Systematic site-directed mutagenesis to characterize subunit interactions in E. coli asparaginase II, an enzyme

L-asparaginases (EC 3.5.1.1) catalyze the hydrolysis of L-asparagine to aspartic acid and ammonia. Asparaginases of bacterial origin have been used for over 40 years in the treatment of acute lymphatic leukemia (ALL). Asparaginase isoenzyme II from Escherichia coli (EcA2) is especially effective in cancer therapy. The enzyme, a homotetramer (138 kDa) made up from equivalent dimers, is also an ideal model compound to study folding/unfolding and associa¬tion/dissociation phenomenon of a large oligomeric protein: It is readily amenable to site-directed mutagenesis, a single tryptophan residue per subunit simplifies spectroscopic analysis of conformational changes, and it spontaneously refolds after chemical denaturation. Our aim was to probe the molecular basis of the interactions between the EcA2 dimers by mutations, and to construct EcA2 variants with improved stability for possible application in leukemia treatment. Using guanidine hydrochloride (Gu.HCl) equilibrium denaturation methods, we analyzed unfolding of wild-type EcA2 and a number of variants with amino acid replacements in the interior of the subunits or at the interfaces between the so-called intimate dimers. Enzymatic activity, fluorescence, and CD spectroscopy were used to monitor chemical denaturation by Gu.HCl. For wild-type EcA2 we found a highly cooperative transition from the folded to the denatured state. By gel filtration and sedimentation equilibrium ultracentrifugation, we showed that unfolding of the wild-type enzyme was preceded by dissociation into dimers and monomers. EcA2(WT) showed little change in stability between pH 5-8 but a rapid loss in stability below pH 5. As compared to wild-type enzyme, variants with amino acid exchanges that weaken dimer-dimer interactions exhibited more complex denaturation profiles with one or two stable intermediate states. So, for instance, variants EcA2(Y176S) and EcA2(W66Y/Y181W) dissociate into fully active dimers at low Gu.HCl concentrations. On the other hand, variant EcA2(Y176F) shows higher structural stability and specific activity than the wild-type protein. Thermal unfolding of EcA2 and its mutants was examined by differential scanning calorimetry (DSC) and CD. Correlation of DSC, CD, and light scattering data showed a single cooperative heat unfolding transition accompanied by irreversible protein aggregation, which precluded quantitative thermodynamic analysis of the excess heat capacity data. DSC data of EcA2 (WT) suggested that the increase in protein stability upon an increase in pH from 5-8 is mainly enthalpy-driven. Consistent with the chemical unfolding data, mutant EcA2(Y176F) showed higher thermal stability (a higher melting temperature, Tm) than wild type enzyme while EcA2(Y176S) and EcA2(W66Y/Y181W) were much more heat-sensitive, indicating the preference for large hydrophobic side chains at this site. In addition, two salt bridges, D156•••R191 and D188 •••R195, markedly contribute to the tetramer stability. Our results demonstrate that even small changes at a subunit interface may markedly enhance or impair EcA2 stability without compromising its catalytic properties. Engineered enzyme variants with enhanced stability, such as EcA2(Y176F), are promising candidates for an improved asparaginase therapy of leukemias

Integration of fuzzy reasoning approach (FRA) and fuzzy analytic hierarchy process (FAHP) for risk assessment in mining industry

Purpose: Mining industry has always been known for its unsafe working environment. This industry is one of the most hazard prone industries. To maintain safety in workplace timely assessment of risk associated with different operations performed to extract ore from the ore body has become necessity. To serve the said purpose, present work demonstrates a robust hybrid risk assessment approach for mining industry. Design/Methodology: Accident data from 1995 to 2012 is reviewed to identify hazards contributed to negative outcomes. The FRA approach is implemented to evaluate the risk levels associated with identified hazard factors. Thereafter AHP pairwise comparison matrix is developed to obtain priority weights for the hazard factors. Final priority of hazards based on severity of level of risk associated with them is obtained considering the outcome of FRA approach in terms of risk score for the hazards, combined with the priority weights obtained from AHP technique. Findings: Defuzzified FAHP weight of hazard factors, this weight gives priority sequence of hazards to be considered for development of plan of mitigation. Originality/Value: Risk assessment is a requirement of the Occupational Health and Safety Act 2000 (Section 7 & 8). The data required to assess the risk is uncertain, and in such case fuzzy approach is well suited to process the data and get the crisp output. The output of fuzzy approach is made robust with its integration to AHP. In this way FAHP can be used as robust technique for risk assessment in this industry and this technique develops an efficient safety management system for the achievement of goal to develop the workplace with zero accident, which many other countries have already achieved.Peer Reviewe

A case report on decidual cast

The mucous membrane lining of uterus is known as decidua once it is implanted with a fertilized egg. A decidual cast forms under the influence of hormones while preparing uterus for implantation and it can shed by taking the shape of uterus. It has well known association with ectopic pregnancy. Also it can occur with incomplete abortion as mentioned in this case report. Other conditions are use of progesterone, standard dose of oral contraceptive pills. Our patient 25 year unbooked gravida 3 parity 2 with 12 week period of gestation presented to emergency department with incomplete abortion with excessive bleeding and anaemia. She was shifted for surgical evacuation in which she passed decidual cast, a very rare finding. The histopathology report suggest retained product of conception with chorionic villi

Deep Learning Techniques in Extreme Weather Events: A Review

Extreme weather events pose significant challenges, thereby demanding techniques for accurate analysis and precise forecasting to mitigate its impact. In recent years, deep learning techniques have emerged as a promising approach for weather forecasting and understanding the dynamics of extreme weather events. This review aims to provide a comprehensive overview of the state-of-the-art deep learning in the field. We explore the utilization of deep learning architectures, across various aspects of weather prediction such as thunderstorm, lightning, precipitation, drought, heatwave, cold waves and tropical cyclones. We highlight the potential of deep learning, such as its ability to capture complex patterns and non-linear relationships. Additionally, we discuss the limitations of current approaches and highlight future directions for advancements in the field of meteorology. The insights gained from this systematic review are crucial for the scientific community to make informed decisions and mitigate the impacts of extreme weather events

Sensory and nutritional evaluation of unleavened flat bread prepared by multigrain flour mixture

The present study was undertaken to develop the value added food product using multigrain flour mixture and to assess its sensory and nutritional composition of unleavened flat bread (Chapatti). It was standardized as Control (T0). Along with control; three variations of Chapatti were prepared by replacing wheat flour with different ratio of multigrain flour mixture which referred as T1, T2, T3 and T4 respectively. They were tested for different attrib-utes (Taste and Flavour, Colour and Appearance, Body and Texture and Overall Acceptability). A food composition table given by Gopalan, et.al, 2007 was used to determine the nutritional composition of Chapatti. Appropriate statis-tical technique was opted for the analysis. The result revealed that the T1 (8.05±0.00) was found most acceptable with regards to its sensory attributes followed by T0 (7.70±0.42), T2 (7.55±0.08), T3 (7.22±0.98) and T4 (6.64±0.46) respectively. Energy (ranging from 388-436 Kcal), Protein (ranging from 22-28 g), fat (ranging from 13-21 g), cal-cium (ranging from145-192 mg), phosphorus (ranging from 466-501 mg), fiber (ranging from 3-4g) and iron (ranging from 6-7 mg) were increased in treatments as compared to control except carbohydrate. Thus, it can be concluded that value added product has good organoleptic and nutritional quality

Systematic site-directed mutagenesis to characterize subunit interactions in E. coli asparaginase II, an enzyme

L-asparaginases (EC 3.5.1.1) catalyze the hydrolysis of L-asparagine to aspartic acid and ammonia. Asparaginases of bacterial origin have been used for over 40 years in the treatment of acute lymphatic leukemia (ALL). Asparaginase isoenzyme II from Escherichia coli (EcA2) is especially effective in cancer therapy. The enzyme, a homotetramer (138 kDa) made up from equivalent dimers, is also an ideal model compound to study folding/unfolding and associa¬tion/dissociation phenomenon of a large oligomeric protein: It is readily amenable to site-directed mutagenesis, a single tryptophan residue per subunit simplifies spectroscopic analysis of conformational changes, and it spontaneously refolds after chemical denaturation. Our aim was to probe the molecular basis of the interactions between the EcA2 dimers by mutations, and to construct EcA2 variants with improved stability for possible application in leukemia treatment. Using guanidine hydrochloride (Gu.HCl) equilibrium denaturation methods, we analyzed unfolding of wild-type EcA2 and a number of variants with amino acid replacements in the interior of the subunits or at the interfaces between the so-called intimate dimers. Enzymatic activity, fluorescence, and CD spectroscopy were used to monitor chemical denaturation by Gu.HCl. For wild-type EcA2 we found a highly cooperative transition from the folded to the denatured state. By gel filtration and sedimentation equilibrium ultracentrifugation, we showed that unfolding of the wild-type enzyme was preceded by dissociation into dimers and monomers. EcA2(WT) showed little change in stability between pH 5-8 but a rapid loss in stability below pH 5. As compared to wild-type enzyme, variants with amino acid exchanges that weaken dimer-dimer interactions exhibited more complex denaturation profiles with one or two stable intermediate states. So, for instance, variants EcA2(Y176S) and EcA2(W66Y/Y181W) dissociate into fully active dimers at low Gu.HCl concentrations. On the other hand, variant EcA2(Y176F) shows higher structural stability and specific activity than the wild-type protein. Thermal unfolding of EcA2 and its mutants was examined by differential scanning calorimetry (DSC) and CD. Correlation of DSC, CD, and light scattering data showed a single cooperative heat unfolding transition accompanied by irreversible protein aggregation, which precluded quantitative thermodynamic analysis of the excess heat capacity data. DSC data of EcA2 (WT) suggested that the increase in protein stability upon an increase in pH from 5-8 is mainly enthalpy-driven. Consistent with the chemical unfolding data, mutant EcA2(Y176F) showed higher thermal stability (a higher melting temperature, Tm) than wild type enzyme while EcA2(Y176S) and EcA2(W66Y/Y181W) were much more heat-sensitive, indicating the preference for large hydrophobic side chains at this site. In addition, two salt bridges, D156•••R191 and D188 •••R195, markedly contribute to the tetramer stability. Our results demonstrate that even small changes at a subunit interface may markedly enhance or impair EcA2 stability without compromising its catalytic properties. Engineered enzyme variants with enhanced stability, such as EcA2(Y176F), are promising candidates for an improved asparaginase therapy of leukemias

Evaluation of echocardiographic systolic parameters in pre eclamptics and normotensives women

Background: Early detection of changes in cardiovascular echocardiographic systolic parameters and their comparison with normotensives women.Methods: This was a hospital based prospective case control study carried out in department of obstetrics and gynaecology in S.M.S Medical College, Jaipur during the period from February 2013 to December 2014. 100 women were enrolled in the study after applying inclusion and exclusion criteria. All women underwent two dimensional echocardiography at rest. Cardiac systolic parameters were recorded and studied.Results: Mean LVEDV in PIH group was 75.71 ± 4.8 ml v/s 71.08 ± 5.6 ml in control group and the difference was statistically significant. Mean stroke volume was higher in PIH group as compared to the control group and the difference was statistically significant (59.83 ± 7.4ml/min v/s 52.48 ± 6.4ml/min). Mean Aortic Root Diameter in PIH group was 2.188 ± 1.04 cm as compared to the 1.866 ± 1.06 cm in the control group which was statistically significant. Mean Left Ventricular outflow Tract diameter was slightly higher in the PIH group as compared to the controls although the P-value was not significant (2.37 ± 1.06 cm and 1.99 ± 1.08 cm). Mean Total Vascular Resistance in PIH group was found to be higher in PIH group as compared to the controls and the difference was statistically significant (1389 ± 57.04 dynes/sec/cm-5 v/s 1286 ± 45.01 dynes/sec/cm-5).Conclusions: Systolic parameters get worsened in pre eclamptics. Early detection of change in these parameters could help to identify high risk women who are prone to develop cardiovascular morbidity in later life

Case control study of supervised and unsupervised medical abortion in first trimester of pregnancy

Background: The MTP act of India 1971 provides for the termination of pregnancy up to 20 weeks of gestation, by a registered medical practitioner, provided all the prerequisites are fulfilled. The objective of this study was to conduct case control study of supervised and unsupervised medical abortion in first trimester of pregnancy.Methods: A case control study was conducted with two groups of patients with period of gestation <9 weeks. Case group consist of 30 patients presenting to emergency department with unsupervised intake of medical abortion pill. Control group consist of 30 patients presenting to family planning outpatient department want medical abortion pill under supervision. The main outcome variables were comparison of success rate, failure rate, complications like pain, fever, heavy bleeding, shock, requirement of blood transfusion. Interventions required were compared in both the groups in terms of medical management, surgical evacuation and laparotomy.Results: In our study, the success rate achieved in terms of complete abortion was 66.7% in the supervised and 13.3% in the unsupervised group with (p value <0.001). The complication rate was 60% in the supervised group in contrast to 93.3% in the unsupervised group. 56.7% patients in unsupervised group required blood transfusion which was nil in supervised groups. 71.4% patients in the supervised group did not require intervention. In the unsupervised group, 13.8% required medical management, 65.5% required surgical evacuation and 6.7% had to undergo laparotomy for ectopic pregnancy.Conclusions: In present study we conclude that first trimester medical abortion under supervision is more effective, safe and convenient as compared to unsupervised medical abortion. We recommend that government must stop over the counter sell of medical abortion pills

To Compare the Perspectives of Teacher Educators and Pupil Teachers for Infrastructure in Teacher’s Training Colleges

Teachers keep the foundation stone of formal learning. This learning should be built up with planning and affection. A great deal of thoughts of teachers is required in a friendly environment. For this, we need well trained teachers. In ITEP, Teacher education colleges are given comprehensive guidelines for infrastructure improvement. Good infrastructure will definitely improve the level of teacher education. A researcher conducted an online survey for a sample of teachers and students. The purpose of this study is to compare the perspectives of the respondents regarding teacher training infrastructure. This study also points out the importance of various features of the teacher training infrastructure from the point of view of the respondents and encourages the respondents to express their opinion on better use of the infrastructure