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The aim of the study was to explore the factors associated with the recall of basic medical physiology knowledge among medical interns and to determine the level of retained basic science knowledge. Two hundred and four interns, 114 women and 90 men, working in two major tertiary medical care centers, King Fahad Medical City (KFMC; 29 students) and King Khalid University Hospital (KKUH; 117 students), in Riyadh city, participated in the study. An anonymous knowledge test with 10 validated multiple-choice questions was developed specifically for this purpose. One hundred and forty-six interns (117 working at KKUH and 29 at KFMC) had graduated from medical schools adopting a conventional instructional system, whereas 58 (3 from KKUH and 55 from KFMC had graduated from schools adopting an integrated system (hybrid problem-based learning). Fifty-two students (26%) gained a score =60%, whereas 152 students (74%) obtained <60% of the score. Higher scores were associated with younger age (P < 0.01), traditional curriculum (P < 0.001), interns from KKUH (P < 0.001), and candidates for postgraduate studies (P < 0.02). There was no significant association between recall of physiology knowledge and all other variables studied, including sex. Multivariate analyses show that age and traditional curriculum are the only significant predictors of knowledge retention. Almost three-fourths of the interns scored <60%, and higher scores were significantly associated with younger interns, traditional curriculum, working in KKUH, and interns preparing for graduate studies. However, the difference between the two curricula disappears when the influence of hospital training is considered.

Glucose-6-phosphate-dehydrogenase deficiency is the most common enzymopathy. Current therapies for G6PD deficiency are unable to treat a broad range of pathogenic variants. In this study, we assess the structural dynamics of six G6PD variants using molecular dynamics simulation to correlate their genotypic and phenotypic attributes. G6PD multimerisation is highly influenced by its ligands G6P and NADP, where the former disrupts dimer formation, and the latter facilitates tetramerisation. Results of our simulation demonstrate that the WT and a relatively stable variant (G131V), were found to have greater NADP binding occupancy and hydrogen bonds between βN sheet of each monomeric subunit, thereby increasing the stability of the dimer interface. G6PD protein structures with high structural integrity at the dimer interface were found to be compact, characterised by low radius of gyration values, and increased surface area or high solvent-accessible surface area at the tetramer salt bridge residues. Using mutational clustering methods, a critical G6PD region at the βK–βL loop was identified and may serve as a potential target for treatment. We further extend this study to identify chemical compounds that induce modulatory effects on the protein using computer aided drug discovery which warrant further studies and future testing. [ABSTRACT FROM AUTHOR]

There are limited data on inflammatory cytokines and chemokines; the humoral immune response; and main clinical laboratory parameters as indicators for disease severity and mortality in patients with critical and mild COVID-19 without comorbidities or immune-mediated diseases in Saudi Arabia. We determined the expression levels of major proinflammatory cytokines and chemokines; C-reactive protein (CRP); procalcitonin; SARS-CoV-2 IgM antibody and twenty-two clinical laboratory parameters and assessed their usefulness as indicators of disease severity and in-hospital death. Our results showed a significant increase in the expression levels of SARS-CoV-2 IgM antibody; IL1-β; IL-6; IL-8; TNF-α and CRP in critical COVID-19 patients; neutrophil count; urea; creatinine and troponin were also increased. The elevation of these biomarkers was significantly associated and positively correlated with in-hospital death in critical COVID-19 patients. Our results suggest that the levels of IL1-β; IL-6; IL-8; TNF-α; and CRP; neutrophil count; urea; creatinine; and troponin could be used to predict disease severity in COVID-19 patients without comorbidities or immune-mediated diseases. These inflammatory mediators could be used as predictive early biomarkers of COVID-19 disease deterioration; shock and death among COVID-19 patients without comorbidities or immune-mediated diseases. [ABSTRACT FROM AUTHOR]

Glutamate dehydrogenase (EC 1.4.1.2-4) from Peptoniphilus asaccharolyticus has been expressed as a selenomethionine-derivatized recombinant protein and diffraction-quality crystals have been grown that are suitable for structure determination. Preliminary structural analyses indicate that the protein assembles as a homohexameric enzyme complex in solution, similar to other bacterial and mammalian enzymes to which its sequence identity varies between 25 and 40%. The structure will provide insight into its preference for the cofactor NADH (over NADPH) by comparisons with the known structures of mammalian and bacterial enzymes. [ABSTRACT FROM AUTHOR]

Abstract: Clostridial glutamate dehydrogenase mutants with the 5 Trp residues in turn replaced by Phe showed the importance of Trp 64 and 449 in cooperativity with glutamate at pH 9. These mutants are examined here for their behaviour with NAD+ at pH 7.0 and 9.0. The wild-type enzyme displays negative NAD+ cooperativity at both pH values. At pH 7.0 W243F gives Michaelis–Menten kinetics, and the same behaviour is shown by W243F and also W310F at pH 9.0, but not by W64F or W449F. W243 and W310 are apparently much more important than W64 and W449 for the coenzyme negative cooperativity, implying that different conformational transitions are involved in cooperativity with the coenzyme and with glutamate. [Copyright &y& Elsevier]

Abstract: Clostridial glutamate dehydrogenase mutants with the 5 Trp residues in turn replaced by Phe showed the importance of Trp 64 and 449 in cooperativity with glutamate at pH 9. These mutants are examined here for their behaviour with NAD+ at pH 7.0 and 9.0. The wild-type enzyme displays negative NAD+ cooperativity at both pH values. At pH 7.0 W243F gives Michaelis–Menten kinetics, and the same behaviour is shown by W243F and also W310F at pH 9.0, but not by W64F or W449F. W243 and W310 are apparently much more important than W64 and W449 for the coenzyme negative cooperativity, implying that different conformational transitions are involved in cooperativity with the coenzyme and with glutamate. [Copyright &y& Elsevier]

Abstract: Glutamate dehydrogenases (EC 1.4.1.2-4) catalyse the oxidative deamination of l-glutamate to α-ketoglutarate using NAD(P) as a cofactor. The bacterial enzymes are hexamers and each polypeptide consists of an N-terminal substrate-binding (Domain I) followed by a C-terminal cofactor-binding segment (Domain II). The reaction takes place at the junction of the two domains, which move as rigid bodies and are presumed to narrow the cleft during catalysis. Distinct signature sequences in the nucleotide-binding domain have been linked to NAD+ vs. NADP+ specificity, but they are not unambiguous predictors of cofactor preferences. Here, we have determined the crystal structure of NAD+-specific Peptoniphilus asaccharolyticus glutamate dehydrogenase in the apo state. The poor quality of native crystals was resolved by derivatization with selenomethionine, and the structure was solved by single-wavelength anomalous diffraction methods. The structure reveals an open catalytic cleft in the absence of substrate and cofactor. Modeling of NAD+ in Domain II suggests that a hydrophobic pocket and polar residues contribute to nucleotide specificity. Mutagenesis and isothermal titration calorimetry studies of a critical glutamate at the P7 position of the core fingerprint confirms its role in NAD+ binding. Finally, the cofactor binding site is compared with bacterial and mammalian enzymes to understand how the amino acid sequences and three-dimensional structures may distinguish between NAD+ vs. NADP+ recognition. [Copyright &y& Elsevier]

The hexameric glutamate dehydrogenase of Clostridium symbiosum has previously been shown to undergo a pH-dependent inactivating conformational change that perturbs the environment of one or more Trp residues and is reversed by glutamate in a highly cooperative fashion with a Hill coefficient of almost 6. Five single mutants have now been made in which each of the Trp residues in turn has been replaced by Phe. All five were successfully over-produced as soluble proteins and purified. Far-UV CD showed that none of the mutations significantly affected secondary structure. All five proteins were active, ranging from 13 U.mg(-1) (W64F) to 20.8 U.mg(-1) (W393F), compared to 20 U.mg(-1) for wild-type, and the kinetic parameters at pH 7 were little changed, except for a five- to six-fold increase in Km for glutamate in W243F. Thermostability was also relatively little changed, although W310F and W393F were somewhat more stable and W64F less stable than the unmutated enzyme. All still showed the characteristic reversible, time-dependent high-pH inactivation. Near-UV CD spectra, reflecting the environment of aromatic residues, were recorded at both pH 7 and 8.8, and four of the mutants showed essentially the same perturbation in the 280 nm region as the wild-type enzyme. W64F, however, showed essentially no change. W64 is thus clearly a passive reporter of the pH-dependent conformational change, and not actually required for the transition to occur. The CD comparisons also suggest that the aromatic CD spectrum is contributed almost entirely by W64 and W449. Consistent with the pH-dependent change, all five mutant proteins also showed a positively cooperative response to glutamate at pH 9, reversing the inactivation. However, the Hill coefficient decreased from > 5 for wild-type to approximately 3 for the active site cleft mutation W243F and to approximately 2 for the interfacial mutants W64F and W449F in which the trimer-trimer interaction may be directly interrupted. W64 of each subunit is in contact with W449 in its dimer partner at the trimer-trimer interface. It seems that, although neither of these two residues is required for the pH-dependent change, together, they are essential in mediating the total cooperativity of the hexameric enzyme's response to glutamate and are presumably directly involved in transmitting conformational information between the two trimers.