Real-time PCR Method Research Articles

Evaluation of the Canonical Wnt Signaling Pathway in the Hearts of Hypertensive Rats of Various Etiologies.

Wnt/β-catenin signaling dysregulation is associated with the pathogenesis of many human diseases, including hypertension and heart disease. The aim of this study was to immunohistochemically evaluate and compare the expression of the Fzd8, WNT1, GSK-3β, and β-catenin genes in the hearts of rats with spontaneous hypertension (SHRs) and deoxycorticosterone acetate (DOCA)-salt-induced hypertension. The myocardial expression of Fzd8, WNT1, GSK-3β, and β-catenin was detected by immunohistochemistry, and the gene expression was assessed with a real-time PCR method. In SHRs, the immunoreactivity of Fzd8, WNT1, GSK-3β, and β-catenin was attenuated in comparison to that in normotensive animals. In DOCA-salt-induced hypertension, the immunoreactivity of Fzd8, WNT1, GSK-3β, and β-catenin was enhanced. In SHRs, decreases in the expression of the genes encoding Fzd8, WNT1, GSK-3β, and β-catenin were observed compared to the control group. Increased expression of the genes encoding Fzd8, WNT1, GSK-3β, and β-catenin was demonstrated in the hearts of rats with DOCA-salt-induced hypertension. Wnt signaling may play an essential role in the pathogenesis of arterial hypertension and the accompanying heart damage. The obtained results may constitute the basis for further research aimed at better understanding the role of the Wnt/β-catenin pathway in the functioning of the heart.

Abstract B029: Assessing gene expression and methylation of KMT2D and IGF2 genes in patients with non-small cell lung cancer

Abstract Background: Aberrant promoter methylation of CpG islands is an important mechanism for regulation of gene expression. Recent data suggest that epigenetic abnormalities may occur very early in lung carcinogenesis. Systemic methylation changes may be a diagnostic marker for tumor development or prognosis. In this study, the expression and methylation of KMT2D and IGF2 genes were investigated in the lung cancer tissue compared to the adjacent normal tissue. Methods: The status of methylation of KMT2D and IGF2 genes were investigated in 30 patients with NSCLC after genomic DNA extraction using bisulfite treatment and MS-HRM method and the expression of these genes were checked by Real-Time PCR method in same samples. Results: For KMT2D gene, the expression and methylation level increased in 46.6% and 6.67% (respectively) for tumor samples comparison with normal samples (P>0.05). Also, for IGF2 gene 50% tumor samples overexpressed and 50% tumor samples showed that reduced expression comparison with the normal samples (P>0.05). In addition, 96.66% of tumor tissues did not show any change in methylation level for IGF2 gene promoter (P>0.05). Conclusion: This study showed that expression and methylation level of KMT2D and IGF2 genes did not change in NSCLC tumor samples compared to normal samples. However, this study was designed as a pilot study, and further investigations are required to confirm our findings. Citation Format: Arash Matinahmadi, Zoofa Zayani, Seyed Hesamoddin Bidooki, Alireza Tavakolpournegari. Assessing gene expression and methylation of KMT2D and IGF2 genes in patients with non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B029.

Abstract B030: Expression patterns of miR181a and miR30d in patients with breast cancer

Abstract Background: One of the important molecular pathways in breast cancer is the PTEN-PI3K-AKT pathway. Any change in the activity of the PTEN gene can alter the PI3K-AKT pathway. Moreover, there are subsets of genes and pathways their expression changes by post-transcriptional regulations. For instance, gene regulation alters by non-coding RNAs such as micro-RNAs as post-transcriptional regulators that prevent the expression of the target transcript. Therefore, it is essential to assess the related alterations in micro-RNA expression patterns to find out the possible causes of conversions in related transcripts and pathways such as the PTENPI3K-AKT pathway in breast cancer. Methods: To determine the expression level of miR-181a and miR-30d in 30 breast tumor samples and 30 adjacent normal samples, the RNA extraction, and cDNA synthesis was performed by RiboEx (GeneAll, Korea). Finally, the Real-Time PCR method was used for quantitative analysis of the expression levels of these miRNAs. all the experimental part of the project in done at Islamic Azad University in 2017. Results: After analyzing comparisons in the expression level of miR-181a and miR-30d in tumor and normal tissues, there was a significant increase in the expression level of miR-181a in tumor samples compared with normal samples. Moreover, the expression level of miR-30d in tumor samples reported a significant decrease in comparison with normal samples (P<0.05). Conclusion: Upregulation of miR-181a may affect the transcription of the PTEN gene resulting in the cell progress to cancer. The Downregulation of miR-30d may also lead to cancer cell growth, due to a reduction in the affecting on the CREB gene transcript. Citation Format: Alireza Tavakolpournegari, Mehrdad Hashemi, Arash Matinahmadi, Seyedeh Safoora Moosavi, Seyed Hesamoddin Bidooki. Expression patterns of miR181a and miR30d in patients with breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B030.

A comparative analysis of real-time quantitative PCR and metabarcoding methods for eDNA-based detection of the toxic dinophyte Alexandrium tamiyavanichii (Dinophyceae)

The marine dinophyte Alexandrium tamiyavanichii is a toxigenic species that produces a group of neurotoxins that is responsible for paralytic shellfish poisoning in humans. Early detection of the species is essential for efficient monitoring. Harmful microalgal monitoring systems have evolved over the years with the advent of environmental DNA (eDNA)-based species detection techniques. In this study, eDNA samples were collected from a large-scale sampling covering the southern South China Sea. The sensitivity and specificity of metabarcoding of the V4 and V9 18S ribosomal DNA barcodes by high-throughput sequencing (HTS) were compared to the species-specific real-time qPCR targeting the A. tamiyavanichii ITS2 region. Environmental samples were screened for A. tamiyavanichii by qPCR (n = 43) and analyzed with metabarcoding (n = 30). Our results revealed a high occupancy profile across samples for both methods; 88% by qPCR, and 80–83% by HTS. When comparing the consistency between the two approaches, only two samples out of 30 were discordant. The V4 and V9 molecular units detected in each sample were positively correlated with the qPCR ITS2 gene copies (V4, rs = 0.67, p < 0.0001; V9, rs = 0.65, p < 0.0001), indicating that metabarcoding could be used as a useful tool for early detection of the species. Our results also revealed that the estimation of A. tamiyavanichii cell abundances based on the HTS read abundances was comparable to that of the qPCR quantification. For long-term monitoring, metabarcoding could serve as a cost-effective screening of detecting not only single HAB species but also simultaneously detecting a multitude of potentially harmful species, which is valuable in informing the subsequent implementation of species-specific monitoring strategies.

The effect of aerobic-resistance training on the population of cardiac multipotent cells, thickness, and diameter of the ventricular wall through the c-kit pathway in male rats during the growth stages

Background. The purpose of the present study was to investigate the effect of aerobic-resistance training on the population of cardiac multipotent cells, thickness, and diameter of the ventricular through the C-Kit pathway of male rats during the growth stages. Methods. Overall, 30 male Wistar rats in three age groups of 2 (n = 10), 8 (n = 10), and 96 (n = 10) weeks were accidentally divided into exercise and control groups. Resistance training programs (resistance ladder, 3 days a week) and aerobics (treadmill running, 3 days a week) were performed for 6 weeks. Hematoxylin-Eosin staining was done the heart tissue and histological images were prepared. Afterward, C-Kit gene expression was examined by the real-time polymerase chain reaction method. The statistical method of one-way analysis of variance (P ≥ 0.05) and Tukey’s test were used at a significant level (P ≥ 0.01). Results. The trained neonatal group had higher values in heart weight to body weight index, and the trained neonatal and trained young adult groups had higher values in left ventricular thickness (P ≥ 0.01). The decrease in left ventricular internal diameter in the trained young adult group was significant compared to the control group (P ≥ 0.01). There was a significant increase in C-Kit gene expression in trained young adult and trained old groups (P ≥ 0.01). Conclusion. Aerobic-resistance training can be an effective stimulus for increasing the number of cardiac stem cells and creating structural adaptations of the heart, including increasing thickness and ventricular diameter in neonatal and young adult groups. Practical Implications. Participation in aerobic-resistance training programs leads to the improvement of the cardiac structure and an increase in cardiac stem cells.

Protective effects of silymarin in glioblastoma cancer cells through redox system regulation.

Glioblastoma multiforme, a deadly form of brain tumor, is characterized by aggressive growth and poor prognosis. Oxidative stress, a disruption in the balance between antioxidants and oxidants, is a crucial factor in its pathogenesis. Silymarin, a flavonoid extracted from milk thistle, has shown therapeutic potential in inhibiting cancer cell growth, promoting apoptosis, and reducing inflammation. It also regulates oxidative stress. This study aims to investigate the regulatory effects of silymarin on oxidative stress parameters, especially the transcription factor Nrf2 and its related enzymes in GBM cancer cells, to develop a new anti-cancer compound with low toxicity. First, the cytotoxicity of silymarin on U-87 MG cells was investigated by MTT and the results showed an IC50 of 264.6μM. Then, some parameters of the redox system were measured with commercial kits, and the obtained results showed that silymarin increased the activity of catalase and superoxide dismutase enzymes, as well as the total antioxidant capacity levels; while the malondialdehyde level that is an indicator of lipid peroxidation was decreased by this compound. The expression level of Nrf2 and HO-1 and glutaredoxin and thioredoxin enzymes were checked by real-time PCR method, and the expression level increased significantly after treatment. Our findings suggest that silymarin may exert its cytotoxic and anticancer effects by enhancing the Nrf2/HO-1 pathway through antioxidant mechanisms in U-87 MG cells.

A robust set of qPCR methods to evaluate adulteration in major spices and herbs

Consumers and companies associated with food or pharmaceuticals rely on spices and herbs in various forms. Their intricate supply chains, elevated prices, and low-volume production render them vulnerable to fraudulent practices. However, comprehensive methodologies to detect adulterants remain scarce, impeding national control laboratories from enforcing European and national legislation. In this study, we present quantitative real-time PCR (qPCR) methods designed to identify the top five adulterants of each of six commonly consumed spices and herbs: paprika/chili, turmeric, saffron, cumin, oregano and black pepper. The specificity of each method was confirmed by qPCR analysis of a large collection of relevant plant species. Each authentic sample was combined with its respective top five adulterants as identified in the European Union-wide coordinated control plan on herbs and spices in 2021 or in the existing literature. These binary mixtures were used to evaluate the method's performance with respect to sensitivity, linearity and trueness at four levels of adulterants concentration. Detection was also investigated in multi-adulterated samples. These SYBR™ Green-based qPCR methods enable the specific detection of adulterants, and their sensitivity allows for the distinction between inadvertent contamination and deliberate adulteration. Altogether, these methods contribute to safeguard the authenticity of these high-value commodities.

Determination of Serum Vitamin D Levels in Patients with COVID-19

Background: In 2019, the first cases of an acute respiratory infectious disease were announced in the city of Wuhan, China. In places where the speed of transmission and the resulting high prevalence and death from this virus is high, it is important to find things that help prevent and reduce the symptoms and complications of the disease, one of these things. Things are serum levels of vitamin D. As a result of this study, serum levels of vitamin D were measured in patients with Covid-19. Methods: From December to March of 1400, 100 samples of people hospitalized in Khurshid laboratory were examined to identify the RNA of the Covid-19 virus by Real Time PCR method and at the same time to check the serum levels of vitamin D. The data was statistically analyzed using SPSS software. Results: The average age of the patients was between 12.45 and 40.8 years. Comparison the two Covid-19 positive and negative groups in terms of symptoms, it was found that the patients with RH positive had more positive PCR percentage and a significant difference was reported between RH positive and Covid-19 (p=0.006). In comparing the relationship between disease symptoms and the rate of PCR positive reports of gastrointestinal symptoms, history of significant disease, cough, fever, a significant difference was reported (p&lt;0.001). Also, 50% of the samples were PCR positive. Based on the t-test, a significant difference was reported between the serum levels of vitamin D and Covid-19 (p&lt;0.001). Conclusion: The results showed that the vitamin D is an acceptable protective factor against Covid19 infection and its deficiency increases the probability of infection.

Expression level of interferon-stimulated genes PKR, OAS1, MX1, and ISG15 in peripheral blood mononuclear cells of COVID-19 patients: A retrospective study

Objective: To explore expression level of interferon-stimulated genes PKR, OASI, MX1, and ISG15 in peripheral blood mononuclear cells of COVID-19 patients. Methods: In this study, changes in the expression of four interferon-stimulated genes (ISGs), including PKR, OASI, MX1, and ISG15, in peripheral blood mononuclear cells of 45 COVID-19 patients with different severities were evaluated by real-time PCR method. Results: OASI, MX1, PKR, and ISG15 were differently expressed in COVID-19 patients with different severity. The results showed that the expression of OASI, MX1, PKR, and ISG15 genes was significantly (P=0.001) lower in severe patients. Conclusions: Weak and defective IFN response and subsequent disruption of ISGs may be associated with COVID-19 severity.

Development and validation of a ddPCR assay to detect and quantify tobacco DNA in smoke and smokeless tobacco and tobacco-free products

The last decade, smoke and smokeless products claiming to be tobacco-free, including herbal cigarettes and herbal shisha, became available on the European market and gained popularity. This study proposes a new digital droplet PCR (ddPCR) method, designed based on a previously developed real-time PCR (qPCR) method being currently used by the U.S. Food and Drug Administration (FDA) to specifically detect the presence of tobacco DNA in targeting a sequence from the Nicotiana tabacum nia-1 gene. To ensure a harmonized and reliable control by enforcement laboratories, both of these qPCR and ddPCR methods were then evaluated and validated for their compliance to an international standard. First, the performance of these PCR-based methods was successfully assessed as specific and sensitive, and in line with minimum performance requirements from international standard. Secondly, the transferability to external laboratory was confirmed for these PCR-based methods. Finally, the applicability of these PCR-based methods was demonstrated using 7 ground tobacco reference materials from the Tobacco Research Center (TRC) Toronto University as well as 6 commercial smokeless and tobacco-free smoke and smokeless products. Based on this study, the previously developed qPCR method was confirmed as complying with international standard, ensuring a efficient and harmonize use by enforcement laboratories for tobacco control on the European market. Moreover, this study proposed to enforcement laboratories the possibility to use a ddPCR method, enabling the simultaneous detection and absolute quantification of tobacco DNA as well as a limited impact of PCR inhibitors.

Optimization of Annealing Temperature and Primer Concentration for Detection of Mycobacterium Tuberculosis Isoniazid Resistance Real-Time PCR Method

Indonesia has the second highest number of TB cases in the world according to the WHO report in 2022. Eradication of the National TB Program is difficult due to the presence of Mycobacterium tuberculosis (M. Tb) bacteria that are resistant to various anti-tuberculosis drugs (OAT) known as Multidrug- Resistant Tuberculosis (MDR-TB). Therefor, currently, a nucleic acid-based diagnostic kit has been developed that is designed to directly detect the presence of resistance to INH with mutations of the KatG gene codon 315 (S315T, S315N, S315I, S315R, S315G, R463L) and rifampicin sensitive M. Tb using the Real-Time PCR method. PCR components that are important for development include annealing temperature and primer concentration. This study aims to determine the optimum annealing temperature and primer concentration. The type of research is the Quasi Experiment. The treatment in this study is by adding each primer that varies in concentration, namely 200nM-400nM while varying the annealing temperature with a range of 52-62°C according to the Tm of each primer. The data obtained from this research are Ct values ​​from the target genes S315T, S315N, S315I, S315R, S315G, R463L and M. Tb Rifampicin sensitive.

Alterations in the Expression of Calcium Channels and Neurotrophic Factors in the Cerebellum Are Linked to the Induction of Morphine Dependence and Withdrawal

Background: Recent studies have shown that the cerebellum directly interacts with the ventral tegmental area, a critical component of the reward system. Objectives: This study aimed to explore potential changes in the expression of neurotrophic factors and various types of voltage-gated calcium channels in the cerebellum following morphine dependence and withdrawal. Methods: This study involved three groups of male Wistar rats. For ten consecutive days, the second and third groups were administered morphine (10 mg/kg), while the first group received saline (1 mL/kg). Analgesic responses were assessed using a hotplate test on days 1 and 10 of the repeated injections and after a 30-day withdrawal period. Rats were sacrificed on day 10 of the injections or on day 30 of withdrawal, and their cerebella were dissected for analysis. Gene expression was analyzed using the real-time PCR method. Results: The study found that morphine analgesia decreased during the 10 days of repeated injections but partially recovered after a 30-day withdrawal period. Morphine dependence led to a decrease in the expression of Cav1.1, which increased after withdrawal. The expression of Cav1.2 in the cerebellum consistently rose after both morphine dependence and withdrawal. There were no significant changes in the expression of Cav2.2 due to morphine dependence or withdrawal. An increase in the expression of Cav3.1 was observed following morphine dependence, which decreased after withdrawal. There were significant reductions in the mRNA levels of neurotrophic factors (BDNF, GDNF, and NGF) and their receptors (TrkB, GFRA1, and NGFR) following morphine dependence. However, the expression of almost all neurotrophic factors increased after morphine withdrawal. Conclusions: The findings suggest that changes in neurotrophic factors, their receptors, and specific types of voltage-gated calcium channels in the cerebellum play roles in the processes of morphine dependency and withdrawal.

Predicting role of Myc-induced nuclear antigen 53 in determining the development and severity of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) as an autoimmune disease can relate to an imbalance between regulatory T cells (Tregs) and Th17 cells. Previous reports have shown that Myc-induced nuclear antigen (Mina) 53 protein is involved in the developments of Tregs and Th17 cells. Therefore, the current study focused on determining whether Mina53 level is correlated to the severity of SLE. The blood samples were collected from 60 patients with SLE (30 cases with mild SLE and 30 cases with severe SLE) and 30 healthy subjects. The serum concentration of Mina53 was measured using enzyme-linked immunosorbent assay (ELISA). The expression of Mina53 gene was assessed using real-time PCR method after extracting RNA from isolated peripheral blood mononuclear cells and synthesizing cDNA. Patients with SLE showed significant increases in the serum level and gene expression of Mina53 compared to healthy subjects (P<0.001). Furthermore, serum level and gene expression of Mina53 showed significant effects on SLE disease and its severity (P<0.01). There was the highest sensitivity and maximum specificity in the cut-off point of Mina53 serum level equal to 125.4 (area under the curve (AUC)=0.951) and Mina53 expression level equal to 8.5 (AUC=0.88) for SLE diagnosis. The cut-off point of Mina53 serum level equal to 139.5 (AUC=0.854) and the cut-off point of Mina53 expression level equal to 8.5 (AUC=0.788) had the highest sensitivity and maximum specificity determining severe forms of SLE. Our results showed that the changes in serum and expression levels of Mina53 have significant effects on SLE disease and its severity. These levels may be considered as diagnostic and predictive markers for SLE.

Microsatellite instability and somatic gene variant profile in solid organ tumors

IntroductionAbsence of mismatch repair (MMR) genes in tumor cells or errors in the replication repair process may lead to DNA-MMR deficiency and microsatellite instability (MSI) formation. Specific tumor environments where gene variations are observed are believed to be conducive to the formation of MSI. This study aimed to determine the MSI status, MMR protein expression, and somatic mutation profile in solid organ tumors.Material and methodsIn this study, the records of 192 patients with solid organ tumors who were referred to the Molecular Pathology Laboratory between January 2018 and December 2022 were reviewed retrospectively. The MSI profiles of the patients were evaluated using real-time polymerase chain reaction (PCR) and immunohistochemical (IHC) methods. Somatic variations in the patients were detected using an NGS colon cancer panel.ResultsIn the IHC evaluation, 22 cases showed MMR-deficient (dMMR) or high MSI (MSI-H), and 170 cases showed MMR-proficient (pMMR) or microsatellite stable (MSS). Real-time PCR results on the 22 dMMR cases revealed that 11 cases had MSI-H and 11 cases had MSS status. Among the 170 cases with pMMR, 160 cases were found to have MSS status, while 10 cases had low MSI (MSI-L). NGS analysis revealed that the three most frequent pathogenic variants in all cases were BLM exon 7 c.1544delA, MSH3 exon 7 c.1148delA, and MLH3 exon 2 c.1755delA. MSI-H cancer patients had a higher variation burden compared to MSS cancer patients. The most frequently observed pathogenic variant in both MSI-H and MSS cancer patients was BLM exon 7 c.1544delA.ConclusionsOur study covers not only colorectal cancer patients but also other solid tumor types, providing the first data from the Turkish population on the MSI-H/dMMR status and somatic mutation profile in the presence of this condition.

Development of a novel Colorimetric Assay for the rapid diagnosis of Coronavirus disease 2019 from nasopharyngeal samples.

Emergence of Coronavirus disease 2019 (COVID-19) pandemic has posed a huge threat to public health. Rapid and reliable test to diagnose infected subjects is crucial for disease spread control. We developed a colorimetric test for COVID-19 detection using a Colorimetric Assay based on thiol-linked RNA modified gold nanoparticles (AuNPs) and oligonucleotide probes. This method was conducted on RNA from 200 pharyngeal swab samples initially tested by Real-Time polymerase chain reaction (RT-PCR) as gold standard. A specific oligonucleotide probe designed based on ORF1ab of COVID-19 was functionalized with AuNPs-probe conjugate. The exposure of AuNP-probe to isolated RNA samples was tested using hybridization. In this comparative study, the colorimetric functionalized AuNPs assay exhibited a detection limit of 25copies/µL. It was higher in comparison to the RT-PCR method, which could only detect 15copies/µL. The results demonstrated 100% specificity and 96% sensitivity for the developed method. Herein, we developed an incredibly rapid, simple and cost-effective Colorimetric Assay lasting approximately 30min which could process considerably higher number of COVID-19 samples compared to the RT-PCR. This AuNP-probe conjugate colorimetric method could be considered the optimum alternatives for conventional diagnostic tools especially in over-populated and/or low-income countries.

Study on producing a LAMP kit for diagnosis of small liver fluke Clonorchis sinensisinfection in humans

Background: Clonorchis sinensis is one of the three species of small liver flukes that cause infections in humans. C. sinensis is found mainly in several Asian countries and Northern, Vietnam. The Loop-mediated isothermal amplification (LAMP) with a sensitivity and specificity comparable to that of the PCR method, which requires less complex equipment and facilities for performance. Methods:The research design describes the laboratory experiment and evaluates a LAMP kit for the diagnosis of C. sinensis in the laboratory and the small-scale field assessment, using 150 human stool samples from fascioliasis endemic areas. Results: The LAMP primer sets have been designed and selected; the conditions and components of the LAMP reaction for C. sinensisdiagnosis have been optimised. Evaluation results of the LAMP kit in the laboratory showed a sensitivity of the test was 98.63% (95% CI: 91.92-99.99%); a specificity was 100% (95% CI: 85.25-100%); the limit of detection was 26 gene copies/μl. The LAMP test kit was found to be stable within 12 months at -20±5oC, and 6 months after opening the tubes. The field assessment of the LAMP kit in Ninh Binh province showed a C. sinensis rate of 6.66%, which was similar to the rate obtained from the studies using the real-time PCR method. Conclusions: The research has successfullyproduced a LAMP kit for the diagnosis of C. sinensis that is sensitive &gt;95% and highly specific &gt;95%. The LAMP kit is stable within 12 months at -20±5oC and for 6 months after opening the tubes

Establishment and preliminary application of duplex fluorescence quantitative PCR for porcine circoviruses type 2 and type 3

Porcine circovirus types 2 (PCV2) and 3 (PCV3) are the two most prevalent porcine circoviruses in China, all of which can infect swine herds and cause serious diseases. To detect coinfection with PCV2 and PCV3, primers and probes for duplex PCV2 and PCV3 real-time PCR were designed to target their cap genes based on the constructed plasmids pUC57-PCV2 and pUC57-PCV3. The established duplex PCV2 and PCV3 real-time PCRs were specific to PCV2 and PCV3 and showed no cross-reactions with other porcine viral pathogens. The limit of detection was 5 and 50 copies for the PCV2 and PCV3 plasmids, respectively. The intra- and interassay repeatability had coefficients of variation below 3 %. The established methods were used to analyze clinical samples from Liaoning and Jilin provinces of China. The coinfection rates of PCV2 and PCV3 in pigs extensively fed in Liaoning and Jilin, large-scale farmed pigs in Liaoning and large-scale farmed pigs in Jilin were 15.0 % (6/40), 36.7 % (11/30) and 35.4 % (62/175), respectively. This study established a useful duplex PCV2 and PCV3 real-time PCR method that can be used for the detection of PCV2 and PCV3 in local clinical samples.

Epithelial-mesenchymal Transition (EMT) and the Effect of Atorvastatin on it in ARPE-19 cells

Proliferative vitreoretinopathy (PVR) develops after an unsuccessful or complicated recovery from rhegmatogenous retinal detachment (RRD) surgery. Intraocular scar formation with the contribution of epithelial-mesenchymal transition (EMT) in RPE cells is prominent in the pathology of PVR. In the present study, the EMT process was experimentally induced in human retinal pigment epithelium (RPE; ARPE-19) cells, and the effect of atorvastatin on the process was studied. The mRNA and protein levels of mesenchymal markers actin alpha 2 (ACTA2) / alpha-smooth muscle actin (α-SMA) and fibronectin (FN), and epithelial markers occludin (OCLN) and zonula occludens-1 (ZO-1) were measured using quantitative real-time PCR (qRT-PCR) and western blot methods, respectively. In addition, α-SMA and FN were visualized using immunofluorescence staining. Cells were photographed under a phase contrast light microscope. Changes in the functionality of cells following the EMT process were studied using the IncuCyte scratch wound cell migration assay and the collagen cell invasion assay with confocal microscopy. The induction of EMT in ARPE-19 cells increased the expression of mesenchymal markers ACTA2/α-SMA and fibronectin and reduced the expression of epithelial marker OCLN both at mRNA and protein levels. The mRNA levels of ZO-1 were lower after EMT, as well. Increased levels of α-SMA and FN were confirmed by immunofluorescence staining. Atorvastatin further increased the mRNA levels of mesenchymal markers ACTA2 and FN as well as the protein levels of α-SMA and reduced the mRNA levels of epithelial markers OCLN and ZO-1 under the EMT process. EMT promoted wound closure and cell invasion into the 3D collagen matrix when compared to untreated control cells. These data present cellular changes upon the induction of the EMT process in ARPE-19 cells and the propensity of atorvastatin to complement the effect. More studies are needed to confirm the exact influence of the EMT process and atorvastatin treatment on the PVR development after RRD surgery.

Frequency of Herpes Simplex Virus Types 1 and 2 and Cytomegalovirus in Perilymph and Peripheral Blood Samples of Cochlear-Implanted Children Using the Polymerase Chain Reaction Method

Background and Aim: Inner ear infection with some viruses may be one of the possible causes of Sensorineural Hearing Loss (SNHL). This study aims to determine the frequency of Herpes Simplex Virus 1 and 2 (HSV-1, HSV-2) and Cytomegalovirus (CMV) in perilymph and peripheral blood samples of cochlear-implanted children. Methods: In this cross-sectional study, 30 children with severe-to-profound SNHL (aged 1.1–5 years) underwent cochlear implantation surgery. During surgery, their perilymph and peripheral blood samples were collected. The samples were analyzed separately for the presence of herpes HSV-1, HSV-2, and CMV by real-time Polymerase Chain Reaction (PCR) method. The load of IgG and IgM antibodies against these viruses was determined using the Enzyme-Linked Immunosorbent Assay (ELISA) method. Results: The frequency of CMV in perilymph samples was 16.7% (5 patients) and in peripheral blood samples was 3.3% (1 patient). The IgG antibody against CMV and HSV- 1 was positive in 80% and 46.7% of the patients, respectively. The IgM antibody against CMV was positive in 10%. The mean IgM serum antibody load against HSV-1, HSV-2, and CMV was 2.70, 1.70, and 5.47, respectively, and the mean IgG antibody load against these viruses was 56.07, 2.50, and 23.67, respectively. Conclusion: The IgG test is positive in cochlear-implanted children with CMV in their perilymph samples, and the CMV genome is not present in their peripheral blood. This may indicate the previous presence of this virus in the ear and its role in hearing loss. Keywords: Sensorineural hearing loss; perilymph fluid; cytomegalovirus; herpes simplex

Real-Time PCR (qtPCR) to Discover the Fate of Plant Growth-Promoting Rhizobacteria (PGPR) in Agricultural Soils.

To optimize the application of plant growth-promoting rhizobacteria (PGPR) in field trials, tracking methods are needed to assess their shelf life and to determine the elements affecting their effectiveness and their interactions with plants and native soil microbiota. This work developed a real-time PCR (qtPCR) method which traces and quantifies bacteria when added as microbial consortia, including five PGPR species: Burkholderia ambifaria, Bacillus amyloliquefaciens, Azotobacter chroococcum, Pseudomonas fluorescens, and Rahnella aquatilis. Through a literature search and in silico sequence analyses, a set of primer pairs which selectively tag three bacterial species (B. ambifaria, B. amyloliquefaciens and R. aquatilis) was retrieved. The primers were used to trace these microbial species in a field trial in which the consortium was tested as a biostimulant on two wheat varieties, in combination with biochar and the mycorrhizal fungus Rhizophagus intraradices. The qtPCR assay demonstrated that the targeted bacteria had colonized and grown into the soil, reaching a maximum of growth between 15 and 20 days after inoculum. The results also showed biochar had a positive effect on PGPR growth. In conclusion, qtPCR was once more an effective method to trace the fate of supplied bacterial species in the consortium when used as a cargo system for their delivery.