Human G6PD variant structural studies: Elucidating the molecular basis of human G6PD deficiency

Glucose-6-phosphate dehydrogenase deficiency is by far the most prevalent human enzymopathy and is gener�ated by a series of point mutations in the X-linked gene encoding G6PD. The severity of the deficiency relies on the various mutational sites in the gene, affecting the protein structure and function in at least two ways: by disrupting the entire protein fold or by altering the functional groups. Thus, the modified enzyme should be identified structurally and functionally to recognize the sequelae of each mutation. Understanding the molecular basis of G6PD deficiency is also essential to determine how mutations influence enzyme structure, stability, and activity. In characterizing 34 G6PD variants selected from Class I, II, and III, we reviewed and compared structural and molecular characterizations. These studies have shown that these mutations can influence the G6PD enzyme's local and global stability by changing the features of the mutant amino acids or by modifying their interactions (lost, increased, or decreased distances). Furthermore, the relationship between the changes in the enzyme structure and the severity of the disease was also reviewed. Overall, their results showed that Class I had the strongest influence on the protein's stability, activity, and function, which correlated with chronic non�spherocytic hemolytic anemia. Furthermore, there have been no drugs available to treat G6PD deficiency until now

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

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

Crystallization and preliminary structural analyses of glutamate dehydrogenase from Peptoniphilus asaccharolyticus

Selenomethionine-derivatized glutamate dehydrogenase from P. asaccharolyticus has been crystallized. Diffraction data were collected to 3.5 Å resolution from crystals belonging to the rhombohedral space group H32 and structure determination is in progress

Amplicon and metagenomic analysis of MERS-CoV and the microbiome in patients with severe Middle East respiratory syndrome (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 spill overs 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 co-incident in the Middle East and a rapid way is required of sequencing MERS-CoV to derive genotype information for molecular epidemiology. Additionally, complicating factors in MERS-CoV infections are co-infections that require clinical management. The ability to rapidly characterise these infections would be advantageous. To rapidly sequence MERS-CoV, we developed an amplicon-based approach coupled to Oxford Nanopore long read length sequencing. The advantage of this approach is that insertions and deletions can be identified – which are the major drivers of genotype change in coronaviruses. This and a metagenomic approach were evaluated on 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. Whereas, the metagenomic analysis provided information of the background microbiome

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