Association between inflammatory cytokines/chemokines, clinical laboratory parameters, disease severity and in-hospital mortality in critical and mild COVID-19 patients without comorbidities or immune-mediated diseases.

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-ß; 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-ß; 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.

Development and Validation of ScriptTaq COVID PCR: An In-House Multiplex rRT-PCR for Low-Cost Detection.

The COVID-19 pandemic necessitated an extensive testing for active SARS-CoV-2 infection. However, securing affordable diagnostic tests is a struggle for low-resource settings. We report herein the development and validation of an in-house multiplex real-time RT-PCR diagnostic test for the detection of active COVID-19 infection (ScriptTaq COVID PCR). Furthermore, we describe two methods for RNA extraction using either an in-house silica column or silica-coated magnetic beads to replace commercial RNA extraction kits. Different buffer formulations for silica column and silica-coated magnetic beads were tested and used for RNA isolation. Taq polymerase enzyme and thermostable reverse transcriptase enzyme were purified from bacterial clones. Primers/probes sequences published by the WHO and CDC were used for the qualitative detection of the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes, respectively. ScriptTaq COVID PCR assay was able to detect up to 100 copies per reaction of the viral RdRP and N genes. The test demonstrated an overall agreement of 95.4%, a positive percent agreement (PPA) of 90.2%, and a negative percent agreement (NPA) of 100.0% when compared with two commercially available kits. ScriptTaq COVID PCR diagnostic test is a specific, sensitive, and low-cost alternative for low-resource settings.

Telomere length and telomere repeat-binding protein in children with sickle cell disease.

BACKGROUND: This study aimed to assess the telomere length and plasma telomere repeat-binding factor 2 (TRF2) levels in addition to other inflammatory markers in children with sickle cell disease (SCD). METHODS: We enrolled 106 children (90 SCD and 26 controls) aged 1-15 years from the Hematology unit of King Fahad Medical City (KFMC), Saudi Arabia. Genomic DNA extracted from blood and leukocyte TL was determined using quantitative reverse transcription PCR, whereas TRF2, C-reactive protein, interleukin-6, and DNA oxidative damage were determined by using respective commercially available assays. RESULTS: Leukocyte TL was inversely correlated with age in the SCD patients (r = -0.24, P = 0.02) and the controls (r = -0.68, P < 0.0001). In addition, SCD patients had significantly shorter TL (7.74 ± 0.81 kb) (P = 0.003) than controls (8.28 ± 0.73 kb). In contrast, no significant difference in TL among the SCD genotypes (HbSS and HbSß0) has been observed. A modest, positive correlation was seen between TL and reticulocyte % (r = 0.21; P = 0.06). There were no significant differences in the TL and TRF2 concentrations between subjects with HbSS and HbSß0 genotypes. CONCLUSIONS: Short leukocyte TL was significantly associated with SCD. An inverse association was observed between TL and hemoglobin. Hydroxyurea treatment revealed no impact on TL. IMPACT: This study explored the TL and plasma TRF2 in Saudi children with SCD. This is the first documentation that SCD children have shorter TL than their healthy counterparts, and no association between TL and TRF2 has been observed. Hydroxyurea treatment showed no impact on TL in children with SCD. This study is the first of its kind in children with SCD. It will pave the way for another study with a larger sample size in a diverse population to scrutinize these findings better.

Association of single-nucleotide polymorphisms in tumour necrosis factor and human leukocyte antigens genes with type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterized by progressive destruction of insulin-producing pancreatic beta cells. This multifactorial disease has a strong genetic component associated with the human leukocyte antigens (HLA) and non-HLA regions. In this study, we compared frequencies of HLA-DRB1 alleles and single-nucleotide polymorphisms (SNPs) associated the genes coding for: toll-like receptors (TLRs), tumour necrosis factor (TNF), interleukin-1 (IL-1), interleukin-1 receptor type 1 (IL-1R1), interleukin-1 receptor antagonist (IL-1RN), interleukin-2 (IL-2) and interleukin-12B (IL-12B), between T1D patients and healthy controls. The aim was to identify frequency differences and linkage between these genetic markers in T1D patients and healthy controls. Twelve SNPs were investigated as follows: rs16944 (IL-1B), rs1143634 (IL-1B), rs1800587 (IL-1A), rs2069762 (IL-2), rs3212227 (IL-12B), rs2234650 (IL-1R1), rs315952 (IL-1RN), rs3804099 (TLR2), rs4986790 (TLR4), rs4986791 (TLR4), rs1800629 (TNF) and rs361525 (TNF). TaqMan genotype assay method was used for SNPs genotyping. HLA-DRB1* genes were typed by Sequence Specific Oligonucleotide Probe (SSOP). SPSS and SNPStats programs were used for the statistical analysis. Significant differences between T1D and control groups were found for the dominant model of rs361525 and rs1800629A:rs361525G genotypes for TNF. Increased frequencies of DRB1*03 and DRB1*04 and decreased frequencies of DRB1*07, DRB1*11 and DRB1*13 and DRB1*15 were observed in T1D patients compared with controls. However, the genotype, DRB1*07 with rs1800629A/G was associated with T1D. We have confirmed that DRB1*03 and DRB1*04 are associated with increased risk and DRB1*07, DRB1*11 and DRB1*13 and DRB1*15 with decreased risk of T1D. Also, the dominant model of rs361525A, and the rs1800629G:361525A genotype were associated with increased risk. The simultaneous presence of DRB1*07 and rs1800629A/G genotypes in 23 out of 27 DRB1*07 positive T1D patients implied that islet cell peptide processing may have been biased towards autoimmunity by upregulation of TNF associated intronic SNPs.

Recall of physiology knowledge among medical interns: an exploratory study in Riyadh, Saudi Arabia.

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.

Computational analysis of dimer G6PD structure to elucidate pathogenicity of G6PD variants.

An inherent genetic enzyme disorder in humans, known as glucose-6-phosphate dehydrogenase (G6PD) deficiency, arises due to specific mutations. While the prevailing approach for investigating G6PD variants involves biochemical analysis, the intricate structural details remain limited, impeding a comprehensive understanding of how different G6PD variants of varying classes impact their functionality. This study 22 examined the dynamic properties of G6PD wild types and six G6PD variants from 23 different classes using molecular dynamic simulation (MDS). The wild-type and variant 24 G6PD structures unveil high fluctuations within the amino acid range of 274-515, the structural NADP+ binding site, pivotal for enzyme dimerization. Specifically, two variants, G6PDZacatecas (R257L) and G6PDDurham (K238R), demonstrate compromised structural stability at the dimer interface, attributable to the disruption of a salt bridge involving Glu 206 and Lys 407, along with the disturbance of hydrogen bonds formed by Asp 421 at the ßN-ßN sheets. Consequently, this impairment cascades to affect the binding affinity of crucial interactions, such as Lys 171-Glucose-6-Phosphate (G6P) and Lys 171-catalytic NADP+, leading to diminished enzyme activity. This study underscores the utility of computational in silico techniques in predicting the structural alterations and flexibility of G6PD variants. This insight holds promise for guiding future endeavors in drug development targeted at mitigating the impacts of G6PD deficiency.

Development and validation of an instrument to assess the knowledge and perceptions of predatory journals.

Objective: The main aim of this study is to develop a valid and reliable instrument to assess levels of knowledge and perceptions of predatory journals. Methods: The current study employed successive methods framework including (1) item generation through a literature review and theoretical framework development, (2) validity testing in terms of face, content, and construct validity for perceptions construct as well as item analysis for knowledge scale, and (3) reliability testing in terms of Cronbach's alpha, Kuder-Richardson (KR-20), item-to-total correlations, corrected item-to-total correlations, Cronbach's alpha if item deleted, and test-retest reliability. A total of 304 participants were recruited from King Fahad Medical City (KFMC) in Riyadh, Saudi Arabia to evaluate its construct validity and reliability. This was established using exploratory factor analysis (EFA) with principal axis factoring (PFA) and varimax rotation as well as confirmatory factor analysis (CFA) for perception construct. Results: An instrument was developed from this study called the "Predatory Journals KP Assessment Questionnaire". The results of EFA and CFA confirmed the construct validity of the perception construct. Item analysis confirmed the construct validity of the knowledge scale. The internal consistency and test-retest reliability were achieved for the knowledge scale items, consisting of 13 items. The results of EFA confirmed the measured constructs of perceptions toward predatory journals. The results of EFA and CFA for perception construct resulted in only one factor with 9 items. Conclusion: This study has successfully developed a valid and reliable questionnaire to measure knowledge and perceptions of predatory journals among researchers in the clinical and health disciplines. This instrument serves as a valuable guide for future studies that aim to assess researcher's knowledge and perceptions about predatory journals and examine the differences in these measured constructs according to their demographic and professional characteristics.

A computational study of structural analysis of Class I human glucose-6-phosphate dehydrogenase (G6PD) variants: Elaborating the correlation to chronic non-spherocytic hemolytic anemia (CNSHA).

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect that affects more than 500 million people worldwide. Individuals affected with G6PD deficiency may occasionally suffer mild-to-severe chronic hemolytic anemia. Chronic non-spherocytic hemolytic anemia (CNSHA) is a potential result of the Class I G6PD variants. This comparative computational study attempted to correct the defect in variants structure by docking the AG1 molecule to selected Class I G6PD variants [G6PDNashville (Arg393His), G6PDAlhambra (Val394Leu), and G6PDDurham (Lys238Arg)] at the dimer interface and structural NADP+ binding site. It was followed by an analysis of the enzyme conformations before and after binding to the AG1 molecule using the molecular dynamics simulation (MDS) approach, while the severity of CNSHA was determined via root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), hydrogen bonds, salt bridges, radius of gyration (Rg), solvent accessible surface area analysis (SASA), and principal component analysis (PCA). The results revealed that G6PDNashville (Arg393His) and G6PDDurham (Lys238Arg) had lost the direct contact with structural NADP+ and salt bridges at Glu419 - Arg427 and Glu206 - Lys407 were disrupted in all selected variants. Furthermore, the AG1 molecule re-stabilized the enzyme structure by restoring the missing interactions. Bioinformatics approaches were also used to conduct a detailed structural analysis of the G6PD enzyme at a molecular level to understand the implications of these variants toward enzyme function. Our findings suggest that despite the lack of treatment for G6PDD to date, AG1 remains a novel molecule that promotes activation in a variety of G6PD variants.

Crystal structure of NAD+-dependent Peptoniphilus asaccharolyticus glutamate dehydrogenase reveals determinants of cofactor specificity.

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.

Single nucleotide polymorphism rs 2070874 at Interleukin-4 is associated with increased risk of type 1 diabetes mellitus independently of human leukocyte antigens.

INTRODUCTION: Type 1 diabetes mellitus (T1DM) is characterized by autoimmune destruction of insulin-producing pancreatic beta (ß-) cells. Previous studies suggested an imbalance between and pro- and anti-inflammatory cytokines exacerbates T1DM development. OBJECTIVES: We aimed to test the hypothesis that patients with T1DM carry a higher frequency of regulatory genes associated with low levels of the anti-inflammatory cytokines interleukin-4 (IL-4), its receptor (IL-4R), and interleukin-10 (IL-10). METHODS: Accordingly, we compared frequencies of five different single nucleotide polymorphisms (SNPs) in T1DM patients and healthy controls who had been typed for HLA-DRB1, HLA-DQA1, and HLA-DQB1 genes. RESULTS: The frequencies of rs2070874 (IL-4) alleles C and T differed between T1DM patients and controls (cp = 0.0065), as did their codominant (cp = 0.026) and recessive (cp = 0.015) models. Increased frequencies were observed in T1DM patients for HLA alleles: DRB1*03 (pc < 0.0013), DRB1*04 (cp = 0.0169), DQA1*03 (cp = 0.0222), DQA1*05 (cp < 0.0006), DQB1*02 (cp = 0.0005), and DQB1*06 (cp < 0.0005). And lower frequencies were observed for: DRB1*07 (cp = 0.0078), DRB1*11 (cp = 0.0013), DRB1*13 (cp < 0.0364), DRB1*15 (cp < 0.0013), DQA1*01 (cp < 0.0006), and DQA1*02 (cp = 0.0348). Certain DRB1: DQA1: DQB1 haplotypes showed greater frequencies, including, 03:05:02 (p < 0.0001) and 04:03:03 (p = 0.0017), whereas others showed lower frequencies, including, 07:02:02 (p = 0.0032), 11:05:03 (p = 0.0007), and 15:01:06 (p = 0.0002). Stratification for the above HLA haplotypes with rs2070874 C/C exhibited no significant differences between T1DM patients overall and controls. However, when stratified for the vulnerable HLA haplotype (03:05:02/04:03:03), young patients in whom T1DM began at ≤13 years had a higher frequency of the SNP (rs2070874 C/C); a gene associated with low IL-4 production (p < 0.024). CONCLUSION: This study suggests that possession of the rs2070874 C/C genotype, which is associated with low production of IL-4, increases the risk of T1DM in young individuals carrying vulnerable HLA alleles/haplotypes.

Contract Cheating and Ghostwriting among University Students in Health Specialties.

Contract cheating and ghostwriting are forms of misconduct that are unethical and a serious academic issue, especially among healthcare professionals, as they directly impact patient health. To date, research on this area in the Middle East has been limited. Therefore, we used a validated self-administered questionnaire to investigate the awareness, perceptions, and reasons for these behaviors among 682 students in health specialties at five universities in Riyadh, Saudi Arabia. The majority of the students (60.1%) were unaware of the terms "contract cheating" and "ghostwriting," and 69.5% had not received any prior training on integrity. However, having prior training had a positive effect on awareness levels, and respondents attending private universities were significantly more aware than those attending public universities. The factors that contributed to contract cheating behavior included poor time management, English language difficulties, and a lack of writing skills. These findings emphasize the need for integrity training at the national level to raise awareness.

Preliminary Study of Structural Changes of Glucose-6-Phosphate Dehydrogenase Deficiency Variants.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency disorder affecting over 400 million individuals worldwide. G6PD protects red blood cells (RBC) from the harmful effects of oxidative substances. There are more than 400 G6PD mutations, of which 186 variants have shown to be linked to G6PD deficiency by decreasing the activity or stability of the enzyme. Different variants manifest different clinical phenotypes which complicate comprehending the mechanism of the disease. In order to carry out computational approaches to elucidate the structural changes of different G6PD variants that are common to the Asian population, a complete G6PD monomer-ligand complex was constructed using AutoDock 4.2, and the molecular dynamics simulation package GROMACS 4.6.7 was used to study the protein dynamics. The G410D and V291M variants were chosen to represent classes I and II respectively and were created by in silico site-directed mutagenesis. Results from the Root mean square deviation (RMSD), Root mean square fluctuation (RMSF) and Radius of gyration (Rg) analyses provided insights on the structure - function relationship for the variants. G410D indicated impaired dimerization and structural NADP binding while the impaired catalytic activity for V291M was indicated by a conformational change at its mutation site.

An Eluate of the Medicinal Plant Garcinia kola Displays Strong Antidiabetic and Neuroprotective Properties in Streptozotocin-Induced Diabetic Mice.

Materials and Methods: G. kola methanolic extract was fractionated using increasingly polar solvents. Fractions were administered to streptozotocin (STZ)-induced diabetic mice until marked motor signs developed in diabetic controls. Fine motor skills indicators were measured in the horizontal grid test (HGT) to confirm the prevention of motor disorders in treated animals. Column chromatography was used to separate the most active fraction, and subfractions were tested in turn in the HGT. Gas chromatography-mass spectrometry (GC-MS) technique was used to assess the components of the most active subfraction. Results: Treatment with ethyl acetate fraction and its fifth eluate (F5) preserved fine motor skills and improved the body weight and blood glucose level. At dose 1.71 mg/kg, F5 kept most parameters comparable to the nondiabetic vehicle group values. GC-MS chromatographic analysis of F5 revealed 36 compounds, the most abundantly expressed (41.8%) being the ß-lactam molecules N-ethyl-2-carbethoxyazetidine (17.8%), N,N-dimethylethanolamine (15%), and isoniacinamide (9%). Conclusions: Our results suggest that subfraction F5 of G. kola extract prevented the development of motor signs and improved disease profile in an STZ-induced mouse model of diabetic encephalopathy. Antidiabetic activity of ß-lactam molecules accounted at least partly for these effects.

The Relationship Between Thyroid Function and Body Composition, Leptin, Adiponectin, and Insulin Sensitivity in Morbidly Obese Euthyroid Subjects Compared to Non-obese Subjects.

BACKGROUND/OBJECTIVES: Thyroid function tests (TFTs) changes in obese people have been studied with increasing interest, however, studies have been inconsistent hence it remains poorly understood. We compared the TFTs of morbidly obese euthyroid Saudi subjects with non-obese controls and then we examined the influence of leptin, adiponectin, and insulin resistance on TFTs. SUBJECTS/METHODS: Fifty-five euthyroid obese subjects attending bariatric surgery clinic and 52 non-obese age-and gender-matched controls were recruited. We measured body weight, BMI, body composition, thyroid-stimulating hormone (TSH), Free T4 (FT4), Free T3(FT3), thyroid antibodies, fasting leptin, adiponectin, and lipid profile. Insulin resistance was quantified by HOMA-IR. Data are presented as mean ± SEM. RESULTS: Mean BMI was 45.6 ± 1.5 and 23.2 ± 0.5 kg/m2, for the obese and non-obese controls, respectively, P value < 0.001. Mean TSH was 2.7 ± 0.18 mIU/L in obese subjects and 1.7 ± 0.13 mIU/L (0.27-4.2) in the non-obese controls, respectively, P value .014. Mean FT3 was 3.9 ± 0.1 pmol/L (3.1-6.8) in obese subjects compared to 5.0 ± 0.1 pmol/L in non-obese controls, respectively, P value 0.001, however, FT4 was similar in the 2 groups. In the whole group (N = 107), BMI correlated positively with TSH and negatively with FT3. Leptin correlated negatively with both FT4 and FT3 in the non-obese group only while none of the TFTs correlated with HOMA-IR or adiponectin in either group. Binary logistic regression showed that each 1 unit increase in TSH increased the odds of becoming obese by 12.7, P value 0.009, 95 C.I. (1.9-85.0). Conversely, each - unit increase in FT3 decreased the odds of becoming obese by 0.2, P value 0.023, 95% C.I. (0.05-0.80). CONCLUSIONS: We report a small increase in TSH and a small decrease in FT3 within the normal range in obese subjects compared to non-obese controls. We also report a positive correlation between TSH and BMI with increased odds ratio of becoming obese with the increase in TSH and decrease in FT3. These changes may be either causally related or adaptive to the obesity state. FT4 and FT3 seem to correlate with leptin (but not with adiponectin or HOMA-IR) in the non-obese controls only. Larger mechanistic studies are needed to further elucidate the interesting association between obesity and TFTs.

Amplicon and Metagenomic Analysis of Middle East Respiratory Syndrome (MERS) Coronavirus and the Microbiome in Patients with Severe MERS.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional zoonotic spill over into humans. The severity of infection in humans is influenced by numerous factors, and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are coincident in the Middle East and thus a rapid way of sequencing MERS-CoV to derive genotype information for molecular epidemiology is needed. Additionally, complicating factors in MERS-CoV infections are coinfections that require clinical management. The ability to rapidly characterize these infections would be advantageous. To rapidly sequence MERS-CoV, an amplicon-based approach was developed and coupled to Oxford Nanopore long read length sequencing. This and a metagenomic approach were evaluated with clinical samples from patients with MERS. The data illustrated that whole-genome or near-whole-genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants, including deletion mutants. The metagenomic analysis provided information of the background microbiome. The advantage of this approach is that insertions and deletions can be identified, which are the major drivers of genotype change in coronaviruses. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in late 2012 in Saudi Arabia. The virus is a serious threat to people not only in the Middle East but also in the world and has been detected in over 27 countries. MERS-CoV is spreading in the Middle East and neighboring countries, and approximately 35% of reported patients with this virus have died. This is the most severe coronavirus infection so far described. Saudi Arabia is a destination for many millions of people in the world who visit for religious purposes (Umrah and Hajj), and so it is a very vulnerable area, which imposes unique challenges for effective control of this epidemic. The significance of our study is that clinical samples from patients with MERS were used for rapid in-depth sequencing and metagenomic analysis using long read length sequencing.

Crystallization and preliminary structural analyses of glutamate dehydrogenase from Peptoniphilus asaccharolyticus.

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.

Evaluation of metabolic, antioxidant and anti-inflammatory effects of Garcinia kola on diabetic rats.

Garcinia kola (G. kola), is a plant characterized by its hypoglycemic properties. We recently reported our findings on the extracts of G. kola, in which we found that it prevented the loss of inflammation-sensible neuronal populations in streptozotocin (STZ)-induced rat models of type 1 diabetes mellitus (T1DM). In the present study we assessed the effect of G. kola bioactive compounds extracted successively with water, hexane, methylene chloride, ethyl acetate, and butanol. through analyzing biochemical markers of oxidative stress, inflammation, and metabolic function in STZ-induced diabetic animals. Animals made diabetic by a single injection with STZ (60 mg/kg, i.p.), were treated daily with either vehicle solution, insulin, or G. kola extracts and its fractions from the first to the 6th-week post-injection. Biochemical markers; glucose, insulin, C-peptide, neuron-specific enolase (NSE), creatinine kinase, glutathione peroxidase, malondialdehyde (MDA), resistin, soluble E-selectin (SE-Selectin), and C-reactive proteins (CRP) levels in the sera were determined in the study groups. A marked increase in blood glucose (209.26% of baseline value), and a decrease in body weight (-12.37%) were observed in diabetic control animals but not in animals treated with either insulin or G. kola extracts and its fractions. The sub-fraction F5, G. kola ethyl acetate had the highest bioactive activities, with a maintenance of blood sugar, malondialdehyde, C-peptide, E-selectin, C-reactive protein (CRP) and neuron-specific enolase (NSE) to levels and responses comparable to healthy non-diabetic vehicle group and the positive control diabetic insulin-treated group. Our findings suggest that G. kola may have a strong therapeutic potential against T1DM and its microvascular complications.

Homotropic allosteric control in clostridial glutamate dehydrogenase: different mechanisms for glutamate and NAD+?

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.

The contribution of tryptophan residues to conformational changes in clostridial glutamate dehydrogenase--W64 and W449 as mediators of the cooperative response to glutamate.

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.

Garcinia kola seeds may prevent cognitive and motor dysfunctions in a type 1 diabetes mellitus rat model partly by mitigating neuroinflammation.

Background We reported recently that extracts of seeds of Garcinia kola, a plant with established hypoglycemic properties, prevented the loss of inflammation-sensible neuronal populations like Purkinje cells in a rat model of type 1 diabetes mellitus (T1DM). Here, we assessed G. kola extract ability to prevent the early cognitive and motor dysfunctions observed in this model. Methods Rats made diabetic by single injection of streptozotocin were treated daily with either vehicle solution (diabetic control group), insulin, or G. kola extract from the first to the 6th week post-injection. Then, cognitive and motor functions were assessed using holeboard and vertical pole behavioral tests, and animals were sacrificed. Brains were dissected out, cut, and processed for Nissl staining and immunohistochemistry. Results Hyperglycemia (209.26 %), body weight loss (-12.37 %), and T1DM-like cognitive and motor dysfunctions revealed behavioral tests in diabetic control animals were not observed in insulin and extract-treated animals. Similar, expressions of inflammation markers tumor necrosis factor (TNF), iba1 (CD68), and Glial fibrillary acidic protein (GFAP), as well as decreases of neuronal density in regions involved in cognitive and motor functions (-49.56 % motor cortex, -33.24 % medial septal nucleus, -41.8 % /-37.34 % cerebellar Purkinje /granular cell layers) were observed in diabetic controls but not in animals treated with insulin or G. kola. Conclusions Our results indicate that T1DM-like functional alterations are mediated, at least partly, by neuroinflammation and neuronal loss in this model. The prevention of the development of such alterations by early treatment with G. kola confirms the neuroprotective properties of the plant and warrant further mechanistic studies, considering the potential for human disease.