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ABSTRACT: Cellulitis is an important disease in commercial turkey farms associated with significant economic loss. Although the etiology of cellulitis is not fully elucidated, Clostridium septicum (C. septicum) is one of the main causes of this infectious disease. In this study, we report the development of a quantitative real-time PCR (qRT PCR) assay targeting the alpha-toxin gene (csa), which involves a prior 15-cyle PCR using a nested pair of primers to increase the detection sensitivity. Additionally, the TaqMan probe was employed to increase the target-specificity of the assay. The performance of our nested qRT-PCR assay was evaluated using Clostridium isolates from turkey farms, representing both septicum and non-septicum species, as well as sponge swab samples from turkey farms. Our step-by-step development of the assay showed that the csa gene is a suitable target for specific detection of C. septicum strains and that the inclusion of nested PCR step significantly increased the detection sensitivity of the final qRT PCR assay. The performance of the assay was also validated by a high correlation of the threshold cycle numbers of the qRT PCR assay with the relative abundance of C. septicum read counts in 16S rRNA gene microbiota profiles of the C. septicum-containing samples from turkey farms.

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Abstract Background Quantitative real time PCR (qPCR) is a powerful tool to evaluate mRNA expression level. However, reliable qPCR results require normalization with validated reference gene(s). In this study, we investigated stable reference genes in seven tissues according to four developmental stages in minipigs. Six candidate reference genes and one target gene (ACE2) were selected and qPCR was performed. BestKeeper, geNorm, NormFinder, and delta Ct method through the RefFinder web-based tool were used to evaluate the stability of candidate reference genes. To verify the selected stable genes, relative expression of ACE2 was calculated and compared with each other. Results As a result, HPRT1 and 18S genes had lower SD value, while HMBS and GAPDH genes had higher SD value in all samples. Using statistical algorithms, HPRT1 was the most stable gene, followed by 18S, β-actin, B2M, GAPDH, and HMBS. In intestine, all candidate reference genes exhibited similar patterns of ACE2 gene expression over time, whereas in liver, lung, and kidney, gene expression pattern normalized with stable reference genes differed from those normalized with less stable genes. When normalized with the most stable genes, the expression levels of ACE2 in minipigs highly increased in intestine and kidney at PND28, which is consistent with the ACE2 expression pattern in humans. Conclusions We suggest that HPRT1 and 18S are good choices for analyzing all these samples across the seven tissues and four developmental stages. However, this study can be a reference literature for gene expression experiments using minipig because reference gene should be validated and chosen according to experimental conditions.

Background: Quantitative real time PCR (qPCR) is a powerful tool to evaluate mRNA expression level. However, reliable qPCR results require normalization with validated reference gene(s). In this study, we investigated stable reference genes in seven tissues according to four developmental stages in minipigs. Six candidate reference genes and one target gene (ACE2) were selected and qPCR was performed. BestKeeper, geNorm, NormFinder, and delta Ct method through the RefFinder web-based tool were used to evaluate the stability of candidate reference genes. To verify the selected stable genes, relative expression of ACE2 was calculated and compared with each other. Results: As a result, HPRT1 and 18S genes had lower SD value, while HMBS and GAPDH genes had higher SD value in all samples. Using statistical algorithms, HPRT1 was the most stable gene, followed by 18S, β-actin, B2M, GAPDH, and HMBS. In intestine, all candidate reference genes exhibited similar patterns of ACE2 gene expression over time, whereas in liver, lung, and kidney, gene expression pattern normalized with stable reference genes differed from those normalized with less stable genes. When normalized with the most stable genes, the expression levels of ACE2 in minipigs highly increased in intestine and kidney at PND28, which is consistent with the ACE2 expression pattern in humans. Conclusions: We suggest that HPRT1 and 18S are good choices for analyzing all these samples across the seven tissues and four developmental stages. However, this study can be a reference literature for gene expression experiments using minipig because reference gene should be validated and chosen according to experimental conditions. [ABSTRACT FROM AUTHOR]

Cellulitis is an important disease in commercial turkey farms associated with significant economic loss. Although the etiology of cellulitis is not fully elucidated, Clostridium septicum ( C. septicum ) is one of the main causes of this infectious disease. In this study, we report the development of a quantitative real-time PCR (qRT PCR) assay targeting the alpha-toxin gene ( csa ), which involves a prior 15-cyle PCR using a nested pair of primers to increase the detection sensitivity. Additionally, the TaqMan probe was employed to increase the target-specificity of the assay. The performance of our nested qRT-PCR assay was evaluated using Clostridium isolates from turkey farms, representing both septicum and non- septicum species, as well as sponge swab samples from turkey farms. Our step-by-step development of the assay showed that the csa gene is a suitable target for specific detection of C. septicum strains and that the inclusion of nested PCR step significantly increased the detection sensitivity of the final qRT PCR assay. The performance of the assay was also validated by a high correlation of the threshold cycle numbers of the qRT PCR assay with the relative abundance of C. septicum read counts in 16S rRNA gene microbiota profiles of the C. septicum -containing samples from turkey farms. [ABSTRACT FROM AUTHOR]

Sanghuangporus baumii (Pilát) (Zhou and Dai), a traditional Chinese medicinal fungus, has been confirmed to produce triterpenoids, which possess a good anti-cancer effect. However, the triterpenoids yield of S. baumii is low and its biosynthetic mechanism is not clearly understood. To investigate the triterpenoids biosynthetic mechanism, the whole cDNA sequence of the HMG-CoA reductase (HMGR) gene was obtained. The open reading frame of HMGR gene cDNA named SbHMGR, comprised 4170 bp, encoding a polypeptide of 1389 amino acids with a predicted protein molecular weight of 147.53 kDa. The HMGR gene includes four exons and three introns. Subsequently, the triterpenoids content was determined at different developmental stages of S. baumii. It showed a dynamic tendency—increased first, then decreased and peaked on 14th day in the mycelium stage. In addition, the changing trend of SbHMGR enzyme activity and transcript level of SbHMGR gene were measured. They were nearly consistent with the triterpenoids content, suggesting that SbHMGR gene may play a key regulatory role in triterpenoids biosynthetic pathway. Similarly, changes in the transcript level of downstream key genes (SbMVD, SbIDI, SbLSD) of SbHMGR gene were also positively correlated with the triterpenoids content, indicating those three genes were also involved in triterpenoids biosynthetic pathway. Moreover, SbHMGR gene had been observed to be expressed in prokaryote. The results will be valuable for elucidation of the biosynthetic pathway of triterpenoids in S. baumii, lay a foundation for genetic modifications to improve triterpenoids yield.

Relative quantification is a popular analysis in gene expression studies using quantitative real‐time PCR (qPCR). However, the calculation steps using the major algorithms for this analysis are rather complicated. In this study, we developed an easy‐to‐use spreadsheet‐based method for relative quantification. The inputs from end‐users are the efficiencies of both target and reference genes and the Cq values of those genes from cases and controls. This method performed normalization (with one or more reference genes), calculation of fold change of gene expression, and statistical analysis to analyze the difference between the groups in a step‐by‐step manner, which would allow the end‐users to understand how the analysis arrived at the conclusion. Four previously published data sets with different experimental designs were used as examples. The calculated results were concordant with the results computed by the Relative Expression Software Tool (REST) 2009, a popular tool for relative quantification. Altogether, our method, which offers easy‐to‐understand calculation steps and does not require specialized instruments, software, or expertise to operate, would be a useful tool for students, educators, and scientists in the field of molecular biology. [ABSTRACT FROM AUTHOR]

Abstract Objective Long non-coding RNAs (lncRNAs) are emerging as novel biomarkers for a variety of chronic conditions including autoimmune disease. PAXgene Blood RNA tubes are routinely used in clinical research and molecular diagnostic development to capture RNA profiles in peripheral whole blood. While the stability of mRNA expression profiles captured using PAXgene tubes has been documented previously, no previous work has determined the stability of lncRNA expression profiles observed in PAXgene tubes stored at − 80 °C. Here we sought to determine the effects on lncRNA expression profiles following − 80 °C storage of total RNA templates, cDNA synthesized using fresh or frozen total RNA template, and the impact of freeze–thaw cycles on both total RNA and cDNA obtained from PAXgene tubes. Results We find that storage of whole blood in PAXgene tubes, total RNA and cDNA for up to 1 year at − 80 °C or up to ten total RNA or cDNA freeze–thaw cycles do not significantly alter lncRNA expression profiles compared to baseline. As monthly expression profiles were determined, some month to month lncRNA expression variability was observed. However, all monthly observations fell within the 95% confidence interval calculated at baseline.

Oral Squamous Cell Carcinoma (OSCC) has varied etiology. Among the etiological factors, genetic predisposition or the presence of oncogenic viruses can cause impairment in the physiological mechanisms of cellular proliferation control. The present study was chosen to determine the association of Epstein- Barr virus (EBV) with oral squamous cell carcinoma and oral potentially malignant disorders (OPMD). Oral exfoliated cells were collected from the subjects diagnosed with OSCC (n = 19), OPMD (n = 23) and healthy subjects without any deleterious condition (n = 39). The DNA extraction was performed with DNeasy Blood & Tissue Kits (Qiagen, Germany). Quantitative Real-Time PCR was then carried out with QuantiNova® SYBR® Green PCR Kit (Qiagen, Germany). Statistical analysis was performed using Chi-square test. EBV DNA was detected in all samples of all the three groups. Compared to healthy subjects, very high EBV load was observed in the oral exfoliated cells of OSCC and OPMD patients. The presence of EBV DNA in all the subjects of all the three groups reveals that EBV is an oral resident. Besides its presence, the abundance of EBV DNA among OSCC and OPMD suggests it's association to these pathologies. The high odds and risk ratio obtained for EBV copy number further supports a strong association of this virus to OSCC and OPMD. This may be the first study to quantify EBV DNA in oral exfoliated epithelial cells of OSCC and OPMD.

Real‐time quantitative PCR is a technique used to monitor the PCR reaction in real time. RT‐qPCR is broadly classified into two types based on its purpose: absolute and relative quantification. Absolute quantification is used in a wide array of fields such as microbiology, food technology, and biotechnology to quantify the microbiological load/adulterants in a commodity/copy numbers respectively, whereas Relative quantification is used in the field of genomics and functional transcriptomics to perform gene expression analysis in biological experiments. A laboratory work that covers the basic principles involved in RT‐qPCR and data analysis using the manual as well as the software methods are incorporated. The laboratory experiment was designed to provide insights on certain important principles such as primer characteristics, PCR efficiency, and melt curve analysis. This laboratory exercise provides all the significant components of RT‐qPCR, which can be useful while performing an experiment in an undergraduate and graduate laboratory. [ABSTRACT FROM AUTHOR]

Sanghuangporus baumii (Pilát) (Zhou and Dai), a traditional Chinese medicinal fungus, has been confirmed to produce triterpenoids, which possess a good anti-cancer effect. However, the triterpenoids yield of S. baumii is low and its biosynthetic mechanism is not clearly understood. To investigate the triterpenoids biosynthetic mechanism, the whole cDNA sequence of the HMG-CoA reductase (HMGR) gene was obtained. The open reading frame of HMGR gene cDNA named SbHMGR, comprised 4170 bp, encoding a polypeptide of 1389 amino acids with a predicted protein molecular weight of 147.53 kDa. The HMGR gene includes four exons and three introns. Subsequently, the triterpenoids content was determined at different developmental stages of S. baumii. It showed a dynamic tendency—increased first, then decreased and peaked on 14th day in the mycelium stage. In addition, the changing trend of SbHMGR enzyme activity and transcript level of SbHMGR gene were measured. They were nearly consistent with the triterpenoids content, suggesting that SbHMGR gene may play a key regulatory role in triterpenoids biosynthetic pathway. Similarly, changes in the transcript level of downstream key genes (SbMVD, SbIDI, SbLSD) of SbHMGR gene were also positively correlated with the triterpenoids content, indicating those three genes were also involved in triterpenoids biosynthetic pathway. Moreover, SbHMGR gene had been observed to be expressed in prokaryote. The results will be valuable for elucidation of the biosynthetic pathway of triterpenoids in S. baumii, lay a foundation for genetic modifications to improve triterpenoids yield. [ABSTRACT FROM AUTHOR]

Recurrent miscarriage (RM) is a complex clinical problem. However, specific diagnostic biomarkers and candidate regulatory targets have not yet been identified. To explore RM-related biological markers and processes, we performed a genome-wide DNA methylation analysis using the Illumina Infinium HumanMethylation450 array platform. Methylation variable positions and differentially methylated regions (DMRs) were selected using the Limma package in R language. Thereafter, gene ontology (GO) enrichment analysis and pathway enrichment analysis were performed on these DMRs. A total of 1,799 DMRs were filtered out between patients with RM and healthy pregnant women. The GO terms were mainly related to system development, plasma membrane part, and sequence-specific DNA binding, while the enriched pathways included cell adhesion molecules, type I diabetes mellitus, and ECM–receptor interactions. In addition, genes, including ABR, ALCAM, HLA-E, HLA-G, and ISG15, were obtained. These genes may be potential candidates for diagnostic biomarkers and possible regulatory targets in RM. We then detected the mRNA expression levels of the candidate genes. The mRNA expression levels of the candidate genes in the RM group were significantly higher than those in the control group. However, additional research is still required to confirm their potential roles in the occurrence of RM.

The present study aimed to observe the expression of MYCN in lungs of mice following chronic exposure of 2,4-D with or without lipopolysaccharide (LPS). 2,4-D was administered orally dissolved in corn oil at high and low dose (1/10th and 1/20th of LD50) for 90 days. After 90 days of exposure, animals from each group were challenged with LPS/normal saline solution at 80 μg/animal. The lung tissues were processed for microarray and real time studies. LPS resulted decrease (–0.173 fold) in m-RNA expression level of MYCN as compared to control, while High dose of 2,4-D alone and in combination with LPS resulted 0.949-fold change and 1.656-fold change increase in expression of MYCN m-RNA, respectively, as compared to control. Similarly, Low dose of 2,4-D alone or in combination with LPS also altered MYCN expression. The microarray data when validated by Real Time PCR was found to be in concordance with the Real Time PCR data. The data taken together suggest that, high and low exposure of 2,4-D alone or in combination with LPS alters expression of MYCN at m-RNA level.

Respiratory sample staining is a standard tool used to diagnose Pneumocystis jirovecii pneumonia (PjP). Although molecular tests are more sensitive, their interpretation can be difficult due to the potential of colonization. We aimed to validate a Pneumocystis jirovecii (Pj) real-time PCR (qPCR) assay in bronchoscopic bronchoalveolar lavage (BAL) and oropharyngeal washes (OW). We included 158 immunosuppressed patients with pneumonia, 35 lung cancer patients who underwent BAL, and 20 healthy individuals. We used a SYBR green qPCR assay to look for a 103 bp fragment of the Pj mtLSU rRNA gene in BAL and OW. We calculated the qPCR cut-off as well as the analytical and diagnostic characteristics. The qPCR was positive in 67.8% of BAL samples from the immunocompromised patients. The established cut-off for discriminating between disease and colonization was Ct 24.53 for BAL samples. In the immunosuppressed group, qPCR detected all 25 microscopy-positive PjP cases, plus three additional cases. Pj colonization in the immunocompromised group was 66.2%, while in the cancer group, colonization rates were 48%. qPCR was ineffective at diagnosing PjP in the OW samples. This new qPCR allowed for reliable diagnosis of PjP, and differentiation between PjP disease and colonization in BAL of immunocompromised patients with pneumonia.

Background: The existence of two distinct forms within a species that differ in one or more characteristics is known as dimorphism. The gonads are the primary organs in teleost to show sexual dimorphism. Lepidocephalus thermalis belongs to the Cobitidae family. No expression study on the developmental stages was done on this species. Since there is no specific primers reported for L. thermalis, the study has been carried out with the help of the specific primers of catfish. Methods: qRT- PCR is an acknowledged method for gene expression analysis due to its precision and reproducibility. In the current study, the expression of 14 different transcription factors involved in sex differentiation of Indian spiny loach during different developmental stages was analyzed using qRT- PCR and has been compared among the different stages of gonadal development for that transcription factor. Result: Gene expression patterns have been obtained from the total RNA isolated from MSG (Meso nephric gonadal complex), medium stage ovary, medium stage testis, large stage ovary and large stage testis. From the study, it has been analyzed that only sf1 has higher expression in testis and all the other transcription factors has shown higher expression in ovary. [ABSTRACT FROM AUTHOR]

We aimed to study the relation between both bacterial colonization of the uterine endometrium & endometrial miR-17-5p levels and endometriosis, and then to evaluate endometrial miR-17-5p as a biomarker of endometriosis. A comparative observational study was carried over 51 endometriosis patients and 51 controls admitted into Obstetrics and Gynecology department, Mansoura Faculty of Medicine. Endometrial tissue samples were collected and aimed for bacterial culture and identification of resulting organisms besides estimation of tissue levels of microRNA-17-5p by quantitative real time PCR. G. vaginalis, S. agalactiae, S. aureus, Mobiluncus and E. coli were associated with endometriosis. MicroRNA-17-5p was up-regulated in endometriosis patients (P value was <0.0001*). Its sensitivity and specificity were 90% and 76.5%. MiR-17-5p showed higher results in culture positive than negative cases. On studying the relation between the positivity of endometrial tissue culture and miR-17-5p and so endometriosis, P value was <0.0001*. We concluded that G. vaginalis, S. agalactiae, S. aureus, Mobiluncus and E. coli were associated with development of endometriosis. Endometrial miR-17-5p was elevated in association to positive detection of bacterial species. MiR-17-5p might be a bio- marker of endometriosis. CFU/ml: Colony Forming Unit per Milliliter; miR-17-5p: MicroRNA-17-5p; qRT PCR: Quantitative Real Time Polymerase Chain Reaction. [ABSTRACT FROM AUTHOR]

We aimed to evaluate apoptosis and myogenesis related genes expression in embryos and fetuses from two divergent genetic groups of pigs: Piau breed and a commercial line. Thirty females (15 Piau and 15 commercial line) were selected at 120 days of age. Estrous cycle was observed and on the third estrus females were considered sexually mature. Gilts were inseminated with semen from males of the respective breed. Three females from each breed were slaughtered at five different gestational ages: 15, 30, 45, 60 and 90 days. Whole embryos (15 and 30 d) and samples of longissimus dorsi muscle from fetuses (45, 60 and 90 d) were collected for RNA extraction. Expression of apoptosis and myogenesis related genes (BAX, BCL2, FGF4, IHH, HHIP, SHH, SOX2, WNT1 and WNT4) were evaluated by quantitative real time PCR. There was significant effect of interaction between breeds and gestational ages for all genes evaluated (P < 0.05). The BCL2 gene expression differed throughout pregnancy in Piau group with lower expression on day 15. The IHH gene expression throughout pregnancy was lower on days 15 and 60 in Piau group and lower on day 90 in commercial line group; and the SHH gene expression throughout pregnancy was higher on day 30 and lower on day 60 in Piau group and lower on day 45 in commercial group. The WNT1 gene expression along pregnancy was lower on day 15 and higher on days 30 and 45 in Piau group, and it was higher on day 30 than days 15 and 60 in commercial group. WNT4 gene expression throughout pregnancy was lower on day 15 in Piau group, and it was lower on day 30 in commercial pigs. Piau group presented higher expression of the FGF4 gene on days 45, 60 and 90, and commercial group showed higher expression on day 15 and 90. SOX2 gene expression was lower on day 15 in Piau pigs and it was constant throughout pregnancy in commercial group. Overall, the expression of IHH, SHH, WNT1, WNT4 and FGF4 genes were higher in commercial than Piau pigs on day 15, besides expression of BCL2 gene in Piau embryos was lower on day 15; these results might indicate that the muscle precursor cells are allowed to proliferate for a longer time in commercial than in Piau embryos by the balance of proliferative and apoptotic genes. Therefore, the expression differential between breeds can stimulate proliferation and differentiation of cells in different ways, explaining the postnatal differences in the muscularity between pigs from Piau breed and a commercial line. • Piau and commercial line embryos and fetuses were compared for apoptosis and myogenesis related genes expression. • Expression of IHH, SHH, WNT1, WNT4, FGF4 and BCL2 genes were higher in commercial than Piau embryos on day 15. • Gene expression in muscle precursor cells leads them to proliferate for a longer time in commercial than in Piau embryos. [ABSTRACT FROM AUTHOR]

Quantitative real time PCR (qRT-PCR) is a nucleic acid quantitative technique and is also considered as a validation tool. The Cry1Ia36 protein isolated from Bacillus thuringiensis (Bt) strain YC-10 has high nematicidal activity against nematodes. Caenorhabditis elegans is one of the major model organisms and a readily accessible source of biological material for gene expression studies. To evaluate the expression stability of 12 candidate reference genes of C. elegans for exposing to different concentrations of Cry1Ia36 protein and different treat time, five statistical approaches (the comparative delta-Ct method, BestKeeper, NormFinder, Genorm and RefFinder) were used to evaluate each individual candidate reference gene. The results indicated that cdc-42 and F35G12.2 were the best reference genes for performing reliable gene expression normalization in the impact of Cry1Ia36 protein. In addition, when C. elegans was exposed to Cry1Ia36 protein and other nematicides, avermectin and 5-aminolevulinic acid, cdc-42 was recommended as the most reliable reference genes. Y45F10D.4 was the least stable reference genes in our experimental settings. Therefore, cdc-42 was reliable reference gene for gene expression studies in C. elegans exposed to Cry1Ia36 protein and other nematicides. [ABSTRACT FROM AUTHOR]

Gene silencing is the epigenetic regulation of any gene in order to prevent gene expression at the transcription or translation levels. Among various gene silencing techniques, RNA silencing (RNAi) is notable gene regulation technique that involves sequence-specific targeting and RNA degradation. However, the effectiveness of transgene-induced RNAi in F1 generation of chrysanthemum has not been studied yet. In the current study, we used RNAi-constructed CmTFL1 (white-flowered) and CmSVP overexpressed (yellow flowered) transgenic plants of previously conducted two studies for our experiment. Cross hybridization was performed between these intergeneric transgenic and non-transgenic plants of the winter-growing chrysanthemum selection “37” (light pink flowered). The transgene CmSVP was confirmed in F1 hybrids by RT-PCR analysis, whereas hybrids of CmTFL1 parental plants were non-transgenic. Besides this, quantitative real-time PCR (qPCR) was used to explain the molecular mechanism of flower development using reference genes. Intergeneric and interspecific hybrids produced different colored flowers unlike their respective parents. These results suggest that generic traits of CmSVP overexpressed plants can be transferred into F1 generations when crossed with mutant plants. This study will aid in understanding the breeding phenomenon among intergeneric hybrids of chrysanthemum plants at an in vivo level, and such transgenics will also be more suitable for sustainable flower yield under a low-light production system.

Background: MicroRNAs (miRNAs) are small non-coding RNAs, whose role in regulating diverse immune functions, suggests they might play a role as biomarkers for immune mediated disorders. Studies showed that miRNA-146a (miR-146a) expression is increased by proinflammatory cytokines and is an important modulator of differentiation and function of cells of innate and adaptive immunity. Aim of the work: The current study aimed to evaluate the expression of miR-146a as a potential biomarker for diagnosis of rheumatoid arthritis (RA) and to explore its association with disease activity. Subjects and methods: The study enrolled 50 Egyptian subjects divided into a patient group, which comprised 25 RA patients, and a control group which comprised 25 healthy individuals. The disease activity for the patients’ group was determined by simplified disease activity index. Relative quantification of miR-146a expression in whole blood was determined using reverse transcriptase quantitative real time polymerase chain reaction. Results: There were highly significant statistical differences between patients and healthy controls as regards miR-146a relative expression, erythrocyte sedimentation rate (ESR) and anti-cyclic citrullinated peptide (anti-CCP) (p

Background: Tumor necrosis factor-α (TNFα) is a crucial physiologic regulator of immune responses, and several disorders have been associated with its dysregulation., Objective: This study aimed to understand TNFα gene expression in adult patients with liver and pancreas disorders and examine the impact of TNFα-238 genotypes on this population., Methods: At the Ibn Al-Baladi Hospital in Baghdad, blood samples were collected from forty patients who were diagnosed with beta thalassemia together with pancreatic disease, forty patients who were diagnosed with thalassemia together with liver disorder, and forty patients who were diagnosed with thalassemia without pancreas or liver disorder. For the purpose of establishing a control group, forty samples were collected from persons who were of the same age and gender and seemed to be in good health. All of these individuals were deemed to be older than 18 years old. Through the utilization of real-time polymerase chain reaction (PCR), the level of TNF-α gene expression was investigated and assessed. The T-ARMS-PCR method was performed for detection and genotyping of TNFα-238 in thalassemia patients and healthy control samples., Results: The result showed that TNF α gene expression assessment showed that group B (thalassemia patients with liver disorder) had higher folding than other groups while the lowest gene expression was in group D (as control group). Furthermore, the relationship between TNFα gene expressions folding with TNFα-238 genotypes in beta thalassemia major patients, discovered a considerable increase at GA genotype patients in TNFα gene expression level, followed by AA genotype compared to the GG genotype. Furthermore, the results of the current study showed an association between the presence of the mutant (A) allele whether heterozygous (GA) and homozygous (AA) with the TNF-α gene expression in thalassemia patients with liver and pancreatic disorders., Conclusion: Based on the results, it can be concluded that there is a relationship between the presence of the mutant (A) allele, whether heterozygous (GA) or homozygous (AA) of TNF-α 238, and TNF-α gene expression in liver and pancreatic diseases as well as in patients with thalassemia.

Aim: This study aimed to investigate the in vitro antitumor activity of new series of 2-thiohydanotin derivatives ( 7 and 9 ) against two cancer cell lines. Materials & methods: A new series of 2-thioxoimidazolidine derivatives ( 3-9 ) were synthesized and investigated for its structure through spectral analysis and also tested against (HepG-2) and (HCT-116) cell line. Results: Among the synthesized compounds, compound 7 halted liver cancer cells at the G0/G1 phase and triggered apoptosis of liver cancer. Contrarily, compound 9 caused colon cancer cells to be arrested at the S phase and trigger apoptosis. Also, they had a good inhibitory effect on (Nrf2). Conclusion: Both compounds had attractive lead molecules for the creation of colon and liver cancer medications.

Background: This study was conducted to compare the levels of maternal serum soluble endoglin (sEng) and cell-free fetal DNA (cffDNA) in pregnant females with PE to normotensive pregnant ones, together with relating these levels to preeclampsia (PE) severity and onset. Method of the study: It was a comparative study in Mansoura University Hospital, Egypt, to detect the levels of serum sEng by ELISA besides the levels of cffDNA by quantitative real-time polymerase chain reaction in 80 pregnant females suffering from PE in addition to 80 normotensive pregnant ones that were included as control. Results: Levels of serum sEng and cffDNA were higher in PE cases than control (p < 0.0001٭ both) and were significantly related to the severity of the disease. Levels were also higher in early than late onset PE (p < 0.003٭ and <0.002٭, respectively). Sensitivities, specificities, positive, and negative predictive values in addition to accuracy of serum sEng and cffDNA were 97.5%, 98.8%, 98.7%, 97.5%, and 98.1% and 97.5%, 93.8%, 94.0%, 97.4%, and 95.6%, respectively. Conclusion: Maternal serum sEng and cffDNA can be good markers for diagnosis of PE in Egyptian patients. They are positively related to the disease severity. Abbreviations: cffDNA; Cell-Free Fetal DNA, sEng; soluble Endoglin, PE; preeclampsia, qRT PCR; Quantitative real-time polymerase chain reaction. [ABSTRACT FROM AUTHOR]

The molecular mechanisms responsible for disease progression of CML are not conclusive. The main functional changes associated with disease evolution in CML was high proliferation rate, decreased apoptosis, blockade of differentiation, and strong resistance to chemotherapeutic agents. The current study analyzed the relative expressional profiles of genes related with proliferation, apoptosis, differentiation, and resistance to chemotherapeutic agents such as c-MYC , BAD , BCL-2 , C/EBPα/-β and ABCB1 respectively in different clinical stages of CML by SYBR Green I quantitative real-time (qRT) PCR. We selected a total of 183 CML patients and 30 healthy control samples. The study populations were classified into four groups, including de novo CML-CP (50/183), CML-AP (32/183), CML-BC (51/183) and Imatinib Mesylate or IM resistant CML-CP (50/183) groups. qRT PCR analysis revealed that significant overexpression of c-MYC , ABCB1 and BCL-2 was observed in advanced phases and IM resistant CP of CML compared to healthy controls. Likewise, the mean expression level of BAD , C/EBPα/-β genes were found to be significantly down regulated. Present study concluded that the complex interplay of several candidate genes like overexpression of c-MYC , ABCB1 , BCL-2 and down regulation of BAD , C/EBPα /- β played a significant role in the disease evolution and development of drug resistant in CML. • The molecular mechanisms responsible for disease progression of CML are not conclusive. • Functional changes associated with disease evolution in CML was high proliferation rate and strong resistance to chemotherapeutic agents. • Complex interplay of several candidate genes play a significant role in the disease evolution of CML. [ABSTRACT FROM AUTHOR]

Porcine circovirus type 3 (PCV3) was described in different clinical cases and healthy pigs. However, little is known about its circulation in pig farms. In order to assess PCV3 prevalence in 21 Polish farms, serum, feces, and oral fluid samples were examined by quantitative real-time PCR. In total, 1451 pairs of serum and feces from the same animals, as well as 327 samples of oral fluids were analyzed. The results showed that PCV3 is more commonly detected in oral fluids (37.3% positives) than in serum (9.7% positives) or feces (15.0% positives) samples. The viral loads detected in these materials ranged from 102.5–107.2 genome equivalent copies/mL. Although in most farms PCV3 was detected post weaning, in nine farms, the virus was also found in groups of suckling piglets, and in six of them viremia was detected. In four farms with reproductive failure, fetal materials were also obtained. PCV3 was detected in 36.0% of fetuses or stillborn piglets (9/25) with viral loads of 103.1–1010.4 genome equivalent copies/mL. In summary, the virus circulation may show different patterns, and congenital or early infection is not uncommon. Precise quantification of PCV3 loads in clinical materials seems to be necessary for the study and diagnosis of the infection.

Most of the toxic algal blooms in coasts of the Mexican Pacific are attributed to planktonic dinoflagellates. Recently, some new records of dinoflagellates producers of emergent toxins have been documented. The genus Alexandrium encompasses several toxic species which produce saxitoxin. In this work, the abundance and distribution of the potentially toxic species Alexandrium tamiyavanichii from coasts of the central Mexican Pacific were studied, following the method of quantitative real-time PCR. During the oceanographic cruise “MareaR IX” (19–30 April, 2017), carried out along coasts of the Mexican Pacific, hydrographic, and environmental variables were measured, and net and bottle samples were collected and preserved in modified saline ethanol buffer for analysis in the laboratory. In order to perform the qPCR method, the molecular target used was the ITS2 of the rDNA of A. tamiyavanichii. From 45 samples analyzed, 14 yielded positive results, showing the presence and abundance of the species in fixed stations of Cabo Corrientes, Manzanillo and Acapulco, with low densities (less than 40 cells/m3), which is an evidence of the sensitiveness of the method. On the other hand, chains of cells of the species were found in net samples, in stations where its presence was detected by qPCR, confirming results by the method. General distribution showed presence of the species in two zones where upwellings were detected, but not at coastal stations, except in Acapulco where a more stratified water column was found. Vertical distribution indicated that highest densities were found at subsurface layers, in association with the chlorophyll a maxima (between 11 and 30 m depth). The results show the importance of assessing the abundance and distribution of a species which may be systematically monitored, and that the method of the qPCR may be very useful.

Background: The World Health Organization (WHO) algorithm for the diagnosis of tuberculosis in seriously ill HIV-infected patients recommends that treatment for Pneumocystis jiroveciipneumonia (PJP) should be considered without giving clear guidance on selecting patients for empiric PJP therapy. PJP is a common cause of hospitalisation in HIV-infected patients in resource-poor settings where diagnostic facilities are limited. Methods: We developed clinical prediction rules for PJP in a prospective cohort of HIV-infected inpatients with WHO danger signs and cough of any duration. The reference standard for PJP was > 1000 copies/mL of P. jirovecii DNA on real-time sputum polymerase chain reaction (PCR). Four potentially predictive variables were selected for regression models: dyspnoea, chest X-ray, haemoglobin and oxygen saturation. Respiratory rate was explored as a replacement for oxygen saturation as pulse oximetry is not always available in resource-poor settings. Results: We enrolled 500 participants. After imputation for missing values, there were 56 PJP outcome events. Dyspnoea was not independently associated with PJP. Oxygen saturation and respiratory rate were inversely correlated. Two clinical prediction rules were developed: chest X-ray possible/likely PJP, haemoglobin ≥ 9 g/dL and either oxygen saturation < 94% or respiratory rate. The area under the receiver operating characteristic curve of the clinical prediction rule models was 0.761 (95% CI 0.683–0.840) for the respiratory rate model and 0.797 (95% CI 0.725–0.868) for the oxygen saturation model. Both models had zero probability for PJP for scores of zero, and positive likelihood ratios exceeded 10 for high scores. Conclusion: We developed simple clinical prediction rules for PJP, which, if externally validated, could assist decision-making in the WHO seriously ill algorithm.

Organic vinasse, a residue produced during bioethanol production, increases nitrous oxide (N2O) emissions when applied with inorganic nitrogen (N) fertilizer in soil. The present study investigated the role of the ammonia-oxidizing bacteria (AOB) community on the N2O emissions in soils amended with organic vinasse (CV: concentrated and V: non-concentrated) plus inorganic N fertilizer. Soil samples and N2O emissions were evaluated at 11, 19, and 45 days after fertilizer application, and the bacterial and archaea gene (amoA) encoding the ammonia monooxygenase enzyme, bacterial denitrifier (nirK, nirS, and nosZ) genes and total bacteria were quantified by real time PCR. We also employed a deep amoA amplicon sequencing approach to evaluate the effect of treatment on the community structure and diversity of the soil AOB community. Both vinasse types applied with inorganic N application increased the total N2O emissions and the abundance of AOB. Nitrosospira sp. was the dominant AOB in the soil and was correlated with N2O emissions. However, the diversity and the community structure of AOB did not change with vinasse and inorganic N fertilizer amendment. The results highlight the importance of residues and fertilizer management in sustainable agriculture and can be used as a reference and an input tool to determine good management practices for organic fertilization.

[Ｏｂｊｅｃｔｉｖｅｓ]Ｔｈｅ ａｉｍ ｏｆ ｔｈｉｓ ｓｔｕｄｙ ｉｓ ｔｏ ｉｎｖｅｓｔｉｇａｔｅ ｔｈｅ ｅｖｏｌｕｔｉｏｎａｒｙ ｒｅｌａｔｉｏｎｓｈｉｐｓꎬｓｔｒｕｃｔｕｒａｌ ｆｅａｔｕｒｅｓꎬｃｈｒｏｍｏｓｏｍａｌ ｌｏｃａｔｉｏｎ ｏｆ ｔｈｅ ｃｙｃｌｉｃ ｎｕｃｌｅｏｔｉｄｅ ｇａｔｅｄ ｉｏｎ ｃｈａｎｎｅｌ ｇｅｎｅｓ ｉｎ Ｃｈｉｎｅｓｅ ｃａｂｂａｇｅ( ＢｒＣＮＧＣ) ａｎｄ ｔｏ ａｎａｌｙｚｅ ｔｈｅｉｒ ｅｘｐｒｅｓｓｉｏｎ ｐａｒｔｅｒｎｓ ｕｎｄｅｒ ｄｉｆｆｅｒｅｎｔ ｔｒｅａｔｍｅｎｔｓ. [ Ｍｅｔｈｏｄｓ] Ｔｈｅ ｂｉｏｉｎｆｏｒｍａｔｉｃｓ ｔｅｃｈｎｉｑｕｅｓ ｗｅｒｅ ｕｓｅｄ ｔｏ ａｎａｌｙｚｅ ｔｈｅ ｍｏｌｅｃｕｌａｒ ｅｖｏｌｕｔｉｏｎꎬ ｇｅｎｅｓ ｓｔｒｕｃｔｕｒｅꎬ ｃｈｒｏｍｏｓｏｍａｌ ｌｏｃａｔｉｏｎ ａｎｄ ｃｏｎｓｅｒｖｅｄ ｍｏｔｉｆｓ ｏｆ ＢｒＣＮＧＣ ｉｎ Ｃｈｉｎｅｓｅ ｃａｂｂａｇｅ. Ｔｈｅ ｅｘｐｒｅｓｓｉｏｎ ｌｅｖｅｌ ｏｆ ＢｒＣＮＧＣ ｗａｓ ａｎａｌｙｚｅｄ ｂｙ ｑｕａｎｔｉｔａｔｉｖｅ ＲＴ￣ＰＣＲ ｕｎｄｅｒ ｄｉｆｆｅｒｅｎｔ ｔｒｅａｔｍｅｎｔｓ:ａｂｓｃｉｓｉｃ ａｃｉｄ( ＡＢＡ) ＋Ｔｕｒｎｉｐ ｍｏｓａｉｃ ｖｉｒｕｓ( ＴｕＭＶ)ꎬｓａｌｉｃｙｌｉｃ ａｃｉｄ( ＳＡ) ＋ＴｕＭＶꎬ ｍｅｔｈｙｌ ｊａｓｍｏｎａｔｅ(ＭｅＪＡ)＋ＴｕＭＶꎬａｎｄ ａｓｃｏｒｂｉｃ ａｃｉｄ(ＡｓＡ) ＋ＴｕＭＶ. [Ｒｅｓｕｌｔｓ] Ｔｈｅ ｒｅｓｕｌｔｓ ｓｈｏｗｅｄ ｔｈａｔ ｔｈｅｒｅ ａｒｅ ２６ ＢｒＣＮＧＣ ｇｅｎｅｓ ｉｎ Ｃｈｉｎｅｓｅ ｃａｂｂａｇｅ ａｎｄ ｃａｎ ｂｅ ｃｌａｓｓｉｆｉｅｄ ｉｎｔｏ ｆｏｕｒ ｍａｊｏｒ ｇｒｏｕｐｓ( ⅠꎬⅡꎬⅢꎬⅣ) ａｎｄ ｔｗｏ ｓｕｂｇｒｏｕｐｓ( Ⅳａ ａｎｄ Ⅳｂ)ꎬｔｈｅｙ ｗｅｒｅ ｐｒｅｄｉｃｔｅｄ ｔｏ ｂｅ ｌｏｃａｔｅｄ ｏｎ ９ ｃｈｒｏｍｏｓｏｍｅｓꎬｔｈｅ ｃｈｒｏｍｏｓｏｍｅ １ ｃｏｎｔａｉｎｅｄ ｆｉｖｅ ＢｒＣＮＧＣꎬｗｈｉｌｅ ｔｈｅ ｃｈｒｏｍｏｓｏｍｅ ５ ｏｎｌｙ ｃｏｎｔａｉｎｅｄ ｏｎｅ ＢｒＣＮＧＣ ａｎｄ ｎｏ ＢｒＣＮＧＣ ｏｎ ｃｈｒｏｍｏｓｏｍｅ ８. Ｇｅｎｅ ｓｔｒｕｃｔｕｒｅ ａｎａｌｙｓｉｓ ｓｈｏｗｅｄ ｔｈａｔ １０ ｋｉｎｄｓ ｏｆ ｍｏｔｉｆｓ ｗｅｒｅ ｉｄｅｎｔｉｆｉｅｄ ｉｎ ｔｈｅｓｅ ｇｅｎｅｓꎬ １４ ＢｒＣＮＧＣ ｐｒｏｔｅｉｎｓ ｃｏｎｔａｉｎｅｄ ａｌｌ ｏｆ １０ ｍｏｔｉｆｓꎬａｎｄ ｔｈｅ ｇｅｎｅｓ ｔｈａｔ ｃａｍｅ ｆｒｏｍ ｔｈｅ ｓａｍｅ ｇｒｏｕｐ ｓｈａｒｅｄ ｓｉｍｉｌａｒ ｍｏｔｉｆ ｃｏｍｐｏｓｉｔｉｏｎ. Ｔｈｅ ｒｅｓｕｌｔｓ ｏｆ ｑｕａｎｔｉｔａｔｉｖｅ ＲＴ￣ＰＣＲ ｓｈｏｗｅｄ ｔｈａｔ ｍｏｓｔ ＢｒＣＮＧＣ ｇｅｎｅｓ ｕｐ￣ｒｅｇｕｌａｔｅｄ ａｎｄ ｆｅｗ ＢｒＣＮＧＣ ｇｅｎｅｓ ｄｏｗｎ￣ｒｅｇｕｌａｔｅｄ ｂｙ ｔｈｅ ｉｎｔｅｒａｃｔｉｏｎ ｂｅｔｗｅｅｎ ｐｌａｎｔ ｇｒｏｗｔｈ ｒｅｇｕｌａｔｏｒ ａｎｄ ＴｕＭＶ. [ Ｃｏｎｃｌｕｓｉｏｎｓ] Ｔｈｅ ｓｔｒｕｃｔｕｒｅ ｏｆ ＢｒＣＮＧＣ ｉｎ Ｃｈｉｎｅｓｅ ｃａｂｂａｇｅ ｉｓ ｈｉｇｈｌｙ ｃｏｎｓｅｒｖｅｄ ａｎｄ ｔｈｅｙ ｐｌａｙ ａ ｒｏｌｅ ｉｎ ｔｈｅ ｐｒｏｔｅｃｔｉｏｎ ａｇａｉｎｓｔ ＴｕＭＶ ｉｎｆｅｃｔｉｏｎ. Ｔｈｉｓ ｆｉｎｄｉｎｇ ｌａｉｄ ｔｈｅ ｆｏｕｎｄａｔｉｏｎ ｆｏｒ ｔｈｅ ｆｕｒｔｈｅｒ ｓｔｕｄｙ ｏｎ ｔｈｅ ｆｕｎｃｔｉｏｎ ｏｆ ＢｒＣＮＧＣ ｇｅｎｅｓ. [ABSTRACT FROM AUTHOR]

Chagas disease, caused by the kinetoplastid protozoan Trypanosoma cruzi, affects millions of people, most of them neglected populations. The different phases of the disease, the transmission mode and the high genetic variability of the parasite determine that molecular detection methods display different degree of success. Molecular diagnostic tests may be employed during epidemiological surveys of transmission, for early diagnosis of congenital transmission and acute infections due to oral transmission, transfusion or transplantation routes, reactivation due to immunosuppression and monitoring of treatment response in chronically infected patients receiving trypanocidal chemotherapy. This manuscript summarizes the most widely used molecular tools to detect T. cruzi infection in different epidemiological and clinical scenarios. [ABSTRACT FROM AUTHOR]

Background: Legionella pneumophila (L. pneumophila) is gram-negative bacterium, which causes Legionnaires' disease as well as Pontiac fever. Objective: To determine the frequency of Legionella pneumophila in pneumonic patients, to determine the clinical utility of diagnosing Legionella pneumonia by urinary antigen testing (LPUAT) in terms of sensitivity and specificity, to compares the Results: obtained from patients by urinary antigen test with q Real Time PCR (RT PCR) using serum samples and to determine the frequency of serogroup 1 and other serogroups of L. pneumophila. Methods: A total of 100 pneumonic patients (community acquired pneumonia) were enrolled in this study during a period between October 2016 to April 2017; 92 samples were collected from patients attended and admitted to Al-Imamein Al-Kadhimein Medical City and 8 samples from those in the (Center of Kidney Diseases and Transplantation) in the Medical City of Baghdad. All patients were under therapy with antibiotics. Serum and urine specimens were obtained from all patients; urine samples were processed for urinary antigen test (rapid test). Serum samples were collected and submitted to DNA extraction for detection of L. pneumophila mip gene by q RT PCR assay. Results: The percentage of L. pneumophila in two hospitals in Bagdad was 30%. Of these 26% was serogroup 1 detected by urinary antigen testing (UAT). In the other hand, 23% of samples were positive by q RT PCR based mip gene, of these 19 % were serogroup 1 and 4% were another serogroup. The sensitivity of UAT is high (P value < 0.001), which means statistically highly significance than q RT PCR. Conclusion: LPUAT is a rapid tool for early diagnosis of Legionella infection, which highlights the need of using this test in hospitals and health institutions and there is a high prevalence of L. pneumophila in Iraq that refer to the necessity of considering this microorganism point of view in future studies for detection and treatment in pneumonic patients. [ABSTRACT FROM AUTHOR]