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The primary objectives of this study were to investigate the suitability of a 6-probe cocktail (caffeine, tolbutamide, omeprazole, dextromethorphan, midazolam, and digoxin) to be used as a tool for assessing the activity of drug metabolizing enzymes and transporters, and examine differences in the way drugs are handled among groups with different genetic regulation of these processes. This was a single-center, open-label, phase I clinical study involving 20 young, healthy Chinese volunteers (equal gender distribution). The subjects were administered a single, oral dose of the 6-probe cocktail and serum samples were collected to assess the disposition of the different probe substrates and produced metabolites. The serum samples were analyzed using ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry technology. The DNA samples were subjected to whole exome sequencing. Nineteen healthy volunteers completed the study. The 6-probe cocktail was safe and well-tolerated by all the subjects. The parent substrates and metabolites-caffeine (paraxanthine), dextromethorphan (dextrorphan), digoxin, midazolam (1-hydroxy-midazolam), omeprazole (5-hydroxy-omeprazole), and tolbutamide (4-hydroxy-tolbutamide)-were within the detectable window. Genetic variations known to alter drug metabolism (CYP2D6*10, CYP2C19*2, CYP2C19*3, and CYP2C9*3) were identified and generally correlated with phenotypic status. The 6-probe cocktail appeared to be suitable for assessing drug metabolizing activities. This, in conjunction with individual genetics, will pave the way for the implementation of personalized medicine in clinical practice. This will hopefully improve efficacy and reduce the incidence of adverse drug reactions.

Background Community-acquired pneumonia (CAP) is one of the most common infectious diseases and is a significant cause of mortality and morbidity globally. A microbial cause was not determined in a sizable percentage of patients with CAP; there are increasing data to suggest regional differences in bacterial aetiology. We devised a multiplex real-time PCR assay for detecting four microorganisms (Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae and Burkholderia pseudomallei) of relevance to CAP infections in Asia. Methods Analytical validation was accomplished using bacterial isolates (n=10–33 of each target organism for analytical sensitivity and n=117 for analytical sensitivity) and clinical validation using 58 culture-positive respiratory tract specimens. Results The qPCR assay exhibited 100% analytical sensitivity and analytical specificity, and 100% clinical sensitivity and 94–100% clinical specificity. The limit of detection and efficiency for the multiplex PCR assay were 3–33 CFU/mL and 93–110%, respectively. The results showed that the PCR-based method had higher sensitivity than traditional culture-based methods. The assay also demonstrated an ability to semiquantify bacterial loads. Conclusion We have devised a reliable laboratory-developed multiplex qPCR assay, with a turnaround time of within one working day, for detection of four clinically important CAP-associated microorganisms in Asia. The availability of a test with improved diagnostic capabilities potentially leads to an informed choice of antibiotic usage and appropriate management of the patient to achieve a better treatment outcome and financial savings.

Prolonged surgery is a known risk of pressure ulcer formation. Pressure ulcers affect the quality of life, are a significant cause of morbidity and mortality, and pose a burden on the healthcare system. This study aimed to compare the effectiveness of an alternating pressure (AP) overlay with Gel pad against the Gel pad in reducing interface pressure (IP) during prolonged surgery.A total of 180 participants from a tertiary hospital were randomized to AP overlay with Gel pad group (n = 90) and Gel pad group (n = 90). Patients were placed supine on the pressure redistributing surfaces, and IP data under the sacrum and ischial tuberosities were collected at an interval of 30 min from 0 min up to a maximum of 570 min.Based on data from 133 participants, the average IPs during all the deflation cycles of the AP overlay (with Gel pad) were significantly lower than the average continuous IP recorded for Gel pad throughout the measuring period (p 0.001). Only three patients (2.26% of study participants) - Gel pad group (n = 2; 2.99%) and AP overlay with Gel pad group (n = 1; 1.52%) developed post-operative pressure ulcer (p = 0.5687).The lower IP during deflation cycles of the AP overlay (with Gel pad) suggests its potential effectiveness in preventing pressure ulcer formation in patients undergoing prolonged surgery. The prevention and reduction of pressure ulcers will have a considerable impact on the improved quality of life and cost savings for the patient. The study findings may facilitate the formulation of policies for preventing pressure ulcer development in the perioperative setting.

Purpose Education in airway management is a fundamental component of anesthesiology training programs. There has been a shift towards the use of simulation models of higher fidelity for education in airway management. The goal of this study was to create a novel cadaveric model of a simulated parapharyngeal abscess with features of a difficult airway such as distorted anatomy and narrow airway passages presenting as stridor. The model was further assessed for its suitability for enhanced experiential learning in the management of difficult airways. Methods Cadaver heads were modified surgically to simulate parapharyngeal abscess. Airtight torso of the cadaver was connected to an Oxylog ventilator to simulate respiratory movements—the opening and closing of air channels with breaths in a patient with parapharyngeal abscess. Advanced airway workshop facilitators conducted directed one-to-one learning, and provided feedback to participants. A paper-based feedback was obtained from 72 participants on their confidence level, and the realism, attractiveness, beneficial, and difficulty levels of the simulated cadaveric models. Results The modified cadavers were reliable in simulating difficult airways. The majority of participants (91%) reported an increase in confidence level for management of the difficult airway after the experience with the modified cadavers and found the models realistic (93%), attractive (92%), beneficial (93%), and difficult (85%). Conclusions Surgical modifications of cadavers to simulate difficult airways such as parapharyngeal abscess with edema and stridor can be incorporated into advanced airway management courses to enhance experiential learning in airway management by awake fibreoptic intubation, and promote patient safety. Supplementary Information The online version contains supplementary material available at 10.1007/s00540-021-02904-0.

Purification of bacteria from human blood samples is essential for rapid identification of pathogens by molecular methods, enabling faster and more accurate diagnosis of bloodstream infection than conventional gold standard blood culture methods. The inertial microfluidic method has been broadly studied to isolate biological cells of interest in various biomedical applications due to its label-free and high-throughput advantages. However, because of the bacteria's tininess, which ranges from 0.5 μm to 3 μm, they are challenging to be effectively focused and sorted out in existing inertial microfluidic devices that work well with biological cells larger than 10 μm. Efforts have been made to sort bacterial cells by utilizing extremely small channel dimensions or employing a sheath flow, which thus results in limitations on the throughput and ease of operation. To overcome this challenge, we develop a method that integrates a non-Newtonian fluid with a novel channel design to allow bacteria to be successfully sorted from larger blood cells in a channel dimension of 120 μm × 20 μm without the use of sheath flows. The throughput of this device with four parallel channels is above 400 μL per minute. The real-time polymerase chain reaction (qPCR) analysis indicates that our inertial sorting approach has a nearly 3-fold improvement in pathogen recovery compared with the commonly used lysis-centrifugation method at pathogen abundances as low as 102 cfu mL-1. With the rapid and simple purification and enrichment of bacterial pathogens, the present inertial sorting method exhibits an ability to enhance the fast and accurate molecular diagnosis of bloodstream bacterial infection.

Introduction Failure of airway management remains a significant source of morbidity and mortality. Advanced airway management has been addressed effectively by simulation-based training. However, simulation of difficult airways in manikins is limited by the pre-set conditions provided by the manufacturer. Life-like conditions in the form of the softness of the tissue and true anatomy as seen in cadaver models are needed to create simulated models with a closer resemblance to real patients. The goal of this study was to determine the feasibility of simulating difficult airway from submandibular abscess in cadaver models by surgical modification of the cadaver heads for use in enhanced experiential learning of the management of difficult airways. Methods The cadaver heads were modified surgically to simulate a submandibular abscess. The models were used in an airway course where participants provided feedback on the realistic nature of the model and its benefits for difficult airway training. The ease of tracheal intubation of the models with the assistance of video laryngoscopy was assessed. Results The modified cadavers were acceptable in simulating difficult airway as demonstrated by the feedback from the participants. All participants (100% [95% confidence interval = 89.1%-100%]) found the models to be realistic and beneficial for difficult airway training. A good proportion (56.3%) felt that the intubation technique was made easier with the video laryngoscopy. Conclusions Cadavers can be modified to simulate pathologies associated with difficult airways. These models can be used to enhance experiential learning and the management of difficult airways.

Acute gastroenteritis (AGE) is caused by a wide range of pathogens. Culture methods for the detection of bacterial pathogens is time consuming and labour intensive. This study compared a same-day-to-result commercial molecular method using BD Max™ Enteric Bacterial Panel against conventional culture and laboratory-developed PCR assays (LDTs), and characterised the epidemiology of bacterial AGE in Singapore.PCRs for Campylobacter spp., Salmonella spp., Shigella spp./Enteroinvasive Escherichia coli (EIEC) and Shiga toxin-producing E. coli (STEC)/Shigella dysenteriae were performed on the BD Max™ platform. Concurrent routine bacterial culture ("reference standard") was performed for Campylobacter, Salmonella, Shigella, Vibrio and Aeromonas spp. In the event of a discrepancy, an "expanded reference standard" (bacterial culture with LDT) was used.There were 299 stool specimens in the study, with no bacterial pathogens detected in 190 samples (63.5%). The positive samples (n = 109,36.5%) were detected with Salmonella (n = 57,19.1%), Campylobacter (n = 28,9.4%), Vibrio parahaemolyticus (n = 6,2.0%), Shigella/EIEC (n = 6,2.0%), ETEC (n = 4,1.3%), STEC (n = 2,0.7%), Aeromonas (n = 2,0.7%), Plesiomonas shigelloides (n = 1,0.3%) and 3(1.0%) co-infections. Compared to the "expanded reference standard", conventional culture missed 38/112 (33.9%) pathogens. Conversely, testing by BD Max™ alone failed to detect 17 pathogens. BD Max™ reported seven (2.3%) false-positive results.BD Max™ increased the detection rate of bacterial AGE pathogens in the panel, but was limited by the absence of detection capability for Vibrio and Aeromonas spp.

Background: Breast cancer is the top cancer suffered by women worldwide and has been identified to be the greatest killer for women living in Singapore. Unfortunately, most of breast cancer cases were detected only at later stage of disease development. This has crippled the effort of breast cancer therapy. As early detection of breast cancer could greatly improve the outcome of breast cancer therapy, it is of utmost importance to identify relevant biomarkers at the primitive stage of breast cancer development before the transformation of normal breast cells into cancerous cells. These biomarkers provide important clues leading to an efficient and targeted treatment approach in breast cancer preventive care.Methods: 455 breast cancer patients were consented to join this study. Buccal swabs were collected for genotyping on CYP2B6*6, CYP2C19 *2 & CYP2C19*3. The genotyping data were then compared to data collected from healthy individuals. Clinical data were collected from patient notes and analysed. All the statistical analyses were done using SPSS statistical software, version 19.0. Chi-square or Fisher’s Exact test were performed to examine the difference between subject’s characteristics for categorical variables and One-Way Anova was performed to assess age difference across alleles of CYP2B6*6, CYP2C19*2 and CYP2C19*3. Binary logistics regression was performed to identify demographic factors associated with breast cancer.Results: We reported thatCYP2B6*6 could be a risk factor leading to earlier onset of breast cancer among Indian population with OR found to be 1.69 (95% C.I.= 0.549-5.191, p=0.359). In the case of CYP2C19*2, OR is 1.57 for Malay (95% C.I. = 0.696-3.522, p=0.278); 1.15 for Chinese population (95% C.I. =0.862-1.545, p=0.335) and 1.03 for Indian (95% C.I. =0.301-3.496, p=0.968). CYP2C19*3 OR in Chinese population is 1.34 (95% C.I. =0.830-2.155, p=0.231) and 0.77 (95% C.I. =0.172-3.394, p=0.724) for Malay population. No CYP2C19*3 was detected in both cohorts of Indian patients and healthy controls. Conclusions: CYP2B6*6 and CYP2C19*2 polymorphisms may confer a risk for breast cancer development in Singaporean breast cancer patients. This is an exploratory study to identify potential breast cancer susceptibility gene polymorphisms, a bigger sample size study could be done to corroborate these findings in future studies.

Background: Healthcare education models have recently shifted from the traditional reliance on the apprenticeship model, where trainees learn on real patients in actual clinical settings, to one based on simulation models. Education in airway management is a fundamental component of anesthetic training programs, and airway modification to simulate difficult airways increases the fidelity of airway management training. Objective: The study goal was to determine the feasibility of simulating difficult airways such as mandibular fracture and post-tonsillectomy bleed in cadaver models by surgical modification for the use in enhanced experiential learning of difficult airway management. Methods: Two cadaver heads were modified surgically to simulate a mandibular fracture and post-tonsillectomy bleed. Workshop facilitators conducted directed one-to-one learning and provided feedback to participants. A paper-based feedback was obtained from the participants on their confidence level, and the realism, attractiveness, beneficial levels, and difficulty levels of the simulation models used. Results: The modified cadavers were reliable in simulating difficult airways. The majority of participants (83.3% for fractured mandible and 87.1% for post-tonsillectomy bleed) reported an increase in confidence level for management of the difficult airway after the experience with the modified cadavers, and found both models realistic, attractive, and beneficial for difficult airway training. Conclusions: Surgical modifications of cadavers to simulate difficult airways such as fractured mandible and post-tonsillectomy bleed may be incorporated into advanced airway management courses to enhance experiential learning.

Introduction: Onychomycosis is a debilitating, difficult-to-treat nail fungal infection with increasing prevalence worldwide. The main etiological agents are dermatophytes, which are common causative pathogens in superficial fungal mycoses. Conventional detection methods such as fungal culture have low sensitivity and specificity and are time-consuming.Aim: The main objective of this study was to design, develop and validate a real-time probe-based multiplex qPCR assay for the detection of dermatophytes and Fusarium species.Methodology: The performance characteristics of the qPCR assays were evaluated. The multiplex qPCR assays targeted four genes (assay 1: pan-dermatophytes/Fusarium spp.; assay 2: Trichophyton rubrum/Microsporum spp.). Analytical validation was accomplished using 150 fungal isolates and clinical validation was done on 204 nail specimens. The performance parameters were compared against the gold standard (fungal culture) and expanded gold standard (culture in conjunction with sequencing).Results: Both the single-plex and multiplex qPCR assays performed well especially when compared against the expanded gold standard. Among the 204 tested nail specimens, the culture method showed that 125 (61.3 %) were infected with at least one organism, of which 40 yielded positive results for dermatophytes and Fusarium spp. These target organisms detected include 20 dermatophytes and 22 Fusarium spp. The developed qPCR assays demonstrated excellent limit of detection, efficiency, coefficient of determination, analytical and clinical sensitivity and specificity.Conclusion: The multiplex qPCR assays were reliable for the diagnosis of onychomycosis, with shorter turn-around time as compared to culture method. This aids in the planning of treatment strategies to achieve optimal therapeutic outcome.

Background and Aim Perturbance in the composition of human gut microbiota has been associated with metabolic disorders such as obesity, diabetes mellitus, and insulin resistance. The objectives of this study are to examine the effects of ethnicity, central obesity, and recorded dietary components on potentially influencing the human gut microbiome. We hypothesize that these factors have an influence on the composition of the gut microbiome. Methods Subjects of Chinese (n = 14), Malay (n = 10), and Indian (n = 11) ancestry, with a median age of 39 years (range: 22–70 years old), provided stool samples for gut microbiome profiling using 16S rRNA sequencing and completed a dietary questionnaire. The serum samples were assayed for a panel of biomarkers (interleukin‐6, tumor necrosis factor alpha, adiponectin, cleaved cytokeratin 18, lipopolysaccharide‐binding protein, and limulus amebocyte lysate). Central obesity was defined by waist circumference cut‐off values for Asians. Results There were no significant differences in Shannon alpha diversity for ethnicity and central obesity and no associations between levels of inflammatory cytokines and obesity. The relative abundances of Anaerofilum (P = 0.02), Gemellaceae (P = 0.02), Streptococcaceae (P = 0.03), and Rikenellaceae (P = 0.04) were significantly lower in the obese group. From principle coordinate analysis, the effects of the intake of fiber and fat/saturated fat were in contrast with each other, with clustering of obese individuals leaning toward fiber. Conclusion The study demonstrated that there were differences in the gut microbiome in obese individuals. Certain bacterial taxa were present in lower abundance in the group with central obesity. Fiber and fat/saturated fat diets were not the key determinants of central obesity., The objectives of this study are to examine the effects of ethnicity, central obesity, and dietary components on the human gut microbiome. Asian subjects (n = 35) provided stool samples for gut microbiome profiling using 16S rRNA sequencing and completed a dietary questionnaire. The relative abundances of Anaerofilum (P = 0.02), Gemellaceae (P = 0.02), Streptococcaceae (P = 0.03), and Rikenellaceae (P = 0.04) were significantly lower in the obese group.

The use of total intravenous anaesthesia (TIVA) is limited by concerns of disconnections of the tubing, resulting in accidental awareness. We designed a sensor device to detect leakages at the patient end and notify the medical personnel, thereby allowing immediate intervention in preventing awareness. For moisture detection, resistive sensing was selected as the working principle. The prototype was in proximity to the tubing from the TIVA pump and the patient���s intravenous cannula, and able to detect leakages in all potential leakage sites and activate an alarm. Our device consists of a disposable bandage (sensor), attached to a reusable clamp that is directly coupled to a central module (SparkFun MicroView, a small microcontroller with built-in Organic Light-Emitting Diode (OLED) display). The disposable bandage is wrapped around the possible leakage sites. Crucially, the disposable bandage is integrated with two separate moisture sensing threads. When moisture is present, the central module detects a drop in resistance across the moisture sensing threads and activates a flashing LED and buzzer. We have successfully created a functional leak detection device, comprising a moisture sensing bandage and an audio and visual alert system, to address the problem of undetected TIVA leakages at the patient end.

Inertial microfluidics has been proven to be a powerful tool for high-throughput, size-based cell sorting in diverse biomedical applications. In the case of

Purpose The emergence of antiseptic resistance and/or antiseptic-resistance genes in methicillin-resistant Staphylococcus aureus (MRSA) may result in failure of decolonization treatments. Plasmid-encoded efflux pump genes qacA/B and qacC (smr) confer tolerance to chlorhexidine and quaternary ammonium compounds. The objective of this study was to develop and validate a multiplex real time-PCR assay for detection of antiseptic-resistance genes, apply the assay on 200 MRSA isolates and explore if carriage of these genes was associated with resistance to topical antibiotics. Methodology A SYBR-Green based multiplex real time-PCR assay was developed to detect qacA/B, qacC, and mecA (internal control) simultaneously. The multiplex assay was compared against conventional single-plex PCR followed by agarose gel electrophoresis , using DNA from the first 73 MRSA isolates, followed by multiplex testing of the remaining 127 MRSA isolates. All 200 MRSA isolates were tested for susceptibility to mupirocin , retapamulin , neomycin , bacitracin and octenidine. The genetic diversity of the isolates was investigated by s pa-typing. Results The concordance between multiplex and conventional PCR, in assignments of qacA/B and qacC status were 99%(72/73) and 100%(73/73) respectively. Among 200 MRSA isolates, 48(24%) and 44(23%) were found to harbour qacA/B and qacC genes, respectively. These isolates remained susceptible to many common decolonization agents, except mupirocin. The predominant spa-types were t020 and t1081 (41 and 32 isolates respectively). Conclusion The real-time assay performed acceptably for the detection of qac genes. A high prevalence of antiseptic-resistance genes were detected in the MRSA isolates in our population and appeared to be associated with spa-type t1081.

Highlights • Performance of FilmArray, AnyplexII, and Argene platforms was comparable. • Nasopharyngeal swabs in UTM and endotracheal aspirates are suitable sample types. • Test volume is the main determinant for the selection of platform. • FilmArray is most automated, of lowest-throughput and has highest reagent cost., Background Respiratory infections are common reasons for hospital admission, and are associated with enormous economic burden due to significant morbidity and mortality. The wide spectrum of microbial agents underlying the pathology renders the diagnosis of respiratory infections challenging. Molecular diagnostics offer an advantage to the current serological and culture-based methods in terms of sensitivity, coverage, hands-on time, and time to results. Objectives This study aimed to compare the clinical performance of three commercial kits for respiratory viral detection. Study design The performance of FilmArray Respiratory Panel, AnyplexII RV16, and Argene was compared using clinical respiratory samples (n = 224, comprising 189 nasopharyngeal swabs in Universal Transport Medium (UTM) and 35 endotracheal aspirates), based on common overlapping targets across the platforms. Influenza A “equivocal” and “no-subtype” samples by FilmArray were further compared to a laboratory-developed Influenza A/B test. Results and Conclusions The overall performance of all three platforms appeared to be comparable with regards to sensitivities (95.8–97.9%) and specificities (96.1–98.0%), detection of coinfections, and distinguishment of influenza from non-influenza cases. “Equivocal” and “no-subtype” samples by FilmArray mostly represented weak Influenza A by laboratory-developed test. Lower respiratory tract samples had comparable final-run success-rates and discordant-rates as compared to UTM. Coronavirus HKU1, which was not targeted by AnyplexII RV16, were detected as OC43. The expected test volume would be the main determinant for the selection of platform. Among the platforms, the FilmArray is the most automated but is of the lowest-throughput and has the highest reagent cost.

Given increasing antimicrobial resistance, we aimed to determine antibiotic susceptibility and presence of resistance genes in uropathogens in primary care, factors associated with resistance to commonly prescribed antibiotics, and effect of treatment on early symptom resolution. We conducted a prospective study of primary care patients with urinary tract infection (UTI) symptoms and culture-confirmed UTI in Singapore from 2015 to 2016., Given increasing antimicrobial resistance, we aimed to determine antibiotic susceptibility and presence of resistance genes in uropathogens in primary care, factors associated with resistance to commonly prescribed antibiotics, and effect of treatment on early symptom resolution. We conducted a prospective study of primary care patients with urinary tract infection (UTI) symptoms and culture-confirmed UTI in Singapore from 2015 to 2016. Cohort characteristics and antimicrobial susceptibility of cultured isolates were analyzed. Among Enterobacteriaceae isolates, early symptom resolution (within 3 days) according to antibiotic prescribed and isolate susceptibility and factors associated with antibiotic resistance were evaluated. Of 695 symptomatic patients, 299 were urine culture positive; of these 299 patients, 259 (87%) were female. Escherichia coli was the most common uropathogen (76%). Enterobacteriaceae isolates (n = 283) were highly susceptible to amoxicillin-clavulanate (86%), nitrofurantoin (87%), and fosfomycin (98%), but >20% were resistant to ciprofloxacin and co-trimoxazole. Isolates resistant to appropriate indicator antibiotics were further tested to determine proportions positive for blaCTX-M (14/26, 54%), plasmid-mediated ampC (12/24, 50%), qnr (7/69, 10%), and fos (1/6, 17%) resistance genes. A total of 67% of patients given antibiotics with susceptible isolates reported early resolution versus 45% given antibiotics with nonsusceptible isolates (P = 0.001) and 27% not treated (P = 0.018). On multivariable analysis, Indian ethnicity and diabetes mellitus were associated with amoxicillin-clavulanate resistance. Genitourinary abnormalities, UTI in the past 12 months, and hospitalization in the past 6 months were associated with ciprofloxacin and co-trimoxazole resistance. Patients given active empirical antibiotics were most likely to report early symptom resolution, but correlation with in vitro susceptibility was imperfect. Factors associated with resistance may guide the decision to obtain initial urine culture.

BACKGROUND & AIMS We compared the effects of weight loss induced with the glucagon-like peptide-1 agonist liraglutide, with that of lifestyle modification, followed by weight maintenance after discontinuing intervention, in obese adults with non-alcoholic fatty liver disease (NAFLD). METHODS Thirty obese (mean age 40.7 ± 9.1 years, BMI 33.2 ± 3.6 kg/m2 , 90% male) adults with NAFLD defined as liver fat fraction (LFF) > 5% on magnetic resonance imaging without other causes of hepatic steatosis were randomized to a supervised programme of energy restriction plus moderate-intensity exercise to induce ≥ 5% weight loss (DE group, n = 15), or liraglutide 3 mg daily (LI group, n = 15) for 26 weeks, followed by 26 weeks with only advice to prevent weight regain. RESULTS Diet and exercise and LI groups had significant (P

Drug transporters are differentially expressed in many polarized tissues. The varied distribution and expression of transporters determines the net transcellular transport and influences the disposition of many clinically used drugs. ATP-binding cassette (ABC) and Solute Carrier (SLC) transporters interact dynamically to mediate the passage of drugs across cells. The variable expression of drug transporters could be attributed to genetic and non-genetic factors, which accounts for the differences in drug response among individuals, in terms of both efficacy and adverse effects.The authors provide the background of ABC and SLC transporters, and highlight the fact that their expression is cell-specific and the study of a transporter in isolation is not an adequate measure of its function. The technologies and approaches to characterize the function of transporters, as well as the genetic and non-genetic factors underlying their variable expression in specific cells and among individuals were reviewed.Many studies have utilized tissue homogenization techniques to isolate mRNA for quantifying transporter expression levels. We highlight that transporter expression is cell-specific and mRNA expression does not always reflect its total functionality. In addition, transporter expression in immortalized cell lines may not mirror its expression in the target tissue site.

Background Brugada syndrome (BrS) is an inherited primary arrhythmia disorder leading to sudden cardiac arrest. SCN 5A , encoding the α‐subunit of the cardiac sodium channel (Nav1.5), is the most common pathogenic gene of BrS. An implantable cardioverter defibrillator ( ICD ) is the standard treatment for secondary prevention. This study aimed to evaluate association of the SCN 5A variant with this cardiac conduction disturbance and appropriate ICD shock therapy in Thai symptomatic BrS patients with ICD implants. Methods and Results Symptomatic BrS patients diagnosed at university hospital were enrolled from 2008 to 2011. The primary outcome of the study was an appropriate ICD shock defined as having non‐pacing‐associated ICD shock after the occurrence of ventricular tachycardia or ventricular fibrillation. Associations between SCN 5A polymorphisms, cardiac conduction disturbance, and potential confounding factors associated with appropriate ICD shock therapy were analyzed. All 40 symptomatic BrS patients (median age, 43 years) with ICD implantations were followed for 24 months. There were 16 patients (40%) who had the appropriate ICD shock therapy after ICD treatment. An independent factor associated with appropriate ICD shock therapy was SCN 5A‐ R1193Q with an adjusted hazard ratio of 10.550 (95% CI , 1.631–68.232). Conclusions SCN 5A‐ R1193Q is associated with cardiac conduction disturbances. It may be a genetic marker associated with ventricular arrhythmia leading to appropriate ICD shock therapy in symptomatic BrS patients with ICD treatment. Because of the small sample size of study population and the appropriate ICD shock outcome, further large studies are needed to confirm the results of this study.

The major histocompatibility complex (MHC) containing the classical human leukocyte antigen (HLA) Class I and Class II genes is among the most polymorphic and diverse regions in the human genome. Despite the clinical importance of identifying the HLA types, very few databases jointly characterize densely genotyped single nucleotide polymorphisms (SNPs) and HLA alleles in the same samples. To date, the HapMap presents the only public resource that provides a SNP reference panel for predicting HLA alleles, constructed with four collections of individuals of north-western European, northern Han Chinese, cosmopolitan Japanese and Yoruba Nigerian ancestry. Owing to complex patterns of linkage disequilibrium in this region, it is unclear whether the HapMap reference panels can be appropriately utilized for other populations. Here, we describe a public resource for the Singapore Genome Variation Project with: (i) dense genotyping across ∼ 9000 SNPs in the MHC; (ii) four-digit HLA typing for eight Class I and Class II loci, in 96 southern Han Chinese, 89 Southeast Asian Malays and 83 Tamil Indians. This resource provides population estimates of the frequencies of HLA alleles at these eight loci in the three population groups, particularly for HLA-DPA1 and HLA-DPB1 that were not assayed in HapMap. Comparing between population-specific reference panels and a cosmopolitan panel created from all four HapMap populations, we demonstrate that more accurate imputation is obtained with population-specific panels than with the cosmopolitan panel, especially for the Malays and Indians but even when imputing between northern and southern Han Chinese. As with SNP imputation, common HLA alleles were imputed with greater accuracy than low-frequency variants.

Novel organic cation transporter 2 (OCTN2) is a multispecific, bidirectional, pH-dependent organic cation transporter. It can function as a carnitine co-transporter with higher affinity for carnitine than OCTN1 but also functions as a uniporter for other cations. Drugs such as verapamil, pyrilamine and beta-lactam antibiotics have been characterized as substrates of OCTN2 and/or inhibitors of carnitine transport. This study identified variants of the SLC22A5 gene in two distinct ethnic groups of the Singaporean population (n=192) by DNA sequencing. Twenty-eight genetic variants of SLC22A5, including 13 that were novel, were found: 14 were located in the coding exons, 10 in the introns, 1 in the promoter region, 2 in the 5'-untranslated region and 1 in the 3'-untranslated region. Among the novel nonsynonymous variants, Asp122Tyr was predicted to be functionally significant. Functional nonsynonymous variants detected include Ser467Cys and Arg254X; the latter resulted in a premature stop codon and is predicted to result in a truncated protein that is less than half the molecular mass of wild-type OCTN2. These data constitute fundamental information of value for future pharmacogenetic studies in Asian populations on drugs that are substrates of OCTN2.

Ribavirin is found to be absorbed in the intestine through the human concentrative nucleoside transporter 2 (hCNT2). Cellular uptake of ribavirin was strongly inhibited by purine nucleoside in an in vitro study. This study aims to examine the effects of dietary purine on the pharmacokinetics of orally administered ribavirin in vivo. Twenty healthy participants were enrolled in a randomized, 2-period crossover study. Participants were administered a single 600-mg oral dose of ribavirin after either a high-purine meal or a low-purine meal. Serial blood samples were collected predose and over 144 hours after dosing. Ribavirin concentrations were measured by liquid chromatography/tandem mass spectrometry. In comparison with corresponding plasma values of ribavirin following a high-purine meal, C(max), AUC(0-144) and AUC(0-infinity) of ribavirin following a low-purine meal were 136% (90% confidence internal [CI]: 120%-155%), 134% (90% CI: 118%-153%), and 139% (90% CI: 120%-159%), respectively. This study indicates that dietary purines have an effect on ribavirin absorption. Dosage regimens of ribavirin might need to be adjusted according to the purine content of the meal.

The novel organic cation transporter 1 (OCTN1) is a multispecific, bidirectional and pH-dependent organic cation transporter with low carnitine transport activity. It is a transporter of the physiological substance ergothioneine and mediates the transport of a variety of organic cations such as tetraethylammonium, pyrilamine and quinidine. This study identifies genetic variations of the SLC22A4 gene in two distinct ethnic groups of the Singaporean population (n=192) by DNA sequencing. Twenty four genetic variants of SLC22A4, including 14 found to be novel. 16 in the coding exons (10 nonsynonymous and 6 synonymous variations) and 8 in the introns. Among the novel nonsynonymous variations, Arg63His, Arg83Pro, Met344Lys and Ile500Asn were predicted to be functionally significant. These data should provide fundamental and useful information for pharmacogenetic studies on drugs that are substrates of OCTN1 in Asians.

MRP2 is a drug transporter that is responsible for the gastrointestinal absorption and biliary excretion of a wide variety of endogenous and xenobiotic compounds, including many clinically used drugs. This study aims to identify genetic variations of ABCC2 gene in three distinct ethnic groups of the Singaporean population (n = 288). The coding region of the gene encoding the transporter protein was screened for genetic variations in the study population by denaturing high-performance liquid chromatography and DNA sequencing. Twenty-two genetic variations of ABCC2, including 8 novel ones, were found: 1 in the 5' untranslated region, 10 in the coding exons (8 nonsynonymous and 2 synonymous variations), and 11 in the introns. Three novel nonsynonymous variations: 2686G > A (Glu896Lys), 4240C > T (His1414Tyr) and 4568A > C (Gln1523Pro) were detected in single heterozygous Malay, Chinese, and Indian subjects, respectively. Among the novel nonsynonymous variations, 4240C > T and 4568A > C were predicted to be functionally significant. These data would provide fundamental and useful information for pharmacogenetic studies on drugs that are substrates of MRP2 in Asians.

OATP1B1 is a liver-specific transporter that mediates the uptake of various endogenous and exogenous compounds including many clinically used drugs from blood into hepatocytes. This study aims to identify genetic variations of SLCO1B1 gene in three distinct ethnic groups of the Singaporean population (n=288). The coding region of the gene encoding the transporter protein was screened for genetic variations in the study population by denaturing high-performance liquid chromatography and DNA sequencing. Twenty-five genetic variations of SLCO1B1, including 10 novel ones, were found: 13 in the coding exons (9 nonsynonymous and 4 synonymous variations), 6 in the introns, and 6 in the 3' untranslated region. Four novel nonsynonymous variations: 633AG (Ile211Met), 875CT (Ala292Val), 1837TC (Cys613Arg), and 1877TA (Leu626Stop) were detected as heterozygotes. Among the novel nonsynonymous variations, 633AG, 1837TC, and 1877TA were predicted to be functionally significant. These data would provide fundamental and useful information for pharmacogenetic studies on drugs that are substrates of OATP1B1 in Asians.

Introduction: Long QT syndrome (LQTS), an inherited cardiac arrhythmia, is a disorder of ventricular repolarisation characterised by electrocardiographic abnormalities and the onset of torsades de pointes leading to syncope and sudden death. Genetic polymorphisms in 5 wellcharacterised cardiac ion channel genes have been identified to be responsible for the disorder. The aim of this study is to identify disease-causing mutations in these candidate genes in a LQTS family. Materials and Methods: The present study systematically screens the coding region of the LQTS-associated genes (KCNQ1, HERG, KCNE1, KCNE2 and SCN5A) for mutations using DNA sequencing analysis. Results: The mutational analysis revealed 7 synonymous and 2 nonsynonymous polymorphisms in the 5 ion channel genes screened. Conclusion: We did not identify any clear identifiable genetic marker causative of LQTS, suggesting the existence of LQTSassociated genes awaiting discovery. Key words: Arrhythmia, Ion channels, Long QT syndrome

What is already known about this subject • Genetic variability of the FcγRIIIa 176 F/V polymorphism has been widely studied in patients with systemic lupus erythomatosus and rheumatoid arthritis, as well as the general population of various White groups. • Its implications in disease pathogenesis and response to therapeutics have been well documented. • This study aimed to profile its polymorphism pattern in the Asian population, thus it serves as useful reference controls in disease association studies and tailoring therapy to ethnic-specific populations. What this study adds • In this study, we have established the genetic variability profile of the FcγRIIIa 176 F/V polymorphism in three distinct Asian groups (Chinese, Malays and Indians) and also supplemented existing data based on ethnic-specific healthy controls. • The polymorphism pattern of Malays was found to differ significantly from Chinese and Indians, which was also extended to almost all other healthy controls of various ethnic groups published elsewhere. • Such differences could have implications in disease susceptibility and pathogenesis, as well as response to drug therapeutics. Aims To determine and compare the distribution of the FcγRIIIa 176 F/V polymorphism across three ethnically distinct populations (Chinese, Asian Indians and Malays) in Singapore. Methods The FcγRIIIa 176 F/V polymorphism was genotyped by direct sequencing from genomic DNA samples obtained from normal healthy Chinese, Asian Indians and Malays (n = 192 from each population). Results The allelic frequencies of the high binding affinity FcγRIIIa 176 V allele for Chinese, Asian Indians and Malays were 35%, 33% and 46%, respectively (F allele frequencies were 65%, 67% and 54%, respectively). Genotype distributions were found to conform to the Hardy–Weinberg law (P > 0.05) in each group. χ2 comparisons revealed significant differences in the genotype distributions of the FcγRIIIa 176 V/F polymorphism of Malays from the other two populations (Chinese and Asian Indians). However, no significant difference in the genotype distributions of the FcγRIIIa 176 V/F polymorphism was observed between Chinese and Asian Indian populations. Conclusions The genotype distributions of the FcγRIIIa 176 V/F polymorphism in healthy Malays are significantly different from both Chinese and Indians. These observations provide the fundamentals on which future disease associations may be built and also present important implications for the design of therapeutic regimens amongst various ethnic groups.

We analyzed the coding regions of the cardiac calcium-handling genes, ryanodine receptor 2 (RyR2) and calsequestrin 2 (CASQ2) for genetic variants in a healthy Chinese population (n = 95) and in a cohort of 28 sudden unexplained death victims. Mutations in RyR2 and CASQ2 have been shown to alter calcium homeostasis during excitation–contraction coupling and predispose individuals to fatal cardiac arrhythmias. The genetic screening was accomplished by denaturing high-performance liquid chromatography and DNA sequencing methods. Genetic analysis revealed the following nonsynonymous genetic variations: two reported RyR2 polymorphisms; 5654G>A (G1885E) and 5656G>A (G1886S), two reported CASQ2 polymorphisms; 196A>G (T66A) and 226G>A (V76M) and one novel CASQ2 mutation; 529G>C (E177Q). The functional significance of the novel CASQ2 mutation has not been evaluated and characterized. This study shows that multiple genetic variations of the RyR2 and CASQ2 genes exist in the two study populations. The inter-individual genetic variability may underlie the different susceptibility of individuals to developing ventricular tachycardia. The research results will be valuable for which future work involving clinical and forensic samples can be based upon to distinguish potential disease-associated mutations from common polymorphisms.

Drug resistance is a major hurdle to the success of chemotherapy. The permeability glycoprotein (P-gp) is an important factor dictating drug access to the cells, as it controls the efflux of chemotherapeutic agents against the concentration gradient. Pmd1, a P-gp-like protein, was recently isolated as a doxorubicin resistance gene in fission yeast. Although the null mutant of pmd1 (Δ pmd1) exhibited sensitivity to doxorubicin, it showed an unexpectedly high resistance to the drug at relatively high concentrations. The data presented here suggest that this is due to the presence of cooperative processes that can complement and counteract drug cytotoxicity in the absence of Pmd1. One such factor, Rav1, is an essential factor in controlling the assembly of the pH-regulating transporter vacuolar-ATPase (V-ATPase) in fission yeast. The simultaneous disruption of Pmd1 and Rav1 resulted in a prominent accumulation of doxorubicin in the cytoplasm of cells, accompanied by a decline in cell viability. With concurrent treatment of pharmacological inhibitors in human cervical cancer cells, P-gp and V-ATPase were further shown to act synergistically to sensitize cells to doxorubicin also in the human cells. Furthermore, a novel Cornichon-like protein SPAC2C4.05 (herein named as Cor1) was demonstrated for the first time to be involved in the interaction with P-gp and V-ATPase to counteract doxorubicin-dependent cytotoxicity. Therefore this study identified a molecular cooperation between multiple membrane transporter proteins that confers chemoresistance to cells against the chemical insult of doxorubicin. Interestingly, this network exhibited differential effects to doxorubicin as compared with its close epimeric analog epirubicin, suggestive of the intricacy of the drug response regulated by this synergistic interaction. A model is discussed on how the versatility of this network can differentiate closely related chemical drug structures yet allow for the robustness to counteract a vast range of drugs.

Atrial fibrillation (AF) is the most common sustained arrhythmia with poor prognosis, reaching epidemic proportions in the ageing population. The condition affects more than five million people worldwide and is a significant clinical problem. Of particular interest is a subset of younger patients (

The sudden unexplained death syndrome is a tragic and distressing event, which may arise due to an underlying fatal cardiac arrhythmia, which may be associated with a defective ion channel. These electrical abnormalities of the heart do not present with gross structural changes, rendering it a challenging task to the forensic pathologist in ascertaining the cause of death during autopsies. The attempts to identify genetic variations in the key ion channels known to cause arrhythmias have only been able to reveal the presence of putative mutations in a small percentage of the cases. It is thus imperative to look beyond the discovery of single-nucleotide polymorphisms to other forms of genetic variations (such as copy number variations and structural rearrangements), as well as alternative mechanisms that may underlie the molecular pathogenesis of arrhythmias and electrocardiographic abnormalities causing sudden death. Additionally, the various technological platforms for genetic analysis are also described in this article. Key Concepts: Sudden unexplained death syndrome is an important clinical problem. Sudden death may be the result of a fatal cardiac arrhythmia. Arrhythmias are associated with genetic mutations in ion channel genes. Genetic mutations often exhibit incomplete penetrance. Genetic mutations may have functional impact on the ion channel. Single-nucleotide polymorphisms only account for a fraction of the cases. Copy number variations and chromosomal aberrations may play a role in sudden death pathology. The role of alternative mechanisms such as epigenetics in arrhythmias/sudden death remains to be ascertained. The technology for identifying genetic variations is advancing rapidly. Genome-wide sequencing is expected to become increasingly feasible. Keywords: sudden unexplained death syndrome; arrhythmias; genetic variants; ion channels; genome-wide association studies; candidate gene sequencing; next-generation sequencing

The human concentrative nucleoside transporter 2 (hCNT2) plays a major role in the intestinal absorption of naturally occurring nucleosides as well as some nucleoside analog drugs. To determine if single nucleotide polymorphisms (SNPs) in the promoter region of hCNT2 affect gene expression, we examined approximately 1 kb upstream the hCNT2 transcription start site. Ninety Chinese samples were screened and seven SNPs were identified: -115T>G, -146T>A, -264A>G, -564G>A, -861A>C, -880T>C and -906C>T. Based on these seven variants and their relative positions, eight haplotypes were identified using PHASE v2.1.1. Three naturally occurring haplotypes were cloned into the pGL3-Basic vector and transfected into HEK293 cells. Dual luciferase assay revealed that haplotype 4 (GTAGACC) and 7 (GAGAACT) exhibited significantly lower expression levels compared to the published haplotype 1 (TTAGATC). Results from our in-vitro study showed that the hCNT2 promoter region haplotype may modulate gene expression and cause different drug responses.

Sudden cardiac death (SCD) can be caused by a number of reasons. Previous works have identified the genetic causes, such as alterations in the DNA sequence, for many of these diseases. We hypothesize that some patients may show genomic imbalances and changes in the gene copy number leading to genetic instability. To clarify this, we analysed DNA samples from SCD victims using comparative genomic hybridization (CGH), a molecular cytogenetic technique that permits the genome-wide screening of chromosomal imbalances, and telomere length measurement. DNA derived from peripheral blood and heart tissue of 14 SCD cases and six apparently healthy control individuals were subjected to CGH analysis. Telomere length measurements were done by the Southern blotting method. Eight out of 14 SCD cases exhibited changes in DNA/gene copy number. CGH analysis showed variation in the gene copy number of some of the genes associated with potassium (KCNAB1, KCNH2, and KCNA4) and calcium (RyR2, ATP2A2) ions which are involved in maintaining the ionic balance of the heart. Alterations in TERC and TERT genes were also detected in SCD victims. In nine SCD victims shorter telomeres were detected. This might have resulted from excessive cellular proliferation and/or oxidative stress in these individuals. Copy number changes observed and telomere shortening detected in SCD cases would possibly explain at least some of the causes of SCD at early ages in humans. Identification of biomarkers of SCD is of great importance and thus the present study will facilitate the identification of some of the biomarkers.

The human concentrative nucleoside transporter (hCNT2), also known as SLC28A2, plays an important role in the cellular uptake across intestinal membrane of some naturally occurring nucleosides and nucleoside analogs. This study aims to determine the genetic variability of hCNT2 (SLC28A2) in three major Asian ethnic groups residing in Singapore: Chinese, Malay and Indian, and functionally characterize the variants of hCNT2. Healthy participants (n=96) from each group were screened for genetic variations in the exons of hCNT2 (SLC28A2) using denaturing high performance liquid chromatography and sequencing analyses. A total of 23 polymorphisms were identified in the exonic and flanking intronic regions, and ethnic differences in single nucleotide polymorphism frequencies were evident. Five novel nonsynonymous variants (L12R, R142H, E172D, E385K, M612T) were constructed by mutagenesis and functionally characterized in U-251 cells. Expression of these variants in U-251 cells revealed that all except E385K can uptake various substrates of hCNT2: inosine, ribavirin and uridine.

1. Pharmacogenetics refers to the study of genetically controlled variations in drug response. Functional variants caused by single nucleotide polymorphisms (SNPs) in genes encoding drug-metabolising enzymes, transporters, ion channels and drug receptors have been known to be associated with interindividual and interethnic variation in drug response. Genetic variations in these genes play a role in influencing the efficacy and toxicity of medications. 2. Rapid, precise and cost-effective high-throughput technological platforms are essential for performing large-scale mutational analysis of genetic markers involved in the aetiology of variable responses to drug therapy. 3. The application of a pharmacogenetics approach to therapeutics in general clinical practice is still far from being achieved today owing to various constraints, such as limited accessibility of technology, inadequate knowledge, ambiguity of the role of variants and ethical concerns. 4. Drug actions are determined by the interplay of several genes encoding different proteins involved in various biochemical pathways. With rapidly emerging SNP discovery technological platforms and widespread knowledge on the role of SNPs in disease susceptibility and variability in drug response, the pharmacogenetics approach to therapeutics is anticipated to take off in the not-too-distant future. This will present profound clinical, economic and social implications for health care.

Aims To determine the genetic variability of long QT syndrome (LQTS)-associated genes (KCNQ1, HERG, KCNE1 and KCNE2) among three distinct ethnic groups in the Singapore population. Methods Genomic DNA samples from up to 265 normal healthy Chinese, 118 Malay and 139 Indian volunteer subjects were screened for genetic variations in the coding region of the LQTS-associated genes using denaturing high-performance liquid chromatography and sequencing analyses. Results In total, 37 single nucleotide polymorphisms (SNPs) were identified in the coding exons of the LQTS-associated potassium ion channel genes, seven of which were novel nonsynonymous polymorphisms. SNPs 356GA (exon 1 of KCNQ1), 2624CT and 2893GA (exon 11 of HERG), 3164GA, 3322CG and 3460GA (exon 14 of HERG), and 79CT (exon 3 of KCNE2) resulted in Gly119Asp, Thr875Met, Gly965Arg, Arg1055Gln, Leu1108Val, Gly1154Ser and Arg27Cys amino acid substitutions, respectively. In addition, 16 intronic variants were detected. The functional consequence of these variants has not been studied and their association with risk of LQTS is unclear. Conclusions There exist multiple genetic polymorphisms of the LQTS-associated genes in the three distinct Asian populations. Though the functional significance of many of these SNPs is unknown, this interindividual and interethnic genetic variability may underlie the different susceptibilities of individuals to developing LQTS.