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  1. Loh GOK, Wong EYL, Tan YTF, Lee YL, Pang LH, Chin MC, et al.
    PMID: 32905988 DOI: 10.1016/j.jchromb.2020.122337
    A simple, rapid, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method was developed to determine sitagliptin in human plasma. Diphenhydramine HCl was used as internal standard (IS). The chromatographic separation was achieved using Agilent Poroshell 120 EC-C18 - Fast LC column (100 × 2.1mmID, 2.7) fitted with UHPLC Guard Poroshell 120 EC-C18 (5 × 2.1mmID, 2.7 µm). The mobile phase consisted of 0.1% v/v formic acid and methanol (45:55, v/v) run at a flow rate of 0.45 mL/min at 30 °C. Methanol produced relatively cleaner plasma sample as deproteinization agent. Polytetrafluoroethylene membrane was preferred over nylon membrane as the former produced clear plasma samples. The standard calibration curve was linear over the concentration range of 5-500.03 ng/mL. The within-run precision was 0.53-7.12% and accuracy 87.09-105.05%. The between-run precision was 4.74-11.68% and accuracy 95.02-97.36%. The extended run precision was 3.60-6.88% and accuracy 93.18-95.82%. The recovery of analyte and IS was consistent. Sitagliptin in plasma was stable at benchtop (short term) for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h (post-preparative), after 7 freeze-thaw cycles (-20 ± 10 °C), and 62 days in the freezer (-20 ± 10 °C). Both sitagliptin (analyte) and IS stock solutions were stable for 62 days when kept at room temperature (25 ± 4 °C) and in chiller (2-8 °C). The validated method was successfully applied to a bioequivalence study of two sitagliptin formulations involving 26 healthy Malaysian volunteers.
    Matched MeSH terms: Chromatography, High Pressure Liquid/methods*; Tandem Mass Spectrometry/methods*
  2. Loh GOK, Wong EYL, Tan YTF, Ong LM, Ng RS, Wee HC, et al.
    PMID: 33429127 DOI: 10.1016/j.jchromb.2020.122517
    A simple, fast and sensitive LC-MS/MS method was developed to quantify terazosin in human plasma. The mobile phase consisted of acetonitrile-0.1% (v/v) formic acid (70:30, v/v). Prazosin was used as internal standard (IS). As deproteinization agent, acetonitrile produced a clean sample. A higher response intensity with more symmetrical peak was obtained using Agilent Poroshell 120 EC-C18 - Fast LC column (100 × 2.1mmID, 2.7 μm) compared with Kinetex XB-C18 (100 × 2.1 mm, 2.6 µm) column. The response of terazosin and IS were approximately two times in citrate phosphate dextrose (CPD) plasma compared with dipotassium ethylenediaminetetraacetic acid (K2EDTA) plasma. Plasma calibration curve was linear from 1.0 to 100.0 ng/mL, with coefficient of determination r2 ≥ 0.99. The within-run and between-run precision values (CV, %) were <5.2% and <7.8%, while accuracy values were 102.8-112.7% and 103.4-112.2%. The extended run accuracy was 98.6-102.8% and precision (CV, %) 4.3-10.4%. The recovery of analyte was >98% and IS >94%. Terazosin in plasma kept at benchtop was stable for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h, for 7 freeze-thaw cycles and in freezer for 140 days. Terazosin and IS stock standard solutions were stable for 140 days at room temperature and in the chiller. The high throughput method was successfully utilized to measure 935 samples in a bioequivalence study of terazosin.
    Matched MeSH terms: Chromatography, High Pressure Liquid/methods*; Tandem Mass Spectrometry/methods*
  3. Wan Raihana WA, Gan SH, Tan SC
    PMID: 21147046 DOI: 10.1016/j.jchromb.2010.10.037
    Amphetamine-type stimulants (ATS) are a group of chiral amine drugs which are commonly abused for their sympathomimetic and stimulant properties. ATS are extensively metabolised by hepatic cytochrome P450 enzymes. As metabolism of ATS has been shown to be highly stereospecific, stereoselective analytical methods are essential for the quantitative determination of ATS concentrations for both in vivo and in vitro studies of ATS metabolism. This paper describes a new stereoselective method for the simultaneous determination of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), 4-hydroxy-3-methoxyamphetamine (HMA), 3,4-hydroxymethamphetamine (HHMA) and 3,4-hydroxyamphetamine (HHA) in human urine samples validated according to the United States Food and Drug Administration guidelines. In this method, analytes are simultaneously extracted and derivatized with R-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (R-MTPCl) as the chiral derivatization reagent. Following this, the analytes were subjected to a second derivatization with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) which targets the hydroxyl groups present in HMMA, HMA, HHMA and HHA. The derivatized analytes were separated and quantified using gas chromatography-mass spectrometry (GC-MS). The method was evaluated according to the established guidelines for specificity, linearity, precision, accuracy, recovery and stability using a five-day protocol. Intra-day precision ranged from 0.89 to 11.23% RSD whereas inter-day precision was between 1.03 and 12.95% RSD. Accuracy values for the analytes ranged from -5.29% to 13.75%. Limits of quantitation were 10 μg/L for AM, MA, MDMA, HMA and HMMA and 2μg/L for MDA, HMA and HHA. Recoveries and stability values were also within accepted values. The method was applied to authentic ATS-positive samples.
    Matched MeSH terms: Chemical Fractionation/methods*; Gas Chromatography-Mass Spectrometry/methods*
  4. Chua LS, Amin NA, Neo JC, Lee TH, Lee CT, Sarmidi MR, et al.
    J Chromatogr B Analyt Technol Biomed Life Sci, 2011 Dec 15;879(32):3909-19.
    PMID: 22119436 DOI: 10.1016/j.jchromb.2011.11.002
    A number of three LC-MS/MS hybrid systems (QTof, TripleTof and QTrap) has been used to profile small metabolites (m/z 100-1000) and to detect the targeted metabolites such as quassinoids, alkaloids, triterpene and biphenylneolignans from the aqueous extracts of Eurycoma longifolia. The metabolite profiles of small molecules showed four significant clusters in the principle component analysis for the aqueous extracts of E. longifolia, which had been collected from different geographical terrains (Perak and Pahang) and processed at different extraction temperatures (35°C and 100°C). A small peptide of leucine (m/z 679) and a new hydroxyl methyl β-carboline propionic acid have been identified to differentiate E. longifolia extracts that prepared at 35°C and 100°C, respectively. From the targeted metabolites identification, it was found that 3,4ɛ-dihydroeurycomanone (quassinoids) and eurylene (squalene-type triterpene) could only be detected in the Pahang extract, whereas canthin-6-one-3N-oxide could only be detected in the Perak extract. Overall, quassinoids were present in the highest concentration, particularly eurycomanone and its derivatives compared to the other groups of metabolites. However, the concentration of canthin-6-one and β-carboline alkaloids was significantly increased when the roots of the plant samples were extracted at 100°C.
    Matched MeSH terms: Chromatography, Liquid/methods*; Tandem Mass Spectrometry/methods*
  5. Mohamad Hanapi NS, Sanagi MM, Ismail AK, Wan Ibrahim WA, Saim N, Wan Ibrahim WN
    PMID: 28142101 DOI: 10.1016/j.jchromb.2017.01.028
    The aim of this study was to investigate and apply supported ionic liquid membrane (SILM) in two-phase micro-electrodriven membrane extraction combined with high performance liquid chromatography-ultraviolet detection (HPLC-UV) for pre-concentration and determination of three selected antidepressant drugs in water samples. A thin agarose film impregnated with 1-hexyl-3-methylimidazolium hexafluorophosphate, [C6MIM] [PF6], was prepared and used as supported ionic liquid membrane between aqueous sample solution and acceptor phase for extraction of imipramine, amitriptyline and chlorpromazine. Under the optimized extraction conditions, the method provided good linearity in the range of 1.0-1000μgL(-1), good coefficients of determination (r(2)=0.9974-0.9992) and low limits of detection (0.1-0.4μgL(-1)). The method showed high enrichment factors in the range of 110-150 and high relative recoveries in the range of 88.2-111.4% and 90.9-107.0%, for river water and tap water samples, respectively with RSDs of ≤7.6 (n=3). This method was successfully applied to the determination of the drugs in river and tap water samples. It is envisaged that the SILM improved the perm-selectivity by providing a pathway for targeted analytes which resulted in rapid extraction with high degree of selectivity and high enrichment factor.
    Matched MeSH terms: Chromatography, High Pressure Liquid/methods; Liquid Phase Microextraction/methods*
  6. Wong EH, Subramaniam G, Navaratnam P, Sekaran SD
    Indian J Med Microbiol, 2007 Oct;25(4):391-4.
    PMID: 18087092
    Fluorescent in situ hybridization (FISH) was carried out using two different oligonucleotide probes specific for Pseudomonas spp. and Acinetobacter spp. These probes were tested against different organisms and were found to be highly specific. Sensitivity testing showed that the probes were able to detect as low as 10 3 CFU/mL. In addition, FISH was carried out directly on positive blood culture samples and the detection of microorganisms took less than 2 h. We believe that FISH is a rapid method that can be used as a routine laboratory diagnostic technique for the detection of Acinetobacter spp. and Pseudomonas spp. in clinical samples.
    Matched MeSH terms: Bacteriological Techniques/methods; In Situ Hybridization, Fluorescence/methods*
  7. Fok D, Aris IM, Ho J, Lim SB, Chua MC, Pang WW, et al.
    Birth, 2016 09;43(3):247-54.
    PMID: 27018256 DOI: 10.1111/birt.12233
    BACKGROUND: Confinement (restrictions placed on diet and practices during the month right after delivery) represents a key feature of Asian populations. Few studies, however, have focused specifically on ethnic differences in confinement practices. This study assesses the confinement practices of three ethnic groups in a multi-ethnic Asian population.

    METHODS: Participants were part of a prospective birth cohort study that recruited 1,247 pregnant women (57.2% Chinese, 25.5% Malay, and 17.3% Indian) during their first trimester. The 1,220 participants were followed up 3 weeks postpartum at home when questionnaires were administered to ascertain the frequency of adherence to the following confinement practices: showering; confinement-specific meals; going out with or without the baby; choice of caregiver assistance; and the use of massage therapy.

    RESULTS: Most participants reported that they followed confinement practices during the first 3 weeks postpartum (Chinese: 96.4%, Malay: 92.4%, Indian: 85.6%). Chinese and Indian mothers tended to eat more special confinement diets than Malay mothers (p < 0.001), and Chinese mothers showered less and were more likely to depend on confinement nannies during this period than mothers from the two other ethnic groups (p < 0.001 for all). Malay mothers tended to make greater use of massage therapy (p < 0.001), whilst Indian mothers tended to have their mothers or mothers-in-law as assistant caregivers (p < 0.001).

    CONCLUSION: Most Singapore mothers follow confinement practices, but the three Asian ethnic groups differed in specific confinement practices. Future studies should examine whether ethnic differences persist in later childrearing practices.

    Matched MeSH terms: Infant Care/methods*; Postnatal Care/methods*
  8. Ramadas A, Chan CKY, Oldenburg B, Hussein Z, Quek KF
    BMC Public Health, 2018 06 08;18(1):716.
    PMID: 29884161 DOI: 10.1186/s12889-018-5640-1
    BACKGROUND: Increasing prevalence and disease burden has led to an increasing demand of programs and studies focused on dietary and lifestyle habits, and chronic diseases such as type 2 diabetes mellitus (T2DM). We evaluated the effects of a 6-month web-based dietary intervention on Dietary Knowledge, Attitude and Behaviour (DKAB), Dietary Stages of Change (DSOC), fasting blood glucose (FBG) and glycosylated haemoglobin (HbA1c) in patients with uncontrolled HbA1c (> 7.0%) in a randomised-controlled trial (myDIDeA) in Malaysia.

    METHODS: The e-intervention group (n = 62) received a 6-month web-delivered intensive dietary intervention while the control group (n = 66) continued with their standard hospital care. Outcomes (DKAB and DSOC scores, FBG and HbA1c) were compared at baseline, post-intervention and follow-up.

    RESULTS: While both study groups showed improvement in total DKAB score, the margin of improvement in mean DKAB score in e-intervention group was larger than the control group at post-intervention (11.1 ± 0.9 vs. 6.5 ± 9.4,p 

    Matched MeSH terms: Health Promotion/methods*; Patient Education as Topic/methods*
  9. Naylor RL, Kishore A, Sumaila UR, Issifu I, Hunter BP, Belton B, et al.
    Nat Commun, 2021 Sep 15;12(1):5413.
    PMID: 34526495 DOI: 10.1038/s41467-021-25516-4
    Numerous studies have focused on the need to expand production of 'blue foods', defined as aquatic foods captured or cultivated in marine and freshwater systems, to meet rising population- and income-driven demand. Here we analyze the roles of economic, demographic, and geographic factors and preferences in shaping blue food demand, using secondary data from FAO and The World Bank, parameters from published models, and case studies at national to sub-national scales. Our results show a weak cross-sectional relationship between per capita income and consumption globally when using an aggregate fish metric. Disaggregation by fish species group reveals distinct geographic patterns; for example, high consumption of freshwater fish in China and pelagic fish in Ghana and Peru where these fish are widely available, affordable, and traditionally eaten. We project a near doubling of global fish demand by mid-century assuming continued growth in aquaculture production and constant real prices for fish. Our study concludes that nutritional and environmental consequences of rising demand will depend on substitution among fish groups and other animal source foods in national diets.
    Matched MeSH terms: Food Supply/methods; Aquaculture/methods
  10. Zaborowski MP, Lee K, Na YJ, Sammarco A, Zhang X, Iwanicki M, et al.
    Cell Rep, 2019 Apr 02;27(1):255-268.e6.
    PMID: 30943406 DOI: 10.1016/j.celrep.2019.03.003
    Analysis of cancer-derived extracellular vesicles (EVs) in biofluids potentially provides a source of disease biomarkers. At present there is no procedure to systematically identify which antigens should be targeted to differentiate cancer-derived from normal host cell-derived EVs. Here, we propose a computational framework that integrates information about membrane proteins in tumors and normal tissues from databases: UniProt, The Cancer Genome Atlas, the Genotype-Tissue Expression Project, and the Human Protein Atlas. We developed two methods to assess capture of EVs from specific cell types. (1) We used palmitoylated fluorescent protein (palmtdTomato) to label tumor-derived EVs. Beads displaying antibodies of interest were incubated with conditioned medium from palmtdTomato-expressing cells. Bound EVs were quantified using flow cytometry. (2) We also showed that membrane-bound Gaussia luciferase allows the detection of cancer-derived EVs in blood of tumor-bearing animals. Our analytical and validation platform should be applicable to identify antigens on EVs from any tumor type.
    Matched MeSH terms: Flow Cytometry/methods*; Immunoassay/methods
  11. Qasim SSB, Nogueria LP, Fawzy AS, Daood U
    AAPS PharmSciTech, 2020 Jun 16;21(5):173.
    PMID: 32548717 DOI: 10.1208/s12249-020-01708-x
    Innovative strategies for periodontal regeneration have been the focus of research clusters across the globe for decades. In order to overcome the drawbacks of currently available options, investigators have suggested a novel concept of functionally graded membrane (FGM) templates with different structural and morphological gradients. Chitosan (CH) has been used in the past for similar purpose. However, the composite formulation of composite and tetracycline when cross-linked with glutaraldehyde have received little attention. Therefore, the purpose of the study was to investigate the drug loading and release characteristics of novel freeze gelated chitosan templates at different percentages of glutaraldehyde. These were cross-linked with 0.1 and 1% glutaraldehyde and loaded with doxycycline hyclate. The electron micrographs depicted porous morphology of neat templates. After cross-linking, these templates showed compressed ultrastructures. Computerized tomography analysis showed that the templates had 88 to 92% porosity with average pore diameter decreased from 78 to 44.9 μm with increasing concentration. Fourier transform infrared spectroscopy showed alterations in the glycosidic segment of chitosan fingerprint region which after drug loading showed a dominant doxycycline spectral composite profile. Interestingly, swelling profile was not affected by cross-linking either at 0.1 and 1% glutaraldehyde and template showed a swelling ratio of 80%, which gained equilibrium after 15 min. The drug release pattern also showed a 40 μg/mL of release after 24 h. These doxycycline-loaded templates show their tendency to be used in a functionally graded membrane facing the defect site.
    Matched MeSH terms: Guided Tissue Regeneration, Periodontal/methods*; Spectroscopy, Fourier Transform Infrared/methods
  12. Chai LC, Lee HY, Ghazali FM, Abu Bakar F, Malakar PK, Nishibuchi M, et al.
    J Food Prot, 2008 Dec;71(12):2448-52.
    PMID: 19244897
    Campylobacter jejuni was found to occur at high prevalence in the raw salad vegetables examined. Previous reports describe cross-contamination involving meat; here we investigated the occurrence of cross-contamination and decontamination events in the domestic kitchen via C. jejuni-contaminated vegetables during salad preparation. This is the first report concerning quantitative cross-contamination and decontamination involving naturally contaminated produce. The study was designed to simulate the real preparation of salad in a household kitchen, starting with washing the vegetables in tap water, then cutting the vegetables on a cutting board, followed by slicing cucumber and blanching (heating in hot water) the vegetables in 85 degrees C water. Vegetables naturally contaminated with C. jejuni were used throughout the simulation to attain realistic quantitative data. The mean of the percent transfer rates for C. jejuni from vegetable to wash water was 30.1 to 38.2%; from wash water to cucumber, it was 26.3 to 47.2%; from vegetables to cutting board, it was 1.6 to 10.3%; and from cutting board to cucumber, it was 22.6 to 73.3%. The data suggest the wash water and plastic cutting board as potential risk factors in C. jejuni transmission to consumers. Washing of the vegetables with tap water caused a 0.4-log reduction of C. jejuni attached to the vegetables (most probable number/gram), while rapid blanching reduced the number of C. jejuni organisms to an undetectable level.
    Matched MeSH terms: Cooking/methods*; Food Handling/methods*
  13. Anang DM, Rusul G, Radu S, Bakar J, Beuchat LR
    J Food Prot, 2006 Aug;69(8):1913-9.
    PMID: 16924917
    Oxalic acid was evaluated as a treatment for reducing populations of naturally occurring microorganisms on raw chicken. Raw chicken breasts were dipped in solutions of oxalic acid (0, 0.5, 1.0, 1.5, and 2.0%, wt/vol) for 10, 20, and 30 min, individually packed in oxygen-permeable polyethylene bags, and stored at 4 degrees C. Total plate counts of aerobic bacteria and populations of Pseudomonas spp. and Enterobacteriaceae on breasts were determined before treatment and after storage for 1, 3, 7, 10, and 14 days. The pH and Hunter L, a, and b values of the breast surface were measured. Total plate counts were ca. 1.5 and 4.0 log CFU/g higher on untreated chicken breasts after storage for 7 and 14 days, respectively, than on breasts treated with 0.5% oxalic acid, regardless of dip time. Differences in counts on chicken breasts treated with water and 1.0 to 2.0% of oxalic acid were greater. Populations of Pseudomonas spp. on chicken breasts treated with 0.5 to 2.0% oxalic acid and stored at 4 degrees C for 1 day were less than 2 log CFU/g (detection limit), compared with 5.14 log CFU/g on untreated breasts. Pseudomonas grew on chicken breasts treated with 0.5% oxalic acid to reach counts not exceeding 3.88 log CFU/g after storage for 14 days. Counts on untreated chicken exceeded 8.83 log CFU/g at 14 days. Treatment with oxalic acid caused similar reductions in Enterobacteriaceae counts. Kocuria rhizophila was the predominant bacterium isolated from treated chicken. Other common bacteria included Escherichia coli and Empedobacter brevis. Treatment with oxalic acid caused a slight darkening in color (decreased Hunter L value), retention of redness (increased Hunter a value), and increase in yellowness (increased Hunter b value). Oxalic acid has potential for use as a sanitizer to reduce populations of spoilage microorganisms naturally occurring on raw chicken, thereby extending chicken shelf life.
    Matched MeSH terms: Food Handling/methods*; Food Preservation/methods
  14. Malcolm TTH, Chang WS, Loo YY, Cheah YK, Radzi CWJWM, Kantilal HK, et al.
    Int J Food Microbiol, 2018 Nov 02;284:112-119.
    PMID: 30142576 DOI: 10.1016/j.ijfoodmicro.2018.08.012
    Kitchen mishandling practices contribute to a large number of foodborne illnesses. In this study, the transfer and cross-contamination potential of Vibrio parahaemolyticus from bloody clams to ready-to-eat food (lettuce) was assessed. Three scenarios were investigated: 1) direct cross-contamination, the transfer of V. parahaemolyticus from bloody clams to non-food contact surfaces (hands and kitchen utensils) to lettuce (via slicing), was evaluated; 2) perfunctory decontamination, the efficacy of two superficial cleaning treatments: a) rinsing in a pail of water, and b) wiping with a kitchen towel, were determined; and 3) secondary cross-contamination, the microbial transfer from cleaning residuals (wash water or stained kitchen towel) to lettuce was assessed. The mean of percent transfer rates through direct contact was 3.6%, and an average of 3.5% of total V. parahaemolyticus was recovered from sliced lettuce. The attempted treatments reduced the transferred population by 99.0% (rinsing) and 94.5% (wiping), and the relative amount of V. parahaemolyticus on sliced lettuce was reduced to 0.008%. V. parahaemolyticus exposure via secondary cross-contamination was marginal. The relative amount of V. parahaemolyticus recovered from washed lettuce was 0.07%, and the transfers from stained kitchen towel to lettuce were insubstantial. Our study highlights that V. parahaemolyticus was readily spread in the kitchen, potentially through sharing of non-food contact surfaces. Results from this study can be used to better understand and potentially raising the awareness of proper handling practices to avert the spread of foodborne pathogens.
    Matched MeSH terms: Food Handling/methods*; Food Microbiology/methods
  15. Mokti K, Md Isa Z, Sharip J, Abu Bakar SN, Atil A, Hayati F, et al.
    Medicine (Baltimore), 2021 Aug 06;100(31):e26841.
    PMID: 34397855 DOI: 10.1097/MD.0000000000026841
    Smear-positive pulmonary tuberculosis (SPPTB) is the major contributor to the spread of tuberculosis (TB) infection, and it creates high morbidity and mortality worldwide. The objective of this study was to determine the predictors of delayed sputum smear conversion at the end of the intensive phase of TB treatment in Kota Kinabalu, Malaysia.This retrospective study was conducted utilising data of SPPTB patients treated in 5 TB treatment centres located in Kota Kinabalu, Malaysia from 2013 to 2018. Pulmonary TB (PTB) patients included in the study were those who had at least completed the intensive phase of anti-TB treatment with sputum smear results at the end of the 2nd month of treatment. The factors associated with delayed sputum smear conversion were analyzed using multiple logistic regression analysis. Predictors of sputum smear conversion at the end of intensive phase were evaluated.A total of 2641 patients from the 2013 to 2018 periods were included in this study. One hundred eighty nine (7.2%) patients were identified as having delayed sputum smear conversion at the end of the intensive phase treatment. Factors of moderate (advanced odd ratio [aOR]: 1.7) and advanced (aOR: 2.7) chest X-ray findings at diagnosis, age range of >60 (aOR: 2.1), year of enrolment 2016 (aOR: 2.8), 2017 (aOR: 3.9), and 2018 (aOR: 2.8), smokers (aOR: 1.5), no directly observed treatment short-course (DOTS) supervisor (aOR: 6.9), non-Malaysian citizens (aOR: 1.5), and suburban home locations (aOR: 1.6) were associated with delayed sputum smear conversion at the end of the intensive phase of the treatment.To improve sputum smear conversion success rate, the early detection of PTB cases has to be fine-tuned so as to reduce late or severe case presentation during diagnosis. Efforts must also be in place to encourage PTB patients to quit smoking. The percentage of patients assigned with DOTS supervisors should be increased while at the same time ensuring that vulnerable groups such as those residing in suburban localities, the elderly and migrant TB patients are provided with proper follow-up treatment and management.
    Matched MeSH terms: Aftercare/methods; Radiography, Thoracic/methods
  16. Kato T, Azegami J, Yokomori A, Dohra H, El Enshasy HA, Park EY
    BMC Genomics, 2020 Apr 23;21(1):319.
    PMID: 32326906 DOI: 10.1186/s12864-020-6709-7
    BACKGROUND: Ashbya gossypii naturally overproduces riboflavin and has been utilized for industrial riboflavin production. To improve riboflavin production, various approaches have been developed. In this study, to investigate the change in metabolism of a riboflavin-overproducing mutant, namely, the W122032 strain (MT strain) that was isolated by disparity mutagenesis, genomic analysis was carried out.

    RESULTS: In the genomic analysis, 33 homozygous and 1377 heterozygous mutations in the coding sequences of the genome of MT strain were detected. Among these heterozygous mutations, the proportion of mutated reads in each gene was different, ranging from 21 to 75%. These results suggest that the MT strain may contain multiple nuclei containing different mutations. We tried to isolate haploid spores from the MT strain to prove its ploidy, but this strain did not sporulate under the conditions tested. Heterozygous mutations detected in genes which are important for sporulation likely contribute to the sporulation deficiency of the MT strain. Homozygous and heterozygous mutations were found in genes encoding enzymes involved in amino acid metabolism, the TCA cycle, purine and pyrimidine nucleotide metabolism and the DNA mismatch repair system. One homozygous mutation in AgILV2 gene encoding acetohydroxyacid synthase, which is also a flavoprotein in mitochondria, was found. Gene ontology (GO) enrichment analysis showed heterozygous mutations in all 22 DNA helicase genes and genes involved in oxidation-reduction process.

    CONCLUSION: This study suggests that oxidative stress and the aging of cells were involved in the riboflavin over-production in A. gossypii riboflavin over-producing mutant and provides new insights into riboflavin production in A. gossypii and the usefulness of disparity mutagenesis for the creation of new types of mutants for metabolic engineering.

    Matched MeSH terms: Genomics/methods*; Metabolic Engineering/methods
  17. Bashir S, Teo YY, Naeem S, Ramesh S, Ramesh K
    PLoS One, 2017;12(7):e0179250.
    PMID: 28678803 DOI: 10.1371/journal.pone.0179250
    There has been significant progress in the last few decades in addressing the biomedical applications of polymer hydrogels. Particularly, stimuli responsive hydrogels have been inspected as elegant drug delivery systems capable to deliver at the appropriate site of action within the specific time. The present work describes the synthesis of pH responsive semi-interpenetrating network (semi-IPN) hydrogels of N-succinyl-chitosan (NSC) via Schiff base mechanism using glutaraldehyde as a crosslinking agent and Poly (acrylamide-co-acrylic acid)(Poly (AAm-co-AA)) was embedded within the N-succinyl chitosan network. The physico-chemical interactions were characterized by Fourier transform infrared (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and field emission scanning electron microscope (FESEM). The synthesized hydrogels constitute porous structure. The swelling ability was analyzed in physiological mediums of pH 7.4 and pH 1.2 at 37°C. Swelling properties of formulations with various amounts of NSC/ Poly (AAm-co-AA) and crosslinking agent at pH 7.4 and pH 1.2 were investigated. Hydrogels showed higher swelling ratios at pH 7.4 while lower at pH 1.2. Swelling kinetics and diffusion parameters were also determined. Drug loading, encapsulation efficiency, and in vitro release of 5-fluorouracil (5-FU) from the synthesized hydrogels were observed. In vitro release profile revealed the significant influence of pH, amount of NSC, Poly (AAm-co-AA), and crosslinking agent on the release of 5-FU. Accordingly, rapid and large release of drug was observed at pH 7.4 than at pH 1.2. The maximum encapsulation efficiency and release of 5-FU from SP2 were found to be 72.45% and 85.99%, respectively. Kinetics of drug release suggested controlled release mechanism of 5-FU is according to trend of non-Fickian. From the above results, it can be concluded that the synthesized hydrogels have capability to adapt their potential exploitation as targeted oral drug delivery carriers.
    Matched MeSH terms: Chemistry, Pharmaceutical/methods; Drug Delivery Systems/methods*
  18. Drinkwater R, Schnell IB, Bohmann K, Bernard H, Veron G, Clare E, et al.
    Mol Ecol Resour, 2019 Jan;19(1):105-117.
    PMID: 30225935 DOI: 10.1111/1755-0998.12943
    The application of high-throughput sequencing (HTS) for metabarcoding of mixed samples offers new opportunities in conservation biology. Recently, the successful detection of prey DNA from the guts of leeches has raised the possibility that these, and other blood-feeding invertebrates, might serve as useful samplers of mammals. Yet little is known about whether sympatric leech species differ in their feeding preferences, and whether this has a bearing on their relative suitability for monitoring local mammalian diversity. To address these questions, we collected spatially matched samples of two congeneric leech species Haemadipsa picta and Haemadipsa sumatrana from lowland rainforest in Borneo. For each species, we pooled ~500 leeches into batches of 10 individuals, performed PCR to target a section of the mammalian 16S rRNA locus and undertook sequencing of amplicon libraries using an Illumina MiSeq. In total, we identified sequences from 14 mammalian genera, spanning nine families and five orders. We found greater numbers of detections, and higher diversity of OTUs, in H. picta compared with H. sumatrana, with rodents only present in the former leech species. However, comparison of samples from across the landscape revealed no significant difference in mammal community composition between the leech species. We therefore suggest that H. picta is the more suitable iDNA sampler in this degraded Bornean forest. We conclude that the choice of invertebrate sampler can influence the detectability of different mammal groups and that this should be accounted for when designing iDNA studies.
    Matched MeSH terms: Metagenomics/methods*; DNA Barcoding, Taxonomic/methods*
  19. Ramli MI, Sulong AB, Muhamad N, Muchtar A, Arifin A, Mohd Foudzi F, et al.
    PLoS One, 2018;13(10):e0206247.
    PMID: 30359433 DOI: 10.1371/journal.pone.0206247
    The combination of metallic bio-inert material, stainless-steel 316L (SS316L) and a bio-active material, hydroxyapatite (HA) can produce a composite which has superior properties for orthopaedic applications. The main objective of this study is to investigate the effects of sintering temperature and holding time on the physical and mechanical properties of the sintered part. 50wt.% SS316L and 50wt.% HA were mixed with a binder system of palm stearin (PS) and polyethylene (PE) at 61 vol.% powder loading. Rheological properties show a pseudo-plastic behaviour of the feedstock, where viscosity decreases with increasing shear rate. The feedstock was injection moulded into a tensile bar shape while thermal debinding was carried out at 320°C and 500°C. The brown parts were sintered at 1000, 1100, 1200 and 1300°C, with three different sintering times of 1, 3 and 5 hours in the furnace. It was found that the highest sintered density measured was 95.61% of the theoretical density. In addition, the highest hardness and Young's modulus measured were 150.45 HV and 52.61 GPa respectively, which are higher than those of human bone. The lowest percentage of carbon content was 0.022wt.% given by the sample sintered at 1300°C for 1 hour. Therefore, SS316L/HA composite with good mechanical and physical properties was successfully produced through the PIM process.
    Matched MeSH terms: Electroplating/methods; Metallurgy/methods
  20. Rangel Pedersen N, Tovborg M, Soleimani Farjam A, Della Pia EA
    PLoS One, 2021;16(6):e0251556.
    PMID: 34086701 DOI: 10.1371/journal.pone.0251556
    A diverse range of monocot and dicot grains and their by-products are commonly used in the animal feed industry. They all come with complex and variable cell wall structures which in turn contribute significant fiber to the complete feed. The cell wall is a highly interconnected matrix of various polysaccharides, proteins and lignin and, as such, requires a collaborative effort of different enzymes for its degradation. In this regard, we investigated the potential of a commercial multicomponent carbohydrase product from a wild type fermentation of Trichoderma reesei (T. reesei) (RONOZYME® MultiGrain) in degrading cell wall components of wheat, barley, rye, de-oiled rice bran, sunflower, rapeseed and cassava. A total of thirty-one different enzyme proteins were identified in the T. Reesei carbohydrase product using liquid chromatography with tandem mass spectrometry LC-MS/MS including glycosyl hydrolases and carbohydrate esterases. As measured by in vitro incubations and non-starch polysaccharide component analysis, and visualization by immunocytochemistry and confocal microscopy imaging of immuno-labeled samples with confocal microscopy, the carbohydrase product effectively solubilized cellulolytic and hemicellulolytic polysaccharides present in the cell walls of all the feed ingredients evaluated. The T. reesei fermentation also decreased viscosity of arabinoxylan, xyloglucan, galactomannan and β-glucan substrates. Combination of several debranching enzymes including arabinofuranosidase, xylosidase, α-galactosidase, acetyl xylan esterase, and 4-O-methyl-glucuronoyl methylesterase with both GH10 and GH11 xylanases in the carbohydrase product resulted in effective hydrolyzation of heavily branched glucuronoarabinoxylans. The different β-glucanases (both endo-β-1,3(4)-glucanase and endo-β-1,3-glucanase), cellulases and a β-glucosidase in the T. reesei fermentation effectively reduced polymerization of both β-glucans and cellulose polysaccharides of viscous cereals grains (wheat, barley, rye and oat). Interestingly, the secretome of T. reesei contained significant amounts of an exceptional direct chain-cutting enzyme from the GH74 family (Cel74A, xyloglucan-specific β-1,4-endoglucanase), that strictly cleaves the xyloglucan backbone at the substituted regions. Here, we demonstrated that the balance of enzymes present in the T. reesei secretome is capable of degrading various cell wall components in both monocot and dicot plant raw material used as animal feed.
    Matched MeSH terms: Chromatography, Liquid/methods; Tandem Mass Spectrometry/methods
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