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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
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
Arabidopsis Group IIId ERF proteins positively regulate primary cell wall-type CESA genes

Saelim L, Akiyoshi N, Tan TT, Ihara A, Yamaguchi M, Hirano K, et al.

J Plant Res, 2019 Jan;132(1):117-129.
PMID: 30478480 DOI: 10.1007/s10265-018-1074-1

The cell wall determines morphology and the environmental responses of plant cells. The primary cell wall (PCW) is produced during cell division and expansion, determining the cell shape and volume. After cell expansion, specific types of plant cells produce a lignified wall, known as a secondary cell wall (SCW). We functionally analyzed Group IIId Arabidopsis AP2/EREBP genes, namely ERF34, ERF35, ERF38, and ERF39, which are homologs of a rice ERF gene previously proposed to be related to SCW biosynthesis. Expression analysis revealed that these four genes are expressed in regions related to cell division and/or cell differentiation in seedlings (i.e., shoot apical meristems, the primordia of leaves and lateral roots, trichomes, and central cylinder of primary roots) and flowers (i.e., vascular tissues of floral organs and replums and/or valve margins of pistils). Overexpression of ERF genes significantly upregulated PCW-type, but not SCW-type, CESA genes encoding cellulose synthase catalytic subunits in Arabidopsis seedlings. Transient co-expression reporter analysis indicated that ERF35, ERF38, and ERF39 possess transcriptional activator activity, and that ERF34, ERF35, ERF38, and ERF39 upregulated the promoter activity of CESA1, a PCW-type CESA gene, through the DRECRTCOREAT elements, the core cis-acting elements known to be recognized by AP2/ERF proteins. Together, our findings show that Group IIId ERF genes are positive transcriptional regulators of PCW-type CESA genes in Arabidopsis and are possibly involved in modulating cellulose biosynthesis in response to developmental requirements and environmental stimuli.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Development of a human salivary gland organoid model

Zulaiha A. Rahman, Colin D. Bingle, Lynne Bingle

Malaysian Journal of Medicine and Health Sciences, 2019;15(107):1-1.
MyJurnal

Introduction: Currently, organoid technology provides a useful tool for modelling human organ development and pathologies in vitro. Salivary gland (SG) organoids developed from mice SG cells display self-organizing properties closely mimic the native organ. Thus, this study would like to investigate the potential of this organoid system to de-velop a human salivary gland in vitro. Methods: Organoids were developed from biopsy samples of normal human sublingual gland tissue. Cells were isolated and cultured in Matrigel at an Air Liquid Interface (ALI) for up to 14 days in an enriched media supplementing with Wnt-3A, R-spondin1, EGF, and FGF2. Specific differentiation factors like TGFβ, BMP, and LIMK inhibitors were added to enriched media for further differentiation studies. Haematoxylin and eosin-stained sections of the cultures were used to visualise growth. RT-PCR, immunohistochemistry and immunoflu-orescence were used to determine the differential expression of cell-specific markers. Results: Human SG organoids developed when the cells were grown in Matrigel at ALI in a defined culture system. The addition of TGFβ inhibitor and all the inhibitors (TGFβ, BMP and LIMK) to the culture media affected SG organoids development by displaying distinct characteristics that closely resemble native glands and expressed specific cell-type markers; BPIFA2, AQP5, CK5 and E-cadherin. The inhibition of BMP signalling demonstrated SG organoids growth more into ductal-like struc-tures and expressed ductal cell marker, CK7. While LIM kinase inhibition signalling showed significantly higher of amylase activity assay. Conclusion: This study certainly offers valuable insight into determining the optimal culture conditions for developing human SG organoids.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Echovirus serotypes circulating in Malaysia from 2014 to 2019

Manisya Zauri Abdul Wahid, Tengku Rogayah T. Abd. Rashid, Hariyati Md. Ali, Hamadah Mohd Shafiff, Mohd. Shamsul Samsuddin, Syarifah Nur Aisyatun Syed Mohd Salleh, et al.

Malaysian Journal of Medicine and Health Sciences, 2019;15(106):91-91.
MyJurnal

Introduction:Echoviruses are Enteroviruses (HEVs) that infect millions of people annually worldwide, primarily paediatrics. These viruses are frequently associated with outbreaks and sporadic cases of viral meningitis, enceph-alitis, paralysis, myocarditis, severe systemic infections; and hand-foot-mouth disease. This study is a retrospective study to identify Echovirus serotypes circulating in Malaysia from January 2014 to June 2019, and their roles in outbreak prediction. This study investigated the Echovirus serotypes circulating in Malaysia from January 2014 to June 2019. Methods: A total of 13,855 inpatient samples consisting respiratory secretion, stool, tissue and body fluid from around the country were received by the Virology Unit, Institute for Medical Research between January 2014 and June 2019. The presence of HEV’s RNA was detected by qPCR. The identified positive sample was further isolated by cell culture and identified by Immunofluorescence Assay (IFA). The IFA positive samples were subjected to amplification of partial VP4 gene by RT-PCR, and proceeded to Sanger sequencing for phylogenetic analysis by using ChromasPro and MEGA Software. The sequence generated were analysed by BLAST to confirm the sequence serotypes generated. Results: Echovirus genome was detected in 0.35% (37/10,681) of the patients. The circulating Echovirus subtypes in Malaysia between January 2014 and June 2019 were Echo-11 (43.2%; 16/37), followed by Echo-6 (16.2%; 6/37); 8.1% (3/37) of Echo-7 and Echo-13, respectively. Meanwhile, other types of Echoviruses (24.3%; 9/37) such as Echo 3-5, Echo-14, Echo-16, Echo-18, Echo-25 and Echo-30 were also detected in this study. Conclusion: In this study, it has been found that Echovirus 11 serotype is the most predominant Echovirus serotype circulating in Malaysia between January 2014 and June 2019. It has been reported to cause severe diseases, such as aseptic meningitis. Therefore, the identification of circulating serotypes of Echovirus is critical to predict the Echovi-rus outbreak and to reduce the risk of developing severe disease in Malaysia.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Molecular investigation of feline coronavirus (FCOV) in local pet cats

Hoong, L.W., Yasmin, A.R., Mummoorthy, K., Arshad, S.S., Omar, A.R., Anand, P., et al.

Jurnal Veterinar Malaysia, 2019;31(2):13-18.
MyJurnal

Feline coronavirus (FCoV) infection is a very common in cat population. FCoV is further classified into two biotypes namely feline enteric coronavirus (FECV) and mutated feline infectious peritonitis virus (FIPV), in which FIPV causes a fatal immune complex disease by changing the tropism from enterocytes to monocytes. Previous studies on molecular detection of FCoV in cats were carried out in catteries but limited study investigate the presence of FCoV antigen in local pet cats. By considering this fact, this study aims to detect FCoV antigen via RT-PCR assay in local pet cats and to compare the similarity of the identified FCoV strain with previous related virus by phylogenetic analysis. By using convenience sampling, rectal swabs and buffy coat were collected from 16 clinically ill pet cats and 5 healthy pet cats. Viral RNA was extracted and subjected to one-step RT-PCR, targeting polymerase gene. Only one out of 21 fecal samples was positive for FCoV and none from buffy coat samples. Phylogenetic analysis revealed that the identified positive sample was highly homologous, up to 95%, to FCoV strain from Netherlands and South Korea on partial sequence of polymerase gene. In conclusion, this study detected FCoV antigen in local pet cats from fecal samples while negative detection from fecal and buffy coat samples could not completely rule out the possibilities of FCoV infection due to the complexity of the virus diagnosis that require multiple series of analysis.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Emergence of Getah Virus Infection in Horse With Fever in China, 2018

Lu G, Ou J, Ji J, Ren Z, Hu X, Wang C, et al.

Front Microbiol, 2019;10:1416.
PMID: 31281304 DOI: 10.3389/fmicb.2019.01416

Getah virus (GETV) is a mosquito-borne virus that was first determined in Malaysia in 1955, and can infect humans and multiple other mammals. GETV infection in horses has been reported in Japan and India, and causes great economic losses. In China, GETV has been identified in mosquitoes, pigs, foxes, and cattle with a wide geographical distribution, but has not been detected in horses. In August 2018, a sudden onset of fever was observed in racehorse in an equestrian training center in Guangdong Province in southern China. Blood samples were collected from the sick horse, and PCR/RT-PCR analysis was performed to screen for equine viral pathogens associated with fever. The results indicated that the samples were GETV RNA positive. After RT-PCR, sequencing, and assembly, the genome of the first Chinese horse-derived GETV strain, GZ201808, was obtained. Compared with the genome sequences of other GETV strains, twelve unique nucleotide substitutions were observed in GZ201808. The genome of GZ201808 had the highest genetic identity (99.6%) with AH9192, which was detected in pigs in China in 2017. Phylogenetic analysis indicated that GZ201808 clustered in Group III, and was located in an independent branch distant from other horse-derived GETV strains, indicating a unique evolutionary pattern of GZ201808. This study first determined and described the disease course of horse infected with GETV in China, sequenced and characterized the genome of the field horse-derived GETV strain, and therefore presented an unequivocal report of GETV infection in horses in China.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Milestone of polio Environmental Surveillance in Malaysia conjoining with the Polio Global Eradication Initiative

Kamal Haikal Mat Rabi, Amry Khursany Ismail, Mohd Samsul Samsuddin, Manisha Zauri Abdul Wahid, Zarina Mohd Zamawi, Ravindran Thayan

Malaysian Journal of Medicine and Health Sciences, 2019;15(106):78-78.
MyJurnal

Introduction: Poliomyelitis is an incapacitating and highly infectious disease which effect mostly young children. It is caused by one of the three serotypes of polioviruses (PV) and transmitted through faecal-oral route hence making the disease quite pertinent to the lower and middle class society or under-immunized population. This surveillance is one of the strategy included by WHO in the “Eradication, Integration and Certification: The Endgame Strategy 2019-2023” as a supplement to AFP surveillance by which it could be more sensitive to detect low circulation of WPV and circulating vaccine derived poliovirus (cVDPV). Methods: Routine collection and testing of representative environmental surveillance are carried out in the National Polio Laboratory. The specimens are collected from designated locations draining target populations at increased risk of poliovirus transmission using the grab method once a month and processed according to WHO standard protocol. Polioviruses were identified by real time reverse transcriptase polymerase chain reaction (rRT-PCR) for intratypic differentiation (ITD) and vaccine derived poliovirus (VDPV) whereas non-polio enteroviruses (NPEVs) were identified by PCR and sequencing. Results: From 2012 to 2019, results showed various isolation of PVs and NPEVs. A total of 12 sewage disposal plants located in urban highly populated areas in Kuala Lumpur (3), Selangor (5), Sabah (3 ) and Negeri Sembilan (1) were investigated. A total of 22 Sabin-like PVs were isolated consisting of 3 PV1, 8 PV2 and 11 PV3 thus indicated that in Malaysia even though PVs were existed in environment, but all of them were Sabin-Like viruses and no evidence of imported WPV or VDPV in the sampling sites. Conclusion: Even though Malaysia has been declared as WPV free country in 2000, Environmental Surveillance is very important and crucial in detecting the introduction and silent circulation of WPV and cVDPV before the virus reaches the community.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Detection of all four dengue virus serotypes and identification of four mismatched nucleotides between DENV4 specific primer rTS4 and DENV 4 sequences in Sandakan and Kudat (2016-2017)

Tin Sabai Aung, Amalina Emran, Chua Tock Hing, Tin Tin Thein, Win Win Than, Aye Aye Wynn, et al.

Malaysian Journal of Medicine and Health Sciences, 2019;15(106):49-49.
MyJurnal

Introduction: Dengue is caused by dengue virus (DENV) which is a member of the genus Flavivirus of the family Flaviviridae. The prevalence of dengue has been increasing all over the world especially in Southeast Asia and Western Pacific regions. In 2016 - 2017 dengue outbreaks were reported in Sandakan and Kudat of Sabah, Malay-sia. The aim of this study was to determine the serotypes of dengue viruses circulating in these two sites during the outbreaks. Methods: A total of 200 dengue patients’ sera tested positive with NS1 and IgM & IgG rapid test (PanBio) were collected from Hospital Duchess of Kent Sandakan and Hospital Kudat between June 2016 and December 2017. PCR was done at the Faculty of Medicine and Health Sciences, Universiti Malaysia Sabah. One-Step Reverse transcriptase PCR (RT-PCR) and nested PCR was performed using C-prM amplimers designed by Lanciotti et al and later redesigned by Chien et al, followed by sequencing some of the PCR products. Results: Out of 200 sera tested 128 were PCR positive. All the four dengue serotypes were detected with PCR products with specific sizes in gel electrophoresis. However, in four samples, no serotype-specific band was amplified by the nested PCR, while they were dengue-positive in RT-PCR showing 511 base pair amplicon. Sequencing results revealed all four samples were found to belong to DENV4. The sequences of these samples were aligned with that of DENV 4 reverse primer rTS4. The DENV4 specific primer rTS4 was found to have four mismatched nucleotides to the DENV4 sequences. Conclusion: There was a co-circulation of DENV1 to 4 in Sandakan and Kudat in the study period. DENV1 was the predominant serotype. DENV4 specific C-prM primer rTS4 should be redesigned for the local DENV4 strain in Sabah in future research.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Multiplex real-time RT-PCR assay for diagnosis of Dengue, Zika and Chikungunya infections

Zarina Mohd Zawawi, Tengku Rogayah Tengku Abdul Rashid, Amir Hussien Adiee, Murni Maya Sari, Ravindran Thayan

Malaysian Journal of Medicine and Health Sciences, 2019;15(106):5-5.
MyJurnal

Introduction: Dengue virus (DENV), Zika virus (ZIKV) and Chikungunya virus (CHIKV) are Arboviruses that are transmitted by the same vector, Aedes aegypti. Dengue has become a global problem since the Second World War and is common in more than 110 countries. In Malaysia, dengue is a major disease burden as total economic costs to the country as a result of dengue is close to RM1.05 billion in 2010 and estimated to rise to 1.3 billion by 2020. Apart from Dengue, Zika and Chikungunya are the other important mosquito borne diseases in Malaysia. The aim of this study was to develop a multiplex real-time assay for simultaneous detection of DENV, ZIKV and CHIKV in clinical specimens. Methods: The published singleplex protocols were used with key modifications to implement a triplex assay. A one-step multiplex real-time RT-PCR assay was developed that can simultaneously detect RNA of DENV, ZIKV and CHIKV with good performance for a routine diagnostic use. The assay was evaluated for inter- and intra-reproducibility by mean CT value. The diagnostic sensitivity was tested with 135 archived samples which had been defined positive or negative by routine singleplex assays. Whole blood, plasma and urines were used in this study. Results: Intra- and inter-reproducibility and sensitivity varied from 0.10% to 4.73% and from 0.45% to 5.98% for each virus respectively. The specificity of detection was 100%. The multiplex real-time RT-PCR assay showed concordance with test results performed by routine singleplex assays. No cross reaction was observed for any of the clinical samples. Conclusion: The development of a rapid, sensitive and specific molecular assay for DENV, ZIKV and CHIKV infections will produce a greater diagnostic capacity in our laboratory. This multiplex approach is cost effective and robust with the concurrent detection of 3 viruses of public health concern.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext An inhibited dopamine synthesizing cell model of AADC deficiency

Melati Khalid, Mohamad Aris Mohd Moklas

Life Sciences, Medicine and Biomedicine, 2019;3(2):1-7.
MyJurnal

Aromatic L-amino acid decarboxylase deficiency (AADC) is a rare autosomal recessive pediatric neurotransmitter disease. To date it remains poorly understood mainly due to an absence of a disease model. The dopaminergic neuroblastoma cell SH-SY5Y was chosen to develop our AADC deficiency model. These cells are not native dopamine synthesizers. Objective: To develop a dopamine-producing cellular model of AADC deficiency using SH-SY5Y neuroblastoma cells. Methods: Dopamine pathway proteins were identified with Western Blotting. Dopaminergic differentiation was attempted using all-trans retinoic acid (ATRA) with dopamine detection via HPLC-ECD post alumina extraction. Treatment with L-DOPA provided SH-SY5Y with excess precursor. RT-PCR was used to determine the expression of markers of mature neurons. Results: Western Blot screening identified AADC, dopamine β-hydroxylase and tyrosine hyrdoxylase proteins, indicative of a dopaminergic pathway. ATRA was unsuccessful in producing dopamine from the cells. L-DOPA treatment however, generated dopamine first visible as a HPLC-ECD peak 30 minutes post-incubation. Prior to this, SH-SY5Y dopamine synthesis from L-DOPA has never been documented. This de novo synthesis is then inhibited using benserazide to form our AADC deficiency cell model. RT-PCR showed that SH-SY5Y cells express markers of mature neurons in its ‘native’ state and is not affected by L-DOPA and benserazide treatment. This cell model will potentially benefit many areas of AADC deficiency research. Conclusion: SH-SY5Y cells produced HPLC-ECD measureable amounts of dopamine with the addition of L-DOPA. Our model of AADC deficiency is generated by quelling the dopamine production with Benserazide.

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 A quantitative reverse transcription-polymerase chain reaction for detection of Getah virus

Sam SS, Teoh BT, Chee CM, Mohamed-Romai-Noor NA, Abd-Jamil J, Loong SK, et al.

Sci Rep, 2018 12 05;8(1):17632.
PMID: 30518924 DOI: 10.1038/s41598-018-36043-6

Getah virus (GETV), a mosquito-borne alphavirus, is an emerging animal pathogen causing outbreaks among racehorses and pigs. Early detection of the GETV infection is essential for timely implementation of disease prevention and control interventions. Thus, a rapid and accurate nucleic acid detection method for GETV is highly needed. Here, two TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were developed. The qRT-PCR primers and TaqMan MGB probe were designed based on the conserved region of nsP1 and nsP2 genes of 23 GETV genome sequences retrieved from GenBank. Only the qRT-PCR assay using nsP2-specific primers and probe detected all two Malaysia GETV strains (MM2021 and B254) without cross-reacting with other closely related arboviruses. The qRT-PCR assay detected as few as 10 copies of GETV RNA, but its detection limit at the 95% probability level was 63.25 GETV genome copies (probit analysis, P ≤ 0.05). Further validation of the qRT-PCR assay using 16 spiked simulated clinical specimens showed 100% for both sensitivity and specificity. In conclusion, the qRT-PCR assay developed in this study is useful for rapid, sensitive and specific detection and quantification of GETV.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Molecular surveillance of coxsackievirus A16 reveals the emergence of a new clade in mainland China

Chen L, Yao XJ, Xu SJ, Yang H, Wu CL, Lu J, et al.

Arch Virol, 2018 Nov 29.
PMID: 30498962 DOI: 10.1007/s00705-018-4112-3

Coxsackievirus A16 (CV-A16) of the genotypes B1a and B1b have co-circulated in mainland China in the past decades. From 2013 to 2017, a total of 3,008 specimens from 3,008 patients with mild hand, foot, and mouth disease were collected in the present study. Viral RNA was tested for CV-A16 by a real-time RT-PCR method, and complete VP1 sequences and full-length genome sequences of CV-A16 strains from this study were determined by RT-PCR and sequencing. Sequences were analyzed using a series of bioinformatics programs. The detection rate for CV-A16 was 4.1%, 25.9%, 10.6%, 28.1% and 12.9% in 2013, 2014, 2015, 2016 and 2017, respectively. Overall, the detection rate for CV-A16 was 16.5% (497/3008) in this 5-year period in Shenzhen, China. One hundred forty-two (142/155, 91.6%) of the 155 genotype B1 strains in the study belonged to subgenotype B1b, and 13 (13/155, 8.4%) strains belonged to subgenotype B1a. Two strains (CVA16/Shenzhen174/CHN/2017 and CVA16/Shenzhen189/CHN/2017) could not be assigned to a known genotype. Phylogenetic analysis of these two strains and other Chinese CV-A16 strains indicated that these two CV-A16 strains clustered independently in a novel clade whose members differed by 8.4%-11.8%, 8.4%-12.1%, and 14.6%-14.8% in their nucleotide sequences from those of Chinese B1a, B1b, and genotype D strains, respectively. Phylogenetic analysis of global CV-A16 strains further indicated that the two novel CV-A16 strains from this study grouped in a previously uncharacterized clade, which was designated as the subgenogroup B3 in present study. Meanwhile, phylogenetic reconstruction revealed two other new genotypes, B1d and B4, which included a Malaysian strain and two American strains, respectively. The complete genome sequences of the two novel CV-A16 strains showed the highest nucleotide sequence identity of 92.3% to the Malaysian strain PM-15765-00 from 2000. Comparative analysis of amino acid sequences of the two novel CV-A16 strains and their relatives suggested that variations in the nonstructural proteins may play an important role in the evolution of modern CV-A16.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Functional Analysis of Circular RNAs

Shanmugapriya, Huda HA, Vijayarathna S, Oon CE, Chen Y, Kanwar JR, et al.

Adv Exp Med Biol, 2018 9 28;1087:95-105.
PMID: 30259360 DOI: 10.1007/978-981-13-1426-1_8

Circular RNAs characterize a class of widespread and diverse endogenous RNAs which are non-coding RNAs that are made by back-splicing events and have covalently closed loops with no polyadenylated tails. Various indications specify that circular RNAs (circRNAs) are plentiful in the human transcriptome. However, their participation in biological processes remains mostly undescribed. To date thousands of circRNAs have been revealed in organisms ranging from Drosophila melanogaster to Homo sapiens. Functional studies specify that these transcripts control expression of protein-coding linear transcripts and thus encompass a key component of gene expression regulation. This chapter provide a comprehensive overview on functional validation of circRNAs. Furthermore, we discuss the recent modern methodologies for the functional validation of circRNAs such as RNA interference (RNAi) gene silencing assay, luciferase reporter assays, circRNA gain-of-function investigation via overexpression of circular transcript assay, RT-q-PCR quantification, and other latest applicable assays. The methods described in this chapter are demonstrated on the cellular model.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
Fulltext Clinical manifestations of dengue in relation to dengue serotype and genotype in Malaysia: A retrospective observational study

Suppiah J, Ching SM, Amin-Nordin S, Mat-Nor LA, Ahmad-Najimudin NA, Low GK, et al.

PLoS Negl Trop Dis, 2018 09;12(9):e0006817.
PMID: 30226880 DOI: 10.1371/journal.pntd.0006817

BACKGROUND: Malaysia experienced an unprecedented dengue outbreak from the year 2014 to 2016 that resulted in an enormous increase in the number of cases and mortality as compared to previous years. The causes that attribute to a dengue outbreak can be multifactorial. Viral factors, such as dengue serotype and genotype, are the components of interest in this study. Although only a small number of studies investigated the association between the serotype of dengue virus and clinical manifestations, none of these studies included analyses on dengue genotypes. The present study aims to investigate dengue serotype and genotype-specific clinical characteristics among dengue fever and severe dengue cases from two Malaysian tertiary hospitals between 2014 and mid-2017.
METHODOLOGY AND PRINCIPAL FINDINGS: A total of 120 retrospective dengue serum specimens were subjected to serotyping and genotyping by Taqman Real-Time RT-PCR, sequencing and phylogenetic analysis. Subsequently, the dengue serotype and genotype data were statistically analyzed for 101 of 120 corresponding patients' clinical manifestations to generate a descriptive relation between the genetic components and clinical outcomes of dengue infected patients. During the study period, predominant dengue serotype and genotype were found to be DENV 1 genotype I. Additionally, non-severe clinical manifestations were commonly observed in patients infected with DENV 1 and DENV 3. Meanwhile, patients with DENV 2 infection showed significant warning signs and developed severe dengue (p = 0.007). Cases infected with DENV 2 were also commonly presented with persistent vomiting (p = 0.010), epigastric pain (p = 0.018), plasma leakage (p = 0.004) and shock (p = 0.038). Moreover, myalgia and arthralgia were highly prevalent among DENV 3 infection (p = 0.015; p = 0.014). The comparison of genotype-specific clinical manifestations showed that DENV 2 Cosmopolitan was significantly common among severe dengue patients. An association was also found between genotype I of DENV 3 and myalgia. In a similar vein, genotype III of DENV 3 was significantly common among patients with arthralgia.
CONCLUSION: The current data contended that different dengue serotype and genotype had caused distinct clinical characteristics in infected patients.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext A simple and inexpensive physical lysis method for DNA and RNA extraction from freshwater microalgae

Yee W, Abdul-Kadir R, Lee LM, Koh B, Lee YS, Chan HY

3 Biotech, 2018 Aug;8(8):354.
PMID: 30105179 DOI: 10.1007/s13205-018-1381-1

In this work, a simple and inexpensive physical lysis method using a cordless drill fitted with a plastic pellet pestle and 150 mg of sterile sea sand was established for the extraction of DNA from six strains of freshwater microalgae. This lysis method was also tested for RNA extraction from two microalgal strains. Lysis duration between 15 and 120 s using the cetyltrimethyl ammonium bromide (CTAB) buffer significantly increased the yield of DNA from four microalgalstrains (Monoraphidium griffithii NS16, Scenedesmus sp. NS6, Scenedesmus sp. DPBC1 and Acutodesmus sp. DPBB10) compared to control. It was also found that grinding was not required to obtain DNA from two strains of microalgae (Choricystis sp. NPA14 and Chlamydomonas sp. BM3). The average DNA yield obtained using this lysis method was between 62.5 and 78.9 ng/mg for M. griffithii NS16, 42.2-247.0 ng/mg for Scenedesmus sp. NS6, 70.2-110.9 ng/mg for Scenedesmus sp. DPBC1 and 142.8-164.8 ng/mg for Acutodesmus sp. DPBB10. DNA obtained using this method was sufficiently pure for PCR amplification. Extraction of total RNA from M. griffithii NS16 and Mychonastes sp. NPD7 using this lysis method yielded high-quality RNA suitable for RT-PCR. This lysis method is simple, cheap and would enable rapid nucleic acid extraction from freshwater microalgae without requiring costly materials and equipment such as liquid nitrogen or beadbeaters, and would facilitate molecular studies on microalgae in general.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
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 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
Gene isolation using degenerate primers targeting protein motif: A laboratory exercise

Yeo BPH, Foong LC, Tam SM, Lee V, Hwang SS

Biochem Mol Biol Educ, 2018 01;46(1):47-53.
PMID: 29131478 DOI: 10.1002/bmb.21089

Structures and functions of protein motifs are widely included in many biology-based course syllabi. However, little emphasis is placed to link this knowledge to applications in biotechnology to enhance the learning experience. Here, the conserved motifs of nucleotide binding site-leucine rich repeats (NBS-LRR) proteins, successfully used for the isolation and characterization of many plant resistance gene analogues (RGAs), is featured in the development of a series of laboratory experiments using important molecular biology techniques. A set of previously isolated RGA sequences is used as the model for performing sequence alignment and visualising 3D protein structure using current bioinformatics programs (Clustal Omega and Argusdock software). A pair of established degenerate primer sequences is provided for the prediction of targeted amino acids sequences in the RGAs. Reverse transcription-polymerase chain reaction (RT-PCR) is used to amplify RGAs from total RNA samples extracted from the tropical wild relative of black pepper, Piper colubrinum (Piperaceae). This laboratory exercise enables students to correlate specific DNA sequences with respective amino acid codes and the interaction between conserved motifs of resistance genes with putatively targeted proteins. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):47-53, 2018.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction

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