In all sigma factor-antisigma factor regulatory switches, the relative abundance of the two proteins is critical to the proper functioning of the system. Many sigma-antisigma operons are cotranscribed and translationally coupled, leading to a generic assumption that the sigma and antisigma factors are produced in a fixed 1:1 ratio. In the case of sigR-rsrA, we show instead that the antisigma factor is produced in excess over the sigma factor, providing a buffer to prevent spurious release of sigma activity. This excess arises in part because sigR has an extremely rare noncanonical GTC start codon, and as a result, SigR translation initiation is repressed by IF3. This finding highlights the potential significance of noncanonical start codons, very few of which have been characterized experimentally. It also emphasizes the limitations of predicting start codons using bioinformatic approaches, which rely heavily on the assumption that ATG, GTG, and TTG are the only permissible start codons

Control of mRNA translation by dynamic ribosome modification

Control of mRNA translation is a crucial regulatory mechanism used by bacteria to respond to their environment. In the soil bacterium Pseudomonas fluorescens, RimK modifies the C-terminus of ribosomal protein RpsF to influence important aspects of rhizosphere colonisation through proteome remodelling. In this study, we show that RimK activity is itself under complex, multifactorial control by the co-transcribed phosphodiesterase trigger enzyme (RimA) and a polyglutamate-specific protease (RimB). Furthermore, biochemical experimentation and mathematical modelling reveal a role for the nucleotide second messenger cyclic-di-GMP in coordinating these activities. Active ribosome regulation by RimK occurs by two main routes: indirectly, through changes in the abundance of the global translational regulator Hfq and directly, with translation of surface attachment factors, amino acid transporters and key secreted molecules linked specifically to RpsF modification. Our findings show that post-translational ribosomal modification functions as a rapid-response mechanism that tunes global gene translation in response to environmental signals

Formulation and Evaluation of Ginger Extract Loaded Nanoemulgel for the Treatment of Rheumatoid Arthritis

Ginger is a potent anti-inflammatory; in the present study the nanoemulgel of ginger extract was formulated for treating rheumatoid arthritis through topical application. The main objective of nanoemulgel formulation was to enhance the bioavailability of ginger extract through topical route and also to reduce the dose of ginger extract. First the nanoemulsion was prepared with the help of ternary phase diagram, using water titration method. The Smix (surfactant and co-surfactant) and extract were mixed together then titrated with water. The Smix was used at the ratio of 1:1, 1:2, and 2:1. The isopropyl myristate is used as oil, tween 80 as surfactant, ethanol as co-surfactant and water was used as aqueous phase and, 32 formulations were prepared. The particle size was found in the range of 60.32 to 230.8nm for formulations F1 to F4. The zeta potential was found between -16.6 to -24.4 and the polydispersibility index was found to be 0.687 to 0.892. Nanoemulsion was converted into nanoemulgel by using carbopol 934 as gelling agent in various concentrations. The rheological properties, spreadability, pH, thermodynamic stability and drug release were also determined. On the basis of thermodynamic stability, spreadability and drug release, the nanoemulgel F4* was considered as best formulation. Keywords: nanoemulgel, nanoemulsion, ternary phase diagram, rheumatoid arthritis, ginger extract, co-surfactan

Status of Plasmodium Falciparum and Vivax in Jharkhand: A Five Year (2004-08) Retrospective Study at Rajendra Institute of Medical Sciences, Ranchi

ABSTRACT Malaria is well known for its fatalities worldwide. In India, it is still endemic in many areas where two species of Plasmodium namely Plasmodium vivax and Plasmodium falciparum are reported. P.vivax is widespread, creating lots of morbidities across the country. P. falciparum, on the other hand, though comparatively narrow in its infectious volume, is a serious cause of mortalities in India. A five year survey was conducted from 2004 to 2008 in a high malaria-hit district, Ranchi. Thick and thin blood smears were made at the Department of Clinical Pathology, Rajendra Institute of Medical Sciences (RIMS), where the microscopic examinations were carried out. The overall reported and examined cases at RIMS included 36643 suspected malaria cases, out of which, 21833(59.5%) were found positive. Out of these positive cases, 6842(31.3%) were confirmed as P. falciparum patients and 14991(68.6%) as P. vivax cases respectively. Number of negative cases was 14811 (40.4%). In this study, it was observed that after the year 2005, incidence of malaria suddenly dropped by 50% and remained almost static on the same level in the following years with only some seasonal variations. However, it was observed that P. falciparum steadily became more dangerous. It is therefore highly necessary to take immediate and effective measures to minimize the complications of P. falciparum along with P. vivax to prevent death toll in these areas

Defining the regulon of genes controlled by σE, a key regulator of the cell envelope stress response in Streptomyces coelicolor

The extracytoplasmic function (ECF) σ factor, σE is a key regulator of the cell envelope stress response in Streptomyces coelicolor. Although its role in maintaining cell wall integrity has been known for over a decade, a comprehensive analysis of the genes under its control has not been undertaken. Here, using a combination of chromatin immunoprecipitation‐sequencing (ChIP‐seq), microarray transcriptional profiling and bioinformatic analysis, we attempt to define the σE regulon. Approximately half of the genes identified encode proteins implicated in cell envelope function. 17 novel targets were validated by S1 nuclease mapping or in vitro transcription, establishing a σE binding consensus. Subsequently, we used bioinformatic analysis to look for conservation of the σE target promoters identified in S. coelicolor across 19 Streptomyces species. Key proteins under σE control across the genus include the actin homolog MreB, three penicillin‐binding proteins, two L,D‐transpeptidases, a LytR‐CpsA‐Psr‐family protein predicted to be involved in cell wall teichoic acid deposition, and a predicted MprF protein, which adds lysyl groups to phosphatidylglycerol to neutralize membrane surface charge. Taken together, these analyses provide biological insight into the σE‐mediated cell envelope stress response in the genus Streptomyces