Efficiency of Biosynthesized Silver and Zinc Nanoparticles Against Multi-Drug Resistant Pathogens

Biosynthesis of metallic nanoparticles has acquired particular attention due to its economic feasibility, low toxicity, and simplicity of the process. In this study, extracellular synthesis of silver and zinc nanoparticle was carried out by Pseudomonas hibiscicola isolated from the effluent of an electroplating industry in Mumbai. Characterization studies revealed synthesis of 40 and 60 nm nanoparticles of silver (AgNP) and zinc (ZnNP), respectively, with distinct morphology as observed in TEM and its crystalline nature confirmed by XRD. DLS, zeta potential, NTA, and FTIR studies further characterized nanoparticles giving data about its size, stability, and functional groups. Considering the toxicity of nanoparticles the evaluation of antimicrobial activity was studied in the range of non-toxic concentration for normal cell lines. Silver nanoparticles were found to be the most effective antimicrobial against all tested strains and drug-resistant clinical isolates of MRSA, VRE, ESBL, MDR, Pseudomonas aeruginosa with MIC in the range of 1.25–5 mg/ml. Zinc nanoparticles were found to be specifically active against Gram-positive bacteria like Staphylococcus aureus including its drug-resistant variant MRSA. Both AgNP and ZnNP were found to be effective against Mycobacterium tuberculosis and its MDR strain with MIC of 1.25 mg/ml. The synergistic action of nanoparticles assessed in combination with a common antibiotic gentamicin (590 μg/mg) used for the treatment of various bacterial infections by Checker board assay. Silver nanoparticles profoundly exhibited synergistic antimicrobial activity against drug-resistant strains of MRSA, ESBL, VRE, and MDR P. aeruginosa while ZnNP were found to give synergism with gentamicin only against MRSA. The MRSA, ESBL, and P. aeruginosa strains exhibited MIC of 2.5 mg/ml except VRE which was 10 mg/ml for both AgNPs and ZnNPs. These results prove the great antimicrobial potential of AgNP and ZnNP against drug-resistant strains of community and hospital-acquired infections and opens a new arena of antimicrobials for treatment, supplementary prophylaxis, and prevention therapy

The role of TOR kinase signaling in responses to bacterial infection

Animals in their natural ecology are often exposed to environmental stressors (e.g., starvation, extreme temperature, hypoxia, pathogens) that can affect their physiology, development, and lifespan. An important question in biology is how animals sense these stresses and, in response, adapt their metabolism to maintain homeostasis and survival. In some cases, specific tissues function as stress sensors to control whole body adaptive responses. One well-studied example is the Drosophila intestine. Along with performing absorptive, digestive, and endocrine functions, the intestine also functions as both a stress sensor and signaling hub, to regulate systemic metabolic changes. Upon encountering enteric pathogenic bacteria, Drosophila adults mount organism-wide immune and physiological responses in order to provide infection resistance and promote tolerance. The Drosophila intestine controls both local and systemic anti-bacterial immune responses. Recent work shows that the gut also signals to other tissues to control whole-body metabolic changes to promote infection tolerance. However, the mechanisms underlying how these stress sensing tissues link the stressors such as infection to metabolic adaptations is not well understood. In my thesis, I show that one way by which the fly intestine mediates these adaptive metabolic responses is via induction of target-of-rapamycin (TOR) kinase signaling. TOR is a well-established regulator of metabolism that has classically been shown to be activated by growth cues and suppressed by stress conditions. Interestingly however, I found a rapid increase in TOR activity in the fly gut in response to enteric gram-negative bacterial infection stress, independent of the classic innate immune response. Furthermore, I showed that blocking this TOR induction reduced survival upon infection. My data suggest that these protective effects of gut TOR signaling on organismal survival may be mediated through altered whole-body lipid metabolism. Lipid stores are an important metabolic fuel source. They can be synthesized and stored in specific tissues and then mobilized, transported to other tissues, and used to fuel metabolism, particularly in stress conditions. Infection leads to transient loss of lipids which is perhaps needed to fuel the immune response. This transient loss and restoration of lipids was further exacerbated by TOR inhibition. Infection also induced TOR dependent systemic expression of transcription factors and enzymes that promote de novo lipid biogenesis, indicating one way by which TOR inhibits excess lipid loss is by promoting de novo lipid synthesis upon infection. Moreover, genetic upregulation of intestinal TOR was sufficient to induce the expression of some of these lipid synthesis genes. In addition to systemic effects, enteric infection also induced TOR dependent local intestinal lipolysis and beta oxidation genes, and endocrine signaling peptides which have previously been implicated in whole body lipid homeostasis. I propose a model in which induction of intestinal TOR signaling is an infection stress sensor that leads to local intestinal changes such as lipolysis and secretion of signaling peptides, which perhaps non autonomously signal to the rest of the animal to upregulate lipid synthesis upon infection. TOR upregulation represents a host adaptive response to counteract infection mediated loss of whole-body lipid stores in order to promote survival. While only a handful of studies have investigated a role for TOR signaling upon infection with varying results, my thesis supports the idea of TOR activity being beneficial for the host to survive enteric infection. I propose TOR signaling as a link between infection and metabolic adaptations which contributes to infection tolerance

Short term analysis of healed post-tubercular kyphosis in younger children based on principles of congenital kyphosis

Background: The patients with healed severe progressive tubercular kyphosis may develop late-onset paraplegia. A particular subgroup of these children (Type IB progression) may benefit from the management principles of congenital kyphosis. Self-correction may be observed by selective continued growth of anterior vertebral epiphyseal end-plates over the posterior fused mass. We report a series of cases with posterior fusion of progressive post-tubercular kyphosis with an aim to prevent further progression of kyphosis and to assess if any gradual self correction is seen in followup. Materials and Methods: Twelve children fulfilling inclusion criteria of clinicoradiological, hematological diagnosis of healed spine TB having no or <2 spine at risk signs having documented progression of kyphosis and neural deficit underwent posterior fusion in situ without instrumentation, using autogenous iliac crest grafts as well as allograft donor bone graft. They were followed up to maximum of 5 years. Results: All 12 children had a progressive increase in angle preoperatively. Mean followup was 3.6 years. Post surgery, 66% showed a clinical improvement and correction, 25% had static angle, and worsening in one patient. Thus, overall 91% have a favorable result. Conclusion: The mechanism of correction of deformity in presence of posterior fusion is continued growth of the anterior epiphyseal end plates and hence this leads to selective differential anterior column growth giving gradual correction of kyphosis. This avoids anterior, technically demanding and complex, internal gibbus surgeries. This procedure is simple, safe, and less morbid with good results, avoiding long term disability to the patients in selected group of patients

Ras/ERK-signalling promotes tRNA synthesis and growth via the RNA polymerase III repressor Maf1 in <i>Drosophila</i>

<div><p>The small G-protein Ras is a conserved regulator of cell and tissue growth. These effects of Ras are mediated largely through activation of a canonical RAF-MEK-ERK kinase cascade. An important challenge is to identify how this Ras/ERK pathway alters cellular metabolism to drive growth. Here we report on stimulation of RNA polymerase III (Pol III)-mediated tRNA synthesis as a growth effector of Ras/ERK signalling in <i>Drosophila</i>. We find that activation of Ras/ERK signalling promotes tRNA synthesis both in vivo and in cultured <i>Drosophila</i> S2 cells. We also show that Pol III function is required for Ras/ERK signalling to drive proliferation in both epithelial and stem cells in <i>Drosophila</i> tissues. We find that the transcription factor Myc is required but not sufficient for Ras-mediated stimulation of tRNA synthesis. Instead we show that Ras signalling promotes Pol III function and tRNA synthesis by phosphorylating, and inhibiting the nuclear localization and function of the Pol III repressor Maf1. We propose that inhibition of Maf1 and stimulation of tRNA synthesis is one way by which Ras signalling enhances protein synthesis to promote cell and tissue growth.</p></div

Brf1 is required for intestinal stem cells (ISCs) homeostasis and for Ras-induced cell proliferation.

<p>(A, B) <i>UAS-Brf1 RNAi</i> was expressed adult ISCs and EBs using the <i>esg-GAL4</i>^<i>ts</i> system. Control flies were <i>esg-GAL4</i>^<i>ts</i> flies crossed to <i>w</i>^<i>1118</i>. Flies were then fed with sucrose or sucrose plus DSS (A) or Bleomycin (B) for 2 days. Intestines were then dissected and stained for phospho-histone H3 positive cells. Data represent the mean number of phospho-histone H3 cells per intestine +/ SEM. N >15 intestines per condition. (C) A UAS-Ras^V12 transgene was expressed in adult intestines using the <i>esg-GAL4</i>^<i>ts</i> driver. Control samples (WT) expressed <i>UAS-GFP</i> alone. Total RNA was isolated and levels of pre-tRNAs measured by qRT-PCR. N = 4 independent samples per condition. Data are presented as mean +/- SEM. (D) <i>UAS-Raf</i>^<i>gof</i> and <i>UAS-Brf1 RNAi</i> were expressed, either alone or together, in the adult ISCs and EBs using the <i>esg-Gal4</i>^<i>ts</i> system. <i>esg</i> positive cells are marked with GFP and DNA is stained with Hoechst dye. Knockdown of Brf 1(<i>UAS-Brf RNAi</i>) suppresses the increased proliferation seen with <i>UAS-Raf</i>^<i>gof</i> expression.</p

dMyc is required but not sufficient for Ras-induced tRNA synthesis.

<p>(A) Ras^V12 expression was induced in <i>Drosophila</i> S2 cells for 24 hours in either control cells or dMyc knockdown cells. dMyc was knocked down by incubating cells with dsRNA against <i>dMyc</i>. Control cells were treated with dsRNA to GFP. Total RNA was isolated with Trizol and analyzed by northern blotting using DIG-labelled antisense probes to tRNA^iMet or tRNA^Arg. Ethidium bromide stained 5S rRNA band was used as a loading control. (B, C and D) dMyc expression was induced in S2 cells for 24hrs, and then cells were treated with 10 μM U0126 or DMSO for 2 hours. Total RNA was isolated with Trizol and analyzed by qRT-PCR to measure levels of (B) pre-tRNAs, (C) <i>dMyc</i> mRNA, or (D) mRNA levels of three dMyc target genes—<i>NOP60B</i>, <i>PPAN</i> and <i>NOP5</i>. N = 4 independent samples per condition. Data are presented as mean +/-SEM.</p

Brf1 is required for Ras-induced tRNA synthesis and growth in both wing imaginal discs and adult midgut progenitor cells (AMPs).

<p>(A). Ras^V12 expression was induced in <i>Drosophila</i> S2 cells for 24 hours in either control cells or Brf1 knockdown cells, Brf1 was knocked down by incubating cells with dsRNA against Brf1. Control cells were treated with dsRNA to GFP. Total RNA was isolated with Trizol and analyzed by northern blotting using DIG-labelled antisense probes to tRNA^iMet or tRNA^Arg. Ethidium bromide stained 5S rRNA band was used as a loading control. (B, C) <i>UAS-EGFR</i> and <i>UAS-Brf1 RNAi</i> were expressed, either alone or together, in the dorsal compartment of larval wing imaginal discs using an <i>ap-Gal4</i> driver. Control discs were from <i>ap-Gal4</i> crossed to <i>w</i>^<i>1118</i>. Wing discs were dissected at the wandering L3 larval stage and the area of the GFP-marked dorsal compartment quantified using NIH imaging software (n > 50 wings per genotype, data presented as mean +/- SEM). Representative images are shown in (B), quantification of tissue area is shown in (C). (D) <i>UAS-EGFR</i> and <i>UAS-Brf1 RNAi</i> were expressed, either alone or together, in the <i>Drosophila</i> larval AMPs using the <i>esg-Gal4</i>^<i>ts</i> system. Larvae were shifted to 29°C at 24 hrs of development to induce transgene expression and dissected as L3 larvae. AMPs are marked <i>by UAS-GFP</i> expression. DNA is stained with Hoechst dye (blue). (E) The number of cells in each AMP cluster was quantified for each of the genotypes in D (left), and an additional similar experiment in which the Ras pathway was activated by expression of a <i>UAS-Raf</i>^<i>gof</i> transgene (right). Data are presented as box plots (25%, median and 75% values) with error bars indicating the min and max values.</p

Ras signalling regulates dMaf1 phosphorylation.

<p>(A) <i>Drosophila</i> S2 cells were treated with 10 μM U0126 for 2 hours. Cells were then lysed and processed for SDS-PAGE and western blotting using the phos-tag reagent, as described in the Methods. The blots were then probed with an anti-dMaf1 antibody (top panel), an anti-total ERK antibody (middle panel) or an anti-phospho ERK antibody (lower panel) (B) Ras^V12 expression was induced in <i>Drosophila</i> S2 cells for 24 hours. Cells were then lysed and processed for SDS-PAGE and western blotting using the phos-tag reagent, as described in the Methods. The blots were then probed with an anti-dMaf1 antibody (top panel), an anti-total ERK antibody (middle panel) or an anti-phospho ERK antibody (lower panel). (C) A model for how Ras signalling may regulate Pol III and tRNA synthesis.</p

The Ras/ERK signalling pathway stimulates tRNA synthesis.

<p>(A, B) <i>Drosophila</i> S2 cells were treated with 10 μM U0126 for 2 hours. Total RNA was isolated and levels of either pre-tRNAs (A), or total tRNAs (B) measured by qRT-PCR. N = 15 independent samples per condition. (C, D) Raf was knocked down in <i>Drosophila</i> S2 cells by incubating cells with dsRNAs against <i>Raf</i>. Control cells were treated with dsRNA to GFP. Total RNA was isolated and levels of either pre-tRNAs (C), or total tRNAs (D) measured by qRT-PCR. N = 4 independent samples per condition. (E, F) Ras^V12 expression was induced in <i>Drosophila</i> S2 cells for 24 hours. Total RNA was isolated and levels of either pre-tRNAs (E), or total tRNAs (F) measured by qRT-PCR. N = 9 independent samples per condition. (G) <i>UAS-Raf</i>^<i>gof</i> was expressed in imaginal tissues using the <i>esg-GAL4</i>^<i>ts</i> system. Control flies were <i>esg-GAL4</i>^<i>ts</i> flies crossed to <i>w</i>^<i>1118</i>. Transgenes were induced by shifting larvae to 29°C at 48hrs of larval development, and then discs were dissected from wandering L3 stage larvae. Total RNA was isolated and levels of pre-tRNAs measured by qRT-PCR. N = 4 independent samples per condition. Data are presented as mean +/- SEM.</p