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p16 gene expression in basal cell carcinoma

Eshkoor SA, Ismail P, Rahman SA, Oshkour SA

Arch Med Res, 2008 Oct;39(7):668-73.
PMID: 18760195 DOI: 10.1016/j.arcmed.2008.06.003

Basal cell carcinoma (BCC) develops predominantly in sun-exposed skin in fair-skinned individuals prone to sunburn. BCC typically occurs in adults. High exposure to ultraviolet (UV) radiation increases rate of developing BCC, a slowly growing tumor that occurs in hair-growing squamous epithelium and rarely metastasizes. In genetic studies, BCC patients have cell-cycle abnormalities of different parts of the signaling pathway. Retinoblastoma regulatory pathway is important in cell cycle arrest. In this pathway, p16INK4a, an inhibitor of Rb pathway, binds to CDK4 and CDK6 competitively with cyclin D1 to prevent phosphorylation of tumor suppressor pRB gene. Alteration of this pathway contributes to development of human cancers and also is effective in skin cancers. In this study, we analyzed mRNA expression using in situ RT-PCR and the role of immunohistochemical expression of p16INK4a in BCC.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext miR-145 supports cancer cell survival and shows association with DDR genes, methylation pattern, and epithelial to mesenchymal transition

Manvati S, Mangalhara KC, Kalaiarasan P, Chopra R, Agarwal G, Kumar R, et al.

Cancer Cell Int, 2019;19:230.
PMID: 31516387 DOI: 10.1186/s12935-019-0933-8

Background: Despite several reports describing the dual role of miR-145 as an oncogene and a tumor suppressor in cancer, not much has been resolved and understood.
Method: In this study, the potential targets of miR-145 were identified bio-informatically using different target prediction tools. The identified target genes were validated in vitro by dual luciferase assay. Wound healing and soft agar colony assay assessed cell proliferation and migration. miR-145 expression level was measured quantitatively by RT-PCR at different stages of breast tumor. Western blot was used to verify the role of miR-145 in EMT transition using key marker proteins.
Result: Wound healing and soft agar colony assays, using miR-145 over-expressing stably transfected MCF7 cells, unraveled its role as a pro-proliferation candidate in cancerous cells. The association between miR-145 over-expression and differential methylation patterns in representative target genes (DR5, BCL2, TP53, RNF8, TIP60, CHK2, and DCR2) supported the inference drawn. These in vitro observations were validated in a representative set of nodal positive tumors of stage 3 and 4 depicting higher miR-145 expression as compared to early stages. Further, the role of miR-145 in epithelial-mesenchymal (EMT) transition found support through the observation of two key markers, Vimentin and ALDL, where a positive correlation with Vimentin protein and a negative correlation with ALDL mRNA expression were observed.
Conclusion: Our results demonstrate miR-145 as a pro-cancerous candidate, evident from the phenotypes of aggressive cellular proliferation, epithelial to mesenchymal transition, hypermethylation of CpG sites in DDR and apoptotic genes and upregulation of miR-145 in later stages of tumor tissues.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext mRNA expression of EgCHI1, EgCHI2, and EgCHI3 in oil palm leaves (Elaeis guineesis Jacq.) after treatment with Ganoderma boninense pat. and Trichoderma harzianum Rifai

Naher L, Tan SG, Ho CL, Yusuf UK, Ahmad SH, Abdullah F

ScientificWorldJournal, 2012;2012:647504.
PMID: 22919345 DOI: 10.1100/2012/647504

Basal stem rot (BSR) disease caused by the fungus Ganoderma boninense is the most serious disease affecting the oil palm; this is because the disease escapes the early disease detection. The biocontrol agent Trichoderma harzianum can protect the disease only at the early stage of the disease. In the present study, the expression levels of three oil palm (Elaeis guineensis Jacq.) chitinases encoding EgCHI1, EgCHI2, and EgCHI3 at 2, 5, and 8 weeks inoculation were measured in oil palm leaves from plants treated with G. boninense or T. harzianum alone or both.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext [Investigation of Group A Rotavirus G10, G12 Genotypes Emerging in Patients with Acute Gastroenteritis in a Tertiary Care Hospital]

Kahraman H, Altay Koçak A, Albakkour K, Muftah H, Dalgıç B, Çağlar K, et al.

Mikrobiyol Bul, 2021 Oct;55(4):553-567.
PMID: 34666655 DOI: 10.5578/mb.20219707

Rotaviruses are the most common cause of viral gastroenteritis with the highest mortality and morbidity rates in children aged 0-5 years. The aim of this study was to determine the frequency of rotavirus infection in patients whose stool samples were sent to microbiology laboratory to investigate the etiology of diarrhea, to investigate the rotavirus genotypes that are common in our region and G10, G12 genotypes that have recently become common in the world. Fecal samples of 476 patients aged between 0-92 years who applied between November 2016 and February 2018 were studied via immunochromatographic rapid test and enzyme-linked immunosorbent assay (ELISA) methods. ELISA positive samples were studied by nested reverse transcriptase chain reaction (RT-PCR) and genotyped by agarose gel electrophoresis. Rotavirus was found positive in 18.3% and 17% of stool samples by immunochromatographic test and ELISA, respectively. All ELISA positive samples were also detected as positive by RT-PCR. 18.5% of female patients and 15.7% of male patients were found to be positive and rotavirus positivity was not statistically significant between genders. The frequency of rotavirus in different age groups was 23.5% (6-12 years), 17.3% (13-24 months) and 16% (25-36 months). It was determined that rotavirus cases were most common in the spring. G1, G2, G3, G4, G9, G10, and G12 were detected in 37%, 7.4%, 16.1%, 6.2%, 9.9%, 2.5%, 26% of the samples, respectively. G12 was the most common genotype after G1. The most common G and P genotype combination was G1P[8] (17.2%). This was followed by G12P[8] (11.11%) and G3P[8] (11.11%). P[8] (53%) was found to be the dominant P genotype. In this study, it was observed that rotavirus, which is the cause of childhood diarrhea, can also be encountered in advanced ages and even new genotypes that infect humans worldwide may also be the causative agents. Therefore, we concluded that it is important to investigate new genotypes such as G10 and G12 in molecular epidemiological studies.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Validation of a multiplex RT-PCR assay for screening significant oncogene fusion transcripts in children with acute lymphoblastic leukaemia

Ariffin H, Chen SP, Wong HL, Yeoh A

Singapore Med J, 2003 Oct;44(10):517-20.
PMID: 15024455

In childhood acute lymphoblastic leukaemia (ALL), cytogenetics play an important role in diagnosis, allocation of treatment and prognosis. Conventional cytogenetic analysis, involving mainly karyotyping in our experience, has not been successful in a large proportion of cases due to inadequate metaphase spreads and poor chromosome morphology. Our aim is to develop a highly sensitive and specific method to screen simultaneously for the four most frequent fusion transcripts resulting from specific chromosomal translocations, namely, both the CML- and ALLtype BCR-ABL transcripts of t(9;22), E2A-PBX1 transcript of t(1;19), the MLL-AF4 transcript of t(4;11) and TEL-AML1 (also termed ETV6-CBFA2) of the cryptic t(12;21). A multiplex reverse transcription polymerase chain reaction protocol (RT-PCR) was developed and tested out on archival bone marrow samples and leukaemia cell lines. In all samples with a known translocation detected by cytogenetic techniques, the same translocation was identified by the multiplex-PCR assay. Multiplex RT-PCR assay is an effective, sensitive, accurate and cost-effective diagnostic tool which can improve our ability to accurately and rapidly risk-stratify patients with childhood ALL.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Fulltext Validation and utilization of an internally controlled multiplex Real-time RT-PCR assay for simultaneous detection of enteroviruses and enterovirus A71 associated with hand foot and mouth disease

Thanh TT, Anh NT, Tham NT, Van HM, Sabanathan S, Qui PT, et al.

Virol J, 2015 Jun 09;12:85.
PMID: 26050791 DOI: 10.1186/s12985-015-0316-2

BACKGROUND: Hand foot and mouth disease (HFMD) is a disease of public health importance across the Asia-Pacific region. The disease is caused by enteroviruses (EVs), in particular enterovirus A71 (EV-A71). In EV-A71-associated HFMD, the infection is sometimes associated with severe manifestations including neurological involvement and fatal outcome. The availability of a robust diagnostic assay to distinguish EV-A71 from other EVs is important for patient management and outbreak response.
METHODS: We developed and validated an internally controlled one-step single-tube real-time RT-PCR in terms of sensitivity, linearity, precision, and specificity for simultaneous detection of EVs and EV-A71. Subsequently, the assay was then applied on throat and rectal swabs sampled from 434 HFMD patients.
RESULTS: The assay was evaluated using both plasmid DNA and viral RNA and has shown to be reproducible with a maximum assay variation of 4.41 % and sensitive with a limit of detection less than 10 copies of target template per reaction, while cross-reactivity with other EV serotypes was not observed. When compared against a published VP1 nested RT-PCR using 112 diagnostic throat and rectal swabs from 112 children with a clinical diagnosis of HFMD during 2014, the multiplex assay had a higher sensitivity and 100 % concordance with sequencing results which showed EVs in 77/112 (68.8 %) and EV-A71 in 7/112 (6.3 %). When applied to clinical diagnostics for 322 children, the assay detected EVs in throat swabs of 257/322 (79.8 %) of which EV-A71 was detected in 36/322 (11.2 %) children. The detection rate increased to 93.5 % (301/322) and 13.4 % (43/322) for EVs and EV-A71, respectively, when rectal swabs from 65 throat-negative children were further analyzed.
CONCLUSION: We have successfully developed and validated a sensitive internally controlled multiplex assay for rapid detection of EVs and EV-A71, which is useful for clinical management and outbreak control of HFMD.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*; Reverse Transcriptase Polymerase Chain Reaction/standards
Type-3 dengue viruses responsible for the dengue epidemic in Malaysia during 1993-1994

Kobayashi N, Thayan R, Sugimoto C, Oda K, Saat Z, Vijayamalar B, et al.

Am J Trop Med Hyg, 1999 Jun;60(6):904-9.
PMID: 10403318

To characterize the dengue epidemic that recently occurred in Malaysia, we sequenced cDNAs from nine 1993-1994 dengue virus type-3 (DEN-3) isolates in Malaysia (DEN-3 was the most common type in Malaysia during this period). Nucleic acid sequences (720 nucleotides in length) from the nine isolates, encompassing the precursor of membrane protein (preM) and membrane (M) protein genes and part of the envelope (E) protein gene were aligned with various reference DEN-3 sequences to generate a neighbor-joining phylogenetic tree. According to the constructed tree, the nine Malaysian isolates were grouped into subtype II, which comprises Thai isolates from 1962 to 1987. Five earlier DEN-3 virus Malaysian isolates from 1974 to 1981 belonged to subtype I. The present data indicate that the recent dengue epidemic in Malaysia was due to the introduction of DEN-3 viruses previously endemic to Thailand.

Matched MeSH terms: Reverse Transcriptase 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: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Transcriptional profiling of feline infectious peritonitis virus infection in CRFK cells and in PBMCs from FIP diagnosed cats

Harun MS, Kuan CO, Selvarajah GT, Wei TS, Arshad SS, Hair Bejo M, et al.

Virol J, 2013;10:329.
PMID: 24209771 DOI: 10.1186/1743-422X-10-329

BACKGROUND:
Feline Infectious Peritonitis (FIP) is a lethal systemic disease, caused by the FIP Virus (FIPV); a virulent mutant of Feline Enteric Coronavirus (FECV). Currently, the viruses virulence determinants and host gene expressions during FIPV infection are not fully understood.

METHODS:
RNA sequencing of Crandell Rees Feline Kidney (CRFK) cells, infected with FIPV strain 79-1146 at 3 hours post infection (h.p.i), were sequenced using the Illumina next generation sequencing approach. Bioinformatic's analysis, based on Felis catus 2X annotated shotgun reference genome, using CLC bio Genome Workbench mapped both control and infected cell reads to 18899 genes out of 19046 annotated genes. Kal's Z test statistical analysis was used to analyse the differentially expressed genes from the infected CRFK cells. Real time RT-qPCR was developed for further transcriptional profiling of three genes (PD-1, PD-L1 and A3H) in infected CRFK cells and Peripheral Blood Mononuclear Cells (PBMCs) from healthy and FIP-diseased cats.

RESULTS:
Based on Kal's Z-test, with False Discovery Rate (FDR) <0.05 and >1.99 fold change on gene expressions, a total of 61 genes were differentially expressed by both samples, where 44 genes were up-regulated and the remainder were down-regulated. Most genes were closely clustered together, suggesting a homogeneous expression. The majority of the genes that were significantly regulated, were those associated with monocytes-macrophage and Th1 cell functions, and the regulation of apoptosis. Real time RT-qPCR developed focusing on 2 up-regulated genes (PD-L1 and A3H) together with an apoptosis associated gene PD-1 expressions in FIPV infected CRFK cells and in PBMCs from healthy and FIP diagnosed cats produced concordant results with transcriptome data.

CONCLUSION:
The possible roles of these genes, and their importance in feline coronaviruses infection, are discussed.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
Tracing the origins of genotype VIIh Newcastle disease in southern Africa

Abolnik C, Mubamba C, Wandrag DBR, Horner R, Gummow B, Dautu G, et al.

Transbound Emerg Dis, 2018 Apr;65(2):e393-e403.
PMID: 29178267 DOI: 10.1111/tbed.12771

It is widely accepted that Newcastle disease is endemic in most African countries, but little attention has been afforded to establishing the sources and frequency of the introductions of exotic strains. Newcastle disease outbreaks have a high cost in Africa, particularly on rural livelihoods. Genotype VIIh emerged in South-East Asia and has since caused serious outbreaks in poultry in Malaysia, Indonesia, southern China, Vietnam and Cambodia. Genotype VIIh reached the African continent in 2011, with the first outbreaks reported in Mozambique. Here, we used a combination of phylogenetic evidence, molecular dating and epidemiological reports to trace the origins and spread of subgenotype VIIh Newcastle disease in southern Africa. We determined that the infection spread northwards through Mozambique, and then into the poultry of the north-eastern provinces of Zimbabwe. From Mozambique, it also reached neighbouring Malawi and Zambia. In Zimbabwe, the disease spread southward towards South Africa and Botswana, causing outbreaks in backyard chickens in early-to-mid 2013. In August 2013, the disease entered South Africa's large commercial industry, and the entire country was infected within a year, likely through fomites and the movements of cull chickens. Illegal poultry trading or infected waste from ships and not wild migratory birds was the likely source of the introduction to Mozambique in 2011.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/veterinary
Fulltext Thyroid Hormone Upregulates Hypothalamic kiss2 Gene in the Male Nile Tilapia, Oreochromis niloticus

Ogawa S, Ng KW, Xue X, Ramadasan PN, Sivalingam M, Li S, et al.

Front Endocrinol (Lausanne), 2013;4:184.
PMID: 24324459 DOI: 10.3389/fendo.2013.00184

Kisspeptin has recently been recognized as a critical regulator of reproductive function in vertebrates. During the sexual development, kisspeptin neurons receive sex steroids feedback to trigger gonadotropin-releasing hormone (GnRH) neurons. In teleosts, a positive correlation has been found between the thyroid status and the reproductive status. However, the role of thyroid hormone in the regulation of kisspeptin system remains unknown. We cloned and characterized a gene encoding kisspeptin (kiss2) in a cichlid fish, the Nile tilapia (Oreochromis niloticus). Expression of kiss2 mRNA in the brain was analyzed by in situ hybridization. The effect of thyroid hormone (triiodothyronine, T3) and hypothyroidism with methimazole (MMI) on kiss2 and the three GnRH types (gnrh1, gnrh2, and gnrh3) mRNA expression was analyzed by real-time PCR. Expression of thyroid hormone receptor mRNAs were analyzed in laser-captured kisspeptin and GnRH neurons by RT-PCR. The kiss2 mRNA expressing cells were seen in the nucleus of the lateral recess in the hypothalamus. Intraperitoneal administration of T3 (5 μg/g body weight) to sexually mature male tilapia significantly increased kiss2 and gnrh1 mRNA levels at 24 h post injection (P

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
The transcript level of interleukin-6 in the cartilage of idiopathic osteoarthritis of knee

Moktar NM, Yusof HM, Yahaya NH, Muhamad R, Das S

Clin Ter, 2010;161(1):25-8.
PMID: 20393674

AIMS: The mRNA level for interleukin-6 (IL-6) is an important marker of osteoarthritis (OA). The present study aimed to investigate the level of IL-6 mRNA in the cartilage of OA knee while comparing it to the normal cartilage obtained from the same patient.
MATERIALS AND METHODS: A total of 21 patients who underwent total knee replacement were recruited for this study. Sectioning of the destructive cartilage was performed in the medial part of the proximal tibiofemoral cartilage. The unaffected lateral part of the knee in the same patient, served as a control. The mRNA level for IL-6 was assessed using LightCycler 2.0 quantitative real-time polymerase chain reaction (qRT-PCR). actin mRNA was used as an endogenous control.
RESULTS: Twelve out of 21 patients (57.1%) exhibited up regulation of IL-6 mRNA in the OA cartilage as compared to the normal cartilage. The rest of the patients (42.9%) showed down regulation of IL-6 mRNA. The statistical analysis showed there was insignificant level of IL-6 mRNA in the OA (1.91 +/- 0.45) as compared to the normal cartilage (1.13 +/- 0.44) (p > 0.05). The inter-individual variation in the level of IL-6 mRNA in the cartilage of idiopathic knee was in accordance with previous findings.
CONCLUSIONS: These observations suggest IL-6 could also act as a catabolic agent in some patients or its expression might be influenced by other cytokines.
Study site: Pusat Perubatan Universiti Kebangsaan Malaysia (PPUKM), Kuala Lumpur, Malaysia

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext The role of blood transfusion in HCV infection: a study testing Ab: RNA & genotype

Al-Kubaisy, Waqar A., Niazi, Amjad D.

Int J Public Health Res, 2011;1(2):72-78.
MyJurnal

Introduction Hepatitis C Virus (HCV) recently was identified as a major cause of post transfusion hepatitis world wide. To evaluate the role of blood transfusion on the prevalence of HCV infection, by testing antibody and RNA as well as the genotypes of HCV .Also to detect if Blood transfusion acts as unconfounding risk factor for HCV infection.
Methods Sera from 3491 pregnant women were investigated for the presence of HCV antibodies (anti-HCV) by using third generation enzyme immunoassay (EIA-3) as screening test, followed by immunoblot assay (Lia Tek-III). In addition 94 sera of studied women were subjected to molecular analysis (at laboratories of Sorin BioMedica - Italy) for the detection of viral RNA and genotypes of HCV. Using RT-PCR & DNA Enzyme immunoassay (DEIA) method.
Results Our study revealed, that seroprevalence rate of HCV specific Ab & RNA were significantly higher (16.32 %, 80% respectively) among women with a history of blood transfusion, compared to those (2.53%, 56.5%) with no such history P=0.0001, P=0.01. And there is a significant direct linear correlation between number of blood transfused and the seropositive rate of anti-HCV (r=0.7, p=0.046). Based on multivariate analysis, interestingly, this study confirmed that, blood transfusion significantly acting as unconfounding risk factor for acquiring HCV infection (Adjusted OR=1.938,95% C.I=1.646-2.28). And the risk of exposure is increases with increased number of blood transfused. Although, we found no significant association between, HCV genotypic distribution and history of blood transfusion. However, high proportion of women with a history of blood transfusion were harboring HCV genotype -4 or 1b, 50%,40%, resepctively.
Conclusions Our study shows, evidence that, blood transfusion acts as unconfounding risk factor for acquiring and in a mode of transmission of HCV infection. Therefore strict screening of blood donor for HCV-Abs and / or RNA is highly recommended.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
The mazEF toxin-antitoxin system as a novel antibacterial target in Acinetobacter baumannii

Ghafourian S, Good L, Sekawi Z, Hamat RA, Soheili S, Sadeghifard N, et al.

Mem Inst Oswaldo Cruz, 2014 Jul;109(4):502-5.
PMID: 25004148

Although analysis of toxin-antitoxin (TA) systems can be instructive, to date, there is no information on the prevalence and identity of TA systems based on a large panel of Acinetobacter baumannii clinical isolates. The aim of the current study was to screen for functional TA systems among clinical isolates of A. baumannii and to identify the systems' locations. For this purpose, we screened 85 A. baumannii isolates collected from different clinical sources for the presence of the mazEF, relBE and higBA TA genes. The results revealed that the genes coding for the mazEF TA system were commonly present in all clinical isolates of A. baumannii. Reverse transcriptase-polymerase chain reaction analysis showed that transcripts were produced in the clinical isolates. Our findings showed that TA genes are prevalent, harboured by chromosomes and transcribed within A. baumannii. Hence, activation of the toxin proteins in the mazEF TA system should be investigated further as an effective antibacterial strategy against this bacterium.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
The effect of extrinsic incubation temperature on development of dengue serotype 2 and 4 viruses in Aedes aegypti (L.)

Rohani A, Wong YC, Zamre I, Lee HL, Zurainee MN

Southeast Asian J. Trop. Med. Public Health, 2009 Sep;40(5):942-50.
PMID: 19842378

Dengue 2 and 4 viruses obtained from dengue-infected patients were maintained in a C6/36 Aedes albopictus Skuse cell line and used to infect adult female Aedes aegypti mosquitoes. Each serotype was mixed separately with fresh human erythrocytes and fed to adult female mosquitoes using an artificial membrane feeding technique. Fully engorged mosquitoes were selected and retained at 26 degrees C, 28 degrees C and 30 degrees C to observe dengue virus development in Aedes vectors. Virus detection was carried out by reverse-transcriptase polymerase chain reaction (RT-PCR). The virus was first detected on Day 9 at 26 degrees C and 28 degrees C and on Day 5 at 30 degrees C for both dengue 2 and 4. The study shows the incubation period of the viruses decreased when the extrinsic incubation temperature increases.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
The distribution of important sero-complexes of flaviviruses in Malaysia

Kumar K, Arshad SS, Toung OP, Abba Y, Selvarajah GT, Abu J, et al.

Trop Anim Health Prod, 2019 Mar;51(3):495-506.
PMID: 30604332 DOI: 10.1007/s11250-018-01786-x

Flaviviruses (FVs) are arthropod-borne viruses of medical and veterinary importance. Numerous species of FVs have been isolated from various host; mainly humans, animals, ticks, and mosquitoes. Certain FVs are extremely host-specific; at the same time, some FVs can infect an extensive range of species. Based on published literatures, 11 species of FVs have been detected from diverse host species in Malaysia. In humans, dengue virus and Japanese encephalitis virus have been reported since 1901 and 1942. In animals, the Batu Cave virus, Sitiawan virus, Carey Island, Tembusu virus, Duck Tembusu virus, and Japanese encephalitis viruses were isolated from various species. In mosquitoes, Japanese encephalitis virus and Kunjin virus were isolated from Culex spp., while Zika virus and Jugra virus were isolated from Aedes spp. In ticks, the Langat virus was isolated from Ixodes spp. One of the major challenges in the diagnosis of FVs is the presence of sero-complexes as a result of cross-reactivity with one or more FV species. Subsequently, the distribution of specific FVs among humans and animals in a specific population is problematic to assess and often require comprehensive and thorough analyses. Molecular assays such as quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and digital droplet RT-PCR (ddRT-PCR) have been used for the differentiation of flavivirus infections to increase the accuracy of epidemiological data for disease surveillance, monitoring, and control. In situations where sero-complexes are common in FVs, even sensitive assays such as qRT-pCR can produce false positive results. In this write up, an overview of the various FV sero-complexes reported in Malaysia to date and the challenges faced in diagnosis of FV infections are presented.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
The appearance of a second genotype of Japanese encephalitis virus in the Australasian region

Pyke AT, Williams DT, Nisbet DJ, van den Hurk AF, Taylor CT, Johansen CA, et al.

Am J Trop Med Hyg, 2001 Dec;65(6):747-53.
PMID: 11791969

In mid-January 2000, the reappearance of Japanese encephalitis (JE) virus activity in the Australasian region was first demonstrated by the isolation of JE virus from 3 sentinel pigs on Badu Island in the Torres Strait. Further evidence of JE virus activity was revealed through the isolation of JE virus from Culex gelidus mosquitoes collected on Badu Island and the detection of specific JE virus neutralizing antibodies in 3 pigs from Saint Pauls community on Moa Island. Nucleotide sequencing and phylogenetic analyses of the premembrane and envelope genes were performed which showed that both the pig and mosquito JE virus isolates (TS00 and TS4152, respectively) clustered in genotype I, along with northern Thai, Cambodian, and Korean isolates. All previous Australasian JE virus isolates belong to genotype II, along with Malaysian and Indonesian isolates. Therefore, for the first time, the appearance and transmission of a second genotype of JE virus in the Australasian region has been demonstrated.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext The Role of Semipurified Fractions Isolated from Quercus infectoria on Bone Metabolism by Using hFOB 1.19 Human Fetal Osteoblast Cell Model

Abdullah AR, Hapidin H, Abdullah H

Evid Based Complement Alternat Med, 2018;2018:5319528.
PMID: 29861772 DOI: 10.1155/2018/5319528

Background. Quercus infectoria (QI) is a plant used in traditional medicines in Asia. The plant was reported to contain various active phytochemical compounds that have potential to stimulate bone formation. However, the precise mechanism of the stimulation effect of QI on osteoblast has not been elucidated. The present study was carried out to isolate QI semipurified fractions from aqueous QI extract and to delineate the molecular mechanism of QI semipurified fraction that enhanced bone formation by using hFOB1.19 human fetal osteoblast cell model. Methods. Isolation of QI semipurified fractions was established by means of column chromatography and thin layer chromatography. Established QI semipurified fractions were identified using Liquid Chromatography-Mass Spectrometry (LC-MS). Cells were treated with derived QI semipurified fractions and investigated for mineralization deposition and protein expression level of BMP-2, Runx2, and OPN by ELISA followed gene expression analysis of BMP-2 and Runx2 by RT-PCR. Results. Column chromatography isolation and purification yield Fractions A, B, and C. LC-MS analysis reveals the presence of polyphenols in each fraction. Results show that QI semipurified fractions increased the activity and upregulated the gene expression of BMP-2 and Runx2 at day 1, day 3, and day 7. OPN activity increased in cells treated with QI semipurified fractions at day 1 and day 3. Meanwhile, at day 7, expression of OPN decreased in activity. Furthermore, the study showed that combination of Fractions A, B, and C with osteoporotic drug (pamidronate) further increased the activity and upregulated the gene expression of BMP-2 and Runx2. Conclusions. These findings demonstrated that polyphenols from semipurified fractions of QI enhanced bone formation through expression of the investigated bone-related marker that is its potential role when combined with readily available osteoporotic drug.

Matched MeSH terms: Reverse Transcriptase 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: Reverse Transcriptase Polymerase Chain Reaction
Fulltext The Effect of Oxidative Stress Towards The Expression of Thiamine Biosynthesis Genes (THIC and THI1/THI4) in Oil Palm (Elaeis guineensis)

Idris ZHC, Abidin AAZ, Subki A, Yusof ZNB

Trop Life Sci Res, 2018 Mar;29(1):71-85.
PMID: 29644016 MyJurnal DOI: 10.21315/tlsr2018.29.1.5

Thiamine is known to be an important compound in human diet and it is a cofactor required for vital metabolic processes such as acetyl-CoA biosynthesis, amino acid biosynthesis, Krebs and Calvin cycle. Besides that, thiamine has been shown to be involved in plant protection against stress. In this study, the level of expression of THIC and THI1/THI4, the genes for the first two enzymes in the thiamine biosynthesis pathway were observed when oil palm (Elaeis guineensis) was subjected to oxidative stress. Primers were designed based on the consensus sequence of thiamine biosynthesis genes obtained from Arabidopsis thaliana, Zea mays, Oryza sativa, and Alnus glutinosa. Oxidative stress were induced with various concentrations of paraquat and samplings were done at various time points post-stress induction. The expression of THIC and THI1/THI4 genes were observed via RT-PCR and qPCR analysis. The expression of THIC was increased 2-fold, while THI1/THI4 gene transcript was increased 4-fold upon induction of oxidative stress. These findings showed that oil palm responded to oxidative stress by over-expressing the genes involved in thiamine biosynthesis. These findings support the suggestion that thiamine may play an important role in plant protection against stress.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction

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