Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

PublishedJournal ArticleThis is the final version of the article. Available from American Chemical Society via the DOI in this record.Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.Work is supported by the National Institute of Malaria Research, Indian Council of Medical Research, New Delhi and Dept. of Biotechnology, New Delhi. K.C.P. is a recipient of the Prof. Ramalingaswami Fellowship (Department of Biotechnology, Government of India (BT/HRD/35/02/2006), K.V. is a recipient of UGC Senior Research Fellowship, M.v.d.G. is grateful for support from the University of Exeter and the Wellcome Trust (078566/A/05/Z)

Nairobi Eye: A Clinico-epidemiological Study from A Tertiary Care Center of Central Nepal

Introduction: Periocular paederus dermatitis (Nairobi eye) is characterized by erythematous vesiculobullouslinear plaque with stinging sensation. It commonly occurs during rainy season. It has been describedin people living near agricultural fields due to the potential toxin pederin. This study aims to evaluate thedemographic profile and clinical presentation of patients with periocular paederus dermatitis in a tertiary carecenter. Methods: This was a descriptive, cross-sectional study evaluating patients attending dermatologyand/or ophthalmology department with features consistent with paederus dermatitis involving perioculararea from June to August, 2019. Relevant demographic and clinical data were obtained; clinical photographswere taken and histopathology performed among selected patients. Results: A total of 24 (14.8%) patients hadfeatures of Nairobi eye among 162 patients of paederus dermatitis. Majority of the patients were males (1.4:1)with mean age 29.08±13.38 years. The peak time of presentation was the first week of July (37.5%). Meantime period between onset of symptoms and presentation was 3.41±2.01 days. The lesions were unilateral inall cases, with predominant involvement of the right eye (62%). Burning sensation (80%) and itching (60%)were the predominant symptoms while conjunctival hyperemia (41.6%), seropurulent discharge (20.8%)and chemosis (16.6%) were the ocular findings. Most of the patients (n=17, 70.8%) noticed the lesion whilewaking up in the morning. Conclusion: The finding of the present study has shown that Nairobi eye isa common presentation during rainy season. Periocular findings with significant intraocular signs weredocumented to be presenting features among patients with periocular paederus dermatitis

Lessons to be learnt from Leishmania studies

Leishmaniasis is a disease caused by infection with the protozoan parasite Leishmania, which is responsible for three main types of disease: cutaneous leishmaniasis, visceral leishmaniasis and mucocutaneous leishmaniasis based to the site of infection for the particular species. This presents a major challenge to successful drug treatment, as a drug must not only reach antileishmanial concentrations in infected macrophages, the parasites' host cell, but also reach infected cells in locations specific to the type of disease. In this paper we discuss how studies using Leishmania have contributed to our knowledge on how drug delivery systems can be used to improve drug efficacy and delivery

The Cost of Menstrual Cycle in Young Nepali Women: A Cross-Sectional Study

Introduction: Every woman must have safe access to clean water and menstrual products. They have to spend more or less money to buy menstrual products or to alleviate menstrual symptoms. The cost of a menstrual cycle is often associated with period poverty. We found a research gap on this issue. The objective of this study was to find the cost of menstrual cycle and their perceptions on menstrual cost among young Nepali women. Methods: We conducted a cross sectional study from 15 August 2019 to 15 February 2020. The total enumeration sampling technique was used to enroll 157 female medical students. Ethical clearance was obtained from institutional review committee of Birat Medical College Teaching Hospital. Open data kit (ODK) collect software was used for data collection. The collected data was extracted in Microsoft Excel 2016 and analyzed by SPSS version 23. Results: Majority (33.8%) had menarche at 13 years and 47.8% had regular menstrual cycle. The total average cost of items used in last menstrual period was NPR 480.41 (NPR 40-1850) and lifetime cost was NPR 219066.96. Sanitary pads/ tampons/menstrual cups etc. should cost less money (85.4%) and tax currently put on it should be removed by government (91.1%) were the perceptions of the participants. Conclusion: Menstrual cycle added additional cost in women's life. Participants perceived that cost is high and it should be tax free. Nepal's constitution already passed sanitation as basic fundamental right. So, the cost of sanitary items needs to be minimized by removing the government tax

Search For Heavy Pointlike Dirac Monopoles

We have searched for central production of a pair of photons with high transverse energies in $p\bar p$ collisions at $\sqrt{s} = 1.8$ TeV using $70 pb^{-1}$ of data collected with the D\O detector at the Fermilab Tevatron in 1994--1996. If they exist, virtual heavy pointlike Dirac monopoles could rescatter pairs of nearly real photons into this final state via a box diagram. We observe no excess of events above background, and set lower 95% C.L. limits of $610, 870, or 1580 GeV/c^2$ on the mass of a spin 0, 1/2, or 1 Dirac monopole.Comment: 12 pages, 4 figure

Search for High Mass Photon Pairs in p-pbar --> gamma-gamma-jet-jet Events at sqrt(s)=1.8 TeV

A search has been carried out for events in the channel p-barp --> gamma gamma jet jet. Such a signature can characterize the production of a non-standard Higgs boson together with a W or Z boson. We refer to this non-standard Higgs, having standard model couplings to vector bosons but no coupling to fermions, as a "bosonic Higgs." With the requirement of two high transverse energy photons and two jets, the diphoton mass (m(gamma gamma)) distribution is consistent with expected background. A 90(95)% C.L. upper limit on the cross section as a function of mass is calculated, ranging from 0.60(0.80) pb for m(gamma gamma) = 65 GeV/c^2 to 0.26(0.34) pb for m(gamma gamma) = 150 GeV/c^2, corresponding to a 95% C.L. lower limit on the mass of a bosonic Higgs of 78.5 GeV/c^2.Comment: 9 pages, 3 figures. Replacement has new H->gamma gamma branching ratios and corresponding new mass limit

Development of a biosensor for urea assay based on amidase inhibition, using an ion-selective electrode

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; anion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensorresponse and showed that 30 mu L of cell-free extract containing 7.47 mg protein mL(-1), 2 mu L of glutaraldehyde (5%, v/v) and 10 mu L of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation ofmembranes in urea. The biosensor exhibited a linear response in the range of 4.0-10.0 mu M urea, a detection limit of 2.0 mu M for urea, a response timeof 20 s, a sensitivity of 58.245 % per mu M urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition

Ratio of the Isolated Photon Cross Sections at \sqrt{s} = 630 and 1800 GeV

The inclusive cross section for production of isolated photons has been measured in \pbarp collisions at $\sqrt{s} = 630$ GeV with the \D0 detector at the Fermilab Tevatron Collider. The photons span a transverse energy ($E_T$) range from 7-49 GeV and have pseudorapidity $|\eta| < 2.5$. This measurement is combined with to previous \D0 result at $\sqrt{s} = 1800$ GeV to form a ratio of the cross sections. Comparison of next-to-leading order QCD with the measured cross section at 630 GeV and ratio of cross sections show satisfactory agreement in most of the $E_T$ range.Comment: 7 pages. Published in Phys. Rev. Lett. 87, 251805, (2001

Risk-taking, delay discounting, and time perspective in adolescent gamblers: an experimental study

Previous research has demonstrated that adult pathological gamblers (compared to controls) show risk-proneness, foreshortened time horizon, and preference for immediate rewards. No study has ever examined the interplay of these factors in adolescent gambling. A total of 104 adolescents took part in the research. Two equal-number groups of adolescent non-problem and problem gamblers, defined using the South Oaks Gambling Screen-Revised for Adolescents (SOGS-RA), were administered the Balloon Analogue Risk Task (BART), the Consideration of Future Consequences (CFC-14) Scale, and the Monetary Choice Questionnaire (MCQ). Adolescent problem gamblers were found to be more risk-prone, more oriented to the present, and to discount delay rewards more steeply than adolescent non-problem gamblers. Results of logistic regression analysis revealed that BART, MCQ, and CFC scores predicted gambling severity. These novel finding provides the first evidence of an association among problematic gambling, high risk-taking proneness, steep delay discounting, and foreshortened time horizon among adolescents. It may be that excessive gambling induces shortsighted behaviors that, in turn, facilitate gambling involvement

Fermentation process for alcoholic beverage production from mahua (Madhuca indica J. F. Mel.) flowers

Mahua flowers are rich in sugar (68-72%), in addition to a number of minerals and one of the most important raw materials for alcohol fermentation. The present investigation was for the development of a non-distilled alcoholic beverage from Mahua flowers. Eighteen (18) treatment combinations consisting of two temperatures (25 and 30°C), three pH (4.0, 4.5 and 5.0) and three period of fermentation (7, 14 and 21 days) were used in the fermentation conditions. The maximum yield of ethanol (9.51 %) occurred at 25°C with pH 4.5 after 14 days of  fermentation of Mahua flower juice. The fermented non-distilled alcoholic beverage contained total sugar (8.83 mg/ml), reducing sugar (0.82 mg/ml), total soluble solids (6.37°Brix) titrable acidity (0.65 %), and volatile acidity (0.086%). Methanol was not detected at any stage of fermentation. The developed fermented alcoholic beverage had characteristic flavor and aroma of Mahua flowers with about 7 to 9% alcohol.Keywords: Madhuca indica, ethanol, reducing sugar, fermentation.African Journal of Biotechnology Vol. 12(39), pp. 5771-577