Murine erythroid 5-aminolevulinate synthase: Adenosyl-binding site Lys221 modulates substrate binding and catalysis

Abstract5-Aminolevulinate synthase (ALAS) catalyzes the initial step of mammalian heme biosynthesis, the condensation between glycine and succinyl-CoA to produce CoA, CO2, and 5-aminolevulinate. The crystal structure of Rhodobacter capsulatus ALAS indicates that the adenosyl moiety of succinyl-CoA is positioned in a mainly hydrophobic pocket, where the ribose group forms a putative hydrogen bond with Lys156. Loss-of-function mutations in the analogous lysine of human erythroid ALAS (ALAS2) cause X-linked sideroblastic anemia. To characterize the contribution of this residue toward catalysis, the equivalent lysine in murine ALAS2 was substituted with valine, eliminating the possibility of a hydrogen bond. The K221V substitution produced a 23-fold increase in the KmSCoA and a 97% decrease in kcat/KmSCoA. This reduction in the specificity constant does not stem from lower affinity toward succinyl-CoA, since the KdSCoA of K221V is lower than that of wild-type ALAS. For both enzymes, the KdSCoA value is significantly different from the KmSCoA. That K221V has stronger binding affinity for succinyl-CoA was further deduced from substrate protection studies, as K221V achieved maximal protection at lower succinyl-CoA concentration than wild-type ALAS. Moreover, it is the CoA, rather than the succinyl moiety, that facilitates binding of succinyl-CoA to wild-type ALAS, as evident from identical KdSCoA and KdCoA values. Transient kinetic analyses of the K221V-catalyzed reaction revealed that the mutation reduced the rates of quinonoid intermediate II formation and decay. Altogether, the results imply that the adenosyl-binding site Lys221 contributes to binding and orientation of succinyl-CoA for effective catalysis

Species Substitution and Country of Origin Mislabeling of Catfish Products on the U.S. Commercial Market

Catfish belong to the order Siluriformes and include both the Ictaluridae and Pangasiidae families. However, U.S. labeling laws require only species of the family Ictaluridae to be marketed as catfish. The lower production price of Pangasiidae, combined with changes in regulations over time, have resulted in high potential for species substitution and country of origin mislabeling among catfish products. The objective of this study was to conduct a market survey of catfish products sold at the U.S. retail level to examine species mislabeling and compliance with Country of Origin Labeling (COOL) regulations. A total of 80 catfish samples were collected from restaurants, grocery stores and fish markets in Orange County, CA. DNA was extracted from each sample and tested with real-time polymerase chain reaction (PCR) using the InstantID™ U.S. Catfish Assay Kit for Ictaluridae spp. (InstantLabs). Samples that tested negative for Ictaluridae were tested with real-time PCR using the InstantID Asian Catfish Assay Kit for Pangasiidae spp. DNA barcoding was used as a final test in cases where species could not be identified with either of the real-time PCR assays. Overall, 7 of the 80 catfish products were found to be substituted with Pangasiidae species for a mislabeling rate of 9%. This included 5 of the 40 restaurant samples and 2 of the 32 grocery store samples. Additionally, 59% of grocery store samples were not compliant with COOL regulations. The results of this study reveal the occurrence of catfish mislabeling on the U.S. commercial market and suggest the need for continuous monitoring of these products

Establishment and in-house validation of stem-loop rt pcr method for microrna398 expression analysis

MicroRNAs (miRNAs) belong to the class of small non-coding RNAs which have important roles throughout development as well as in plant response to diverse environmental stresses. Some of plant miRNAs are essential for regulation and maintenance of nutritive homeostasis when nutrients are in excess or shortage comparing to optimal concentration for certain plant species. Better understanding of miRNAs functions implies development of efficient technology for profiling their gene expression. We set out to establish validate the methodology for miRNA gene expression analysis in cucumber grown under suboptimal mineral nutrient regimes, including iron deficiency. Reverse transcription by "stem-loop" primers in combination with Real time PCR method is one of potential approaches for quantification of miRNA gene expression. In this paper we presented a method for "stem loop" primer design specific for miR398, as well as reaction optimization and determination of Real time PCR efficiency. Proving the accuracy of this method was imperative as "stem loop" RT which consider separate transcription of target and endogenous control. The method was verified by comparison of the obtained data with results of miR398 expression achieved using a commercial kit based on simultaneous conversion of all RNAs in cDNAs

Establishment and in-house validation of stem-loop rt pcr method for microrna398 expression analysis

MicroRNAs (miRNAs) belong to the class of small non-coding RNAs which have important roles throughout development as well as in plant response to diverse environmental stresses. Some of plant miRNAs are essential for regulation and maintenance of nutritive homeostasis when nutrients are in excess or shortage comparing to optimal concentration for certain plant species. Better understanding of miRNAs functions implies development of efficient technology for profiling their gene expression. We set out to establish validate the methodology for miRNA gene expression analysis in cucumber grown under suboptimal mineral nutrient regimes, including iron deficiency. Reverse transcription by "stem-loop" primers in combination with Real time PCR method is one of potential approaches for quantification of miRNA gene expression. In this paper we presented a method for "stem loop" primer design specific for miR398, as well as reaction optimization and determination of Real time PCR efficiency. Proving the accuracy of this method was imperative as "stem loop" RT which consider separate transcription of target and endogenous control. The method was verified by comparison of the obtained data with results of miR398 expression achieved using a commercial kit based on simultaneous conversion of all RNAs in cDNAs

Penjabaran Asas-Asas Pembaharuan Agraria Berdasarkan Tap MPR No. IX/MPR/2001 dalam Perundang-undangan di Bidang Pertanahan

This research discusses whether Agrarian Reform principles have been synchronous with the Indonesian land laws and Basic Agrarian Law and how those principles are implemented within government regulations on land law. Results show that, instead of manifesting, numerous government regulations are contradicting the principles therefore should be revoked or amended. Penelitian ini membahas apakah asasasas Pembaruan Agraria telah sejalan dengan hukum pertanahan Indonesia dan UUPA serta bagaimana asas-asas tersebut diterapkan dalam peraturan-peraturan pemerintah di bidang pertanahan. Hasil penelitian menunjukkan bahwa, alih-alih mengejawantahkan, banyak PP yang bertentangan dengan asas Pembaruan Agraria sehingga harus dicabut atau diamandemen

Hypermethylation of p15 Gene in Diffuse - Large B-Cell Lymphoma: Association with Less Aggressiveness of the Disease

In this study, methylation-specific polymerase chain reaction was used to investigate the potential prognostic significance of the methylation status of p15, p16, MGMT, and DAPK genes in 51 specimens of diffuse large B-cell lymphoma (DLBCL). Hypermethylation of p15 gene was significantly more prevalent in patients without relapse (p = 0.001) and there was a trend toward more frequent presence of p15 methylation in patients without death outcome within 5-year follow-up period (p = 0.086) Also, there was a trend toward accumulation of p15 methylation with favorable clinicopathological parameters including: age 60 years (p = 0.091), normal levels of lactate dehydrogenase (p = 0.090), Eastern Cooperative Oncology Group performance status LT 2 (p = 0.095), and low/intermediate low International Prognostic Index (p = 0.076). In the female group and group of the patients without bulky tumor mass, treated with chemotherapeutic regimens including rituximab, methylation of p15 was significantly related to longer overall survival (p = 0.036 and 0.027, respectively). Our results suggest that promoter methylation of p15 gene could have prognostic value in DLBCL patients treated with rituximab when used in combination with gender and tumor size

AN OVERVIEW OF THE INTEGRATED CRATE INTERROGATION SYSTEM (ICIS) FOR USE AT THE SAVANNAH RIVER SITE

ABSTRACT The Integrated Crate Interrogation System (ICIS) was developed for use at the Savannah River Site to assay transuranic waste in large containers. The system comprises a Box Segmented Gamma Scanner (BSGS) providing high resolution gamma spectroscopy, and a Box Neutron Assay System (BNAS) providing passive neutron counting capability. The multi-modality approach is taken where the assay results from the gamma and neutron systems are combined to complement each other in satisfying Waste Isolation Pilot Plant (WIPP) criteria. This paper gives an overview of the system that has been built, factory calibrated, and delivered to the site. The BSGS is similar to a standard Canberra Segmented Gamma Box Counter, but with the addition of a transmission option for ascertaining density and rudimentary fill-height information. This supplements the Multi-Curve approach based on efficiency in energy and density. The BSGS has a moving trolley which travels on rails through a passive emission counting station using large BEGe detectors, and a transmission counting station using NaI detectors in conjunction with high-and low-beam transmission stages. The assay result provides information on the Pu, Am and/or U isotopic ratios using standard isotopic analysis codes, quantitative measurement of Pu and U for low and medium density matrices, and direct measurement of other gamma emitters in the waste that are not identified in the isotopic measurement. It also provides basic positional information to improve the accuracy of both the gamma and the neutron measurement

Клик-химия и методы молекулярного моделирования в компьютерном дизайне и идентификации потенциальных ингибиторов ВИЧ-1

An integrated approach including the click chemistry methodology, molecular docking, quantum mechanics, and molecular dynamics was used to perform the computer-aided design of potential HIV-1 inhibitors able to block the membrane- proximal external region (MPER) of HIV-1 gp41 that plays an important role in the fusion of the viral and host cell membranes. Evaluation of the binding efficiency of the designed compounds to the HIV-1 MPER peptide was performed using the methods of molecular modeling, resulting in nine chemical compounds that exhibit the high-affinity binding to this functionally important site of the trimeric “spike” of the viral envelope. The data obtained indicate that the identified compounds are promising for the development of novel antiviral drugs, HIV fusion inhibitors blocking the early stages of HIV infection.С помощью комплексного подхода, включающего методологию клик-химии, молекулярный докинг, квантовую механику и молекулярную динамику, осуществлен компьютерный дизайн потенциальных ингибиторов ВИЧ-1, способных блокировать мембрано-проксимальную внешнюю область (MPER, Membrane-Proximal External Region) белка gp41, играющую важную роль в процессе слияния мембран вируса и клетки хозяина. Методами молекулярного моделирования выполнена оценка эффективности связывания сконструированных соединений с пептидом MPER ВИЧ-1, в результате которой идентифицированы девять химических соединений, характеризующихся высокой аффинностью связывания с этим функционально важным участком оболочки вируса. Полученные данные свидетельствуют о перспективности использования этих соединений в работах по созданию новых противовирусных препаратов – ингибиторов слияния ВИЧ, блокирующих ранние стадии развития ВИЧ инфекции

A community effort in SARS-CoV-2 drug discovery.

peer reviewedThe COVID-19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small-molecule drugs that are widely available, including in low- and middle-income countries, is an ongoing challenge. In this work, we report the results of an open science community effort, the "Billion molecules against Covid-19 challenge", to identify small-molecule inhibitors against SARS-CoV-2 or relevant human receptors. Participating teams used a wide variety of computational methods to screen a minimum of 1 billion virtual molecules against 6 protein targets. Overall, 31 teams participated, and they suggested a total of 639,024 molecules, which were subsequently ranked to find 'consensus compounds'. The organizing team coordinated with various contract research organizations (CROs) and collaborating institutions to synthesize and test 878 compounds for biological activity against proteases (Nsp5, Nsp3, TMPRSS2), nucleocapsid N, RdRP (only the Nsp12 domain), and (alpha) spike protein S. Overall, 27 compounds with weak inhibition/binding were experimentally identified by binding-, cleavage-, and/or viral suppression assays and are presented here. Open science approaches such as the one presented here contribute to the knowledge base of future drug discovery efforts in finding better SARS-CoV-2 treatments.R-AGR-3826 - COVID19-14715687-CovScreen (01/06/2020 - 31/01/2021) - GLAAB Enric