Identification of Translesion Synthesis Inhibitors that Target Rev7/Rev3 Protein-Protein Interactions

Translesion synthesis (TLS) is a cellular mechanism utilized by cancer cells to tolerate DNA damage caused by chemotherapeutics, like cisplatin, by replicating past unrepaired lesions. This increases the rate of mutations, which leads to the emergence of drug-resistant cancer cells. Preliminary studies have shown that disrupting the protein-protein interactions (PPI) in the TLS heteroprotein complex increases cells’ sensitivity to first-line genotoxic chemotherapy, illustrating how inhibiting TLS assembly and function can significantly increase cancer cell death. These results underscore the therapeutic potential of targeting TLS PPI. Our current work in this area is focusing on inhibitors capable of disrupting the Rev7/Rev3 TLS PPI. This study identified three promising inhibitors that disrupt the Rev7/Rev3 PPI with verification of TLS inhibition through biochemical and cellular studies. Future research can use optimize these inhibitors to develop a new class of anti-cancer agents that can enhance the efficacy of other drugs and treatments that exert their effects through DNA damage

Testing Predicted Specificity Determinants in the Dihydrofolate Reductase Enzyme Family

Dihydrofolate Reductase (DHFR) is an essential enzyme for most organisms, ranging from bacteria to humans. DHFR has essential functions in DNA biosynthesis and cell replication; as a result, cell growth can be inhibited by the inhibition of DHFR. Evaluating the use of well-known DHFR inhibitors is becoming essential in treating infections in the developing world as DHFR is a known target of antibacterial and antiparasitic drugs. Understanding determinants of DHFR inhibitor specificity in terms of amino acid sequence and structure will allow repurposing or designing of new compounds that selectively target DHFR from the pathogenic organism of interest over the Hs DHFR. Previously, a computational analysis was developed to predict allosteric residues involved in ligand discrimination using DHFR as a model system. The approach was based on inhibitor specificity and amino acid composition for sets of protein homolog pairs, predicting eighteen alignment positions. The residues were clustered as follows: three of the residues are found in the active site; four of the residues are proximal to the active site, four of the residues are clustered together in the adenosine binding domain and five of the residues are on the βFβG loop. Many of the predicted residues are located in allosteric region away from the active site. My role in this project was to experimentally validate these predictions using site specific mutations in B. Strearothermophilus DHFR gene as a model system. To ensure all mutants were correctly folded and active, turnover numbers (kcat) and Michaelis constants (KM) were measured for wildtype and mutants. KI values of the 12 single mutants against four DHFR inhibitors, methotrexate, trimethoprim, pyrimethamine, and raltitrexed were determined. Interestingly, comparisons of the ligand binding profiles of the mutants to those of the wildtype enzyme revealed significant changes in ligand specificity, supporting the predictions. In addition, the effects of mutations on KI values are ligand specific

IMPLEMENTATION OF QBD APPROACH TO DEVELOP AND VALIDATE ANALYTICAL METHOD FOR SIMULTANEOUS ESTIMATION OF DULOXETINE HYDROCHLORIDE AND METHYLCOBALAMIN IN PHARMACEUTICAL DOSAGE FORM BY HPTLC METHOD

Objective: To develop and validate High-Performance Thin Layer Chromatography (HPTLC) analytical method for the determination of Duloxetine Hydrochloride (DUL) and Methylcobalamin (MEC) in the standard mixture and pharmaceutical capsule dosage form by implementing Quality by the Design (QbD) approach.Methods: The chromatographic separation was performed on aluminium plates precoated by silica gel 60 F-254 using propanol: water: 25% v/v ammonia solution (9:2:1, v/v/v) as a mobile phase which was optimized with the help of a design expert. Densitometric analysis was carried out in the absorbance mode at 280 nm.Results: Compact spots for Duloxetine HCl and Methylcobalamin were found at retardation factor (Rf) value of 0.77±0.02 and 0.55±0.03, respectively. The linear regression analysis data for the calibration plots showed correlation coefficient 0.9989 and 0.999 with a concentration range of 1200-3600 ng/spot and 60-180 ng/spot for Duloxetine HCl and Methylcobalamin respectively. Limit of detection (LOD) and limit of quantification (LOQ) was found to be 113.39 and 343.62 ng for Duloxetine HCl and 6.68 and 20.24 ng for Methylcobalamin, respectively. The method was validated for precision, accuracy, robustness, LOD and LOQ according to ICH Q2 R1 guidelines.Conclusion: A new, simple, accurate, and precise HPTLC analytical method has been developed and validated for the determination of Duloxetine HCl and Methylcobalamin in pharmaceutical capsule dosage form by QbD concept in favour of fewer trials and error-free experimentation for the optimization process. The method seems to be suitable for the quality control in the pharmaceutical industry because of its sensitivity, simplicity, and selectivity.Keywords: Duloxetine HCl (DUL), Methylcobalamin (MEC), Quality by Design (QbD), HPTLC, Validatio

Studying prevention of parent to child transmission services, interventions, coverage and utilization

Background: Human immunodeficiency virus (HIV) infection is increasing at an alarming rate globally. Apart from heterosexual route, mother to-child transmission is the next most important route of HIV transmission accounting for over 90% of infections in children. The present retrospective study is undertaken to evaluate the effectiveness of implementation of PPTCT programmed in GMERS Medical College, Sola, Ahmedabad, Gujarat, India.Methods: At ICTCs, registered ANCs are counseled and tested for HIV. HIV+ve ANCs are linked to services and followed-up for institutional delivery. Antiretroviral prophylaxis with nevirapine was given to seropositive mother-baby pairs during delivery and children testing. HIV+ve ANCs since 2014 to 2016 subsequently delivered till December 2018 and their exposed children followed up till 18 months for confirmation of their seropositivity status in PPTCT were study at GMERS medical college, sola, Ahmedabad, Gujarat, India.Results: 13595 ANCs registered, 100% were counseled pre-test, and 99.5% of them tested, 79.5% were counseled post-test.  CD4 testing was carried out in all HIV+ve ANCs. 55 ANCs were detected HIV+ve inclusive of 37 new registered, 16 re-pregnancy and 2 unregistered cases. 55 pregnancy outcomes reported institutionally total 85.9% delivered at institute out of them 26.5% were caesarian sections and 62.4% delivered vaginally. Out of 48 live births, 47 sdNVP-MB-Pair were given. 48 children were traced till 18 months, 82.5% of live births were alive, and 68.75% of live births were tested. 1 was found HIV+ve with history of adherence to all prescribed PPTCT guidelines.Conclusions: PMTCT services - counseling and testing should be provided to all ANCs. EDD-based tracking, institutional deliveries, postnatal counseling to be encouraged along with complete MB pair coverage, capacity building of concerned staff regarding delivery of HIV+ve ANCs and exposed children tracking

Study of diagnosis and management of ectopic pregnancy

Background: Diagnosis of ectopic pregnancy was frequently missed. Aim of the study was to determine the clinical presentation, and treatment associated with ectopic pregnancy.Methods: This is a prospective study which was carried out at Obstetrics and Gynaecology department, GMERS SOLA civil hospital from August 2017 to October 2018. Total 416 patients were admitted during study period out of them 50 patients diagnosed with ectopic pregnancy were enrolled in the study and information was collected and analysed.Results: 80% patients were between the age group of 21-30 years. 56% patients were nulliparous. Amenorrhea (92%) with lower abdominal pain (94%) is the most common presenting symptom. 26% of patients show typical triad of amenorrhea, abdominal pain and bleeding per vagina. UPT and USG were most commonly performed investigations. 96% cases showed UPT positive. 100% USG showed adnexal pathology. Serum beta-hCG was done in 37 patients as an aid for diagnosis and to decide the line of management. Conservative medical management with Injection MTX was done in 4 patients of which 1 patients required laparotomy later on. Surgical management was done in 90% of patients. Laparoscopic management was done in 54% of cases.Conclusions: Early diagnosis and timely intervention in the form of conservative or surgical treatment will help in reducing the morbidity and mortality associated with ectopic pregnancy

Obstetric and perinatal outcome in previous one cesarean section

Background: Women with previous cesarean sections constitute a high risk group in obstetrics, with associated complications. The cesarean section carries 3 fold risk of mortality compared with vaginal deliveries. The study was conducted to determine the mode of deliveries after previous one cesarean section, maternal and fetal complication.Methods: This is a prospective observation study. Total 150 patients of previous one caesarean with gestational weeks between 37 to 40 weeks admitted in labour room of Obstetrics and Gynecology Department in Sola Civil Hospital with spontaneous onset of labour, over a period of one year from April 2014 to April 2015 or till the desired sample will reached.Results: In the present study, out of 150 pregnant women with history of previous one LSCS who were subjected to this study, 39(26%) underwent elective LSCS, commonest indication being previous pregnancy bad experience (38.46%). 111(74%) underwent trial of labour after cesarean section out of it 77(69.36%) had successful VBAC and 34(30.63%) underwent repeat emergency LSCS. Maternal complications were higher in Emergency LSCS group than in those had a successful VBAC (17.64% vs.3.89%)). Neonatal complications were also higher in Emergency LSCS group than in those had a successful VBAC (2.95% vs. 0%).Conclusions: With proper case selection, appropriate timing and close supervision trial of labour after prior LSCS is safe and often successful

Live Image Colour Segmentation Using Different Methods of ANN

Machine learning is a new dimension of science since last 2 decade which motivates algorithms that can learn from data by building a model, based on inputs and using that to make predications or decisions, rather than following only explicitly programmed instructions. Machine learning is sometimes conflated with data mining, which focuses more on exploratory data analysis. Data mining is the extraction of interesting (non-trivial, implicit, previously unknow and potential useful) patterns of knowledge from huge amount of data In computer vision image segmentation is the process of partitioning a digital image into multiple segments (set of pixels, also known as super-pixels). The goals of segmentation is to simplify and/or change the representation of an image into something that is more meaningful and easier to analyze. Image segmentation is typically used to locate objects and boundaries (lines, curves, etc.) in images. More precisely, image segmentation is the process of assigning a label to every pixel in an image such that pixels with the same label share certain characteristics. After that by considering region co-ordinates it separates all color in different figure

Pročišćavanje i karakterizacija ekstracelularne dekstran saharaze iz bakterije Pediococcus pentosaceus, izolirane iz tla sjeveroistočne Indije

The extracellular dextransucrase produced from Pediococcus pentosaceus, a new isolate from the soil in Assam, India, was purified and characterized. The enzyme activity of cell-free supernatant was 3.4 U/mL and specific activity was 0.6 U/mg. The crude enzyme was purified by a single-step fractionation using polyethylene glycols of different molecular mass. The specific activity achieved was 18 U/mg with 31-fold purification by PEG 400 and 26 U/mg with 45-fold purification by PEG 1500. The molecular mass of dextransucrase determined by non-denaturing SDS-PAGE was approx. 180 kDa. The dextran formation activity of the enzyme was confirmed by activity staining. Optimum conditions for dextransucrase activity were: pH=5.4, reaction temperature 30 °C, 5 % sucrose and 20 mM sodium acetate buffer. A concentration of 1 mM MgCl2 and 6 mM CaCl2 enhanced dextransucrase activity by 5 and 150 %, respectively. The chaotropic agent urea (7 M) and chelating agent EDTA (1 mM) resulted in the residual enzyme activity of 98 and 80 %, respectively. The organic solvents such as ethanol (50 %), DMSO (90 %), acetone (50 %) and acetonitrile (20 %) decreased the dextransucrase activity by 80, 91, 94 and 80 %, respectively.U radu je pročišćena i okarakterizirana dekstran saharaza iz bakterije Pediococcus pentosaceus, izolirane iz tla u gradu Assamu, Indija. Aktivnost je enzima u supernatantu bila 3,4 U/mL, a njegova je specifična aktivnost iznosila 0,6 U/mg. Sirovi je enzim pročišćen jednostupanjskim frakcioniranjem pomoću polietilen glikola različite molekularne mase. Utvrđena je specifična aktivnost enzima od 18 (pročišćenog 31 put pomoću PEG 400), odnosno 26 U/mg (pročišćenog 45 puta pomoću PEG 1500). Molekularna je masa dekstran saharaze određena pomoću SDS-PAGE, a iznosila je otprilike 180 kDa. Aktivnost je enzima potvrđena bojanjem nastalog dekstrana s Coomasie brilijant plavom bojom. Optimalni su uvjeti za aktivnost enzima bili: pH=5,4; temperatura reakcije od 30 °C; te dodatak 5 %-tne saharoze i acetatnog pufera (20 mM). Dodatak 1 mM MgCl2 i 6 mM CaCl2 povećali su aktivnost enzima za 5, odnosno 150 %. Inaktivirajući agensi, poput uree (7 M) i EDTA (1 mM) smanjili su aktivnost enzima na 98 odnosno 80 %. Organska su otapala također smanjila aktivnost enzima, i to: 50 %-tni etanol na 80 %, 90 %-tni DMSO na 91 %, 50 %-tni aceton na 94 % i 20 %-tni acetonitril na 80 %

Study the role of hepcidin in diagnosis of iron deficiency anemia in young females of northern India

Background: Iron deficiency is one of the most common nutritional disorder. Maintenance of body iron status is an integral part of healthcare in young female of reproductive age group. Thereby early detection could lead to early intervention and reduce its comorbidity.  Indeed, an ideal screening test should be capable of identifying iron deficiency long before developing anemia. Henceforth, the present study was aimed to determine utility of serum hepcidin in iron deficiency and to access the baseline value of hepcidin in young female.Methods: This sectional study was conducted in the Department of biochemistry SGT Medical College Hospital and Research Institute, Budhera, Gurugram. It included non-pregnant female students of age 18-25 years with normal RBC indices and hemoglobin >12 gm%. Estimation of serum hepcidin-25 was by ELISA.Results: The reference range of hepcidin established in this study was 12.14-139.89 ng/ml for females with the mean being 42.4±29.13 ng/ml. It showed higher discriminating power in evaluating iron status in young healthy women (AUC 0.984) with best combination of diagnostic sensitivity (95.7%) and specificity (93.2%) at a cut off of >15.7 ng/ml. Serum hepcidin identified 17% of young healthy females with normal hemoglobin to have functional or storage iron deficiency.Conclusions: The prevention of iron deficiency anemia remains insufficient worldwide especially among underprivileged women and children Therefore, estimation of serum hepcidin may be considered as a valuable tool in assessing iron status in young healthy female population who are the prime target group for iron supplements to reduce comorbidity associated with iron deficiency and anemia