Discovery and Validation of New Regulatory RNA Elements in Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular bacterium that exhibits a unique biphasic developmental cycle that can be disrupted by growth in the presence of IFN-g and b-lactams, giving rise to an abnormal growth state termed persistence. Relatively little is known about the regulatory mechanisms that control temporal gene expression during the developmental cycle or the control of persistence and reactivation. Here we have examined the expression of a newly defined family of non-coding RNAs (ncRNAs) that are differentially expressed during the developmental cycle and the induction of persistence and reactivation (Using IFNγ and Carbenicillin). Non-coding RNAs were initially identified using an intergenic tiling microarray and were confirmed by Northern blotting. A group of 10 ncRNAs were mapped and characterized and compared to the previously described chlamydial ncRNAs (IhtA, pCHL antisense transcripts). The 5’ and 3’ ends of the ncRNAs were determined using an RNA circularization procedure. Promoter predictions indicated that all ncRNAs were expressed from s66 promoters and 9 ncRNAs contained non-templated 3’ poly-A or poly-AG additions. Expression of ncRNAs was studied by Northern blotting during i) the normal developmental cycle, ii) IFN-g-induced persistence, and iii) carbenicillin-induced persistence. Differential temporal expression during the developmental cycle was seen for all ncRNAs and distinct differences in expression were seen during IFN-g and carbenicillin-induced persistence and reactivation. Two of the studied ncRNAs were cis acting antisense molecules (CTIG270 and CTIG153). Expression of CTIG270 in a surrogate E. coli system along with its target gene ftsI proved that it was an antisense RNA, and it effectively controlled ftsI availability. A screening system was developed to determine the targets of potential trans-acting ncRNAs. The screen was composed of two compatible plasmids, a high copy effector plasmid expressing the ncRNA and a low/medium copy target plasmid expressing chlamydial library. Target plasmid contained translational fusion between chlamydial library and a positive/negative selection fusion system (ccdB/CAT fusion/TEV protease). The selection process to identify potential targets for ncRNAs was carried out over two stages. Stage one was to eliminate self ligated plasmids and non translational fusions, in this phase, only CAT+ ccdB resistant cells survived chloramphenicol selection i.e. cells representing true translational fusions survived antibiotic selection. Plasmids enriched for by stage one selection were subjected to stage two selection. In stage two ccdB sensitive cells were double transformed with both target plasmid and effector plasmid. Only in instances when ncRNA inhibits translation of ccdB would cells survive this selection. Stage two selection enriched for target(s) of ncRNA under test. We show in details the construction of this screening system and its functional aspect

Optimization of polysaccharide productio by Lactobacillus kefiranofaciens using response surface methdology

Kefiran is an exopolysaccharides produced by Lactobacillus kefiranofaciens which was isolated from kefir grains. Kefiran has wide applications mainly in food and pharmaceutical industries. Growth and kefiran production of L. kefiranofaciens can be significantly enhanced by using mixed culture technique. Saccharomyces cerevisiae in this study was used to enhance the kefiran production by reducing lactic acid accumulation in the cultivation medium. The statistical analysis was used for optimization study by using response surface methodology based on Box-Behnken design. The interaction was studied between three different levels of variables that were lactose, yeast extract and phosphate. A second order polynomial model was used to correlate the factors. The model was found to be significant. The optimum concentration of lactose, yeast extract and phosphate obtained using statistical media optimization is 59.09 g L-1, 8.69 g L-1and 0.5 g L-1 respectively. Kefiran production in optimized medium was 0.97 g L-1 which gave an increase in kefiran production up to 42.65 % compared to the un-optimized medium which was only 0.68 g L-1 of kefiran

Impact of Culture Media Composition, Nutrients Stress and Gamma Radiation on Biomass and Lipid of the Green Microalga, Dictyochloropsis splendida as a Potential Feedstock for Biodiesel Production

يعتمد إنتاج الديزل الحيوي من الطحالب الدقيقة على إنتاج الكتلة الحيوية ومحتوى الدهون. يتم التحكم في زياده انتاج الكتلة الحيوية و تراكم الدهون بواسطة عدة عوامل.فى هذا العمل تم دراسة تأثيرات ثلاث اوساط غذائيه خاصة بزراعة الطحالب ( BG11, BBM, Urea media) وايضا بعض المغذيات)النيتروجين والفوسفورو المغنيسيوم والكربون) واشعة جاما على نمو وانتاج الدهون لطحالب Dictyochloropsis splendida. تم الحصول علي أعلي انتاج للكتله الحيويه والدهون لطحلب Dictyochloropsis splendida  عندما تم زراعة الطحلب على الوسط الغذائي BG11 . علاوة على ذلك كان اعلى انتاجية للكتله الحيويه عند 3000 مليجرام / لتر نيتروجين او 160 مليجرام / لتر فوسفور او 113 مليجرام/لتر مغنسيوم او 20 مليجرام/ لتر كربونات.  بينما عند غياب المغذيات فان تراكم الدهون زاد. من ناحية اخرى فان المحتوى الدهنى للطحلب وصل الى 18.26% عندما تعرضت خلايا الطحلب لجرعة 25 جراى من اشعة جاما. وكانت  الدهون المستخلصة من الطحلب تتكون من نسبة عالية من الاحماض الدهنية المشبعة (SFAs, 63.33% ) والاحماض الدهنية الغيرمشبعة (UFAs, 37.02%) وكانت السيادة لحمض البالمتيك (C16:0) ثم حمض اللينوليك (C18:2) فحمض بنتاديكانويك (C15:0) وحمض بالميتوليك (C16:1). و تمثل الاحماض الدهنية المستخلصه من طحلب Dictyochloropsis splendida بهذا التركيب مواد خام واعدة لانتاج الديزل الحيوي.Biodiesel production from microalgae depends on the biomass and lipid production. Both biomass and lipid accumulation is controlled by several factors. The effect of various culture media (BG11, BBM, and Urea), nutrients stress [nitrogen (N), phosphorous (P), magnesium (Mg) and carbonate (CO3)] and gamma (γ) radiation on the growth and lipid accumulation of   Dictyochloropsis splendida were investigated. The highest biomass and lipid yield of D. splendida were achieved on BG11 medium. Cultivation of D. splendida in a medium containing 3000 mg L−1 N, or 160 mg L−1 P, or 113 mg L−1 Mg, or 20 mg L-1 CO3, led to enhanced growth rate. While under the low concentrations of nutrients caused a marked increase in the lipid content. Cultures exposure to 25 Gy of γ-rays, led to an increase in lipid content up to 18.26 ± 0.81 %. Lipid profile showed the maximum presence of saturated fatty acids (SFAs, 63.33%), and unsaturated fatty acids (UFAs, 37.02%). Fatty acids (FAs) recorded the predominance of C16:0, C18:2, C15:0 and C16:1, which strongly proved D. splendida is a promising feedstock for biodiesel production.        

Evaluation of outcome of percutaneous K-wires fixation of pediatric both bone forearm fractures

Background: Forearm fractures are the most common fractures in children, representing 40 to 50 percent of all childhood fractures. Objective: To improving outcome of pediatric both bone forearm fractures using K-wires. Patients and Methods: This study included 24 patients aged between 3 and 12 years with closed displaced fractures of shaft of both bones forearm presented to Zagazig General Hospital. On admission all patients were assessed by history taking, clinical examination and radiological evaluation. The K-wires were passed under control of image intensifier across the fracture site as far as the proximal physis in the radius and the distal metaphysis in the ulna taking care not to reach the growth plate. Postoperatively the limb was placed in above elbow plaster cast with the elbow at 90 degrees of flexion and the forearm at mid pronation. After the follow up period (6 - 12) month the results of patients were assessed clinically and radiologically and were classified according to the Price criteria. Results: 9 patients (37.5%) were classified as excellent results while 15 patients (62.5%) were classified as good results and no patients were classified as fair or poor results. Conclusion: Closed reduction percutaneous intramedullary fixation of pediatric displaced diaphyseal forearm fractures by K-wires is a safe, reliable, minimally invasive procedure and effective method of treatment. Based upon this study, it is concluded that displaced fractures of both forearm bones in children are preferred to be intramedullary fixed with K-wires when surgery is indicated with excellent and good results

Optimization of exopolysaccharide production by pleurotus ostreatus using diffrent cultivation strategies

Pleurotus ostreatus or known as oyster mushroom was regarded as one of the most cultivated mushroom around the world. One of the qualities it has is it able to produced exopolysaccharide called pleuran which secreted into the medium during submerged fermentation. The polysaccharide composed mainly of ß-(1/3)-D glucose and ß-(1/6)-D glucose linked by glycosidic bond. It has molecular weight of 2.4 X 104 Da with molecular formula of (C6H10O5)x The importance of pleuran is that it has the immunomodulatory properties that associated in triggering our immune system response. Nowadays, submerged fermentation is considered as the best method in cultivation this kind of mushroom. However, the production process of this kind of mushroom and its exopolysaccharide production especially in term of medium component is still unclear. In this research, the objectives were to optimize the medium composition and to find the optimum carbon to nitrogen (C: N) ratio for high exopolysaccharide production. Eight different media was screened and followed by factor by factor optimization of the medium component. The factors that been studied were ideal concentration of glucose, yeast extract, ammonium sulfate and dipotassium phosphate. Media number six which contain glucose 60.0 g L-1, yeast extract 2.0 g L-1, (NH4)2SO4 5.0 g L-1, MgSO4.7H2O 0.2 g L-1, K2HPO4 1.0 g L-1 was selected as best media production for P. ostreatus cultivation . The experiment then was further with different concentration of each component in the medium six excluding magnesium sulfate heptahydrate which maintained at 0.2 g L-1 throughout all the experiment stage. The range concentration for glucose, yeast extract, ammonium sulfate and dipotassium phosphate was setup between 0 – 120 g L-1, 0 – 4 g L-1, 0 – 5 g L-1 and 0 – 2 g L-1 respectively. In order to get the best C: N ratio for highest exopolysaccharide production, eleven ratio of carbon to nitrogen was experimented ranging from 15:1 to 65:1.Result shown that the optimum concentration for glucose, yeast extract, ammonium sulfate and dipotassium phosphate was 80.0, 4.0, 2.5 and 1.0 g L-1 respectively whiles the optimal C: N ratio recorded was 40: 1. The optimized medium also produced 2.83 g L-1 of exopolysaccharide increasingly up to 49 % when compared with un-optimized medium which only produced 1.9 g L-1 of exopolysaccharide

The Influence of Serum Leptin Level and Body Mass Index on the Prognosis of Patients with Diffuse Large B Cell Lymphoma

Background: More research is ongoing on obesity as a risk factor for cancer occurrence.Cell of origin (COO), stage and National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI) are frequently used for risk evaluation and treatment tailoring in patients with Diffuse large B-cell lymphoma (DLBCL). Relatively, few studies assessed the prognostic role of obesity and leptin level in patients with DLBCL. Aim: The aim of this study was to investigate the effect of obesity and leptin level on response and prognosis in DLBCL. Methods: A single institution prospective study that included patients with DLBCL. For each patient, demographic data, body mass index (BMI), serum leptin level by ELISA, response and survival were determined. Results: Seventeen (24.3%) out of the 70 patients in our cohort were classified as obese (BMI>30). They had a higher serum leptin level (p < 0.001) and less response to R-CHOP chemotherapy (P= 0.003). Forty (57.14%) patients had elevated serum leptin level with B symptoms, Cell of origin and response to chemotherapy were significantly different between the two groups.  There was no significant relationship between BMI and survival. On the other hand, higher serum leptin was associated with worse disease-free survival (p=0.035). Conclusion: The results support a relationship between both BMI and serum leptin level and response to treatment in DLBCL patients. Leptin level like other common prognostic factors is related to disease-free survival