Reverse Transcription-polymerase Chain Reaction Technique Research Articles

UHRF1 Is a Novel Druggable Epigenetic Target in Malignant Pleural Mesothelioma.

Ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1) encodes a master regulator of DNA methylation that has emerged as an epigenetic driver in human cancers. To date, no studies have evaluated UHRF1 expression in malignant pleural mesothelioma (MPM). This study was undertaken to explore the therapeutic potential of targeting UHRF1 in MPM. Microarray, real-time quantitative reverse transcription-polymerase chain reaction, immunoblot, and immunohistochemistry techniques were used to evaluate UHRF1 expression in normal mesothelial cells (NMCs) cultured with or without asbestos, MPM lines, normal pleura, and primary MPM specimens. The impact of UHRF1 expression on MPM patient survival was evaluated using two independent databases. RNA-sequencing, proliferation, invasion, and colony formation assays, and murine xenograft experiments were performed to evaluate gene expression and growth of MPM cells after biochemical or pharmacologic inhibition of UHRF1 expression. UHRF1 expression was significantly higher in MPM lines and specimens relative to NMC and normal pleura. Asbestos induced UHRF1 expression in NMC. The overexpression of UHRF1 was associated with decreased overall survival in patients with MPM. UHRF1 knockdown reversed genomewide DNA hypomethylation, and inhibited proliferation, invasion, and clonogenicity of MPM cells, and growth of MPM xenografts. These effects were phenocopied by the repurposed chemotherapeutic agent, mithramycin. Biochemical or pharmacologic up-regulation of p53 significantly reduced UHRF1 expression in MPM cells. RNA-sequencing experiments exhibited the pleiotropic effects of UHRF1 down-regulation and identified novel, clinically relevant biomarkers of UHRF1 expression in MPM. UHRF1 is an epigenetic driver in MPM. These findings support the efforts to target UHRF1 expression or activity for mesothelioma therapy.

Mandatory preoperative COVID-19 testing for cancer patients-Is it justified?

BackgroundSevere acute respiratory syndrome coronavirus 2 has caused substantial disruptions in routine clinical care. Emerging data show that surgery in coronavirus disease (COVID)‐positive cases can be associated with worsening of clinical outcomes and increased postoperative mortality. Hence, preoperative COVID‐19 testing for all patients before elective surgery was implemented in our institution.Materials and MethodsTwo hundred and sixty‐two asymptomatic cancer patients were preoperatively tested for COVID‐19 using reverse‐transcription polymerase chain reaction technique with nasopharyngeal and oropharyngeal swabbing. All negative patients were operated within 72 hours, and positive patients were quarantined for a minimum 14 days before re‐swabbing.ResultsIn our cohort, 21 of 262 (8.0%) asymptomatic preoperative patients, who were otherwise fit for surgery, tested positive. After adequate quarantine and a negative follow‐up test report, 12 of 21 (57%) had an operation. No major postoperative morbidity due to COVID‐19 was noted during the immediate postoperative period before discharge from the hospital.ConclusionRoutine preoperative COVID‐19 testing was successful in identifying asymptomatic viral carriers. There was no incidence of symptomatic COVID‐19 disease in the postoperative period, and there was no incidence of morbidity attributable to COVID‐19. These data suggested a beneficial role for mandatory preoperative COVID‐19 testing.

COVID-19 anticuerpos IgM/IgG por ensayo inmunocromatográfico (prueba rápida)

Las pruebas serológicas son de gran apoyo para el diagnóstico de la enfermedad por coronavirus 2019 (COVID-19), causada por el virus SARS-CoV-2. La prueba serológica basada en inmunocromatografía (también conocida como prueba rápida), disponible en nuestro medio, detecta la presencia de anticuerpos tipo IgM e IgG, generados como respuesta a la infección. Los anticuerpos IgM comienzan a ser detectables en la sangre después de la primera semana de iniciada la infección (etapa intermedia) y perduran por 2 ó 3 semanas, en tanto que losanticuerpos IgG aparecen en la etapa tardía, que ocurre generalmente después de la segunda semana, y perduran en el tiempo. Debido a que las pruebas moleculares, basadas en la técnica de la reacción en cadena de la polimerasa con transcriptasa reversa (RT-PCR), detectan el genoma viral, estas pueden arrojar resultados negativos en las etapas tardías de la infección, por lo que el uso conjunto de las pruebas moleculares con las serológicas, aumenta de manera significativa la capacidad diagnóstica para COVID-19.

How to perform a nasopharyngeal swab in adults and children in the COVID-19 era

The nasopharyngeal swab is currently the main testing method used to diagnose COVID-19. The principle is to collect respiratory cells infected by the virus and to use the RT-PCR (Reverse Transcription - Polymerase Chain Reaction) technique to detect the RNA of the virus. The false negative rate is high, about 30%, which can mainly be explained by an incorrect execution of the technique may increase the false negative rate and decrease the test's sensitivity. The aim of this note is to help healthcare providers to perform this test correctly in adults and children.

Molecular Detection of Avian Infectious Bronchitis Viruses in Live Bird Markets, Gilan Province.

Coronaviruses (AvCoV) which include infectious bronchitis virus (IBV) and other bird coronaviruses belong to the genus gammacoronavirus, subfamily Coronavirinae. One of the most prominent representatives of gammacoronavirus genus is infectious bronchitis virus (IBV) which is a highly contagious viral pathogen of chickens causing considerable economic losses to the poultry industry. IBVs mostly affect the respiratory, urinary, and reproductive tracts leading to a substantial drop in production. Backyard poultry in the villages usually share their food and water with free flight birds which puts them at serious risk of disease transmission. Furthermore, the poor hygienic measurements which are often used in backyard flocks make them a potential reservoir for diseases that can be transferred to commercial poultry flocks. Live bird markets (LBMs) which receive live poultry to be resold or slaughtered and sold onsite play a significant role in spreading infectious diseases among the different bird species. In the present study, a number of 354 cloacal swab samples were collected from different bird species from LBMs of Gilan province. Subsequently, after RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR) technique was carried out using specific primers of S1 gene to detect coronavirus infectious bronchitis virus. Two samples from backyard chickens were reported to be positive to coronavirus which were named Iran/Backyardchicken 96/2017 and Iran/Backyardchicken 94/2017. The results of the phylogenetic analysis demonstrated that these two isolates are placed in QX and IS-1494 strains, respectively. On a final note, the obtained results highlighted the role of live birds offered in LBMs in the epidemiology of IBV and the transmission of the virus to the industrial flock.

The Concept of Making On-Chip Thermal Cycler for RT-PCR Using Conjugate Heat Transfer in Diverging Microchannel.

Covid-19 is pandemic to which the world is fighting. Various precautionary measures are being imposed all over the world which is affecting the routine life of an individual and also the economy worldwide. Although, a definite vaccine is still not known to medical science but they are able to distinguish Covid-19 from the other types of flu. Presently this is being done by detecting the SARS-CoV-2 virus using RT-PCR technique as recommended by the World Health Organization (WHO) (WHO, Geneva, 2020). Reverse Transcription Polymerase Chain Reaction (RT-PCR) is a nucleic acid amplification test that converts the RNA into DNA and subsequently amplifies the specific DNA targets. This method was already being employed to detect the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (Emery et al. in Emerg Infect Dis 10(2):311, 2004). The entire process of DNA amplification takes place in three steps: denaturation, annealing, and extension for which the sample is required to be maintained at constant temperatures of 95, 55 and 72 °C, respectively. This article introduces the technology to achieve a constant temperature which can be tweaked to develop on-chip RT-PCR.

Optimized integration of fluoxetine and 7, 8-dihydroxyflavone as an efficient therapy for reversing depressive-like behavior in mice during the perimenopausal period

Fluoxetine (FLX) has been considered as an effective anti-depressant drug. Besides, previous studies reported reasonable anti-depressant effects for 7, 8-dihydroxyflavone (7, 8 DHF). However, the combination of FLX and 7, 8 DHF in a well-established depression model has not been explored. In this study, we demonstrate that the 7, 8 DHF can improve the anti-depressant efficacy of FLX in a chronic unpredictable mild stress (CUMS)-induced depression during the perimenopausal period. The corresponding mechanism of FLX+7, 8 DHF therapy and the effect of ANA-12 are also investigated. Moreover, the influences of 7, 8 DHF (5 mg/kg/day), FLX (18 mg/kg/day), and ANA-12 (0.5 mg/kg/day) on a depressive-like behavior are displayed. Inflammatory, autophagic and apoptotic changes of hippocampus and cortex are examined by using western blot, immunofluorescence, and Real-Time Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) techniques. The protein levels of phosphatidylinositol 3 kinase (PI3K)/ protein kinase B (Akt)/mechanistic target of rapamycin (mTOR)/phosphorylated extracellular signal-regulated kinase1/2 (p-ErK 1/2)/brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) of hippocampus and cortex are assessed by western blot. The combined FLX and 7, 8 DHF treatment can significantly improve depressive-like behavior in sucrose preference and forced swimming tests accompanied by a noticeable upregulation of autophagy, neuronal nuclei (NeuN), ionized calcium-binding adaptor molecule 1 (Iba1) expressions, and PI3K/Akt/ mTOR/ p-ErK 1/2 signaling pathways. Besides, an obvious increase of the brain-derived neurotrophic factor (BDNF) and TrkB levels are observed with down-regulated inflammation and apoptosis. These findings suggest that the integrated FLX and 7, 8 DHF holds a potential as an efficient treatment to ameliorate depressive-like behavior in perimenopausal patients.

Reverse transcription loop-mediated isothermal amplification based rapid detection of Sugarcane mosaic virus and Sugarcane streak mosaic virus associated with mosaic disease of sugarcane

Sugarcane mosaic virus (SCMV) and Sugarcane streak mosaic virus (SCSMV) belonging to the family Potyviridae are the causal agents of mosaic disease complex of sugarcane reported from different Asian countries which causes significant economic losses to sugarcane production. In the present study, a sensitive RT-LAMP based assay was developed for rapid diagnosis of SCMV and SCSMV causing mosaic disease of sugarcane. Primers were designed to amplify the six different regions of the coat protein genes of SCMV and SCSMV and both the viruses were successfully detected by this technique. Comparative analysis of RT-LAMP with the conventional reverse transcription polymerase chain reaction (RT-PCR) and quantitative real time PCR (RT-qPCR) showed that RT-LAMP was more sensitive than the other methods due to its rapid visual detection of the viruses. The sensitivity of RT-LAMP assay developed in the present study was tenfold more than that of conventional RT-PCR and RT-qPCR techniques. This is the first report of RT-LAMP based diagnosis of SCMV and SCSMV from India.

Effects of Cigarette Smoke and Opium on the Expression of CD9, CD36, and CD68 at mRNA and Protein Levels in Human Macrophage Cell Line THP-1.

Cigarette smoking and opium use are risk factors for coronary artery disease (CAD). It has been known that scavenger receptors such as CD36 and CD68 play critical roles in the pathogenesis of CAD. CD9, as a member of the tetraspanin, has been shown to interact with scavenger receptors. The aim of this study was to investigate the effects of these risk factors on expression levels of CD9, CD36, and CD68 on the THP-1 cell line. The THP-1 cell line treated with cigarette smoke extract (CSE( and opium, both individually and combinatory, in 24 h incubation. The protein and mRNA levels of CD9, CD36, and CD68 were evaluated by flow cytometry and quantitative reverse transcription-Polymerase Chain Reaction (qRT-PCR) techniques, respectively. CD36 and CD68 mRNA and protein expression levels were significantly increased in the cells treated with cigarette smoke extract compared to the control (p<0.001 in mRNA expression levels and p=0.016 and p=0.012 in protein expression levels, respectively). The CSE increased the level of CD9 protein expression compared to the control group (p=0.041) on the human macrophage cell line THP-1. No significant differences were observed in the CD9, CD36, and CD68 gene expression and at the protein levels between opium-treated THP-1 cells and controls. In conclusion, cigarettes by increasing the levels of CD36, CD68, and CD9 can be a risk factor in the development of many inflammatory diseases, including cardiovascular diseases, chronic obstructive pulmonary disease (COPD) and lung carcinoma.

An Analysis of ESR2 and CYP19A1 Gene Expression Levels in Women With Endometriosis

The analysis of oestrogen receptor (ESR2) and cytochrome P450 family 19 subfamily A member (CYP19A1) gene expression in the context of the risk for endometriosis development. Tissue specimens, collected from patients with endometriosis (n=100) and from control patients (n=100) embedded into paraffin blocks, provided the material for genetic studies, oriented towards the expression of ESR2 and CYP19A1 genes. The gene expression was assessed by the reverse transcription-polymerase chain reaction technique. Higher expression levels of ESR2 gene were demonstrated in the patients with endometriosis in comparison with the healthy controls. The expression intensity of CYP19A1 gene was associated with endometriosis, manifested as abdominal wall nodules. A relationship was observed between CYP19A1 gene expression and the Revised American Society for Reproductive Medicine classification in the group with ovarian endometrioid cysts, as well as in the group with peritoneal endometriosis. This study suggests the significant role of ESR2 and CYP19A1 gene expression in the pathogenesis of endometriosis.

Management perspective of COVID-19 patients from L1 till L3 level hospital: An observational study

Background: The coronavirus (CoV) specifically known as CoVs disease (COVID)-19 spread from Wuhan city of China to the whole world and created havoc worldwide. It is an RNA virus of family beta- CoVs, and it is third CoV infection after Middle East respiratory syndrome and subacute respiratory syndrome. Aim and Objective: The objective of this study was to assess the status of COVID-19 patients in the study area. Materials and Methods: A hospital-based cross-sectional study was conducted in Government Medical College, Azamgarh, on the subjects either suspected or confirmed with COVID 19 tests for March 20, 2020, till the submission of this research. Oral consent was taken from all the patients. The total number of patients included in the study is 81 who were declared as COVID positive patients admitted in the isolation ward after taking throat and nasal swab followed by reverse transcription-polymerase chain reaction technique. Out of 81, 73 (90.12 %) were male and 8 (9.88 %) were female and the age varies from 8 years to 95 years with the mean age of 35.48 years. Out of total patients, 1 was referred due to critical illness, two were shifted to L1 hospital, and 27 were discharged in satisfactory conditions. Results: The majority of patients (61.00%) were asymptomatic and the patients who were having symptoms presented sore throat and cough (32.00%), fever and cough 19%, sore throat, headache and body-ache (16.00%), cough, sore throat and chest pain (12.00%), and vomiting and pain abdomen (0.06%). Radiological investigations have suggested mild acute respiratory distress syndrome (ARDS) in almost (10.00%), and some subjects show changes in chronic obstructive pulmonary disease. There were 2 patients who showed severe changes of ARDS (

Prevalence of Rotavirus diarrhea among under-five hospitalized children in government tertiary hospital, Tirupati

Context: Rotavirus is the most common cause of severe diarrhea requiring hospitalization among infants and young children worldwide. The prevalence of rotavirus diarrhea in India has been found to vary in the range 5-71% in hospitalized children aged under 5 years with acute gastroenteritis. The seasonal variation of rotavirus diarrhea in India varies across different geographical regions, with high incidence in the winter months at low relative humidity in northern India. Aim: This study aimed to estimate the prevalence of rotavirus diarrhea among hospitalized children aged under 5 years and to learn about the genotypic distribution of rotaviruses causing diarrhea. Settings and Design: Study design: hospital-based cross-sectional study. Study setting: the pediatrics department of a tertiary care Government hospital, Tirupati, India. Study period: September 20, 2012-September 19, 2013. Materials and Methods: Study units: children under 5 years of age presenting with diarrhea. Stool specimens from all hospitalized children aged under 5 years who had presented with acute watery diarrhea were collected and tested for rotavirus by the enzyme-linked immunosorbent assay (ELISA). Positive samples were tested for G and P typing by the reverse transcription-polymerase chain reaction (RT-PCR) technique. Statistical Analysis Used: Percentage and chi-square analysis. Results: Among the study sample, 68.7% of children were in the age group between 1-12 months and 25.6% children showed positive result for rotavirus by ELISA. Of the rotavirus positives, 50% were G1P8 viruses. Conclusion: Rotavirus is an important cause of diarrhea in hospitalized children.

COVID-19 pneumonia: When negative RT-PCR testing does not rule out the disease

In diagnosing COVID-19, false negative findings from the biological sample taken from a mucosal swab of the upper respiratory tract and tested with the real-time reverse-transcription polymerase chain reaction (RT-PCR) technique have been reported. This patient has had a proven contact with an infected person, clear symptoms of viral respiratory disease, yet negative test results on the fifth day of self-isolation. On repeated test after 48 hours, on the 7th day of isolation, due to persistence of some symptoms, he tested positive for SARS-CoV-2. The existence of symptoms and characteristic sings after laboratory and radiological analysis of the patient prompted the repetition of the tests, which at the end led to the confirmed diagnosis and the possibility for adequate treatment of the patient as well.

Association of ATP2B1 gene polymorphism with incidence of eclampsia.

The aim of this study was to analyze the association of ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) gene polymorphism with the incidence of eclampsia, and to investigate the possible underlying mechanism. ATP2B1 genotype and allele distributions in umbilical venous blood cells were analyzed in 50 control subjects and 117 eclampsia patients via Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and TaqMan genotyping technique. Meanwhile, the differences in the single nucleotide polymorphisms at rs2681472 and rs17249754 in the case group and control group were analyzed using the χ2-test. The risk factors for eclampsia were analyzed via univariate, multivariate, and Logistic regression analyses. Furthermore, the associations of rs2681472 gene polymorphism with risk factors for eclampsia (hypertension and lower extremity edema) were verified via χ2-test. The statistically significant differences were observed in the gestational week, body mass index, blood pressure, and incidence rates of proteinuria and lower extremity edema of pregnant women between the case group and the control group (p<0.05). Meanwhile, the genotype and allele distributions at rs2681472 in the case group were remarkably different from those of the control group (p<0.05). However, no evident differences were observed at rs17249754 between the two groups (p>0.05). According to univariate, multivariate, and logistic regression analyses, hypertension, and lower extremity edema were significantly associated with the incidence of eclampsia (p<0.05). In addition, the gene polymorphism at rs2681472 showed significant differences among subjects with and without hypertension and lower extremity edema (p<0.05). ATP2B1 gene polymorphism at rs2681472 shows significant differences between eclampsia patients and normal controls. Moreover, its gene polymorphism is closely related to the occurrence of hypertension and lower extremity edema.

Development of a multiplex RT-PCR assay for simultaneous detection of Cucumber green mottle mosaic virus and Acidovorax citrulli in watermelon.

Watermelon (Citrullus lanatus Thunb.) is considered as a popular and nutritious fruit crop worldwide. Watermelon blood flesh disease caused by Cucumber green mottle mosaic virus (CGMMV) and bacterial fruit blotch caused by Acidovorax citrulli, are two major quarantine diseases of watermelon and result in considerable losses to global watermelon production. In this study, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) method was developed for simultaneous detection of CGMMV and A. citrulli in both watermelon leaves and seeds. Two pairs of specific primers were designed based on the conserved sequences of the genomic RNA of CGMMV and the internal transcribed spacer of A. citrulli, respectively. Transcriptional elongation factor-1α from watermelon was added as an internal reference gene to prevent false negatives. No cross-reactivity was detected with other viral or bacterial pathogens infecting watermelon. Moreover, the multiplex RT-PCR showed high sensitivity and could simultaneously detect CGMMV and A. citrulli as little as 102 copies of plasmid DNA. This method was successfully applied to test field-collected watermelon leaves and stored seeds of cucurbitaceous crops. These results suggested that the developed multiplex RT-PCR technique is a rapid, efficient, and sensitive method for simultaneous detection of CGMMV and A. citrulli, providing technical support for monitoring, predicting, and preventing these two quarantine diseases. To our knowledge, this is the first report on simultaneous detection of a virus and a bacterium by multiplex RT-PCR in watermelon.

A study on role and mechanism of TLR4/NF-κB pathway in cognitive impairment induced by cerebral small vascular disease.

To investigate the role and potential mechanism of Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway in cognitive impairment induced by cerebral small vascular disease (CSVD), so as to provide a reference for the clinical treatment of CSVD-induced cognitive impairment. Mice with TLR4 gene knockout (n = 20) and those with wild-type TLR4 gene (n = 40) aged 8-10 weeks old were divided into blank control group (Control group, n = 20), wild-type + CSVD group (WT + CSVD group, n = 20) and TLR4 gene knockout + CSVD group (TLR4 KO + CSVD group, n = 20). Allogeneic thrombosis (particle diameter: 50-70 mm) was injected to the mouse's external carotid artery to create a model of learning and memory dysfunction. Step-down test and Y-type maze test were utilized to examine the learning and memory abilities of the mice. Reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting techniques were adopted to measure the levels of apoptosis-related genes [B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), C-caspase-3 and T-caspase-3] in the brain tissues of mice. Terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method was applied to detect the apoptosis of neuronal cells in the brain tissues. Meanwhile, the levels of oxidative stress markers, including superoxide dismutase (SOD), gp91 and malondialdehyde (MDA), were measured. Finally, the expression level of TLR4/NF-κB pathway was detected. The latency in the step-down test in the WT + CSVD group was remarkably longer than that in the Control group, and the number of errors was evidently larger than that in the Control group (p < 0.05). At the same time, in the WT + CSVD group, the expression levels of pro-apoptotic genes Bax and C-caspase-3 were up-regulated markedly, while the expression level of anti-apoptotic gene Bcl-2 declined notably (p < 0.05). TUNEL results showed that the number of apoptotic cells in the brain tissues in the WT + CSVD group was about 12 times that in the Control group (p < 0.05). Meanwhile, the SOD expression level was lowered, and the MDA expression level was elevated in the brain tissues in the WT + CSVD group. In addition, the TLR4/NF-κB pathway was prominently activated in the mice in the WT + CSVD group (p < 0.05). After TLR4 gene knockout, the cognitive functions of the mice were improved markedly, and the apoptosis of neuronal cells and oxidative stress in the brain tissues were suppressed significantly in the meantime. Moreover, the activation of the TLR4/NF-κB signaling pathway was also inhibited. The TLR4/NF-κB pathway is involved in the occurrence and development of CSVD-induced cognitive impairment through regulating oxidative stress and cell apoptosis.

Effects of lncRNA DANCR on proliferation and differentiation of osteoblasts by regulating the Wnt/β-catenin pathway.

To measure the expression level of long non-coding ribonucleic acids (lncRNAs) differentiation antagonizing non-protein coding RNA (DANCR) in serum of patients with fracture and investigate its influences on the proliferation and differentiation of osteoblasts. Serum samples were collected from 44 fracture patients treated in our hospital and 24 healthy people receiving physical examination in our hospital. Then, reverse transcription-polymerase chain reaction (RT-PCR) technique was used to detect the expression of lncRNA DANCR in the serum of patients with fracture and healthy subjects. MC3T3-E1 mouse osteoblast cell line with stably-knocked out DANCR was further established using small interfering RNAs (siRNAs), and the effect of DANCR knockout on the proliferation of osteoblasts was determined using cell counting kit-8 (CCK-8). At the same time, 5-Ethynyl-2'-deoxyuridine (EdU) staining assay was performed to detect the percentage of EdU-positive cells in osteoblasts in control group and DANCR knockout group. In addition, the mRNA levels of differentiation-related genes including Runt-related transcription factor 2 (Runx2), Collagen1α1, osteocalcin (OC) and osterix (OSX) were detected via RT-PCR, and the protein level of Runx2 was measured through Western blotting. Moreover, osteoblasts were cultured with osteogenic medium for 14 d, and then alizarin red staining and alkaline phosphatase (ALP) staining assays were carried out to examine the differentiation of these osteoblasts. Lastly, Western blotting technique was employed to analyze the expression of the Wnt/β-catenin signaling pathway. The expression of lncRNA DANCR was significantly increased in the serum of fracture patients (p<0.05). The results of in-vitro cell experiments showed that the intervention of DANCR with siRNA was able to clearly promote the proliferation and differentiation of MC3T3-E1 osteoblast cell line. According to the results of Western blotting, DANCR promoted the apoptosis and proliferation, which was mediated by the activated Wnt/β-catenin signaling pathway in osteoblasts. LncRNA DANCR inhibition can facilitate the proliferation and differentiation of osteoblasts by activating the Wnt/β-catenin signaling pathway in osteoblasts. Therefore, DANCR is expected to be a new target promoting fracture healing.

Retracted: Long noncoding RNA LINC00052 inhibits colorectal cancer metastasis by sponging microRNA‐574‐5p to modulate CALCOCO1 expression

The dysregulation of long-chain noncoding ribonucleic acid (lncRNA) is a common phenomenon in many human cancers. Some studies on the biological function of long intergenic non-protein-coding RNA 52 (LINC00052) in cancer indicate that this gene can act as either oncogene or tumor suppressor in some kinds of cancers, such as breast cancer, gastric cancer, liver cancer, and lung cancer. However, the biological function of LINC00052 in colorectal cancer (CRC) has not been studied. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot (WB) techniques were applied to detect the expression levels of LINC00052, miR-574-5p, and calcium-binding and coiled-coil domain 1 (CALCOCO1) in CRC cells and tissues. We authenticated the biological function of LINC00052 and miR-574-5p in CRC, and find some target genes for LINC00052 and miR-574-5p via bioinformatics methods. Dual-luciferase reporter gene assay was performed to identify the interaction between LINC00052 and miR-574-5p or CALCOCO1 and miR-574-5p. The results demonstrated that LINC00052 was downregulated in CRC tissues compared with their adjacent tissues. And LINC00052 could suppress CRC cells metastasis both in vivo and in vitro. Beyond that, miR-574-5p was upregulated in CRC tissues, and as an oncogene, it accelerated CRC cell migration and invasion. More importantly, the results of our research demonstrated that LINC00052 could regulate the expression of CALCOCO1 via sponging miR-574-5p in CRC. Overall, our study illuminated the lncRNA-miRNA functional networks in CRC, and these results might provide a new research direction for the diagnosis and treatment of CRC.

Pollen and seed transmission of Columnea latent viroid in eggplants

Viroid seed transmission data are important for plant production because such information can be very helpful in preventing the movement of viroids worldwide via infected seed. In this study, the reverse transcription-polymerase chain reaction (RT-PCR) technique was used with columnea latent viroid (CLVd) specific primers to investigate viroid seed transmission from three infected eggplant (Solanum melongena L.) genotypes through cross-pollination to seeds and seedlings. Pollen was harvested from CLVd-infected eggplant plants and used to cross-pollinate healthy plants (recipient parents) to produce seeds for the CLVd seed transmission test. CLVd that was localized on the seed coat and in the seedlings was determined using a ‘top-of-paper’ germination technique. The RT-PCR results showed positive results, indicating that CLVd was located in both the seed coat and the seedling. The RT-PCR test performed on 8 week-old seedlings of ‘Farmers Long’, ‘Jamaica’ and ‘RPG’ showed that the seed transmission rates were 82%, 6.4% and 2.3%, respectively. Based on this work, CLVd seed transmission was confirmed in eggplant. Furthermore, these results indicated that CLVd-infected pollen carries the risk of transmitting the viroid, during cross-pollination, from a donor eggplant to its progeny.

Detection Of Hepatitis C Virus (Hcv) Infection And Its Genotype In Patients At Hepatology Outpatient Clinic, Dr Soetomo General Hospital, Surabaya.

Hepatitis C virus (HCV) is an RNA virus that can cause liver inflammation (hepatitis) and has the potential to become chronic and can progress to liver cirrhosis and hepatocellular carcinoma. Detection of HCV RNA infection, and genotype/subtype of HCV was performed on 70 blood sera of patients at the Hepatology Outpatient Clinic of Dr Soetomo General Hospital, Surabaya, Indonesia. Detection of HCV infection was carried out by Anti-HCV determination using enzyme immunoassay (EIA) technique, detection of HCV RNA by Reverse Transcription Polymerase Chain Reaction (RT-PCR) technique based on genome regions NS5b and 5’UTR, followed by electrophoresis with agarose gel. In positive PCR results, HCV genotype/subtype was determined by direct sequencing method using ABI 310 sequencer and sequencing results were analyzed by comparing the products with previously published HCV nucleotides. Sera were obtained from 41 (58.6%) male and 29 (41.4%) female patients. Anti-HCV was found positive in 17/70 (24.29%) patients and 16/17 (94.1%) was proved to contain HCV RNA when determined by RT-PCR technique. Patients with positive HCV RNA have the potential to transmit HCV infection. From the genotype/subtype analysis of sequencing results we obtained 2/16 (12,5%), 3/16 (18,75%) and 6/16 (37,5%), 1/16 (6,25%), 1/16 (6, 25%), 2/16 (12,5%), 1/16 (6,25%) HCV genotypes 1, 2, and HCV subtypes 1b, 1c, 2a, 3a, 3k respectively.Conclusion: In patients who went to the Hepatology Outpatient Clinic, Dr. Soetomo General Hospital Surabaya, we found positive Anti-HCV was 24,29%. In 94,1% of patients with positive Anti-HCV, HCV RNA was still detected and HCV genotype 1 with subtype 1b were still dominant HCV subtypes.