Effect of a single amino acid substitution G98D in a ribosome-associated essential GTPase, CgtA, on the growth and morphology of Vibrio cholerae.

CgtA, a highly conserved 50S ribosome-associated essential GTPase, acts as a repressor of the stringent stress response under nutrient-rich growth conditions to suppress basal levels of the alarmone ppGpp in V. cholerae. To further explore the in vivo functionality of CgtA, we introduced an amino acid substitution, i.e., Gly98Asp, in a conserved glycine residue in the N-terminal domain. The constructed V. cholerae mutant was designated CgtA(G98D). Comparison of cell sizes of the CgtA(G98D)mutant with its isogenic wild-type (Wt) strain N16961 under different phases of growth by Transmission Electron Microscopy (TEM) and statistical analysis suggests that CgtA may control the cell size of V. cholerae. The cell length is significantly reduced, corresponding to the delayed growth in the mid-logarithmic phase. The differences in the cell length of CgtA(G98D) and Wt are indistinguishable in the late logarithmic phase. During the stationary phase, marked by higher OD600, a sub-population of CgtA(G98D) cells outnumbered the Wt cells lengthwise. CgtA(G98D) cells appeared slenderer than Wt cells with significantly reduced cell width. However, the centerline curvature is preserved in CgtA(G98D) cells. We propose that in addition to its multitude of intracellular roles, CgtA may influence the cell size of V. cholerae.

Case Report of Neonatal Sotos Syndrome with a New Missense Mutation in the NSD1 Gene and Literature Analysis in the Chinese Han Population.

Currently, no consensus exists regarding Sotos syndrome in the Chinese population. Here, we present a case of neonatal Sotos syndrome, followed by a retrospective analysis of five cases of neonatal Sotos syndrome, reported in China. The study subject was a twin premature infant, heavier than gestational age, with characteristic facial features, limb shaking, and hypertonia. Transient hypoglycemia, abnormal cranial magnetic resonance imaging, multiple nodules in polycystic kidneys and liver, abnormal hearing, patent ductus arteriosus, and an atrial septal defect were also noted. The subject showed overgrowth and developmental retardation at 3 months of age. Sequencing revealed a novel missense mutation, c.5000C>A, in the nuclear receptor binding the SET domain protein 1 gene, resulting in an alanine-to-glutamate substitution. The bioinformatics analysis suggested high pathogenicity at this site. This study provides insights into diagnosis of neonatal Sotos syndrome based on specific phenotypes. Subsequent treatment and follow-up should focus on developmental retardation, epilepsy, and scoliosis.

Deletion analyses reveal insights into the domain specific activities of an essential GTPase CgtA in Vibrio cholerae.

CgtA is an essential bacterial GTPase protein involved in multiple cellular activities. In the presence of 50S ribosome, its GTPase activity increases significantly. Through sequential deletions of CgtA protein of Vibrio cholerae (CgtAvc) we found that its N terminal Obg domain is essential for ribosome binding and augmenting the ribosome mediated GTPase activity. Strategic deletions of the three glycine rich loops of Obg domain revealed that loop 1 of Obg domain is involved in anchoring the protein into the 50S, whereas, loop 2 & loop 3 are involved in conveying the effect of interaction of the Obg domain with the 50S to the GTPase domain through an interdomain linker, followed by GTP hydrolysis. On the other hand, the non-conserved C-terminal domain (CTD) is not directly involved in ribosome binding but shows negative impact on GTPase activity.

Two conserved amino acids of juxtaposed domains of a ribosomal maturation protein CgtA sustain its optimal GTPase activity.

CgtA is a highly conserved ribosome binding protein involved in ribosome biogenesis and associated with stringent response. It is a 55 KDa GTPase protein consisting of GTPase, Obg and C-terminal domains. The function of the latter two domains was not clear and despite the importance, the mode of action of CgtA is still largely unknown. Knocking out of CgtA gene is lethal and mutations lead to growth, sporulation and developmental defects in bacteria. It was found that a growth defect and pinhole size colony morphology of Bacillus subtilis was associated with a Gly92Asp point mutation on the Obg domain of its CgtA protein, instead of its GTPase domain. CgtA is an important and essential protein of the deadly diarrhea causing bacteria Vibrio cholerae and in order to investigate the mode of action of the V. cholerae CgtA we have utilized this information. We measured the GTPase activity of V. cholerae CgtA (CgtAvc) protein in the presence of purified ribosome. Our results showed 5-fold increased GTP hydrolysis activity compared to its intrinsic activity. Then we explored the GTPase activity of the mutated CgtAvc (Gly98Asp) located at the Obg domain, which reduced the GTP hydrolysis rate to half. The double point mutations (Gly98Asp, and Tyr194Gly) encompassing another conserved residue, Tyr194, located at the diagonally opposite position in the GTPase domain largely restored (about 82%) the reduced GTPase activity, revealing a fine-tuned inter-domain movement readily associated with the GTPase activity of CgtA and thus maintaining the proper functioning of the CgtA protein.

Knockdown Experiment Reveals an Essential GTPase CgtA's Involvement in Growth, Viability, Motility, Morphology, and Persister Phenotypes in Vibrio cholerae.

CgtA is an essential bacterial GTPase consisting of a highly conserved N-terminal Spo0B-associated GTP-binding protein (Obg) domain, a central GTPase domain, and a variable C-terminal domain (CTD). This study reports global changes in the proteome and transcriptome of wild-type (Wt) versus full-length CgtA-depleted Vibrio cholerae in minimal media. Comparative transcriptome sequencing (RNA-Seq), followed by comparative proteomic analyses, revealed that the knockdown of cgtA significantly altered expressions of 311 proteins involved in diverse cellular activities, many of which are associated with the survival of V. cholerae. Various intracellular functional roles of CgtA in growth, viability, motility, morphology, and persister phenotypes in V. cholerae are revealed based on subsequent confirmatory experiments. Furthermore, a more sustained mRNA expression pattern of cgtA in a minimal medium than in a rich medium was also observed for Wt V. cholerae, where the highest level of mRNA expression of cgtA was observed during the logarithmic growth phase. Thereby, we propose that minimal medium-associated reduced growth rate coupled with cgtA depletion aggravates the intracellular stress in V. cholerae, interrupting vital cellular processes. The functional role of the CTD in V. cholerae is not fully understood. Hence, to specifically investigate the intracellular role of the 57-amino-acid-long CTD of CgtAVC, the CTD-only portion of CgtA was deleted. Subsequent proteomics studies revealed an altered expression of 240 proteins in the CgtA(ΔCTD) mutant, having major overlap with the full-length cgtA-deleted condition. Overall, our study reveals an alternative facet of the survival mechanism of V. cholerae during nutritional downshift as per the concomitant consequences of cgtA depletion. IMPORTANCE It is very important that we must find new drug target proteins from multidrug-resistant human-pathogenic organisms like V. cholerae. CgtA is among such potential candidates, and here, we are reporting about some newly identified cellular roles of it that are important for the survival of V. cholerae. Briefly, we knocked down the full-length cgtA gene, as well as did a partial deletion of this gene from the V. cholerae genome followed by RNA-Seq and proteomics studies. Results from our study revealed up- and downregulation of several known and unknown genes and proteins as the effect of the cgtA knockdown experiment. Also, we have presented some interesting observations that are linked with cgtA for growth, viability, motility, morphology, and persister phenotypes in V. cholerae. Our study enhances the importance of CgtA and paves the way for further exploration based on our provided data.

Effect of ITPA Polymorphism on Adverse Drug Reactions of 6-Mercaptopurine in Pediatric Patients with Acute Lymphoblastic Leukemia: A Systematic Review and Meta-Analysis.

6-Mercaptopurine (6-MP) is a cornerstone of the maintenance regimen for pediatric acute lymphoblastic leukemia (ALL). Inosine triphosphate pyrophosphatase (ITPA) is considered a candidate pharmacogenetic marker that may affect metabolism and 6-MP-induced toxicities; however, the findings are inconsistent. Therefore, we attempted to evaluate the effect of ITPA 94C>A polymorphism on 6-MP-induced hematological toxicity and hepatotoxicity through a systematic review and meta-analysis. A literature search for qualifying studies was conducted using the PubMed, Web of Science, and Embase databases until October 2021. Overall, 10 eligible studies with 1072 pediatric ALL patients were included in this meta-analysis. The results indicated that ITPA 94C>A was significantly associated with 6-MP-induced neutropenia (OR 2.38, 95% CI: 1.56−3.62; p = 0.005) and hepatotoxicity (OR 1.98, 95% CI: 1.32−2.95; p = 0.0009); however, no significant association was found between the ITPA 94C>A variant and 6-MP-induced leukopenia (OR 1.75, 95% CI: 0.74−4.12; p = 0.20). This meta-analysis demonstrated that ITPA 94C>A polymorphism could affect 6-MP-induced toxicities. Our findings suggested that ITPA genotyping might help predict 6-MP-induced myelosuppression and hepatotoxicity.

The Association between ABCG2 421C>A (rs2231142) Polymorphism and Rosuvastatin Pharmacokinetics: A Systematic Review and Meta-Analysis

Although several studies have revealed the association between rosuvastatin pharmacokinetics and the ABCG2 421C>A (rs2231142) polymorphism, most studies were conducted with small sample sizes, making it challenging to apply the findings clinically. Therefore, the purpose of this study is to perform a meta-analysis of the relationship between the ABCG2 421C>A polymorphism and rosuvastatin pharmacokinetics. We searched three electronic databases, EMBASE, PubMed, and Web of Science, using search terms related to ABCG2 gene polymorphisms and rosuvastatin. In addition, we reviewed studies published before 12 August 2021, to examine the relationship between the ABCG2 421C>A polymorphism and rosuvastatin pharmacokinetics. To examine the magnitude of the association, the log geometric mean difference (lnGM) and 95% confidence intervals (CIs) were calculated and interpreted as the antilogarithm of a natural logarithm (elnGM). The meta-analysis was performed using Review Manager (version 5.4) and R Studio (version 4.0.2). Subgroup analysis was performed according to race and the types of mean values. Among the 318 identified studies, a total of 8 studies involving 423 patients is included in this meta-analysis. The A allele carriers of ABCG2 421C>A showed 1.5 times higher in both AUC0-∞ (lnGM = 0.43; 95% CI = 0.35−0.50; p < 0.00001) and Cmax (lnGM = 0.42; 95% CI = 0.33−0.51; p < 0.00001) than non-carriers, while there was no significant difference in Tmax and half-life. There was no significance in the pharmacokinetic parameters of the subgroups using either ethnicity or mean values. This meta-analysis demonstrates that subjects carrying the A allele of ABCG2 421C>A show significantly increased AUC0-∞ and Cmax values compared to subjects with the CC genotype. Therefore, information about ABCG2 genotypes might be useful for individualized rosuvastatin therapy.

Clinical Implications of Glyoxalase1 Gene Polymorphism and Elevated Levels of the Reactive Metabolite Methylglyoxal in the Susceptibility of Type 2 Diabetes Mellitus in the Patients from Asir and Tabuk Regions of Saudi Arabia.

Diabetes mellitus constitutes a big challenge to the global health care system due to its socioeconomic impacts and very serious complications. The incidence and the prevalence rate are increased in the Gulf region including the KSA. Type 2 diabetes mellitus (T2DM) is caused by diverse risk factors including obesity, unhealthy dietary habits, physical inactivity, smoking and genetic factors. The molecular genetic studies have helped in the detection of many single nucleotide polymorphisms (SNP) with different diseases including cancers, cardiovascular diseases and T2DM. The glyoxalase 1 (GLO1) is a detoxifying enzyme and catalyzes the elimination of the cytotoxic product methylglyoxal (MG) by converting it to D-lactate, which is not toxic to tissues. MG accumulation is associated with the pathogenesis of different diseases including T2DM. In this study, we have investigated the association of the glyoxalase 1 SNPs (rs2736654) rs4746 C>A and rs1130534 T>A with T2DM using the amplification refractory mutation system PCR. We also measured the concentration of MG by ELISA in T2DM patients and matched heathy controls. Results show that the CA genotype of the GLO rs4647 A>C was associated with T2DM with OR = 2.57, p-value 0.0008 and the C allele was also associated with increased risk to T2DM with OR = 2.24, p-value = 0.0001. It was also observed that AT genotype of the rs1130534 was associated with decreased susceptibility to T2DM with OR = 0.3, p-value = 0.02. The A allele of rs1130534 was also associated with reduced risk to T2DM with PR = 0.27 = 0.006. In addition, our ELISA results demonstrate significantly increased MG concentrations in serum of the T2DM patients. We conclude that the GLO1 SNP may be associated with decreased enzyme activity and a resultant susceptibility to T2DM. Further well-designed studies in different and large patient populations are recommended to verify these findings.

Intrinsic GTPase activity of a ribosomal maturation protein CgtA is associated with its inter-domain movement: insights from MD simulations and biochemical studies.

CgtA is an essential ribosome associated GTPase protein of bacteria. It has three domains, viz., Obg, GTPase, and C-terminal domain. It is a multifunctional protein and it is being considered as a potential drug target against bacterial infections. Despite the importance, CgtA's action mechanisms are not well known which warrants further study. Towards that goal, we are pursuing biochemical and computational studies in Vibrio cholerae CgtA (CgtAvc). Biochemically we found that a single amino acid substitution from Gly98 to Asp98 belonging to the Obg domain caused reduced GTPase activity of CgtAvc. The results from our comparative MD simulations studies revealed that in silico amino acid substitution for Gly98Asp influenced the inter-domain movement between Obg domain and GTPase domain. Moreover, we found significant alteration of intra-domain movements among the P-loop, G4 box, and G5 box of the GTPase domain, implying a potential cause for the reduced GTPase activity.

The bacterial cell cycle checkpoint protein Obg and its role in programmed cell death.

The phenomenon of programmed cell death (PCD), in which cells initiate their own demise, is not restricted to multicellular organisms. Unicellular organisms, both eukaryotes and prokaryotes, also possess pathways that mediate PCD. We recently identified a PCD mechanism in Escherichia coli that is triggered by a mutant isoform of the essential GTPase ObgE (Obg of E. coli). Importantly, the PCD pathway mediated by mutant Obg (Obg*) differs fundamentally from other previously described bacterial PCD pathways and thus constitutes a new mode of PCD. ObgE was previously proposed to act as a cell cycle checkpoint protein able to halt cell division. The implication of ObgE in the regulation of PCD further increases the similarity between this protein and eukaryotic cell cycle regulators that are capable of doing both. Moreover, since Obg is conserved in eukaryotes, the elucidation of this cell death mechanism might contribute to the understanding of PCD in higher organisms. Additionally, if Obg*-mediated PCD is conserved among different bacterial species, it will be a prime target for the development of innovative antibacterials that artificially induce this pathway.

Membrane depolarization-triggered responsive diversification leads to antibiotic tolerance.

Bacterial populations are known to harbor a small fraction of so-called persister cells that have the remarkable ability to survive treatment with very high doses of antibiotics. Recent studies underscore the importance of persistence in chronic infections, yet the nature of persisters remains poorly understood. We recently showed that the universally conserved GTPase Obg modulates persistence via a (p)ppGpp-dependent mechanism that proceeds through expression of hokB. HokB is a membrane-bound toxin that causes the membrane potential to collapse. The resulting drop in cellular energy levels triggers a switch to the persistent state, yielding protection from antibiotic attack. Obg-mediated persistence is conserved in the human pathogen Pseudomonas aeruginosa, making Obg a promising target for therapies directed against bacterial persistence.