Preparation of a bank of cloned genes from the chromosome of Agrobacterium tumefaciens and the isolation of genes involved in DNA repair and genetic recombination

The virulent property of the Agrobacterium tumefaciens is associated with its tumor inducing (Ti) plasmid. Recent studies on the virulence of this bacterium has shown that genes located on its chromosome also contribute to this property. One such gene is thought to produce a protein that has properties similar to that of the recA protein of E. colt. This thesis outlines the techniques that were used to try and isolate the recA-like gene from the chromosome of the Agrobacterium tumefaciens. All the techniques used in this study are outlined in detail and an explanation given for the choice of each technique. For reasons not completely understood, we were unable to isolate the recA-like gene even though a gene bank was successfully constructed and appropriately sized fragments extracted. Attempts were made to explain some of the unexpected results and appropriate steps were proposed to further understand them

Cellular localization, accumulation and trafficking of double-walled carbon nanotubes in human prostate cancer cells

Carbon nanotubes (CNTs) are at present being considered as potential nanovectors with the ability to deliver therapeutic cargoes into living cells. Previous studies established the ability of CNTs to enter cells and their therapeutic utility, but an appreciation of global intracellular trafficking associated with their cellular distribution has yet to be described. Despite the many aspects of the uptake mechanism of CNTs being studied, only a few studies have investigated internalization and fate of CNTs inside cells in detail. In the present study, intracellular localization and trafficking of RNA-wrapped, oxidized double-walled CNTs (oxDWNT–RNA) is presented. Fixed cells, previously exposed to oxDWNT–RNA, were subjected to immunocytochemical analysis using antibodies specific to proteins implicated in endocytosis; moreover cell compartment markers and pharmacological inhibitory conditions were also employed in this study. Our results revealed that an endocytic pathway is involved in the internalization of oxDWNT–RNA. The nanotubes were found in clathrin-coated vesicles, after which they appear to be sorted in early endosomes, followed by vesicular maturation, become located in lysosomes. Furthermore, we observed co-localization of oxDWNT–RNA with the small GTP-binding protein (Rab 11), involved in their recycling back to the plasma membrane via endosomes from the trans-golgi network

A long view of liberal peace and its crisis

The ‘crisis’ of liberal peace has generated considerable debate in International Relations. However, analysis is inhibited by a shared set of spatial, cultural and temporal assumptions that rest on and reproduce a problematic separation between self-evident ‘liberal’ and ‘non-liberal’ worlds, and locates the crisis in presentist terms of the latter’s resistance to the former’s expansion. By contrast, this article argues that efforts to advance liberal rule have always been interwoven with processes of alternative order-making, and in this way are actively integral, not external, to the generation of the subjectivities, contestations, violence and rival social orders that are then apprehended as self-evident obstacles and threats to liberal peace and as characteristic of its periphery. Making visible these intimate relations of co-constitution elided by representations of liberal peace and its crisis requires a long view and an analytical frame that encompasses both liberalism and its others in the world. The argument is developed using a Foucauldian governmentality framework and illustrated with reference to Sri Lanka

Obesity in inflammatory bowel disease: Gains in Adiposity despite high prevalence of Myopenia and Osteopenia

Background: Rising rates of obesity have been reported in patients with inflammatory bowel disease (IBD); however, prospective data is lacking. The aim of this study is to prospectively evaluate body composition in adults with IBD over 24 months. Methods: Whole body dual energy X-ray absorptiometry (DXA) data was performed at 0 months, 12 months, and 24 months. Bone mineral density (BMD), fat mass index (FMI (kg)/height (m2)), appendicular skeletal muscle index (ASMI (kg)/height (m2)), visceral adipose tissue and the visceral adipose height index (VHI, VAT area (cm3)/height (m2)), and clinical and anthropometric assessments were performed at each time point. Multivariable linear mixed effects regression analyses were performed. Results: Initially, 154 participants were assessed at baseline (70% Crohn’s disease, 55% male, median age 31 years), of whom 129 underwent repeated DXA at 12 months, and 110 underwent repeated DXA at 24 months. Amongst those undergoing repeated DXA, their body mass index (BMI) significantly increased over time, such that by 24 months, 62% of patients were overweight or obese (annual change BMI β = 0.43, 95%CI = [0.18, 0.67], p = 0.0006). Gains in BMI related to increases in both FMI and VHI (β = 0.33, 95%CI = [0.14, 0.53], p = 0.0007; β = 0.08, 95%CI = [0.02, 0.13], p = 0.001; respectively), whereas ASMI decreased (β = −0.07, 95%CI = [−0.12, −0.01], p = 0.01) with a concordant rise in rates of myopenia (OR = 3.1 95%CI = [1.2, 7.7]; p = 0.01). Rates of osteopenia and osteoporosis were high (37%), but remained unchanged over time (p = 0.23). Conclusion: Increasing rates of obesity in patients with IBD coincide with decreases in lean muscle mass over time, while high rates of osteopenia remain stable. These previously undocumented issues warrant attention in routine care to prevent avoidable morbidity

Visceral adipose tissue is associated with stricturing Crohn's disease Behavior, fecal calprotectin, and quality of life

BackgroundVisceral adipose tissue (VAT) has been proposed to play a pathogenic role in Crohn’s disease (CD); however, prospective clinical data are lacking. The aim was to evaluate whether VAT, beyond body mass index (BMI), is associated with CD behavior, disease activity, quality of life (QoL), or outcomes.MethodsBody composition data and clinical, anthropometric, disease activity (fecal calprotectin [FC]), and QoL scores were gathered prospectively on adults with CD at 0, 12, and 24 months. BMI and, VAT metrics (visceral adipose tissue volume [cm3]/height [m2] index and VAT:subcutaneous adipose tissue [SAT] ratio) were calculated. Inflammatory bowel disease–related surgery and hospitalization were recorded over extended follow-up (median, 51 months). Multivariable linear mixed effects and logistic regression analyses were performed.ResultsNinety-seven participants were assessed at baseline (55% male; median age, 31 years), 84 at 12 months, and 72 at 24 months. VAT:SAT was positively associated with stricturing disease behavior (log odds ratio [OR], 1.7; 95% confidence interval [CI], 0.32 to 3; P = 0.01) and elevated FC in patients with ileocolonic disease (β, 1.3; 95% CI, 0.32 to 2.3; P = 0.01). VAT:SAT was associated with lower QoL, particularly in those with ileal disease (β, –12; 95% CI, –19 to –4.5; P = 0.05). However, no prospective associations were observed between serial VAT measurements and time to surgery or hospitalization. No correlations were found between BMI and disease behavior, activity, or QoL.ConclusionVAT:SAT, rather than BMI, is associated with stricturing CD behavior, elevated FC, and reduced QoL in a disease distribution–dependent manner. Further studies are required to substantiate the role of VAT as a useful biomarker in CD

Automobile Spare Parts System – Web Solution

“With the right spare parts on hand, your business can continue without ceasing operations even after a major malfuction, allowing you to avoid loss of crucial time and profits as you wait for the right parts to be delivered”. This system which is specialized in trading automobile spare parts claims to provide sufficient spare parts throughout the life cycle of products, to the clients in need, so as to achieve sustainability. This research discusses how such a system can be designed and implemented to assist clients in search of the best automobile parts that fits their purpose. The research paper has been divided in such a manner to explore and comprehend each and every aspect of the system in detail. Initially, the introduction will provide a simple overview of the system with details regarding what the system will achieve; this will be followed by the methodology of the implementation process. The functionalities of the proposed system will then be discussed with the entails of the testing methodologies and the outcomes

Results of a single-arm pilot study of 32P microparticles in unresectable locally advanced pancreatic adenocarcinoma with gemcitabine/nab-paclitaxel or FOLFIRINOX chemotherapy.

BACKGROUND: Unresectable locally advanced pancreatic cancer (LAPC) is generally managed with chemotherapy or chemoradiotherapy, but prognosis is poor with a median survival of ∼13 months (or up to 19 months in some studies). We assessed a novel brachytherapy device, using phosphorous-32 (32P) microparticles, combined with standard-of-care chemotherapy. PATIENTS AND METHODS: In this international, multicentre, single-arm, open-label pilot study, adult patients with histologically or cytologically proven unresectable LAPC received 32P microparticles, via endoscopic ultrasound-guided fine-needle implantation, planned for week 4 of 5-fluorouracil, leucovorin, irinotecan and oxaliplatin (FOLFIRINOX) or gemcitabine/nab-paclitaxel chemotherapy, per investigator's choice. The primary endpoint was safety and tolerability measured using Common Terminology Criteria for Adverse Events version 4.0. The lead efficacy endpoint was local disease control rate at 16 weeks. RESULTS: Fifty patients were enrolled and received chemotherapy [intention-to-treat (ITT) population]. Forty-two patients received 32P microparticle implantation [per protocol (PP) population]. A total of 1102 treatment-emergent adverse events (TEAEs) were reported in the ITT/safety population (956 PP), of which 167 (139 PP) were grade ≥3. In the PP population, 41 TEAEs in 16 (38.1%) patients were possibly or probably related to 32P microparticles or implantation procedure, including 8 grade ≥3 in 3 (7.1%) patients, compared with 609 TEAEs in 42 (100%) patients attributed to chemotherapy, including 67 grade ≥3 in 28 patients (66.7%). The local disease control rate at 16 weeks was 82.0% (95% confidence interval: 68.6% to 90.9%) (ITT) and 90.5% (95% confidence interval: 77.4% to 97.3%) (PP). Tumour volume, carbohydrate antigen 19-9 levels, and metabolic tumour response at week 12 improved significantly. Ten patients (20.0% ITT; 23.8% PP) had surgical resection and median overall survival was 15.2 and 15.5 months for ITT and PP populations, respectively. CONCLUSIONS: Endoscopic ultrasound-guided 32P microparticle implantation has an acceptable safety profile. This study also suggests clinically relevant benefits of combining 32P microparticles with standard-of-care systemic chemotherapy for patients with unresectable LAPC

6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase-2 regulates TP53-dependent paclitaxel sensitivity in ovarian and breast cancers

Purpose: Paclitaxel is an integral component of primary therapy for breast and epithelial ovarian cancers, but less than half of these cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with both diseases. Experimental Design: Twelve kinases that regulate metabolism were depleted in multiple ovarian and breast cancer cell lines to determine whether they regulate sensitivity to paclitaxel in Sulforhodamine B assays. The effects of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2) depletion on cell metabolomics, extracellular acidification rate, nicotinamide adenine dinucleotide phosphate, reactive oxygen species (ROS), and apoptosis were studied in multiple ovarian and breast cancer cell lines. Four breast and ovarian human xenografts and a breast cancer patient-derived xenograft (PDX) were used to examine the knockdown effect of PFKFB2 on tumor cell growth in vivo. Results: Knockdown of PFKFB2 inhibited clonogenic growth and enhanced paclitaxel sensitivity in ovarian and breast cancer cell lines with wild-type TP53 (wtTP53). Silencing PFKFB2 significantly inhibited tumor growth and enhanced paclitaxel sensitivity in four xenografts derived from two ovarian and two breast cancer cell lines, and prolonged survival in a triple-negative breast cancer PDX. Transfection of siPFKFB2 increased the glycolysis rate, but decreased the flow of intermediates through the pentose–phosphate pathway in cancer cells with wtTP53, decreasing NADPH. ROS accumulated after PFKFB2 knockdown, which stimulated Jun N-terminal kinase and p53 phosphorylation, and induced apoptosis that depended upon upregulation of p21 and Puma. Conclusions: PFKFB2 is a novel target whose inhibition can enhance the effect of paclitaxel-based primary chemotherapy upon ovarian and breast cancers retaining wtTP53

Comparative evaluation of INNO-LiPA HBV assay, direct DNA sequencing and subtractive PCR-RFLP for genotyping of clinical HBV isolates

Genotypes (A to H) of hepatitis B virus (HBV) influence liver disease progression and response to antiviral therapy in HBV-infected patients. Several methods have been developed for rapid genotyping of HBV strains. However, some of these methods may not be suitable for developing countries. The performance of INNO-LiPA HBV Genotyping assay (LiPA), direct DNA sequencing and subtractive PCR-RFLP of genotype-specific HBV genome regions were evaluated for accurately determining the HBV genotypes by analyzing sera (n = 80) samples from chronic HBV patients. Both, LiPA and DNA sequencing identified 63, 4 and 13 HBV strains as belonging to genotype D, genotype A and mixed genotype A and D, respectively. On the contrary, the PCR-RFLP-based method correctly identified all 4 genotype A but only 56 of 63 genotype D strains. Seven genotype D strains yielded indeterminate results. DNA sequence comparisons showed that a single nucleotide change in the target region generated an additional restriction site for Nla IV that compromised the accuracy of this method. Furthermore, all the mixed genotype A and D strains were identified only as genotype A strains. The data show that the PCR-RFLP-based method incorrectly identified some genotype D strains and failed to identify mixed genotype infections while LiPA and DNA sequencing yielded accurate results