Development of DNA-Based Lateral Flow Assay for Detection of LDLR Gene Mutation for Familial Hypercholesterolemia.

Background: The techniques for detecting single nucleotide polymorphisms (SNP) require lengthy and complex experimental procedures and expensive instruments that may only be available in some laboratories. Thus, a deoxyribonucleic acid (DNA)-based lateral flow assay (LFA) was developed as a point-of-care test (POCT) diagnostic tool for genotyping. In this study, single nucleotide variation (E101K) in the low-density lipoprotein receptor (LDLR) gene leading to familial hypercholesterolemia (FH) was chosen as a model. Methods: Hypercholesterolemic individuals (n = 103) were selected from the Malaysian Cohort project (UKM Medical Molecular Biology Institute) while the control samples were selected from the Biobank (UKM Medical Molecular Biology Institute). The DNA samples were isolated from whole blood. Polymerase chain reaction (PCR) amplification process was performed using bifunctional labelled primers specifically designed to correspond to the variant that differentiates wild-type and mutant DNA for visual detection on LFA. The variant was confirmed using Sanger sequencing, and the sensitivity and specificity of the LFA detection method were validated using the Agena MassARRAY® technique. Results: Out of 103 hypercholesterolemic individuals, 5 individuals (4.8%) tested positive for E101K, LDLR mutation and the rest, including healthy control individuals, tested negative. This result was concordant with Sanger sequencing and Agena MassARRAY®. These five individuals could be classified as Definite FH, as the DNA diagnosis was confirmed. The sensitivity and specificity of the variant detection by LFA is 100% compared to results using the genotyping method using Agena MassARRAY®. Conclusion: The developed LFA can potentially be used in the POC setting for detecting the E101K variant in the LDLR gene. This LFA can also be used to screen family members with E101K variant in the LDLR gene and is applicable for other SNP's detection.

Body composition and risk factors for cardiovascular disease in global multi-ethnic populations.

BACKGROUND: No large-scale studies have compared associations between body composition and cardiovascular risk factors across multi-ethnic populations. METHODS: Population-based surveys included 30,721 Malay, 10,865 Indian and 25,296 Chinese adults from The Malaysian Cohort, and 413,737 White adults from UK Biobank. Sex-specific linear regression models estimated associations of anthropometry and body composition (body mass index [BMI], waist circumference [WC], fat mass, appendicular lean mass) with systolic blood pressure (SBP), low-density lipoprotein cholesterol (LDL-C), triglycerides and HbA1c. RESULTS: Compared to Malay and Indian participants, Chinese adults had lower BMI and fat mass while White participants were taller with more appendicular lean mass. For BMI and fat mass, positive associations with SBP and HbA1c were strongest among the Chinese and Malay and weaker in White participants. Associations with triglycerides were considerably weaker in those of Indian ethnicity (eg 0.09 [0.02] mmol/L per 5 kg/m2 BMI in men, vs 0.38 [0.02] in Chinese). For appendicular lean mass, there were weak associations among men; but stronger positive associations with SBP, triglycerides, and HbA1c, and inverse associations with LDL-C, among Malay and Indian women. Associations between WC and risk factors were generally strongest in Chinese and weakest in Indian ethnicities, although this pattern was reversed for HbA1c. CONCLUSION: There were distinct patterns of adiposity and body composition and cardiovascular risk factors across ethnic groups. We need to better understand the mechanisms relating body composition with cardiovascular risk to attenuate the increasing global burden of obesity-related disease.

Stability of glycated haemoglobin (HbA1c) measurements from whole blood samples kept at -196°C for seven to eight years in The Malaysian Cohort study.

OBJECTIVE: Glycated haemoglobin (HbA1c) is a standard indication for screening type 2 diabetes that also has been widely used in large-scale epidemiological studies. However, its long-term quality (in terms of reproducibility) stored in liquid nitrogen is still unknown. This study is aimed to evaluate the stability and reproducibility of HbA1c measurements from frozen whole blood samples kept at -196°C for more than 7 years. METHODS: A total of 401 whole blood samples with a fresh HbA1c measurement were randomly selected from The Malaysian Cohort's (TMC) biobank. The HbA1c measurements of fresh and frozen (stored for 7-8 years) samples were assayed using different high-performance liquid chromatography (HPLC) systems. The HbA1c values of the fresh samples were then calculated and corrected according to the later system. The reproducibility of HbA1c measurements between calculated-fresh and frozen samples was assessed using a Passing-Bablok linear regression model. The Bland-Altman plot was then used to evaluate the concordance of HbA1c values. RESULTS: The different HPLC systems highly correlated (r = 0.99) and agreed (ICC = 0.96) with each other. Furthermore, the HbA1c measurements for frozen samples strongly correlate with the corrected HbA1c values of the fresh samples (r = 0.875) with a mean difference of -0.02 (SD: -0.38 to 0.38). Although the mean difference is small, discrepancies were observed within the diabetic and non-diabetic samples. CONCLUSION: These data demonstrate that the HbA1c measurements between fresh and frozen samples are highly correlated and reproducible.

Silencing of ZFP36L2 increases sensitivity to temozolomide through G2/M cell cycle arrest and BAX mediated apoptosis in GBM cells.

Despite the advancements in primary brain tumour diagnoses and treatments, the mortality rate remains high, particularly in glioblastoma (GBM). Chemoresistance, predominantly in recurrent cases, results in decreased mean survival of patients with GBM. We aimed to determine the chemosensitisation and oncogenic characteristics of zinc finger protein 36-like 2 (ZFP36L2) in LN18 GBM cells via RNA interference (RNAi) delivery. We conducted a meta-analysis of microarray datasets and RNAi screening using pooled small interference RNA (siRNA) to identify the druggable genes responsive to GBM chemosensitivity. Temozolomide-resistant LN18 cells were used to evaluate the effects of gene silencing on chemosensitisation to the sub-lethal dose (1/10 of the median inhibitory concentration [IC50]) of temozolomide. ZFP36L2 protein expression was detected by western blotting. Cell viability, proliferation, cell cycle and apoptosis assays were carried out using commercial kits. A human apoptosis array kit was used to determine the apoptosis pathway underlying chemosensitisation by siRNA against ZFP36L2 (siZFP36L2). Statistical analyses were performed using one-way analysis of variance; p > 0.05 was considered significant. The meta-analysis and RNAi screening identified ZFP36L2 as a potential marker of GBM. ZFP36L2 knockdown significantly induced apoptosis (p < 0.05). Moreover, ZFP36L2 inhibition led to increased cell cycle arrest and decreased cell proliferation. Downstream analysis showed that the sub-lethal dose of temozolomide and siZFP26L2 caused major upregulation of BCL2-associated X, apoptosis regulator (BAX). ZFP36L2 has oncogenic and chemosensitive characteristics and may play an important role in gliomagenesis through cell proliferation, cell cycle arrest and apoptosis. This suggests that RNAi combined with chemotherapy treatment such as temozolomide may be a potential GBM therapeutic intervention in the future.

Prevalence of anaemia and associated risk factors amongst The Malaysian Cohort participants.

This study was aimed at determining the prevalence of anaemia amongst the Malaysian Cohort participants and the associated risk factors. This was a cross-sectional study that involved 102,388 participants from The Malaysian Cohort (TMC) aged between 35 and 70 years old recruited from April 2006 to September 2012. Venous blood was taken for the full blood count. The prevalence of anaemia was 13.8% with majority having the microcytic-hypochromic type (59.7%). Comparison between the ethnic groups showed that Indians have the highest prevalence of anaemia (19.9%), followed by Malays (13.1%), and Chinese (12.0%). The prevalence of anaemia was substantially higher in females (20.1%) compared to males (4.9%). Amongst the female participants, the prevalence of anaemia was highest amongst those who were younger than 49 years old and decreased as the age increased. In contrast, the prevalence of anaemia in males increased with age. Gender, ethnicity, age, marital status, presence of platelet disorders and kidney disease were significant risk factors associated with anaemia and contributed to 14.9% of the risk of developing anaemia in this population. The prevalence of anaemia amongst the Malaysian Cohort participants is 13.8% with the majority having the microcytic and hypochromic type implying iron deficiency as the main cause. It is important that those who have anaemia be further investigated and treated.

Connecting Proteomics to Next-Generation Sequencing: Proteogenomics and Its Current Applications in Biology.

Understanding the relationship between genotypes and phenotypes is essential to disentangle biological mechanisms and to unravel the molecular basis of diseases. Genes and proteins are closely linked in biological systems. However, genomics and proteomics have developed separately into two distinct disciplines whereby crosstalk among scientists from the two domains is limited and this constrains the integration of both fields into a single data modality of useful information. The emerging field of proteogenomics attempts to address this by building bridges between the two disciplines. In this review, how genomics and transcriptomics data in different formats can be utilized to assist proteogenomics application is briefly discussed. Subsequently, a much larger part of this review focuses on proteogenomics research articles that are published in the last five years that answer two important questions. First, how proteogenomics can be applied to tackle biological problems is discussed, covering genome annotation and precision medicine. Second, the latest developments in analytical technologies for data acquisition and the bioinformatics tools to interpret and visualize proteogenomics data are covered.

Analyzing the Secretome of Gut Microbiota as the Next Strategy For Early Detection of Colorectal Cancer.

Dysbiosis of gut microbiome can contribute to inflammation, and subsequently initiation and progression of colorectal cancer (CRC). Throughout these stages, various proteins and metabolites are secreted to the external environment by microorganisms or the hosts themselves. Studying these proteins may help enhance our understanding of the host-microorganism relationship or they may even serve as useful biomarkers for CRC. However, secretomic studies of gut microbiome of CRC patients, until now, are scarcely performed. In this review article, the focus is on the roles of gut microbiome in CRC, the current findings on CRC secretome are highlighted, and the emerging challenges and strategies to drive forward this area of research are addressed.

Sudden Cardiac Death (SCD) - risk stratification and prediction with molecular biomarkers.

Sudden cardiac death (SCD) is a sudden, unexpected death that is caused by the loss of heart function. While SCD affects many patients suffering from coronary artery diseases (CAD) and heart failure (HF), a considerable number of SCD events occur in asymptomatic individuals. Certain risk factors for SCD have been identified and incorporated in different clinical scores, however, risk stratification using such algorithms is only useful for health management rather than for early detection and prediction of future SCD events in high-risk individuals. In this review, we discuss different molecular biomarkers that are used for early detection of SCD. This includes genetic biomarkers, where the majority of them are genomic variants for genes that encode for ion channels. Meanwhile, protein biomarkers often denote proteins that play roles in pathophysiological processes that lead to CAD and HF, notably (i) atherosclerosis that involves oxidative stress and inflammation, as well as (ii) cardiac tissue damage that involves neurohormonal and hemodynamic regulation and myocardial stress. Finally, we outline existing challenges and future directions including the use of OMICS strategy for biomarker discovery and the multimarker panels.

Signatures of gene expression, DNA methylation and microRNAs of hepatocellular carcinoma with vascular invasion.

Aim: Micro and macro vascular invasion (VI) are known as independent predictors of tumor recurrence and poor survival after surgical treatment of hepatocellular carcinoma (HCC). Here, we aimed to re-analyze The Cancer Genome Atlas of liver hepatocellular carcinoma datasets to identify the VI-expression signatures. Materials & methods: We filtered The Cancer Genome Atlas liver hepatocellular carcinoma (LIHC) datasets into three groups: no VI (NVI = 198); micro VI (MIVI = 89) and macro VI (MAVI = 16). We performed differential gene expression, methylation and microRNA analyses. Results & conclusion: We identified 12 differentially expressed genes and 55 differentially methylated genes in MAVI compared with no VI. The GPD1L gene appeared in all of the comparative analyses. Higher GPD1L expression was associated with VI and poor outcomes in the HCC patients.

Oxidative stress resistance and fitness-compensatory response in vancomycin-intermediate Staphylococcus aureus (VISA).

In this study, vancomycin-intermediate Staphylococcus aureus (VISA) cells carrying vraS and (or) graR mutations were shown to be more resistant to oxidative stress. Caenorhabditis elegans infected with these strains in turn demonstrated lower survival. Altered regulation in oxidative stress response and virulence appear to be physiological adaptations associated with the VISA phenotype in the Mu50 lineage.

Long noncoding RNAs as biotargets in cisplatin-based drug resistance.

Since its discovery, cisplatin has become the key drug in chemotherapy for cancers. Nevertheless, chemoresistance in cancers has become an impediment in using cisplatin for cancer treatment. The resistance toward cisplatin is multifaceted as it involves multiple cellular pathways. Ever since the knowledge of long noncoding RNAs as modulators of various molecular pathways came to light, the interest in the biological function of lncRNAs as biomarkers has increased dramatically. Numerous studies have reported the link between the dysregulation of lncRNAs and drug resistance in cancers. More importantly, several lncRNAs were found to be vital in regulating cisplatin resistance. Therefore, this review summarizes the recent efforts in linking between cisplatin resistance and different types of lncRNAs.

Global Lung Initiative 2012 spirometry reference values in a large Asian cohort of Malay, Chinese and Indian ancestry.

BACKGROUND AND OBJECTIVE: Although the multi-ethnic European Respiratory Society/Global Lung Initiative (ERS/GLI) 2012 reference values have been developed, the Taskforce has called for further validation specifically on subpopulations that were under represented such as the Malays, Chinese and Indians, in which the two latter ethnic groups represent about one-third of the world population. Thus, the aims of this study were to evaluate the appropriateness of the ERS/GLI 2012 reference values in a healthy adult Malaysian population and to construct a local lung function reference for the Malaysia population specific to the three major ethnic groups. METHODS: Acceptable spirometry data were obtained from 30 281 healthy subjects aged 35-70 years comprising Malays, Chinese and Indians from the Malaysian Cohort. Local reference values were calculated using regression analysis and evaluated using ERS/GLI reference values to obtain GLI Z-scores. RESULTS: The mean (SD) of the forced expiratory volume in 1 s (FEV1 ) for males were 2.67 (0.46), 2.89 (0.48) and 2.60 (0.46) and females were 1.91 (0.36), 2.13 (0.37) and 1.86 (0.35) for Malays, Chinese and Indians, respectively. For forced vital capacity (FVC), the mean (SD) for males were 3.03 (0.53), 3.28 (0.58) and 2.92 (0.53) and females were 2.15 (0.40), 2.38 (0.43) and 2.07 (0.41) for Malays, Chinese and Indians, respectively. The mean GLI Z-scores were less than -0.5 for FEV1 and FVC and more than 0.5 for FEV1 /FVC. A large percentage of subjects in all the three ethnic groups were defined lower than the lower limit of normal. CONCLUSION: This present and large multi-ethnic Asian-based study demonstrates clinically significant deviation from ERS/GLI 2012 equations for spirometry. It highlights the importance of validating predicted equations for spirometry in local populations.

Estimating dengue incidence and hospitalization in Malaysia, 2001 to 2013.

BACKGROUND: Epidemiologic measures of the dengue burden such as prevalence and incidence are important for policy-making and monitoring the progress of disease control. It is a common practice where epidemiologic and economic research estimate dengue burden based on notification data. However, a basic challenge in estimating the incidence of dengue is that a significant proportion of infected population are asymptomatic. It can be overcome by using mathematical models that relate observed prevalence and mortality to incidence. In this study, we estimate the trend of dengue incidence and hospitalization in Malaysia. METHODS: This study is based entirely on the available secondary data sources on dengue in Malaysia. The age-specific incidence of dengue between 2001 and 2013 was estimated using the prevalence and mortality estimates in an incidence-prevalence-mortality (IPM) model. Data on dengue prevalence were extracted from six sero-surveys conducted in Malaysia between 2001 and 2013; while statistics on dengue notification and Case Fatality Rate were derived from National Dengue Surveillance System. Dengue hospitalization data for the years 2009 to 2013 were extracted from the Health Informatics Centre and the volumes of dengue hospitalization for hospitals with missing data were estimated with Poisson models. RESULTS: The dengue incidence in Malaysia varied from 69.9 to 93.4 per 1000 population (pkp) between 2001 and 2013.The temporal trend in incidence rate was decreasing since 2001. It has been reducing at an average rate of 2.57 pkp per year from 2001 to 2013 (p = 0.011). The age-specific incidence of dengue decreased steadily with dengue incidence reaching zero by age > 70 years. Dengue notification rate has remained stable since 2001 and the number of notified cases each year was only a small fraction of the incident cases (0.7 to 2.3%). Similarly, the dengue hospitalization was larger but still a small fraction of the incident cases (3.0 to 5.6%). CONCLUSION: Dengue incidence can be estimated with the use of sero-prevalence surveys and mortality data. This study highlights a reducing trend of dengue incidence in Malaysia and demonstrates the discrepancy between true dengue disease burden and cases reported by national surveillance system. Sero-prevalence studies with representative samples should be conducted regularly to allow better estimation of dengue burden in Malaysia.

Prospective Advances in Circular RNA Investigation.

circRNAs have emerged as one of the key regulators in many cellular mechanisms and pathogenesis of diseases. However, with the limited knowledge and current technologies for circRNA investigations, there are several challenges that need to be addressed for. These include challenges in understanding the regulation of circRNA biogenesis, experimental designs, and sample preparations to characterize the circRNAs in diseases as well as the bioinformatics pipelines and algorithms. In this chapter, we discussed the above challenges and possible strategies to overcome those limitations. We also addressed the differences between the existing applications and technologies to study the circRNAs in diseases. By addressing these challenges, further understanding of circRNAs roles and regulations as well as the discovery of novel circRNAs could be achieved.

10-Year Cardiovascular Disease Risk Estimation Based on Lipid Profile-Based and BMI-Based Framingham Risk Scores across Multiple Sociodemographic Characteristics: The Malaysian Cohort Project.

Cardiovascular disease (CVD) leads to high morbidity and mortality rate worldwide. Therefore, it is important to determine the risk of CVD across the sociodemographic factors to strategize preventive measures. The current study consisted of 53,122 adults between the ages of 35 and 65 years from The Malaysian Cohort project during recruitment phase from year 2006 to year 2012. Sociodemographic profile and physical activity level were assessed via self-reported questionnaire, whereas relevant CVD-related biomarkers and biophysical variables were measured to determine the Framingham Risk Score (FRS). The main outcome was the 10-year risk of CVD via FRS calculated based on lipid profile and body mass index (BMI) associated formulae. The BMI-based formula yielded a higher estimation of 10-year CVD risk than the lipid profile-based formula in the study for both males (median = 13.2% and 12.7%, respectively) and females (median = 4.3% and 4.2%, respectively). The subgroup with the highest risk for 10-year CVD events (based on both FRS formulae) was the Malay males who have lower education level and low physical activity level. Future strategies for the reduction of CVD risk should focus on screening via BMI-based FRS in this at-risk subpopulation to increase the cost-effectiveness of the prevention initiatives.

Profiling of cytokines, chemokines and other soluble proteins as a potential biomarker in colorectal cancer and polyps.

Soluble proteins including cytokines, chemokines and growth factors are small proteins that mediate and regulate immunity. They involved in the pathogenesis of many diseases including cancers. The concentration of these proteins in biological fluids (serum or plasma) and tissues in diseases may suggest pathway activation that leads to inflammatory response or disease progression. Therefore, these soluble proteins may be useful as a tool for screening, diagnosis classification between stages of disease or surveillance for therapy. Enzyme-linked immunosorbent assays (ELISA) and bioassay have been used as a gold standard in cytokine level measurements in clinical practice. However, these methods allow only single cytokine detection at a time and ineffective for screening purposes. Hence, the innovation of multiplexing technology allows measurement of many these soluble proteins simultaneously, thus allowing rapid, cost effective and better efficiency by using a minute amount of sample. In this study, we explored the profiles of key inflammatory cytokines, chemokines and other soluble proteins from the serum derived from colorectal carcinoma (CRC, n=20), colorectal polyps (P, n=20) and healthy volunteers (N, n=20) using multiplexed bead-based immunoassays. We aimed to evaluate if the levels of these soluble proteins can classify these groups of populations and explore the possible application of the soluble proteins as biomarkers in early stage screening and/or surveillance. We observed significant high IL-4, MIP-1ß, FasL and TGF-ß1 levels but lower levels for RANTES in P-derived serum as compared to N-derived serum. Significant high IL-8, VEGF, MIP-1ß, Eotaxin and G-CSF observed in CRC-derived serum when compared to N-derived serum. Between CRC- and P-derived serum, significantly higher levels of IL-8, Eotaxin and G-CSF but lower levels for TGF-ß1 were detected in CRC-derived serum. These preliminary results were obtained from small sample size and could be further validated with larger sample size cohort to produce a panel of biomarkers for CRC and P patients. Our findings might be useful in developing a disease-specific panel for biomarker screening assay. This could be used for early diagnosis and/or treatment surveillance.

Meta-analysis of gene expression in relapsed childhood B-acute lymphoblastic leukemia.

BACKGROUND: Relapsed pediatric B-acute lymphoblastic leukemia (B-ALL) remains as the leading cause of cancer death among children. Other than stem cell transplantation and intensified chemotherapy, no other improved treatment strategies have been approved clinically. Gene expression profiling represents a powerful approach to identify potential biomarkers and new therapeutic targets for various diseases including leukemias. However, inadequate sample size in many individual experiments has failed to provide adequate study power to yield translatable findings. With the hope of getting new insights into the biological mechanisms underpinning relapsed ALL and identifying more promising biomarkers or therapeutic targets, we conducted a meta-analysis of gene expression studies involving ALL from 3 separate studies. METHOD: By using the keywords "acute lymphoblastic leukemia", and "microarray", a total of 280 and 275 microarray datasets were found listed in Gene Expression Omnibus database GEO and ArrayExpress database respectively. Further manual inspection found that only three studies (GSE18497, GSE28460, GSE3910) were focused on gene expression profiling of paired diagnosis-relapsed pediatric B-ALL. These three datasets which comprised of a total of 108 matched diagnosis-relapsed pediatric B-ALL samples were then included for this meta-analysis using RankProd approach. RESULTS: Our analysis identified a total of 1795 upregulated probes which corresponded to 1527 genes (pfp < 0.01; FC > 1), and 1493 downregulated probes which corresponded to 1214 genes (pfp < 0.01; FC < 1) respectively. S100A8 appeared as the top most overexpressed gene (pfp < 0.01, FC = 1.8) and is a potential target for further validation. Based on gene ontology biological process annotation, the upregulated genes were most enriched in cell cycle processes (enrichment score = 15.3), whilst the downregulated genes were clustered in transcription regulation (enrichment score = 12.6). Elevated expression of cell cycle regulators (e.g kinesins, AURKA, CDKs) was the key genetic defect implicated in relapsed ALL, and serve as attractive targets for therapeutic intervention. CONCLUSION: We identified S100A8 as the most overexpressed gene, and the cell cycle pathway as the most promising biomarker and therapeutic target for relapsed childhood B-ALL. The validity of the results warrants further investigation.

Metaproteomic analysis of human gut microbiota: where are we heading?

The human gut is home to complex microbial populations that change dynamically in response to various internal and external stimuli. The gut microbiota provides numerous functional benefits that are crucial for human health but in the setting of a disturbed equilibrium, the microbial community can cause deleterious outcomes such as diseases and cancers. Characterization of the functional activities of human gut microbiota is fundamental to understand their roles in human health and disease. Metaproteomics, which refers to the study of the entire protein collection of the microbial community in a given sample is an emerging area of research that provides informative details concerning functional aspects of the microbiota. In this mini review, we present a summary of the progress of metaproteomic analysis for studying the functional role of gut microbiota. This is followed by an overview of the experimental approaches focusing on fecal specimen for metaproteomics and is concluded by a discussion on the challenges and future directions of metaproteomic research.

Giant Cell Tumor of the Ribs and Aneurysmal Bone Cyst Presenting With Hemothorax in a Child.

Giant cell tumor (GCT) is one of the most common tumors of bone and is the most common precursor of aneurysmal bone cysts (ABC). The clinical behavior of concurrent GCT and ABC can be very aggressive in children. GCT of the ribs, with or without ABC, is rarely seen in children. We report a case of an 8-year-old girl with GCT and associated ABC of the ribs who presented with sudden onset of chest pain and breathlessness due to a hemothorax. The patient was successfully treated by surgical resections and arterial embolization. She has remained well for 4 years after the initial surgery.

Activated ADI pathway: the initiator of intermediate vancomycin resistance in Staphylococcus aureus.

Comparative proteomic profiling between 2 vancomycin-intermediate Staphylococcus aureus (VISA) strains, Mu50Ω-vraSm and Mu50Ω-vraSm-graRm, and vancomycin-susceptible S. aureus (VSSA) strain Mu50Ω revealed upregulated levels of catabolic ornithine carbamoyltransferase (ArcB) of the arginine catabolism pathway in VISA strains. Subsequent analyses showed that the VISA strains have higher levels of cellular ATP and ammonia, which are by-products of arginine catabolism, and displayed thicker cell walls. We postulate that elevated cytoplasmic ammonia and ATP molecules, resulting from activated arginine catabolism upon acquisition of vraS and graR mutations, are important requirements facilitating cell wall biosynthesis, thereby contributing to thickened cell wall and consequently reduced vancomycin susceptibility in VISA strains.