Structure-Function Analysis of Enzymes of the Polyisoprenyl-Phosphate Hexose-1-Phosphate Transferase family

Enzymes of the polyisoprenyl-phosphate hexose-1-phosphate transferase (PHPT) family are integral membrane proteins that initiate the synthesis of glycans by catalyzing the transfer of a hexose-1-phosphate sugar from UDP-hexose to the lipid carrier undecaprenyl phosphate (Und-P). These glycans such as O antigen and exopolysaccharide (EPS) provide bacteria with protection and adaptation to the environment and host immune factors. The role of PHPT proteins in initiation and the absence of any eukaryotic homologues make them an attractive target for novel antimicrobials; however study of these proteins is difficult due to the presence of multiple transmembrane helices. A requirement of the C-terminal domain for catalytic activity has been demonstrated in vitro, but the importance of specific regions and/or residues for the activity of these enzymes was not understood and was investigated in this work. The galactose-1-phosphate transferase WbaP of S. enterica initiates the synthesis of O antigen and was used as a model to study PHPT proteins. In vivo and in vitro functional assays of WbaP allowed us to show that a soluble loop region affects O antigen chain length distribution and the C-terminal domain (WbaPCT) containing one putative transmembrane (TM) domain and tail region is sufficient for transferase activity in vivo. The location of the WbaP tail region was found to be cytoplasmic and to contain highly conserved residues essential for activity. In this work a thioredoxin (TrxA) fusion was used as a tool to improve protein folding, which allowed for the solubilization and purification of WbaPCT. Obtaining a pure WbaPCT sample has allowed for the biochemical characterization of this protein revealing that this region is sufficient to maintain specificity for undecaprenyl phosphate (Und-P). Characterization of two glucose-1-phosphate transferases from Escherichia coli K-12 and Caulobacter crescentus revealed that some members of the PHPT family are able to utilize a second sugar substrate leading to the synthesis of a different glycan. These studies will pave the way to a detailed structural and mechanistic understanding of these proteins

V. NEGUNDO, L. CAMARA AND B. VARIEGATA PLANTS LEAF EXTRACT EXHIBIT CONSIDERABLE IN VITRO ANTIOXIDANT AND ANTICANCER ACTIVITIES

Objective: The study was planned to investigate antioxidant and anticancer activities with the preliminary phytochemical analysis of methanolic extracts of Vitex negundo (V. negundo), Lantana camara (L. camara) and Bauhania variegata (B. variegata) plants leaf extracts.Methods: Phytochemical evaluation was performed for all the extracts, as per the standard methods. In vitro antioxidant activities were performed by using DPPH (2,2-Diphenyl-1-Picrylhydrazyl), ABTS (2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid) and FRAP (Ferric reducing antioxidant power assay) method and compared with standard antioxidants. The anticancer activity of plant extract was assessed using MTT colorimetric assay.Results: The study of preliminary phytochemical proved the existence of alkaloids, flavonoids and phenolic types of phytochemicals in high amount. Methanolic extract of L. camara shows minimum IC50 value for DPPH assay (48.75±2.34 µg/ml) and FRAP assay (274.66±3.65 µg/ml). In ABTS assay B. variegata extract exhibit minimum IC50 value (60.48±3.01 µg/ml). Lower the IC50 value of extract, higher the effectiveness of the plant. Methanolic extract of all plants methanolic extracts showed anticancer activity against SH-SY-5Y cells (human neuroblastoma cell) but V. negundo was more effective against SH-SY-5Y cells with IC50 value (209 µg/ml) compared to remaining extracts.Conclusion: The current finding accomplished the in vitro activities, so that plant could be a superior source of antioxidant and anticancer drugs. But further in vivo assessment was needed before adding it into the pharma industry

Role of immunohistochemistry in the differential diagnosis of malignant small round cell tumor: a study of 38 cases

Background: : Immunohistochemistry play a very important role in modern surgical pathology especially for identification of tumors lacking the evidence of lineage differentiation on the basis of routine Hematoxylin and Eosin (H&E) stain alone. More than 90% of the tumor with diagnostic difficulties by routine H & E stain could be very well classified by using IHC. The aim of present study is to classify and identify MSRCT.Methods: The study was carried out in Department of Pathology, Government Medical College in a period from August 2008 to November 2011. Total 38 cases of MSRCT are selected for Immunohistochemical staining (IHC) and they are classified and categorized accordingly after IHC.Results: Out of 38 cases of  MSRCT, there are 18 cases (47.36%) of non-Hodgkin lymphoma, 3 (7.89%) cases of Neuroblastoma,  3 (7.89%) cases of Synovial Sarcoma, 2 (5.26%) cases of Ewing Sarcoma, 2 (5.26%) cases of Undifferentiated neuroendocrine carcinoma,  2 (5.26%) case of Desmoplastic Small Round Cell Tumor, 1 (2.63%) case of  Primitive Neuroectodermal Tumor, 1 (2.63%) case of Amelanotic Melanoma (Small cell variant), 1 (2.63%)  case of  Anaplastic Dysgerminoma, 1 (2.63%)  case of Osteosarcoma, 1 (2.63 %) case of Wilm's tumor, 1 (2.63%)  case of  Dendritic cell Tumor Testis, 1 (2.63%) case of Undifferentiated Nasopharyngeal Carcinoma, 1 (2.63%) case of  Embryonal rhabdomyosarcoma.Conclusions: IHC is very valuable tool for adequate and accurate categorization of MSRCT.

The Influence of Handgrip and Pedal Cadence During Sustained Cycling Power Outputs

In cycling performance, there has been interest in contribution the upper body (i.e. handgrip) provides during cycling efforts as well as power output during varying cadences. Little has been done pertaining to cardiovascular responses with the aforementioned during sustained power. PURPOSE: To determine the cardiovascular reactions to isometric hand-grip and different pedal cadences during sustained cycling efforts. METHODS: Subjects were nine (n=9) experienced cyclists. Each signed a medical-health and physical readiness questionnaire, and IRB approved informed consent. Resting values of heart rate (b*min.-1), blood pressure (mmHg), height (cm), weight (kg) and age (years) were assessed. A MonarkTM bicycle ergometer was used for testing. Grip was substantiated through the use of a hand grip dynamometer at 20 kg of tension. An exercise test of 20 minutes at 150 Watts was performed four times with random assignment through a Latin Squares Design. Protocols were distinguished by grip/no grip; 50/100 RPM; 3kp/1.5kp resistance. During the 20 minute exercise test, heart rate, blood pressure and calculated Rate Pressure Product (HR * SBP)*100-1 were recorded every minute. Statistical measures included group means (SD) between protocols and use of a Repeated Measures ANOVA to examine variable differences between grip/ no-grip and cadence/resistance protocols. Significance was set at p \u3c 0.05. RESULTS: Statistically significant differences were reported for the variables of heart rate, systolic blood pressure and work of the heart for the following protocols: high cadence/low resistance-grip HR=147.72 (3.24); SBP= 164.59 (6.42); WH=242.58 (23.99) and high cadence/low resistance-no grip HR=150.83 (6.49); SBP=166.05 (5.60); WH=245.57 (25.70). The following protocols showed no significant differences: low cadence/high resistance-grip HR=132.50 (3.24); SBP=160.29 (4.95); WH=211.54 (12.22) and low cadence/high resistance-no grip HR=130.51 (3.36); SBP=156.66 (5.17); WH=204.63 (11.45). Conclusion: This research demonstrated that grip appears to be a transient influence during sustained work. Cadence was a more influential factor on cardiovascular responses during sustained cycling performance

Shifting trends of lung tumours and its diagnosis by lung biopsy: a study of 78 cases

Background: The objective of the study was to study the spectrum of pathological lesions in patients with lung mass and to study correlation between clinical findings, histopathological pattern and immunohistochemical stains in various biopsy specimen for differentiation and typing of tumors.Methods: This retrospective study was done for the period of three years at Department of Pathology, New Civil Hospital, Surat, India, which is a tertiary health care Centre. Here we studied 78 cases of lung biopsy received in formalin, which were subjected to histopathological examination. Immunohistochemistry was performed as and when required.Results: Total 78 lung biopsy specimens were examined. Out of which, 59 cases (75.6%) were neoplastic, 12 cases(15.4%) were non-neoplastic and 7 cases (9%) were inconclusive. The commonest histological type of malignancy was adenocarcinoma which is associated with peripheral mass lesion, female gender and in non-smokers. Commonest non-neoplastic lesion was tuberculosis.  Malignancy was seen quite common in patients presented with lung masses in our institute.Conclusions: Lung tumours are quite common in patients presented with mass lesion. Similar to global trend, adenocarcinoma is the commonest histological type now and associated with change in incidence among women, in non-smokers, molecular alteration and prognosis which need further investigation. Immunohistochemistry is helpful in cases which are not accurately subtyped by histomorphology alone.

An unusual case of labial fusion in a post pubertal girl

Labial fusion is defined as either partial or complete adherence of the labia minora. It is also known as vulvar fusion, labial adhesion, labial agglutination or synechia of the vulva. This condition is common in pre-pubertal girls when estrogen levels are low and commonly resolves spontaneously post-puberty. This condition is usually asymptomatic and can be treated with topical application of estrogen or betamethasone cream or by manual or surgical separation of adhesions. We present a case of labial fusion in post pubertal girl which was managed surgically

Strategies to Prevent Biofilm Infections on Biomaterials: Effect of Novel Naturally-Derived Biofilm Inhibitors on a Competitive Colonization Model of Titanium by Staphylococcus aureus and SaOS-2 Cells

Biofilm-mediated infection is a major cause of bone prosthesis failure. The lack of molecules able to act in biofilms has driven research aimed at identifying new anti-biofilm agents via chemical screens. However, to be able to accommodate a large number of compounds, the testing conditions of these screenings end up being typically far from the clinical scenario. In this study, we assess the potential applicability of three previously discovered anti-biofilm compounds to be part of implanted medical devices by testing them on in vitro systems that more closely resemble the clinical scenario. To that end, we used a competition model based on the co-culture of SaOS-2 mammalian cells and Staphylococcus aureus (collection and clinical strains) on a titanium surface, as well as titanium pre-conditioned with high serum protein concentration. Additionally, we studied whether these compounds enhance the previously proven protective effect of pre-incubating titanium with SaOS-2 cells. Out of the three, DHA1 was the one with the highest potential, showing a preventive effect on bacterial adherence in all tested conditions, making it the most promising agent for incorporation into bone implants. This study emphasizes and demonstrates the importance of using meaningful experimental models, where potential antimicrobials ought to be tested for the protection of biomaterials in translational applications

Strategies to Prevent Biofilm Infections on Biomaterials: Effect of Novel Naturally-Derived Biofilm Inhibitors on a Competitive Colonization Model of Titanium by Staphylococcus aureus and SaOS-2 Cells

Biofilm-mediated infection is a major cause of bone prosthesis failure. The lack of molecules able to act in biofilms has driven research aimed at identifying new anti-biofilm agents via chemical screens. However, to be able to accommodate a large number of compounds, the testing conditions of these screenings end up being typically far from the clinical scenario. In this study, we assess the potential applicability of three previously discovered anti-biofilm compounds to be part of implanted medical devices by testing them on in vitro systems that more closely resemble the clinical scenario. To that end, we used a competition model based on the co-culture of SaOS-2 mammalian cells and Staphylococcus aureus (collection and clinical strains) on a titanium surface, as well as titanium pre-conditioned with high serum protein concentration. Additionally, we studied whether these compounds enhance the previously proven protective effect of pre-incubating titanium with SaOS-2 cells. Out of the three, DHA1 was the one with the highest potential, showing a preventive effect on bacterial adherence in all tested conditions, making it the most promising agent for incorporation into bone implants. This study emphasizes and demonstrates the importance of using meaningful experimental models, where potential antimicrobials ought to be tested for the protection of biomaterials in translational applications

A multicenter comparison between Child Pugh and ALBI scores in patients treated with sorafenib for hepatocellular carcinoma

Background & aims: The ALBI grade was proposed as an objective means to evaluate liver function in patients with Hepatocellular Carcinoma (HCC). ALBI grade 1 vs 2 were proposed as stratification factors within the Child Pugh (CP) A class. However, the original publication did not provide comparison with the sub-classification by points (5 to 15) within the CP classification. Methods: We retrospectively analyzed data from patients treated with sorafenib for HCC from 17 centers in United Kingdom and France. Overall survival (OS) was analyzed with the Kaplan-Meier method and a Cox regression model. Discriminatory abilities of the classifications were assessed with the log likelihood ratio, Harrell’s C statistics and Akaike information criterion. Results: Data from 1,019 patients were collected, of which 905 could be assessed for both scores. 92% of ALBI grade 1 were CP A5 while ALBI 2 included a broad range of CP scores of which 44% were CP A6. Median OS was 10.2, 7.0 and 3.6 months for CP scores A5, A6 and >A6, respectively (P<0.001), Hazard Ratio (HR)=1.60 (95%CI: 1.35-1.89, P<0.001) for A6 vs A5. Median OS was 10.9, 6.6 and 3.0 months for ALBI grade 1, 2 and 3, respectively (P<0.001), HR=1.68 (1.43-1.97, P<0.001) for grade 2 vs 1. Discriminatory abilities of CP and ALBI were similar in the CP A population, but better for CP in the overall population. Conclusions: Our findings support the use CP class A as an inclusion criterion, and ALBI as a stratification factor in trials of systemic therapy

Aspirin as an adjuvant treatment for cancer:feasibility results from the Add-Aspirin randomised trial

BACKGROUND: Preclinical, epidemiological, and randomised data indicate that aspirin might prevent tumour development and metastasis, leading to reduced cancer mortality, particularly for gastro-oesophageal and colorectal cancer. Randomised trials evaluating aspirin use after primary radical therapy are ongoing. We present the pre-planned feasibility analysis of the run-in phase of the Add-Aspirin trial to address concerns about toxicity, particularly bleeding after radical treatment for gastro-oesophageal cancer.METHODS: The Add-Aspirin protocol includes four phase 3 randomised controlled trials evaluating the effect of daily aspirin on recurrence and survival after radical cancer therapy in four tumour cohorts: gastro-oesophageal, colorectal, breast, and prostate cancer. An open-label run-in phase (aspirin 100 mg daily for 8 weeks) precedes double-blind randomisation (for participants aged under 75 years, aspirin 300 mg, aspirin 100 mg, or matched placebo in a 1:1:1 ratio; for patients aged 75 years or older, aspirin 100 mg or matched placebo in a 2:1 ratio). A preplanned analysis of feasibility, including recruitment rate, adherence, and toxicity was performed. The trial is registered with the International Standard Randomised Controlled Trials Number registry (ISRCTN74358648) and remains open to recruitment.FINDINGS: After 2 years of recruitment (October, 2015, to October, 2017), 3494 participants were registered (115 in the gastro-oesophageal cancer cohort, 950 in the colorectal cancer cohort, 1675 in the breast cancer cohort, and 754 in the prostate cancer cohort); 2719 (85%) of 3194 participants who had finished the run-in period proceeded to randomisation, with rates consistent across tumour cohorts. End of run-in data were available for 2253 patients; 2148 (95%) of the participants took six or seven tablets per week. 11 (0·5%) of the 2253 participants reported grade 3 toxicity during the run-in period, with no upper gastrointestinal bleeding (any grade) in the gastro-oesophageal cancer cohort. The most frequent grade 1-2 toxicity overall was dyspepsia (246 [11%] of 2253 participants).INTERPRETATION: Aspirin is well-tolerated after radical cancer therapy. Toxicity has been low and there is no evidence of a difference in adherence, acceptance of randomisation, or toxicity between the different cancer cohorts. Trial recruitment continues to determine whether aspirin could offer a potential low cost and well tolerated therapy to improve cancer outcomes.FUNDING: Cancer Research UK, The National Institute for Health Research Health Technology Assessment Programme, The MRC Clinical Trials Unit at UCL.</p