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Fulltext Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia

Al-Marzooq F, Imad MA, How SH, Kuan YC

Trop Biomed, 2011 Dec;28(3):545-56.
PMID: 22433883 MyJurnal

Establishing a microbial diagnosis for patients with community-acquired pneumonia (CAP) is still challenging and is often achieved in only 30-50% of cases. Polymerase chain reaction (PCR) has been shown to be more sensitive than conventional microbiological methods and it could help to increase the microbial yield for CAP patients. This study was designed to develop, optimize and evaluate multiplex real-time PCR as a method for rapid differential detection of five bacterial causes of CAP namely Streptococcus pneumoniae, Burkholderia pseudomallei and atypical bacterial pathogens, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila. Duplex and triplex real-time PCR assays were developed using five sets of primers and probes that were designed based on an appropriate specific gene for each of the above CAP pathogens. The performance of primers for each organism was tested using SYBR Green melt curve analysis following monoplex realtime PCR amplification. Monoplex real-time PCR assays were also used to optimize each primers-probe set before combining them in multiplex assays. Two multiplex real-time PCR assays were then optimized; duplex assay for the differential detection of S. pneumoniae and B. pseudomallei, and triplex assay for the atypical bacterial pathogens. Both duplex and triplex real-time PCR assays were tested for specificity by using DNA extracted from 26 related microorganisms and sensitivity by running serial dilutions of positive control DNAs. The developed multiplex real-time PCR assays shall be used later for directly identifying CAP causative agents in clinical samples.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
Association between genetic polymorphisms in interferon regulatory factor 5 (IRF5) gene and Malaysian patients with Crohn's disease

Chua KH, Lian LH, Khor WC, Lee WS, Hilmi I, Goh KL, et al.

J Dig Dis, 2015 Apr;16(4):205-16.
PMID: 25564941 DOI: 10.1111/1751-2980.12229

The study aimed to investigate the association between the interferon regulatory factor 5 (IRF5) gene polymorphisms and the onset of Crohn's disease (CD) in a Malaysian cohort.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
In vitro expression of Candida albicans alcohol dehydrogenase genes involved in acetaldehyde metabolism

Bakri MM, Rich AM, Cannon RD, Holmes AR

Mol Oral Microbiol, 2015 Feb;30(1):27-38.
PMID: 24975985 DOI: 10.1111/omi.12064

Alcohol consumption is a risk factor for oral cancer, possibly via its conversion to acetaldehyde, a known carcinogen. The oral commensal yeast Candida albicans may be one of the agents responsible for this conversion intra-orally. The alcohol dehydrogenase (Adh) family of enzymes are involved in acetaldehyde metabolism in yeast but, for C. albicans it is not known which family member is responsible for the conversion of ethanol to acetaldehyde. In this study we determined the expression of mRNAs from three C. albicans Adh genes (CaADH1, CaADH2 and CaCDH3) for cells grown in different culture media at different growth phases by Northern blot analysis and quantitative reverse transcription polymerase chain reaction. CaADH1 was constitutively expressed under all growth conditions but there was differential expression of CaADH2. CaADH3 expression was not detected. To investigate whether CaAdh1p or CaAdh2p can contribute to alcohol catabolism in C. albicans, each gene from the reference strain C. albicans SC5314 was expressed in Saccharomyces cerevisiae. Cell extracts from an CaAdh1p-expressing S. cerevisiae recombinant, but not an CaAdh2p-expressing recombinant, or an empty vector control strain, possessed ethanol-utilizing Adh activity above endogenous S. cerevisiae activity. Furthermore, expression of C. albicans Adh1p in a recombinant S. cerevisiae strain in which the endogenous ScADH2 gene (known to convert ethanol to acetaldehyde in this yeast) had been deleted, conferred an NAD-dependent ethanol-utilizing, and so acetaldehyde-producing, Adh activity. We conclude that CaAdh1p is the enzyme responsible for ethanol use under in vitro growth conditions, and may contribute to the intra-oral production of acetaldehyde.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Nasal epithelial cells can act as a physiological surrogate for paediatric asthma studies

Thavagnanam S, Parker JC, McBrien ME, Skibinski G, Shields MD, Heaney LG

PLoS One, 2014;9(1):e85802.
PMID: 24475053 DOI: 10.1371/journal.pone.0085802

Differentiated paediatric epithelial cells can be used to study the role of epithelial cells in asthma. Nasal epithelial cells are easier to obtain and may act as a surrogate for bronchial epithelium in asthma studies. We assessed the suitability of nasal epithelium from asthmatic children to be a surrogate for bronchial epithelium using air-liquid interface cultures.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
The L-cell isolation from heterogonous population of intestinal cell line using antibiotic selection method

Rasouli M, Abbasi S, Sarsaifi K, Hani H, Ahmad Z, Omar AR

Appl Biochem Biotechnol, 2014 Jan;172(1):394-404.
PMID: 24081707 DOI: 10.1007/s12010-013-0514-6

Enteroendocrine cells are the largest population of hormone-producing cells in the body and play important roles in many aspects of body functions. The enteroendocrine cell population is divided into different subpopulations that secrete different hormones and peptides. Characterization of each subpopulation is particularly useful for analyzing the cellular mechanisms responsible for specific cell types. Therefore, the necessity of a pure cell line for a specific study purpose was the important motivation for the separation of cell lines for each subpopulation of enteroendocrine cells. The present research introduces a method for the isolation of L-cells, one of the important subpopulations of enteroendocrine cells. The antibiotic selection method was conducted in order to isolate the L-cells from a heterogonous population of intestinal cell line. In this method, a neomycin resistance gene (as selected marker) was expressed under the control of a specific promoter of L-cells. After transfection of manipulated plasmid, only the cells which determine the specific promoter and express neomycin resistance protein would be able to survive under Geneticin antibiotic treatment condition. In order to confirm that the isolated cells were L-cells, reverse transcriptase polymerase chain reaction (PCR) and quantitative PCR assays were performed. Based on the results, the isolated cells were pure L-cells that could be able to express specific mRNA of L-cells efficiently. This technique provides a unique method for the isolation and purification of any cell line. The purified isolated L-cells by this method can be used for future studies and for analyzing cellular mechanisms that involve L-cells' functions.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Potentiality of a triple microRNA classifier: miR-193a-3p, miR-23a and miR-338-5p for early detection of colorectal cancer

Yong FL, Law CW, Wang CW

BMC Cancer, 2013 Jun 08;13:280.
PMID: 23758639 DOI: 10.1186/1471-2407-13-280

BACKGROUND: MicroRNAs (miRNAs) are short, non-coding RNA molecules that act as regulators of gene expression. Circulating blood miRNAs offer great potential as cancer biomarkers. The objective of this study was to correlate the differential expression of miRNAs in tissue and blood in the identification of biomarkers for early detection of colorectal cancer (CRC).
METHODS: The study was divided into two phases: (I) Marker discovery by miRNA microarray using paired cancer tissues (n = 30) and blood samples (CRC, n = 42; control, n = 18). (II) Marker validation by stem-loop reverse transcription real time PCR using an independent set of paired cancer tissues (n = 30) and blood samples (CRC, n = 70; control, n = 32). Correlation analysis was determined by Pearson's test. Logistic regression and receiver operating characteristics curve analyses were applied to obtain diagnostic utility of the miRNAs.
RESULTS: Seven miRNAs (miR-150, miR-193a-3p, miR-23a, miR-23b, miR-338-5p, miR-342-3p and miR-483-3p) have been found to be differentially expressed in both tissue and blood samples. Significant positive correlations were observed in the tissue and blood levels of miR-193a-3p, miR-23a and miR-338-5p. Moreover, increased expressions of these miRNAs were detected in the more advanced stages. MiR-193a-3p, miR-23a and miR-338-5p were demonstrated as a classifier for CRC detection, yielding a receiver operating characteristic curve area of 0.887 (80.0% sensitivity, 84.4% specificity and 83.3% accuracy).
CONCLUSION: Dysregulations in circulating blood miRNAs are reflective of those in colorectal tissues. The triple miRNA classifier of miR-193a-3p, miR-23a and miR-338-5p appears to be a potential blood biomarker for early detection of CRC.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Gamma-tocotrienol modulated gene expression in senescent human diploid fibroblasts as revealed by microarray analysis

Makpol S, Zainuddin A, Chua KH, Mohd Yusof YA, Ngah WZ

Oxid Med Cell Longev, 2013;2013:454328.
PMID: 23634235 DOI: 10.1155/2013/454328

The effect of γ -tocotrienol, a vitamin E isomer, in modulating gene expression in cellular aging of human diploid fibroblasts was studied. Senescent cells at passage 30 were incubated with 70 μ M of γ -tocotrienol for 24 h. Gene expression patterns were evaluated using Sentrix HumanRef-8 Expression BeadChip from Illumina, analysed using GeneSpring GX10 software, and validated using quantitative RT-PCR. A total of 100 genes were differentially expressed (P < 0.001) by at least 1.5 fold in response to γ -tocotrienol treatment. Amongst the genes were IRAK3, SelS, HSPA5, HERPUD1, DNAJB9, SEPR1, C18orf55, ARF4, RINT1, NXT1, CADPS2, COG6, and GLRX5. Significant gene list was further analysed by Gene Set Enrichment Analysis (GSEA), and the Normalized Enrichment Score (NES) showed that biological processes such as inflammation, protein transport, apoptosis, and cell redox homeostasis were modulated in senescent fibroblasts treated with γ -tocotrienol. These findings revealed that γ -tocotrienol may prevent cellular aging of human diploid fibroblasts by modulating gene expression.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Identification of Toxoplasma gondii in-vivo induced antigens by cDNA library immunoscreening with chronic toxoplasmosis sera

Amerizadeh A, Idris ZM, Khoo BY, Kotresha D, Yunus MH, Karim IZ, et al.

Microb Pathog, 2013 Jan;54:60-6.
PMID: 23044055 DOI: 10.1016/j.micpath.2012.09.006

Toxoplasmosis is an infection caused by the parasite Toxoplasma gondii. Chronically-infected individuals with a compromised immune system are at risk for reactivation of the disease. In-vivo induced antigen technology (IVIAT) is a promising method for the identification of antigens expressed in-vivo. The aim of the present study was to apply IVIAT to identify antigens which are expressed in-vivo during T. gondii infection using sera from individuals with chronic toxoplasmosis. Forty serum samples were pooled, pre-adsorped against three different preparations of antigens, from each in-vitro grown T. gondii and Escherichia coli XLBlue MRF', and then used to screen a T. gondii cDNA expression library. Sequencing of DNA inserts from positive clones showed eight open reading frames with high homology to T. gondii genes. Expression analysis using quantitative real-time PCR showed that SAG1-related sequence 3 (SRS3) and two hypothetical genes were up-regulated in-vivo relative to their expression levels in-vitro. These three proteins also showed high sensitivity and specificity when tested with individual serum samples. Five other proteins namely M16 domain peptidase, microneme protein, elongation factor 1-alpha, pre-mRNA-splicing factor and small nuclear ribonucleoprotein F had lower RNA expression in-vivo as compared to in-vitro. SRS3 and the two hypothetical proteins warrant further investigation into their roles in the pathogenesis of toxoplasmosis.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
The changes of stemness biomarkers expression in human adipose-derived stem cells during long-term manipulation

Wan Safwani WK, Makpol S, Sathapan S, Chua KH

Biotechnol Appl Biochem, 2011 Jul-Aug;58(4):261-70.
PMID: 21838801 DOI: 10.1002/bab.38

One of the advantages of human adipose-derived stem cells (ASCs) in regenerative medicine is that they can be harvested in abundance. However, the stemness biomarkers, which marked the safety and efficacy of ASCs in accordance with the good manufacturing practice guidelines, is not yet well established. This study was designed to investigate the effect of long-term culture on the stemness properties of ASCs using quantitative real-time polymerase chain reaction and flow cytometry. Results showed the growth rate of ASCs was at its peak when they reached P10 (population doubling; PD = 26) but started to decrease when they were expanded to P15 (PD = 36) and P20 (PD = 46). The ASCs can be culture expanded with minimal alteration in the stemness genes and cluster of differentiation (CD) markers expression up to P10. Expression level of Sox2, Nestin, and Nanog3 was significantly decreased at later passage. CD31, CD45, CD117, and human leukocyte antigen DR, DQ, and DP were lowly expressed at P5 and P10 but their expressions increased significantly at P15 or P20. The differentiation ability of ASCs (adipogenesis, osteogenesis, and neurogenesis) also decreased in long-term culture. Our findings suggested that P10 (PD = 26) should be the "cutoff point" for clinical usage because ASCs at passage 15 onward showed significant changes in the stemness genes, CD markers expression, and differentiation capability.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Does GSTP1 polymorphism contribute to genetic damage caused by ageing and occupational exposure?

Eshkoor SA, Ismail P, Rahman SA, Moin S

Arh Hig Rada Toksikol, 2011 Dec;62(4):291-8.
PMID: 22202462 DOI: 10.2478/10004-1254-62-2011-2088

The aim of our study was to see the effects of GSTP1 polymorphism on biomarkers of ageing, including micronuclei (MN), comet tail length, and relative telomere length in automobile repair workers, who are exposed to a broad spectrum of potential mutagens. The analysis was performed on buccal cells collected from occupationally exposed and non-exposed (control) subjects. Samples were analysed using cytogenetic and molecular methods, including restriction fragment length polymorphism (RFLP), MN test, comet assay, and real-time PCR. The results confirmed the DNA damaging effects of substances used in the mechanical workshops, but did not confirm the influence of GSTP1 gene polymorphism on DNA damage. However, further studies on both occupationally exposed and control populations are needed to understand the relationship between GSTP1 polymorphism and genome damage.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext An insight into the isolation, enumeration, and molecular detection of Listeria monocytogenes in food

Law JW, Ab Mutalib NS, Chan KG, Lee LH

Front Microbiol, 2015;6:1227.
PMID: 26579116 DOI: 10.3389/fmicb.2015.01227

Listeria monocytogenes, a foodborne pathogen that can cause listeriosis through the consumption of food contaminated with this pathogen. The ability of L. monocytogenes to survive in extreme conditions and cause food contaminations have become a major concern. Hence, routine microbiological food testing is necessary to prevent food contamination and outbreaks of foodborne illness. This review provides insight into the methods for cultural detection, enumeration, and molecular identification of L. monocytogenes in various food samples. There are a number of enrichment and plating media that can be used for the isolation of L. monocytogenes from food samples. Enrichment media such as buffered Listeria enrichment broth, Fraser broth, and University of Vermont Medium (UVM) Listeria enrichment broth are recommended by regulatory agencies such as Food and Drug Administration-bacteriological and analytical method (FDA-BAM), US Department of Agriculture-Food and Safety (USDA-FSIS), and International Organization for Standardization (ISO). Many plating media are available for the isolation of L. monocytogenes, for instance, polymyxin acriflavin lithium-chloride ceftazidime aesculin mannitol, Oxford, and other chromogenic media. Besides, reference methods like FDA-BAM, ISO 11290 method, and USDA-FSIS method are usually applied for the cultural detection or enumeration of L. monocytogenes. most probable number technique is applied for the enumeration of L. monocytogenes in the case of low level contamination. Molecular methods including polymerase chain reaction, multiplex polymerase chain reaction, real-time/quantitative polymerase chain reaction, nucleic acid sequence-based amplification, loop-mediated isothermal amplification, DNA microarray, and next generation sequencing technology for the detection and identification of L. monocytogenes are discussed in this review. Overall, molecular methods are rapid, sensitive, specific, time- and labor-saving. In future, there are chances for the development of new techniques for the detection and identification of foodborne with improved features.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Isolation and Functional Characterisation of a fads2 in Rainbow Trout (Oncorhynchus mykiss) with Δ5 Desaturase Activity

Abdul Hamid NK, Carmona-Antoñanzas G, Monroig Ó, Tocher DR, Turchini GM, Donald JA

PLoS One, 2016;11(3):e0150770.
PMID: 26943160 DOI: 10.1371/journal.pone.0150770

Rainbow trout, Oncorhynchus mykiss, are intensively cultured globally. Understanding their requirement for long-chain polyunsaturated fatty acids (LC-PUFA) and the biochemistry of the enzymes and biosynthetic pathways required for fatty acid synthesis is important and highly relevant in current aquaculture. Most gnathostome vertebrates have two fatty acid desaturase (fads) genes with known functions in LC-PUFA biosynthesis and termed fads1 and fads2. However, teleost fish have exclusively fads2 genes. In rainbow trout, a fads2 cDNA had been previously cloned and found to encode an enzyme with Δ6 desaturase activity. In the present study, a second fads2 cDNA was cloned from the liver of rainbow trout and termed fads2b. The full-length mRNA contained 1578 nucleotides with an open reading frame of 1365 nucleotides that encoded a 454 amino acid protein with a predicted molecular weight of 52.48 kDa. The predicted Fads2b protein had the characteristic traits of the microsomal Fads family, including an N-terminal cytochrome b5 domain containing the heme-binding motif (HPPG), histidine boxes (HDXGH, HFQHH and QIEHH) and three transmembrane regions. The fads2b was expressed predominantly in the brain, liver, intestine and pyloric caeca. Expression of the fasd2b in yeast generated a protein that was found to specifically convert eicosatetraenoic acid (20:4n-3) to eicosapentaenoic acid (20:5n-3), and therefore functioned as a Δ5 desaturase. Therefore, rainbow trout have two fads2 genes that encode proteins with Δ5 and Δ6 desaturase activities, respectively, which enable this species to perform all the desaturation steps required for the biosynthesis of LC-PUFA from C18 precursors.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Presence of Cryptosporidium parvum and Giardia lamblia in water samples from Southeast Asia: towards an integrated water detection system

Kumar T, Abd Majid MA, Onichandran S, Jaturas N, Andiappan H, Salibay CC, et al.

Infect Dis Poverty, 2016 Jan 13;5:3.
PMID: 26763230 DOI: 10.1186/s40249-016-0095-z

Access to clean and safe drinking water that is free from pathogenic protozoan parasites, especially Cryptosporidium parvum and Giardia lamblia that cause gastrointestinal illness in humans, is still an issue in Southeast Asia (SEA). This study is the first attempt to detect the aforementioned protozoan parasites in water samples from countries in SEA, using real-time polymerase chain reaction (qPCR) assays.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
A transcriptome study on Macrobrachium rosenbergii hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)

Rao R, Bhassu S, Bing RZ, Alinejad T, Hassan SS, Wang J

J Invertebr Pathol, 2016 05;136:10-22.
PMID: 26880158 DOI: 10.1016/j.jip.2016.01.002

The world production of shrimp such as the Malaysian giant freshwater prawn, Macrobrachium rosenbergii is seriously affected by the white spot syndrome virus (WSSV). There is an urgent need to understand the host pathogen interaction between M. rosenbergii and WSSV which will be able to provide a solution in controlling the spread of this infectious disease and lastly save the aquaculture industry. Now, using Next Generation Sequencing (NGS), we will be able to capture the response of the M. rosenbergii to the pathogen and have a better understanding of the host defence mechanism. Two cDNA libraries, one of WSSV-challenged M. rosenbergii and a normal control one, were sequenced using the Illumina HiSeq™ 2000 platform. After de novo assembly and clustering of the unigenes from both libraries, 63,584 standard unigenes were generated with a mean size of 698bp and an N50 of 1137bp. We successfully annotated 35.31% of all unigenes by using BLASTX program (E-value <10-5) against NCBI non-redundant (Nr), Swiss-Prot, Kyoto Encyclopedia of Genes and Genome pathway (KEGG) and Orthologous Groups of proteins (COG) databases. Gene Ontology (GO) assessment was conducted using BLAST2GO software. Differentially expressed genes (DEGs) by using the FPKM method showed 8443 host genes were significantly up-regulated whereas 5973 genes were significantly down-regulated. The differentially expressed immune related genes were grouped into 15 animal immune functions. The present study showed that WSSV infection has a significant impact on the transcriptome profile of M. rosenbergii's hepatopancreas, and further enhanced the knowledge of this host-virus interaction. Furthermore, the high number of transcripts generated in this study will provide a platform for future genomic research on freshwater prawns.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Transcriptome-wide effect of DE-ETIOLATED1 (DET1) suppression in embryogenic callus of Carica papaya

Jamaluddin ND, Rohani ER, Mohd Noor N, Goh HH

J Plant Res, 2019 Mar;132(2):181-195.
PMID: 30649676 DOI: 10.1007/s10265-019-01086-x

Papaya is one of the most nutritional fruits, rich in vitamins, carotenoids, flavonoids and other antioxidants. Previous studies showed phytonutrient improvement without affecting quality in tomato fruit and rapeseed through the suppression of DE-ETIOLATED-1 (DET1), a negative regulator in photomorphogenesis. This study is conducted to study the effects of DET1 gene suppression in papaya embryogenic callus. Immature zygotic embryos were transformed with constitutive expression of a hairpin DET1 construct (hpDET1). PCR screening of transformed calli and reverse transcription quantitative PCR (RT-qPCR) verified that DET1 gene downregulation in two of the positive transformants. High-throughput cDNA 3' ends sequencing on DET1-suppressed and control calli for transcriptomic analysis of global gene expression identified a total of 452 significant (FDR

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Evaluation of a Recombinant Newcastle Disease Virus Expressing Human IL12 against Human Breast Cancer

Mohamed Amin Z, Che Ani MA, Tan SW, Yeap SK, Alitheen NB, Syed Najmuddin SUF, et al.

Sci Rep, 2019 Sep 30;9(1):13999.
PMID: 31570732 DOI: 10.1038/s41598-019-50222-z

The Newcastle disease virus (NDV) strain AF2240 is an avian avulavirus that has been demonstrated to possess oncolytic activity against cancer cells. However, to illicit a greater anti-cancer immune response, it is believed that the incorporation of immunostimulatory genes such as IL12 into a recombinant NDV backbone will enhance its oncolytic effect. In this study, a newly developed recombinant NDV that expresses IL12 (rAF-IL12) was tested for its safety, stability and cytotoxicity. The stability of rAF-IL12 was maintained when passaged in specific pathogen free (SPF) chicken eggs from passage 1 to passage 10; with an HA titer of 29. Based on the results obtained from the MTT cytotoxic assay, rAF-IL12 was determined to be safe as it only induced cytotoxic effects against normal chicken cell lines and human breast cancer cells while sparing normal cells. Significant tumor growth inhibition (52%) was observed in the rAF-IL12-treated mice. The in vivo safety profile of rAF-IL12 was confirmed through histological observation and viral load titer assay. The concentration and presence of the expressed IL12 was quantified and verified via ELISA assay. In summary, rAF-IL12 was proven to be safe, selectively replicating in chicken and cancer cells and was able to maintain its stability throughout several passages; thus enhancing its potential as an anti-breast cancer vaccine.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Induction of MHC Class I HLA-A2 promoter by dengue virus occurs at the NFκB binding domains of the Class I Regulatory Complex

Othman S, Rahman NA, Yusof R

Virus Res, 2012 Jan;163(1):238-45.
PMID: 22001567 DOI: 10.1016/j.virusres.2011.09.040

Despite aggressive efforts in dengue research, the control of dengue diseases and discovery of therapeutics against them await complete elucidation of its complex immune-pathogenesis. Unlike many viruses that escape the host's immune responses by suppressing the major histocompatibility complex (MHC) Class I pathway, many Flaviviruses up-regulate the cell surface expression of MHC Class I complex. We recently reported MHC Class I HLA-A2 promoter activation by all serotypes of dengue virus (DV). The mechanism by which DV regulates this is further explored here in HepG2 human liver cell line. Using real-time PCR, evidence that, similar to infections by other Flaviviruses, DV infection has the ability to up-regulate the MHC Class I transcription and mRNA synthesis, is presented. The region responsive towards DV infection of all serotypes was mapped to the Class I Regulatory Complex (CRC) of the HLA-A2 promoter. Competition electrophoretic mobility shift assay (EMSA) with NFκB probe established the presence of specific DNA-protein complex in DV-infected nuclear extracts. Antibody-supershift assays identified the MHC Class I promoter activation by DV to occur through binding of p65/p50 heterodimers and p65 homodimers to κB1 and κB2 cis-acting elements, respectively, within the CRC, and not with the interferon consensus sequence (ICS). This study presents evidence of MHC Class I gene modulation by DV, hence providing a better understanding of dengue immune-pathogenesis that would consequently facilitate the discovery of antiviral therapeutics against dengue.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
The effect of adenosine monophosphate deaminase overexpression on the accumulation of umami-related metabolites in tomatoes

Chew BL, Fisk ID, Fray R, Tucker GA, Bodi Z, Ferguson A, et al.

Plant Cell Rep, 2017 Jan;36(1):81-87.
PMID: 27662835 DOI: 10.1007/s00299-016-2058-z

KEY MESSAGE: This study highlights the changes in umami-related nucleotide and glutamate levels when the AMP deaminase gene was elevated in transgenic tomato. Taste is perceived as one of a combination of five sensations, sweet, sour, bitter, salty, and umami. The umami taste is best known as a savoury sensation and plays a central role in food flavour, palatability, and eating satisfaction. Umami flavour can be imparted by the presence of glutamate and is greatly enhanced by the addition of ribonucleotides, such as inosine monophosphate (IMP) and guanosine monophosphate (GMP). The production of IMP is regulated by the enzyme adenosine monophosphate (AMP) deaminase which functions to convert AMP into IMP. We have generated transgenic tomato (Solanum lycopersicum) lines over expressing AMP deaminase under the control of a fruit-specific promoter. The transgenic lines showed substantially enhanced levels of AMP deaminase expression in comparison to the wild-type control. Elevated AMP deaminase levels resulted in the reduced accumulation of glutamate and increased levels of the umami nucleotide GMP. AMP concentrations were unchanged. The effects on the levels of glutamate and GMP were unexpected and are discussed in relation to the metabolite flux within this pathway.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Characterization of Burkholderia pseudomallei from spontaneous melioidosis in a Bornean orangutan

Testamenti VA, Surya M, Saepuloh U, Iskandriati D, Tandang MV, Kristina L, et al.

Vet World, 2020 Nov;13(11):2459-2468.
PMID: 33363342 DOI: 10.14202/vetworld.2020.2459-2468

Background and Aim: Melioidosis is a potentially fatal disease affecting humans and a wide range of animal species; it is often underdiagnosed and underreported in veterinary medicine in Indonesia. This study aimed to characterize morphological and molecular features of Burkholderia pseudomallei, the causative agent of melioidosis which caused the death of a Bornean orangutan.
Materials and Methods: Pulmonary abscess samples were cultured on several types of media, including Ashdown agar, Ashdown broth, and MacConkey agar. Type three secretion system orf 2 real-time polymerase chain reaction (PCR) and latex agglutination tests were performed to identify the bacteria. Morphological characteristics were compared to all previously published morphotypes. Subsequently, the bacteria were characterized by multilocus sequence typing (MLST) and Yersinia-like flagellum/Burkholderia thailandensis-like flagellum and chemotaxis PCR. The results of the genotyping were afterward compared to all genotypes from Southeast Asia.
Results: Multiple morphotypes of B. pseudomallei were perceived during the growth on Ashdown agar. Furthermore, it was identified by MLST that the Type I and Type II morphotypes observed in this study were clones of a single ST, ST54, which is predominantly found in humans and the environment in Malaysia and Thailand, although a very limited number of reports was published in association with animals. Moreover, the E-BURST analysis showed that the ST is grouped together with isolates from Southeast Asian countries, including Malaysia, Thailand, Singapore, and Cambodia. ST54 was predicted to be the founding genotype of several STs from those regions.
Conclusion: B. pseudomallei ST54 that caused the death of a Bornean orangutan has a distant genetic relationship with other STs which were previously reported in Indonesia, implying a vast genetic diversity in Indonesia that has not been discovered yet.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Kisspeptin-1 regulates forebrain dopaminergic neurons in the zebrafish

Abdul Satar NM, Ogawa S, Parhar IS

Sci Rep, 2020 11 09;10(1):19361.
PMID: 33168887 DOI: 10.1038/s41598-020-75777-0

The habenula is a phylogenetically conserved epithalamic structure, which conveys negative information via inhibition of mesolimbic dopamine neurons. We have previously shown the expression of kisspeptin (Kiss1) in the habenula and its role in the modulation of fear responses in the zebrafish. In this study, to investigate whether habenular Kiss1 regulates fear responses via dopamine neurons in the zebrafish, Kiss1 peptides were intracranially administered close to the habenula, and the expression of dopamine-related genes (th1, th2 and dat) were examined in the brain using real-time PCR and dopamine levels using LC-MS/MS. th1 mRNA levels and dopamine levels were significantly increased in the telencephalon 24-h and 30-min after Kiss1 administration, respectively. In fish administered with Kiss1, expression of neural activity marker gene, npas4a and kiss1 gene were significantly decreased in the ventral habenula. Application of neural tracer into the median raphe, site of habenular Kiss1 neural terminal projections showed tracer-labelled projections in the medial forebrain bundle towards the telencephalon where dopamine neurons reside. These results suggest that Kiss1 negatively regulates its own neuronal activity in the ventral habenula via autocrine action. This, in turn affects neurons of the median raphe via interneurons, which project to the telencephalic dopaminergic neurons.

Matched MeSH terms: Real-Time Polymerase Chain Reaction

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