NONLINEAR MIXED MODELS TO EVALUATE EFFECTS OF ENVIRONMENT CONDITIONS, HAIR COAT, AND ANCHOR LENGTH ON BODY TEMPERATURE DURING AFTERNOON MILKING OF HOLSTEIN COWS IN HAWAII

A hot thermal environment is one factor that causes loss of activity, productivity and even death in cows. One reason may be the effect of hair coat (black or white) on body temperature during different activities and environment conditions. In this study, we attempt to characterize the relationship between body temperature over time using activity, physiological and environmental effects with a nonlinear regression model. The fixed and mixed versions of models are examined. We also examine the effect of the measuring device, anchor length (long or short). Environmental effects, such as, air temperature, thermal heat index (THI), relative humidity, solar radiation, and wind speed, For PM Milking activity, differences in hair coat and anchor length tested as treatment effects. We present a detailed approach focusing on the rate of change in animal body temperature using the nonlinear regression model and investigate how the dynamics between the random-effects changes

Structure–activity relationship of ipglycermide binding to phosphoglycerate mutases

Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex

Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM-1

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data

A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases

Carbapenem-resistant Enterobacteriaceae threaten human health, since carbapenems are last resort drugs for infections by such organisms. Metallo-β-lactamases (MβLs) are the main mechanism of resistance against carbapenems. Clinically approved inhibitors of MBLs are currently unavailable as design has been limited by the incomplete knowledge of their mechanism. Here, we report a biochemical and biophysical study of carbapenem hydrolysis by the B1 enzymes NDM-1 and BcII in the bi-Zn(II) form, the mono-Zn(II) B2 Sfh-I and the mono-Zn(II) B3 GOB-18. These MβLs hydrolyse carbapenems via a similar mechanism, with accumulation of the same anionic intermediates. We characterize the Michaelis complex formed by mono-Zn(II) enzymes, and we identify all intermediate species, enabling us to propose a chemical mechanism for mono and binuclear MβLs. This common mechanism open avenues for rationally designed inhibitors of all MβLs, notwithstanding the profound differences between these enzymes' active site structure, β-lactam specificity and metal content.Fil: Lisa, María Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Instituto Pasteur de Montevideo; UruguayFil: Palacios, Antonela Rocio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Aitha, Mahesh. Miami University; Estados UnidosFil: Gonzalez, Mariano Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Moreno, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Crowder, Michael W.. Miami University; Estados UnidosFil: Bonomo, Robert A.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Department of Veterans Affairs Medical Center; Estados UnidosFil: Spencer, James. University Walk; Reino Unido. University of Bristol; Reino UnidoFil: Tierney, David L.. Miami University; Estados UnidosFil: Llarrull, Leticia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; ArgentinaFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; Argentina. Case Western Reserve University; Estados Unido

Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as ‘transition state analogue’ inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs

Concrete mix design using Java Eclipse IDE

Concrete mix design is the process of selecting the proper components and figuring out the proper ratios to achieve the desired workability, strength, and durability. As a result, an attempt has been made to create a Java program for concrete mix design by IS 10262:2019 and IS 456:2000. Computer software is created in this way to proportion input variables to a set of output variables, which avoids time-consuming computations in real time. The produced program was carefully checked. The results were compared to make sure they were accurate after being put through a series of manually check issues. The concrete mix design produced by the Java program was exact, time-saving, and computationally efficient

Mechanistic and Spectroscopic Studies of Metallo-β-lactamase NDM-1

In an effort to biochemically characterize metallo-β-lactamase NDM-1, we cloned, overexpressed, purified, and characterized several maltose binding protein (MBP)–NDM-1 fusion proteins with different N-termini (full-length, Δ6, Δ21, and Δ36). All MBP–NDM-1 fusion proteins were soluble; however, only one, MBP–NDM-1Δ36, exhibited high activity and bound 2 equiv of Zn­(II). Thrombin cleavage of this fusion protein resulted in the truncated NDM-1Δ36 variant, which exhibited a <i>k</i>_cat of 16 s^–1 and a <i>K</i>_m of 1.1 μM when using nitrocefin as a substrate, bound 2 equiv of Zn­(II), and was monomeric in solution. Extended X-ray absorption fine structure studies of the NDM-1Δ36 variant indicate the average metal binding site for Zn­(II) in this variant consists of four N/O donors (two of which are histidines) and 0.5 sulfur donor per zinc, with a Zn–Zn distance of 3.38 Å. This metal binding site is very similar to those of other metallo-β-lactamases that belong to the B1 subclass. Pre-steady-state kinetic studies using nitrocefin and chromacef and the NDM-1Δ36 variant indicate that the enzyme utilizes a kinetic mechanism similar to that used by metallo-β-lactamases L1 and CcrA, in which a reactive nitrogen anion is stabilized and its protonation is rate-limiting. While they are very different in terms of amino acid sequence, these studies demonstrate that NDM-1 is structurally and mechanistically very similar to metallo-β-lactamase CcrA

Probing the interaction of Aspergillomarasmine A (AMA) with metallo-β-lactamases NDM-1, VIM-2, and IMP-7

Metallo-β-lactamases (MBLs) are a growing threat to the continued efficacy of β-lactam antibiotics. Recently, aspergillomarasmine A (AMA) was identified as an MBL inhibitor, but the mode of inhibition was not fully characterized. Equilibrium dialysis and metal analysis studies revealed that 2 equiv of AMA effectively removes 1 equiv of Zn(II) from MBLs NDM-1, VIM-2, and IMP-7 when the MBL is at micromolar concentrations. Conversely, 1H NMR studies revealed that 2 equiv of AMA remove 2 equiv of Co(II) from Co(II)-substituted NDM-1, VIM-2, and IMP-7 when the MBL/AMA are at millimolar concentrations. Our findings reveal that AMA inhibits the MBLs by removal of the active site metal ions required for β-lactam hydrolysis among the most clinically significant MBLs.Microbial Biotechnolog