Reverse Transcriptase-polymerase Chain Reaction Technique Research Articles

Severe acute respiratory syndrome coronavirus-2 detection in domestic animals as a reservoir for the virus transmission to humans in Yogyakarta, Indonesia.

The coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that attacks the respiratory and digestive tract. The SARS-CoV-2 showed systemic characteristics with various clinical symptoms from subclinical to fatal (causing death). Transmission of SARS-CoV-2 has been reported to occur from humans to pets (cats, dogs, tigers, ferrets, and poultry). Knowledge about the role of domestic animals in the transmission of SARS-CoV-2 to humans, and as reservoirs of this virus needs to be investigated further. This study aimed to detect the presence of SARS-CoV-2 in domestic animals such as dogs, cats, pigs, cows, birds, and bats that are often in contact with humans. A total of 157 samples, which included nasopharyngeal and oropharyngeal swabs, along with sera samples from domestic animals such as cats, pigs, cows, birds, and bats, were taken from Veterinary Hospitals, Veterinary Clinics, and farms around the Yogyakarta region. Detection of the virus was done using rapid detection of viral antigens, antibodies, and reverse transcriptase-polymerase chain reaction (RT-PCR) technique. The results showed that 5/157 (3.1%) samples found positive against the COVID-19 virus using a rapid antibody test; however, the results were negative on the rapid antigen and RT-PCR tests. Antibody-positive samples came from animals that had a history of household COVID-19 human infection. Thus, findings of the present study conclude that there is a potential for transmission of the COVID-19 virus between animals and humans.

Molecular Diagnosis of Hepatitis C Viruses; Technologies and Their Clinical Applications

Hepatitis-C is one of the most common viral diseases caused by hepatitis C virus (HCV). It is responsible for millions of deaths each year in the developing world. The common dissemination paths of HCV include the use of contaminated water and transfusion of infected blood. Control of this virus has become a challenge for scientists and health professionals due to its versatility and adaptability in different host environments. Along with other problems, lack of efficient diagnosis, quantification and genotyping of viral strains are the major hindrances in a management of this notorious epidemic. The knowledge of HCV genotype and an amount of virus in patient’s blood are pre-requisites to determine the duration and method of treatment. In this review, we discuss the implications of HCV molecular diagnostic methods and their clinical applications. We conclude that while, several commercial and home-brewed methods are available for this purpose, and there is a visible vacuum for cost effective, robust, sensitive assays that can detect multiple viral genotypes in a single reaction. We are of the view that the level of sensitivity offered by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) technique is unequivocal as compared to other techniques. Therefore, researchers may explore further possibilities using this technique in the management of HCV.
 Key words: Hepatitis C virus, Genotyping, Mixed infection, Fluorescence melting curve analysis, Viral Load, Quantification

Occurrence of cherry viruses in South Tyrol (Italy) by comparing growth periods in two consecutive years

Sweet cherries (Prunus avium L.) are important as fruit crops, and can be affected by numerous viruses. An investigation on the occurrence of the three most common viruses of sweet cherry was carried out in commercially managed orchards in South Tyrol (Italy). The incidence of apple chlorotic leaf spot virus (ACLSV), Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) was investigated using enzyme-linked immunosorbent assays (ELISA) and the reverse transcriptase-polymerase chain reaction technique (RT-PCR) in spring 2018 and 2020, and during the summer and autumn of 2020. All three viruses were detected in the surveyed orchards. Comparative analyses showed that detection was more effective with RT-PCR than with ELISA, especially for detecting PNRSV and PDV. Mixed infections were detected in all the surveyed orchards. The results also showed clear differences between and during host growth periods, likely due to a variable virus concentration in the host trees.

Knowledge, Attitude and Practices Regarding Utility of CBNAAT in the Diagnosis of SARSCoV-2 Disease among Medical Interns: A Cross-sectional Study

Introduction: Coronavirus Disease-2019 (COVID-19) causes respiratory tract infections in human beings ranging from mild illnesses like common cold to severe disease like pneumonia. Currently, nucleic acid amplification tests Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), Cartridge Based Nucleic Acid Amplification Test (CBNAAT) and Truenat) and rapid antigen detection tests are approved for diagnostic purpose by Indian Council of Medical Research (ICMR). Medical Interns, the primary contact health-care personnel, need to be sensitised regarding proper utilisation of CBNAAT, so that rapid and accurate diagnosis of Severe Acute Respiratory Syndrome Corona Virus 2 (SARSCoV-2) can be made in comparison to the more commonly used RT-PCR technique. Aim: To evaluate the Knowledge, Attitude and Practices (KAP) of Bachelor of Medicine and a Bachelor of Surgery (MBBS) interns towards accessibility of CBNAAT in SARS-CoV-2 infection in a tertiary care hospital. Materials and Methods: A cross-sectional study was performed through questionnaire shared via online platform amongst 102 Medical interns working at College of Medicine and Sagore Dutta Hospital from 22nd August 2021 to 21st September 2021. Based on their response, KAP was assessed by using a threepoint Likert Scale. The collected data was entered in Microsoft excel, and reported as frequencies and percentages. Results: Among 102 internees, 84 interns responded. Among 84 medical interns 31% had good, 50% had average whereas 19% had poor level of knowledge. About 58 (69%) agreed that CBNAAT can be used as a method for rapid diagnosis of SARS-CoV-2. About 75 (89.3%) answered that they are sending samples for COVID-19 testing. Only 56 (66.7%) agreed that CBNAAT should be recommended. Conclusion: This study revealed that majority of the MBBS interns had positive attitude towards different aspects of CBNAAT utility but most of them had gaps in their KAP. This demands extra efforts to sensitise and train them adequately.

The Impact of DNA Methylation on IL6 mRNA Levels in Hematinic Deficiency and Atopy-Associated Recurrent Aphthous Stomatitis Patients.

Objective To investigate the DNA methylation using pyrosequencing and its effects on the upregulation of IL6 mRNA in patients with recurrent aphthous stomatitis (RAS) in connection with hematinic deficiency and atopy. Material and Methods. This cross-sectional study was conducted at Dr. Hasan Sadikin Hospital, Bandung, from January–March 2019 and was approved by the Health Research Ethics Committee of Universitas Padjadjaran (Ethics No. 990/UN6.KEP/EC/2018). Furthermore, the subjects had RAS ulcers with a history of at least twice a year along with atopy and dietary imbalance with no history of recurrent intraoral herpes or any systemic diseases. This study was performed on 23 RAS patients and 21 healthy subjects, and the sampling was carried out consecutively. The blood samples were collected from all the subjects, and then, the DNA and RNA were extracted from the peripheral blood mononuclear cells (PBMCs). Consequently, the bisulfite-modified DNA was used to confirm the methylation status of the IL6 gene promoter through the pyrosequencing method. The methylation levels of the IL6 promoter were assessed by a reverse transcriptase-polymerase chain reaction technique. The gene expression of RAS and the control group was analyzed by the 2−ΔΔCT method. The statistical analysis using the Mann–Whitney U test was conducted to evaluate IL6 mRNA levels and DNA methylation with p value <0.05 considered to be statistically significant. Result The IL6 mRNA levels were approximately 1.88-fold in RAS patients, and there was a significant relationship between the expression of the IL6 gene and the increased risk of RAS (p < 0.001). It was reported that four out of six sites in the cytosine phosphate guanine (CpG) island IL6 promoter had a lower degree of methylation, and two other sites in patients with RAS had greater methylation compared with control, but not statistically significant. Conclusion This study showed the upregulation of IL6 mRNA levels in RAS patients compared to control. DNA methylation in the present study is at sites 566–658, whereas the location of the IL6 promoter is at sites 1–1684. Thus, it would be necessary conducting some research at other CpG sites of IL6 promoter islands to determine the status of DNA methylation.

Differential clinic in children infected by SARS-CoV-2, traceability of contacts and cost-effectiveness of diagnostic tests: Cross-sectional observational study

IntroductionGiven the possible coexistence of infection by the SARS-CoV-2 with other seasonal infections, the aim is to identify differential symptoms. The role of children in intrafamily contagion and the sensitivity of reverse transcriptase polymerase chain reaction (RT-PCR) in an area with low community transmission has been studied. Material and methodsCross-sectional observational study. Patients between 0–15 years studied by RT-PCR technique due to clinical suspicion of infection by SARS-CoV-2 virus in the months of March-May 2020. Survey on symptoms and contacts. Determination of Anti-SARS-CoV-2 antibodies at least 21 days after the RT-PCR test. Results126 patients were included, 33 with confirmed infection and age mean 8.4 years (95% CI 6.8–10.5) higher than not infected. Fever was the most common and with greater sensitivity. The differences found were a greater frequency of anosmia (P=.029) and headache (P=.009) among children infected with a specificity of 96.7% and 81.5% respectively. There were no differences in the duration of the symptoms. 81.8% of those infected were probably infected in the nucleus 85.2% by a parent who worked outside the home. The sensitivity of RT-PCR was 70.9% and its negative predictive value 91.1%. ConclusionsThe clinical picture is nonspecific and the symptoms more specific difficult to detect in younger children. Children had a reduced role in the intrafamily transmission. The sensitivity of RT-PCR could be related to a less contagiousness in children after one week of infection.

Deciphering the role of nanostructured materials in the point-of-care diagnostics for COVID-19: a comprehensive review.

The infamous COVID-19 outbreak has left a crippling impact on the economy, healthcare infrastructure, and lives of the general working class, with all the scientists determined to find suitable and efficient diagnostic techniques and therapies to contain its ramifications. This article presents the complete outline of the diagnostic platforms developed using nanoparticles in the detection of SARS-CoV-2, delineating the direct and indirect use of nanomaterials in COVID-19 diagnosis. The properties of nanostructured materials and their relevance in the development of novel point-of-care diagnostic approaches for COVID-19 are highlighted. More importantly, the advantages of nanotechnologies over conventional reverse transcriptase-polymerase chain reaction technique and few other methods used in the detection of SARS-CoV-2 along with the viewpoints are discussed. Also, the future perspectives highlighting the commercial aspects of the nanotechnology-based diagnostic tools developed to combat the COVID-19 pandemic are presented.

A Comprehensive Review on Covid-19 Evolution and Development

The emerging pandemic causing flu-like symptoms in patients is caused by the novel Coronavirus that was previously called 2019-nCoV. The mutated gene of SARS-CoV was transformed to SARS-CoV-2 in animals and further transmitted to humans in the month of December 2019 in Wuhan city of China. Eventually, the novel coronavirus spread all around the globe as a highly contagious disease. COVID-19 (the illness caused by Coronavirus) has affected 213 countries and territories around the world. There are about 18,852,205 new positive cases, 707,451 casualties, 12,032,508 recoveries till date that is, 05 th August 2020. This virus resulted in Acute Respiratory Distress Syndrome (ARDS) and multi-organ dysfunction. Human transmissions are caused due to inhaling or by contact with viral droplets. The infected patient seems to show an incubation period of 2–14 days. Due to this ominous situation, several countries got shut down to contain the spread of novel coronavirus. This review article includes the etiology, epidemiology, detection and diagnosis, histopathology, pathophysiology of novel coronavirus. To control the virus, many drugs are in use. Short-term testing kits have been discovered by many countries using the Reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Vaccines from numerous research laboratories are at the stage of human trails. This review article can be preferred for further investigation on coronavirus.

Consistent Detection of TLS/FUS-ERG Chimeric Transcripts in Acute Myeloid Leukemia With t(16; 21)(p11; q22) and Identification of a Novel Transcript

Abstract16; 21 translocation is a recurrent primary abnormality in acute myeloid leukemia (AML). The genes involved in this translocation are ERG on chromosome 21 and TLS/FUS on chromosome 16. The rearrangement of the two chromosomes forms the TLS/FUS-ERG fusion gene and produces a consistent chimeric transcript on the der (21) chromosome. In this study, we analyzed the clinical characteristics of 19 patients with t(16; 21)-AML, including 2 patients who evolved from myelodysplastic syndrome, and detected the chimeric transcripts of the TLS/FUS-ERG fusion gene in the patients during various clinical stages by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. We found that the patients with t(16; 21) are characterized by a relatively younger age (median age, 22 years old), involvement of various subtypes of French-American-British classification and a poor prognosis: 18 of the 19 patients died of the disease (median survival was 16 months). Four types of TLS/FUS-ERG chimeric transcripts including a novel type were noted in the RT-PCR analysis. The novel transcript contained an additional 138 nucleotides consisting of TLS/FUS exon 8 and ERG exons 7 and 8 and had an in-frame fusion. These chimeric transcripts were consistently detectable in the samples obtained not only at diagnosis and relapse but also in short and long complete remission, suggesting that t(16; 21)-AML is resistant to conventional chemotherapy. Thus, we recommend that t(16; 21) should be monitored by RT-PCR even in clinical remission and the patients should be treated by other more powerful modality like stem-cell transplantation in the first remission.

Isolation and Characterization of Coronavirus and Rotavirus Associated with Calves in Central Part of Oromia, Ethiopia.

Background Coronavirus and rotavirus are most commonly associated etiologies for calves' diarrhoea, resulting in loss of productivity and economy of farmers. However, various facets of diarrheal disease caused by coronavirus and rotavirus in calves in Ethiopia are inadequately understood. A cross-sectional study was conducted with the aim of isolation and molecular characterization of coronavirus and rotavirus from calves in the central part of Oromia (Bishoftu, Sebata, Holeta, and Addis Ababa), Ethiopia, from November 2018 to May 2019. The four study areas were purposively selected and faecal samples were collected by simple random sampling for diagnosis of coronavirus and rotavirus infection by using the antigen detection enzyme-linked immunosorbent assay (Ag-ELISA) kit. In addition, this study was carried out to have insight in prevalence and associated risk factors of coronavirus and rotavirus infection in calves. Result During the study, 83 diarrheic and 162 nondiarrheic faecal samples collected from calves less than 4 weeks of age were screened for coronavirus and rotavirus. Of the 83 diarrheic samples, 1 sample (1.2%) was positive for coronavirus antigen and 6 samples (7.2%) were found to be positive for rotavirus antigen by Ag-ELISA. All the nondiarrheic samples were negative for both coronavirus and rotavirus Ag. The overall prevalence of coronavirus and rotavirus infection in calves was estimated at 0.4% (1/245) and 2.45% (6/245), respectively. All samples (7) of ELISA test positive of both coronavirus and rotavirus were propagated in Madin-Darby bovine kidney (MDBK) cells. After 3 subsequent passages, progressive cytopathic effect (CPE), i.e., rounding, detachment, and the destruction of monolayer cell of five samples (1 sample of coronavirus and 4 samples of rotavirus) (71.4%) were observed. At the molecular stage, reverse transcriptase polymerase chain reaction (RT-PCR) technique was used to determine the presence of coronavirus and rotavirus nucleic acid by using specific primers. The 5 samples that were coronavirus and rotavirus antigen positive by ELISA and develop CPE on cell culture were also positive on RT-PCR technique. The prevalence of infection peaked at 1st and 2nd weeks of age in male calves. Conclusion Diarrheal disease caused by coronavirus and rotavirus has a great health problem in calves that interrupts production benefits with reduced weight gain and increased mortality and its potential for zoonotic spread. So, the present findings show coronavirus and rotavirus infection in calves in Ethiopia that needs to be addressed by practising early colostrum feeding in newborn calves, using vaccine, or improving livestock management.

Annona senegalensis extract demonstrates anticancer properties in N-diethylnitrosamine-induced hepatocellular carcinoma in male Wistar rats

BackgroundHepatocellular carcinoma (HCC) is a common and leading cancer around the globe. This study investigated the anticancer properties of extract of Annona senegalensis in N-diethylnitrosamine (DEN) - induced hepatocellular carcinoma in male Wistar rats. MethodsRats were simultaneously induced with a combination of 100 mg/kg b.wt of DEN and 0.5 mL/kg of carbon tetrachloride (CCl4) intraperitoneally once a week for three weeks in a row. Thereafter, animals were treated with 100 mg/kg and 200 mg/kg b.wt of A. senegalensis extract daily for 21days. Analysis using gas chromatography-mass spectrometry (GC–MS) was carried out to discover the phytoconstituents contained in the n-hexane extract of A. senegelensis. The levels of liver function parameters and antioxidant enzyme activities were determined via spectrophotometric analysis. Reverse transcriptase-polymerase chain reaction technique was used to assess the gene expression patterns of BCL-2, P53, P21, IL-6, FNTA, VEGF, HIF, AFP, XIAP, and EGFR mRNAs. ResultsTreatment of DEN-induced hepatocellular carcinoma Wistar rats with the extract caused significant (p < 0.05) decrease in the activities of ALT and AST. It also resulted in a reduction of the concentration of MDA and a significant increase (p < 0.05) in SOD and GSH activities. IL-6, BCL-2, VEGF, EGFR, XIAP, FNTA, and P21 mRNAs expressions were significantly (p < 0.05) downregulated after treatment. Histopathological analysis revealed that the extract improved the liver architecture. ConclusionA. senegelensis n-hexane extract demonstrates its anticancer properties by improving the liver architecture, increasing the antioxidant defense systems, downregulating the pro-inflammatory, anti-apoptotic, angiogenic, alpha-fetoprotein and farnesyl transferase mRNAs expression and hitherto up-regulate the expression of tumor suppressor (P21 and P53) mRNAs.

Optimization of Real-time Reverse Transcriptase Polymerase Chain Reaction for Detection of Dengue Virus

Aims: This study was set to optimize conditions for real time reverse transcriptase polymerase chain reaction (RT-PCR) for detection of dengue virus by using rapid and simple nucleic acid extraction method.&#x0D; Methodology: One step and two step real time RT-PCR were evaluated in different PCR thermocyclers. Extraction of viral RNA was done by using a simple boom method.&#x0D; Results: The real time RT-PCR technique was successfully optimized using simple and rapid method for purification of nucleic acid, ‘boom method’. The technique works better when performed in a two-step procedure and can works well with all range of real time PCR machines. The optimized real time RT-PCR used in the present study is a valuable and reliable technique for routine diagnosis of dengue. Further investigation on the cost effectiveness in adopting this technique for routine screening and monitoring of the dengue infection should be done.

Geospatial clustering, seasonal trend and forecasting of Kyasanur Forest Disease in the state of Goa, India, 2015\u20132018

IntroductionFive states in India are reporting sporadic outbreaks of Kyasanur Forest Disease (KFD). Goa experienced an outbreak of KFD in 2015. It remains as an important differential diagnosis for tropical fever in the endemic regions. Few studies among neighboring two states (Karnataka and Kerala) have described the epidemiological characteristics of KFD. However, there is no study which describes the same among cases in the state of Goa. Hence, we planned to understand the epidemiology (time, place, and person distribution) of the disease including seasonal pattern with forecasting using zero-inflated negative binomial regression and time series models. We also explored geo-spatial clustering of KFD cases in Goa during 2015–2018 which would help design effective intervention to curb its transmission in Goa.ResultsBlood samples of all suspected cases of KFD during 2015 to 2018 were tested using reverse transcriptase-polymerase chain reaction technique. Reports of these results were periodically shared with the state surveillance unit. Records of 448 confirmed cases of KFD available at the State Integrated Disease Surveillance Programme were analyzed. The mean (SD) age of the patients was 41.6 (14.9) years. Of 143 cases with documented travel history, 135 (94.4%) had history of travel to forest for cashew plucking. Two thirds of cases (66.3%) did not receive KFD vaccine prior to the disease. Case fatality rate of 0.9% was reported. Seasonal peaks were observed during January to April, and forecasting demonstrated a peak in cases in the subsequent year also during January–April persisting till May. Around 40 villages located along the Western Ghats had reported KFD, and affected villages continued to report cases in the subsequent years also. Case density-based geographic maps show clustering of cases around the index village.ConclusionMost of the confirmed cases did not receive any vaccination. KFD cases in Goa followed a specific seasonal pattern, and clustering of cases occurred in selected villages located in North Goa. Most of the patients who had suffered from the disease had visited the forest for cashew plucking. Planning for public health interventions such as health education and vaccination campaigns should consider these epidemiological features.

Pathological effect of infectious bronchitis disease virus on broiler chicken trachea and kidney tissues.

Aim:This study aimed to investigate the pathological effects of the infectious bronchitis virus (IBV) on chicken trachea and kidney tissues and also desired to diagnose the virus genome using a molecular tool.Materials and Methods:Twenty trachea and kidney samples collected from one broiler farm contain 10,000 chickens at Tikrit city. The chickens showed signs of gasping and mortality (20%) at early ages (20 days old), the presence of IBV investigated using conventional reverse transcriptase-polymerase chain reaction technique with routine histopathological study to tracheal and renal tissue.Results:Postmortem lesion showed severe respiratory inflammation with abscesses at tracheal bifurcation lead to airway blog. Molecular results showed two genotypes of IBV, one of them not included in primer designer research. The histological study showed different stages of inflammation, degeneration, and necrosis to the renal and tracheal tissues.Conclusion:The respiratory and renal pathological effect of the virus responsible for the symptoms appeared on the affected chicks that caused mortality, with a high probability of presence of a new viral genotype added to the untranslated region.

Mineralisation Influence of Betamethasone on Lipopolysaccharide-Stimulated Dental Pulp Cells.

To evaluate the mineralisation response of lipopolysaccharide (LPS)-induced dental pulp cells (DPCs) to betamethasone and the potential benefit of betamethasone application on the recovery of injured dental pulp. The proliferation influence of betamethasone on DPCs was analysed through the cell counting kit-8 assay. To assess the anti-inflammatory effects of betamethasone, the expression levels of inflammatory factors IL-6, IL-1ß and TNF-∂ were determined by real-time polymerase chain reaction (PCR). Mineralisation was investigated through the detection of the mineralisation-related biomarkers alkaline phosphatase (ALP), dentine sialophosphoprotein (DSPP) and osteocalcin (OCN) through the ALP activity assay, immunohistochemistry staining, Alizarin Red and tissue nonspecific alkaline phosphatase (TNAP) staining, the reverse transcriptase PCR technique and western blot. A low concentration of betamethasone (1 µ/mL) promoted the proliferation of DPCs. The real-time PCR results demonstrated that inflammatory cytokines were downregulated by betamethasone treatment. The mineralisation outcome in DPCs treated with betamethasone was better than in those treated without betamethasone. Betamethasone promoted the proliferation of DPCs. Betamethasone enhanced mineralisation in LPS-stimulated DPCs.

Frequency and Molecular Typing of Human Metapneumovirus in Children with Acute Respiratory Tract Infection in Bojnurd, Northeastern Iran

Background: Acute respiratory infection plays a major role in hospitalization of children worldwide and viruses are one of the most important causative pathogens of acute respiratory tract infections (ARTIs). Human metapneumovirus (hMPV) is a respiratory pathogen responsible for ARTIs in infants and children around the world. The epidemiology of hMPV in the northeast of Iran is poorly described. This study aims to evaluate the prevalence and molecular types of hMPV in children under the age of 12 along with clinical relevance in northeast Iran. Methods: In this cross sectional study, from autumn 2015 to spring 2016 (nine months), 150 nasopharyngeal swabs were collected from children with ARTI and were evaluated for the presence of hMPV by reverse transcriptase-polymerase chain reaction (RT-PCR) technique; after that hMPV typing on positive samples was performed using PCR method. Results: Among the 150 patients that were included the study, 31 (20.6%) were positive for the hMPV genome. We also found that 96.8% (n = 30 cases) of positive cases belonged to genotype A; whereas no B genotype was detected. One of our hMPV isolates was non typable (3.2%). Conclusions: This study, for the first time, reports the existence of Human metapneumovirus virus in children with respiratory symptoms in northeastern Iran. We have also shown that the genotype A is the predominant circulating type among our patients.

Cyclooxygenase inhibitors for treating preterm labour? What is the evidence?

BackgroundPreterm labour, a condition characterized by regular uterine contractions and cervical dilation prior to 37 weeks of pregnancy can result in premature birth of babies that may not be fully developed and/or possess serious health problems such as cerebral palsy and learning disabilities. Is associated with an increase in uterine prostaglandins (PGs) synthesis by cyclooxygenase (COX) enzymes which promotes inflammatory and contractile processes that drive labour. For this reason, COX inhibitors (e.g. indomethacin) have been used to treat/prevent preterm labour. Despite limited success, tocolysis by COX‐inhibition carries serious adverse fetal effects due to inhibition of prostaglandin‐dependent developmental homeostatic functions. The more recent discovery of a second, inducible COX isoform, COX‐2, presented an opportunity to spare homeostatic prostaglandin by selective inhibition of COX‐2. However, subsequent clinical studies have called into question the appropriateness of COX‐2 selective inhibition. This study reviewed the molecular evidence concerning the role of COX‐2 in human parturition that underpins the rationale of use of COX‐2 inhibitors in the treatment of preterm labour and presents evidence of COX‐2 gene expression in term and preterm human myometrium.MethodsFor our systematic literature review, we searched MEDLINE and EMBASE databases to identify papers reporting molecular evidence of COX enzyme expression and activity in gestational tissues at term and preterm. Two independent reviewers evaluated abstracts, and qualifying references were reviewed in full text. Data were extracted using a standardized questionnaire. COX‐2 gene expression analysis was conducted on samples of myometrium and decidua from women at term (after 37 weeks) and preterm (below 37 weeks) of gestation, with and without labour using reverse transcriptase‐polymerase chain reaction technique (RT‐PCR).ResultsThirty‐five studies were included in the review. Eight studies on fetal membranes (amnion and chorion) reported significantly higher COX expression or activity with labour. However, in the myometrium and decidua, which are the most relevant therapeutic target tissues for uterine contraction, nine reported no labour‐associated differences COX expression. In the myometrium, four studies reported higher levels of COX mRNA with labour, but did not consider protein expression. Our data show spatial (upper versus lower segment) and temporal (labour versus non‐labour) demonstrate differences in COX‐2 expression; at term, expression of COX‐2 is higher in lower segment myometrium and decidua samples from women in term labour (n=16) compared to term non‐labour (n=18), but no significant differences were observed for upper segment samples. Similarly, COX‐2 expression was higher in lower segment preterm labourThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RT-PCR for detection of bovine parainfluenza virus type 3 (bPIV-3)

The RT-PCR technique has been frequentely used for detection of the human parainfluenza virus type 3 (hPIV-3) but the literature is scarce in relation to the bovine parainfluenza virus type 3 (bPIV-3). The aim of this study was to describe a reverse transcriptase polymerase chain reaction (RT-PCR) for detection of bovine parainfluenza virus type 3 (bPIV-3) using degenerate oligonucleotides targeting a conserved region of hemagglutinin-neuraminidase (HN) gene. Reference strain SF-4 and three different brazilian bPIV-3 isolates, besides five viral strains from different sources, were included in this study. Viruses were cultured in MDBK cells under standard conditions. Hemagglutination (HA) test was used for viral titration and a direct immunofluorescence test (DFAT) for isolate screening. In RT-PCR all bPIV-3 isolates showed amplification of an expected 1009 bp fragment of HN gene, as oposed to non PIV-3 viral samples where no amplification was detected. Using SF-4 as positive control, sensitivity of 95 pg cDNA wasachieved. In spite of the low number of bPIV-3 isolates tested, the results obtained in this study point out the potential use of this technique for detection of bPIV-3 in bovine clinical specimens.

Prognostic Implication of BCR-ABL Fusion Transcript Variants in Chronic Myeloid Leukemia (CML) Treated with Imatinib. A First of Its Kind Study on CML Patients of Kashmir

Background:The prognostic significance of the common BCR-ABL transcripts like e13a2 (b2a2) and e14a2 (b3a2) in Chronic myeloid leukemia (CML) has been reported from patients treated with different tyrosine kinase inhibitors but its impact on clinical response and overall survival remains still unexplored. The aim of this study was to evaluate the prognostic significance of different transcript types in a cohort of CML patients treated with imatinib.Methods:A total 42 confirmed cases of Chronic Myeloid Leukemia (CML) patients were recruited into our cohort study and a multiplex Reverse Transcriptase-Polymerase Chain Reaction technique (RT-PCR) was used to detect 3 main transcript types ‘e1a2’, ‘e13a2’, and ‘e14a2’ found in CML.Results:Only two types of transcripts e13a2 (b2a2) and e14a2 (b3a2) were detected in our CML patients and none had the e1a2 type. All the patients were RT-PCR positive for either e13a2 or e14a2 fusion transcript demonstrating 100% concordance with their Ph+ve cytogenetic status at baseline. TLC count (range of 201-600x103/µl) and platelet count (range of 201-900x103/µl) at baseline were found to be associated more with the e14a2 (b3a2) than the e13a2 (b2a2) transcript type (p-value: 0.001). The two transcripts found did not relate significantly towards sex, age-group or indicated spleen size ranges as well as percentage ranges of blast cells.Conclusion:We conclude that there is no overall prognostic implication of either the e13a2 or the e14a2 transcript type across the spectrum of indicated clinical parameters evaluated. Even the overall survival analysis of the two transcript types revealed no prognostic association whatsoever.

Isolation and Identification of Short Term Drought-Induced Genes in Zea mays L. Leaves

Drought is one of the detrimental factors that impair plant growth and productivity. In this study, we applied annealing control primer (ACP)-based reverse transcriptase PCR (polymerase chain reaction) technique to identify differentially expressed genes (DEGs) in maize leaves in response to drought stress. Two-week-old maize seedlings were exposed to drought (DT) by suspending water supply. DEGs were screened after 3 days of DT-treated samples using the ACP-based technique. Several DEGs encoding 16.9 protein, antimicrobial protein, hypothetical protein NCLIV_068840, thioredoxin M-type were identified in maize leaves under drought stress. These genes have putative functions in plant defense response, growth and development. These identified genes would be useful for predictive markers of plant defense, and growth responses under drought stress in plants.