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• Foliar proline combats lead-induced crop stress. • Enhanced wheat crop health with external proline. • Lead stress impacts biomass, chlorophyll, and yield. • Foliar proline reduces reactive oxygen species. • TaGSK1 gene expression upregulation under proline stress. The application of proline as foliar spraying has emerged as a promising solution to combat lead-induced crop stress. This study highlights the effectiveness of external proline in enhancing wheat crop health under lead stress. We reveal how lead stress negatively impacts key factors like biomass, chlorophyll levels, and crop yield, particularly in 100-grain weight. Gas exchange parameters, including stomatal conductance, CO 2 levels, photosynthesis, water-use efficiency, and transpiration, also suffer under lead stress. Significantly, exposure to lead stress triggers the activation of antioxidant enzymes and elevates levels of proline, calcium, and protein. Nonetheless, the application of foliar proline proves highly effective in alleviating these detrimental effects by reducing reactive oxygen species and enhancing proline and protein levels in wheat. Additionally, our findings demonstrate a significant upregulation of TaGSK1 gene expression at different proline concentrations. Compared to controls, TaGSK1 expression increases by 1.7-fold under 50 mM proline stress and an impressive 4.7-fold under 200 mM proline stress in wheat. This finding holds promise in alleviating the adverse effects of lead stress and strengthening crop resilience for a more secure and sustainable future in wheat cultivation amid various stressors. [ABSTRACT FROM AUTHOR]

Drought and high temperature stress affect chickpea growth and productivity. Often these stresses occur simultaneously in the field and lead to a wide range of molecular and metabolic adaptations. Two chickpea varieties; GPF2 (heat sensitive) and PDG4 variety (heat tolerant) were exposed to 35 °C for 24 h individually and along with drought stress. Five heat responsive signalling genes and 11 structural genes were analyzed using qPCR along with untargeted metabolites analysis using GC MS. Expression of antioxidant genes (CaSOD and CaGPX, CaAPX and CaCAT), transcription factors (CaHSFB2, CaHSFB2A, CaHSFB2B, CaHSP17.5 and CaHSP22.7) and signalling genes (CaCAM, CaGAD, and CaMAPK) were upregulated in GPF2 as compared to PDG4 variety. Principal component analysis (PCA), partial least-square discriminant analysis (PLS-DA), and heat map analysis were applied to the metabolomics data to identify the differential response of metabolites in two chickpea varieties. GC-MS analysis identified 107 and 83 metabolites in PDG4 and GPF2 varieties respectively. PDG4 variety accumulated more sugars, amino acids, sugar alcohols, TCA cycle intermediates which provided heat resistance. Additionally, the differential metabolic pathways involved in heat tolerance were alanine, aspartate, and glutamate metabolism, pantothenate CoA biosynthesis, fructose and mannose metabolism and pentose phosphate pathway in PDG4 variety. There was less accumulation of metabolites in the primed plants of both varieties as compared to the non-primed plants indicating less damage due to heat stress. The present study gives an overview of the molecular changes occurring in response to heat stress in sensitive and tolerant chickpea. • Chickpea is exposed to terminal heat and drought stress. • Five heat signaling genes and 11 structural genes were analyzed using qPCR along with GC-MS analysis. • High temperature and combinatorial stress enhanced the expression of almost all genes in GPF2. • In PDG4 only selective genes and metabolites were upregulated. • Priming modulates the transcriptional, metabolic activity and maintains cellular homeostasis. [ABSTRACT FROM AUTHOR]

RT-PCR is the gold standard for diagnosis of COVID-19. All RT-PCR kits are based on RNA extraction from the clinical sample. There was a sudden increase in demand of these kits, both RNA extraction and COVID-19 RT-PCR kits during the pandemic. This sudden spurt in global demand created a situation of shortage of consumables, especially the RNA extraction kits. Hence, this study was carried out to evaluate and compare COVID-19 RT-PCR without RNA extraction step using buffer R3. Sensitivity, specificity and accuracy of RT-PCR kit without RNA extraction were 89.16 %, 100% and 89.6% respectively. This approach saved more than 50 % time compared to the RT-PCR kit with RNA extraction approach allowing enhanced daily sample processing capability. RT-PCR kit without RNA extraction help in managing a greater number of samples, reduces cost and turnaround time. [ABSTRACT FROM AUTHOR]

The most commonly used molecular diagnostic technique is the polymerase chain reaction (PCR). PCR detects a short section of genetic code of interest, a cancer gene, human mRNA or a pathogen's genome. It is used by every specialty in medicine and surgery, with increasing frequency and importance. In this article, the history, steps of the cycle, uses, forms, advantages and disadvantages of PCR are discussed. With the SARS coronavirus-2 pandemic having such an enormous impact on the delivery of elective surgery, decisions to proceed or defer are made by surgeons on a daily basis, based on PCR results. An understanding of these results is provided, what they tell us, what they do not and what other information is required to make these decisions. It is imperative to also look beyond PCR results, seeing the patient within the context of their symptoms, other pathology and imaging results, with the assistance of a medical virologist or microbiologist, in complex cases. [ABSTRACT FROM AUTHOR]

We quantified viable hepatocyte-like cells (HLCs) administered via portal or tail veins in the livers and lungs of immunodeficient rats using real-time reverse transcription polymerase chain reaction (RT-PCR) and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers. Immunodeficient rats were infused with HLCs via portal or tail veins. mRNA was quantified based on the route of cell administration and the presence of liver injury. Human - specific GAPDH mRNA primers detected 0.1 pg human RNA in 100 ng (1:106) of rat liver RNA. When infused into the portal vein, the quantity of HLC mRNA reduced to 5% 3 h after infusion. Most HLCs were entrapped in the lungs when infused via the tail vein and decreased to approximately 10% 6 h after infusion. A small number of HLCs made it to the liver but disappeared rapidly, regardless of liver injury. 24 h after infusion, viable HLCs were detected only in the lungs of rats with liver injury (P < 0.05). The quantity of viable human cells in immunodeficient rats was estimated using real-time RT-PCR and primers specific to human mRNA. [ABSTRACT FROM AUTHOR]

[Display omitted] The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread throughout the globe, causing a pandemic. In Egypt over 115,000 individuals were infected so far. In the present study, the objective is to perform a complete genome sequence of SAR-CoV2 isolated from Egyptian coronavirus disease (COVID-19) patients. Nasopharyngeal swabs were collected from 61 COVID-19 patients who attended at National Cancer Institute, Kasr Al-Aini Hospital and the army hospital. Viral RNA was extracted and whole genomic sequencing was conducted using Next Generation Sequencing. In all cases, the sequenced virus has at least 99% identity to the reference Wuhan 1. The sequence analysis showed 204 distinct genome variations including 114 missense mutations, 72 synonymous mutations, 1 disruptive in-frame deletion, 7 downstream gene mutations, 6 upstream gene mutations, 3 frame-shift deletions, and 1 in-frame deletion. The most dominant clades were G/GH/GR/O and the dominant type is B. The whole genomic sequence of SARS-CoV2 showed 204 variations in the genomes of the Egyptian isolates, where the Asp614Gly (D614G) substitution is the most common among the samples (60/61). So far, there were no strikingly variations specific to the Egyptian population, at least for this set of samples. [ABSTRACT FROM AUTHOR]

Out of all non-communicable diseases, cancer is the leading cause of death in the 21st century. Oral cancer ranks in top ten cancers in the world and oncogenic viruses contribute to its development and are a major preventable risk factor for it. In the present study we intend to find the incidence of HPV and EBV by PCR in 108 sporadic oral cancer patients and study the clinico-pathological variables in agreement to their presence or absence. We found that the incidence of EBV in oral cancer is much higher (30/108; 27.8%) than HPV 16 (14/108; 13%) and, a complete absence of HPV 18 (0/108) was reported by real time PCR. Co-infection by EBV and HPV was reported in 5.6% cases (6/108). However, on comparing this data with the corresponding clinico-pathological cofactors (age, gender, grade of tumour, tumour size, nodal status, metastasis, DOI, TIL, ENE) it was found that there was no statistical significance between the two. Hence, we conclude that these oncogenic viruses are independent cofactors to oral cancer development and none of the clinico-morphologic variable is associated with the viral etiology. [ABSTRACT FROM AUTHOR]

This study aimed to develop a rapid multiplex high resolution melt-curve (HRM) qPCR with an internal amplification control (IAC) for the simultaneous detection of Shiga-toxin producing Escherichia coli (STEC) and bla CTX-M -carrying E. coli in beef. Specific primer pairs targeting eae A, uid A, stx 1 , stx 2 and bla CTX-M genes were designed. The specificity of the optimized assay was validated using genomic DNA from Gram-positive, Gram-negative, and STEC (non-O157 and O157:H7) strains. The accuracy of the assay for detecting bla CTX-M was confirmed using genomic DNA from bacteria previously reported to carry this gene. Beef products were inoculated with a cocktail of E. coli O157:H7 and E. coli M-1, an isolate reported to harbor bla CTX-M , at concentrations of 10, 100, and 1000 CFU/mL. Among non-STEC DNA samples (n = 45) tested, no target genes were detected in 32 isolates. However, the assay was able to detect uid A and bla CTX-M in seven and six bacterial isolates, respectively. Among the STEC bacterial isolates (n = 57), at least two or more target genes were detected in 50 STEC isolates. All isolates carried the eae A gene, 55 had stx 1 , 26 had stx 2 , and 26 had both stx genes. The uid A gene was detected in all non-O157:H7 isolates, whereas no E. coli O157:H7 isolates was found to be positive for uid A. The bla CTX-M gene was not detected in any of the STEC isolates tested but it was detected in 20 of 23 DNA samples from isolates previously verified as positive for the gene, thus demonstrating the technique's accuracy. Enrichment times ranging from 4 to 8 h were required to achieve a detection limit of 10 CFU/mL in 325 g of inoculated beef. The multiplex HRM real-time PCR assay can be used as a rapid, yet specific, tool for routine examination of STEC and bla CTX-M -harboring bacteria that are present in low concentrations in beef products. • A HRM qPCR was developed to detect Shiga-toxin producing E. coli and bla CTX-M -harboring E. coli. • The assay targeted st x1, stx 2, eae A, uid A and bla CTX-M genes. • A detection limit of 10 CFU/mL in 325 g of beef was obtained within 8 h of enrichment. • The assay can be applied to detect STEC and ESBL-producing pathogens in beef. [ABSTRACT FROM AUTHOR]

Field sowthistle, a plant in the Asteraceae family, contains high levels of chlorogenic and chicoric acids, which have anticancer properties. In the current investigation, the expression of genes responsible for chlorogenic acid biosynthesis was studied in S. arvensis in drought stress conditions. The following genes were investigated: phenylalanine ammonia-lyase (PAL), cinnamate 4 hydroxylase (C 4 H), 4-hydroxycinnamoyl CoA ligase (4CL), hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) and p-coumaroyl ester 3'-hydroxylase (C 3' H). As part of a research study, the levels of several anticancer compounds and their correlation with the transcriptional activity of the surveyed genes were investigated under drought stress conditions. The study also involved the identification and submission of the partial coding sequences of the C 4 H , HCT , C 3' H , and actin genes in S. arvensis to the GenBank database. The plants' middle leaves exposed to 50% field capacity (FC) exhibited the highest expression of all genes. The highest amount of chicoric, chlorogenic, and 2,5 dihydroxybenzoic (2,5 DHB) acids was observed in the middle leaves under 50% FC. In contrast, the highest content of gallic acid and catechin was found in the lower leaves at 75% FC. A strong association between HQT expression and chlorogenic acid content proposed the HQT -dependent metabolic pathway as the primary route for its production in S. arvensis. Furthermore, the consistency between the patterns of gene expression and the levels of compounds found in the middle leaves at 50% FC, except for catechin, showed that the production of chlorogenic acid in S. arvensis could potentially be enhanced by stimulating the expression of the genes that are responsible for its biosynthesis. [Display omitted] • C 4 H , HCT and C 3' H gene sequences in Sonchus arvensis was released in GenBank. • Sequence of actin, a reference gene, in S. arvensis was released in GenBank. • The highest content of chlorogenic acid was observed in the middle leaves at 50% FC. • HQT -dependent pathway is the major for chlorogenic acid biosynthesis in S. arvensis. [ABSTRACT FROM AUTHOR]

Within the context of an investigation focused at improving the effectiveness of test-and-removal for the control of ovine paratuberculosis, we recently conducted a preliminary study using ELISA and real time PCR to assess whether parturition affects test-positivity, in animals with subclinical paratuberculosis. Samples of faeces and blood were collected from 42 adult female animals, before (PP1) and after parturition (PP2), and before mating (PP3). In the preliminary stage of the analysis, only one of the animals tested reacted positively to ELISA (2.38%, 1 of 42), which corresponds to 2.8% of PCR-reactors (1 of 36). Therefore, the final stage the investigation was conducted using only real time PCR, which was applied to test samples of faeces collected from 85 animals, in 5 periods of sampling: 4–15/1–3 days (FP1/FP2) before and after parturition (FP3/FP4), and before mating (FP5). The result of the preliminary analysis indicated that PCR-positivity in terms of the number of shedders and the amount of MAP, is statistically significantly lower before parturition (PP1), whereas that of the final, higher in FP4 compared to FP5. Significantly higher levels of positivity in PP2 and FP4 were also recorded in connection with animals reacting positively to PCR more than once. In conclusion, in sheep with subclinical paratuberculosis, the period of 4–15 days postpartum is more suitable for the application of test-and-removal aiming to the control of the disease, using real time PCR. The use of ELISA for the same purpose is not recommended in the specific category of animals, due to low sensitivity. [ABSTRACT FROM AUTHOR]

We herein report the case of a patient with advanced lung adenocarcinoma who presented a heterogeneous distribution of EGFR mutation. A 74-year-old Moroccan male former smoker was diagnosed with advanced lung adenocarcinoma, harboring S768I exon 20 substitution mutation confirmed by Real Time PCR and Pyrosequencing, but not detected by direct sequencing despite 70% of tumor cells. The present report describes a case of minor histologic intratumoral heterogeneity with heterogeneous distribution of EGFR mutation. Both sensitivity and specificity of molecular methods can provide evidence of intratumoral heterogeneity, which may explain the mismatch between the validation of oncology biomarkers and predicting therapeutic response to targeted therapy. [ABSTRACT FROM AUTHOR]

Paraoxonase-1 (PON1) has been shown to protect low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) against oxidative-modification and thereby might protect against coronary-artery-disease (CAD). Here we explored the relationship of a genetic variant (a substitution (R) Arg with (Q) Gln at position 192) of PON1 in 250 patients with/without CAD. Genotyping of PON1 Q192R was carried out using Real-Time-PCR TaqMan-based-probe. Demographic-characteristics and biochemical-analyses, including fasting blood sugar (FBS), HDL, LDL, triglycerides (TG) and C-reactive protein (CRP) were evaluated. Univariate/multivariate analyses were performed to determine the association of the genetic polymorphism and CAD as well as with clinical-characteristics of population. Our findings showed that RR-genotype was more frequent in CAD-patients, compared to the wild-type genotype. Moreover, CAD patients with RR-genotype had an odd ratio of 5.0 (95% CI: 1.3–18.6; p = 0.017), versus wild-type genotype, in multivariate-analysis. Of note we also observed that CAD-patients with QQ-genotype had a significantly lower Hs-CRP level, compared to the RR-genotype. we demonstrate that PON1-Q192R-polymorphism was associated with CRP and FBS levels; R-allele of PON1-Q192R may be an independent risk factor for CAD. Further studies are warranted to determine the value of this marker as a surrogate marker in CAD patients. [ABSTRACT FROM AUTHOR]

Abstract Internet gaming addiction (IGA) has been associated with many negative health outcomes, especially for youth. In particular, the potential association between IGA and leukocyte telomere length (LTL) has yet to be examined. In this study we compared LTL in Korean male adolescents with and without IGA and examined the association between LTL and autonomic functions. Specifically, plasma catecholamine, serum cortisol, and psychological stress levels were measured as autonomic functions. Data were collected using participant blood samples analyzed for LTL, catecholamine, and cortisol levels and a set of questionnaires to assess IGA and psychological stress levels of the participants. The LTL measurements were made using a qPCR-based technique, and the relative LTL was calculated as the telomere/single copy (T/S) ratio. T/S ratio was significantly shorter in the IGA group than in the non-IGA group (150.43 ± 6.20 and 187.23 ± 6.42, respectively; p <.001) after adjusting for age. In a univariate regression analysis, age, daily Internet gaming time, IGA score, and catecholamine level (epinephrine and norepinephrine) were significantly associated with T/S ratio. However, duration of Internet gaming exposure, dopamine, cortisol, and psychological stress levels were not found to be associated with T/S ratio. In the final multiple linear regression model, age, daily Internet gaming time, and epinephrine level showed statistically significant relationships with T/S ratio. Our results indicate that in addition to age, involvement in excessive Internet gaming may induce LTL shortening in male adolescents, which may be partially attributable to changes in autonomic function such as catecholamine level. These findings further understanding of the health effects of IGA and highlight the need for screening and intervention strategies for male adolescents with IGA. Highlights • Internet gaming addiction (IGA) can induce various changes in physiological states. • Plasma epinephrine level differs significantly between the IGA and non-IGA groups. • Male adolescents with IGA have shorter telomere length than those without. • Internet gaming time, epinephrine, and age are associated with telomere shortening. [ABSTRACT FROM AUTHOR]

Abstract In the United States, Shiga toxin-producing Escherichia coli (STEC) O157 and six non-O157 serogroups O26, O45, O103, O111, O121 and O145 are considered adulterants in non-intact beef. Further, Salmonella is responsible for one of the highest numbers of foodborne infections worldwide. Multiple foods, especially meats, are routinely tested for these pathogens using methods like PCR. However, with such a large group of organisms, multiplexing using probe-based PCR assays is expensive due to the need for differently labeled oligonucleotide probes and sophisticated instrumentation. The aim of this study was to design low-cost multiplex real-time PCR assays for the detection of seven STEC serogroups, stx 1 , stx 2 genes and virulent Salmonella. Two multiplex real-time PCR melt curve assays with internal amplification controls (IAC) were standardized. The first assay detected E. coli O121, E. coli O145, E. coli O157, stx 1 , and stx 2. The second assay targeted E. coli O26, E. coli O111, E. coli O103, E. coli O45, and Salmonella. Ground beef and beef trim inoculated with 5–27 CFU/325 g of STEC and 9–36 CFU/325 g of Salmonella could be detected following an 8–10 h enrichment at 40 °C ± 2 °C in buffered peptone water containing 8 mg/L vancomycin. The assays showed reproducible results for beef products with different fat contents. These assays do not rely on fluorescent-labeled probes or immunomagnetic beads, yet accurately detect seven STEC serogroups, seven stx gene subtypes and Salmonella , making them suitable for routine testing of STEC and Salmonella in beef. Highlights • Multiplex melting curve PCR to detect STEC, stx subtypes and Salmonella were developed. • The assays targeted E. coli O121, O145, O157, O26, O111, O103, O45, Salmonella , stx 1 , stx 2. • Beef samples inoculated with 5–27 CFU/325 g of beef could be detected after 8–12 h enrichment. • The assays complied with USDA, FSIS recommendations for these pathogens. [ABSTRACT FROM AUTHOR]

Abstract Background Invasive fungal diseases (IFDs) are difficult to diagnose and associated with high mortality rates, especially in the immunosuppressed. Species of Aspergillus and Candida are the cause of majority of invasive fungal disease however IFDs are also caused by Fusarium , Zygomycetes, Trichosporon , etc. Early detection is crucial for appropriate antifungal therapy. Blood cultures usually fail to isolate filamentous fungi, while detection of circulating beta- d -glucan or galactomannan antigens show variable sensitivity and specificity. There is a need of reliable, sensitive and specific diagnostic tests for IFDs. Methods A real-time Polymerase Chain Reaction (PCR) assay with a universal primer/molecular beacon system was developed for detecting and speciating most of the pathogenic fungi implicated in IFD. A single-reaction assay was designed targeting a carefully selected region of the ITS2 and ITS5 subunits of the fungal rDNA gene along with four molecular beacons capable of differential hybridization to the amplicons of different species. This generated a signature set of melting temperatures using the standard strains. The assay was tested on clinical specimens from patients with suspected invasive fungal disease. Results The assay was tested on 72 clinical samples and 72 healthy controls. Of these, 22 clinical samples (6/8 proven; 13/29 probable; 3/35 possible IFD, classified by the EORTC/MSG criteria) were positive by PCR and generated a set of melting temperatures enabling identification of the causative fungus. The assay was negative in all healthy controls. Conclusion The molecular beacon assay is a promising tool providing a rapid method for detection and monitoring of invasive fungal disease in immunosuppressed patients. [ABSTRACT FROM AUTHOR]

Highlights • PC3 cell line demonstrates two types of grow, adherent and in suspension. • Cells from each type of growing pattern present different ultrastructure. • Zona pellucida proteins expression was found on both prostate cancer and PC3 cells. • Pavement-cells (adherent) have a higher expression of ZP1, ZP3 and ZP4 mRNA. • A greater expression of ZP3 was found on both types of PC3 cells. Abstract The human oocyte zona pellucida (ZP) is made of four glycoproteins, ZP1-ZP4. Recently, the prostate adenocarcinoma and prostate cancer PC3 cell-line were shown to express the human oocyte ZP3 glycoprotein, which was evaluated in a single report subject to patent. To further clarify whether oocyte zona pellucida glycoproteins are expressed in prostate cancer tissue and PC3-cells, in this report we evaluated protein expression of the four ZP glycoproteins in normal prostate tissue, prostate adenocarcinoma tissue and PC3-cells, and performed quantitative mRNA expression of the four ZP glycoproteins in the PC3 cell-line. Furthermore, as PC3-cells have not yet been studied in detail regarding their ultrastructural characteristics, in the present report we bring forward the detailed ultrastructure of PC3-cells. PC3-cells were divided into pavement and aggregated cells. We observed new ultrastructural features in pavement and aggregated cells, with the later exhibiting two different cell types. In prostate carcinoma tissue and PC3-cells we found protein expression of the four oocyte glycoproteins, ZP1, ZP2, ZP3 and ZP4. In addition, mRNA expression studies revealed expression of ZP1, ZP3 and ZP4 glycoproteins, but not of ZP2. Interestingly, the ZP1 mRNA product exhibited intron retention. [ABSTRACT FROM AUTHOR]

Abstract Background LATS2 , a presumed tumor suppressor gene located on chromosome 13q11–12 is involved in cell growth related activity like regulation of cell cycle at G1/S. The reduced expression of LATS2 has been reported in many tumors; including tumors of Breast, which is to the best of our knowledge has not been studied in north Indian female breast cancer population. Objective Here, we looked upon the expression pattern and methylation status of the LATS2 gene in north Indian female breast cancer cases to further strengthen its role as a tumor suppressor gene and more importantly as a cancer biomarker. Methods mRNA expression level was determined by real time PCR in 140 Breast cancer patients, Protein expression was studied by Immunohistochemistry and Promoter methylation was studied by Methylation specific PCR. All findings were correlated with clinicopathological features. Results LATS2 mRNA expression was remarkably downregulated in 67.85% (95/140) cases. The expression of Large Associated Tumor Suppressor 2 at protein level was also absent in 67.85% (95/140) cases. The absence of LATS2 protein strongly correlated with promoter hypermethylation where 91 out of a total of 107 hyper methylated cases showed absence of protein (91/107, 85%). The absence of LATS2 protein was strongly significant with HER2 neu status (0.01), TNM staging (0.009) and Molecular subtype (0.024). Conclusion The decreased expression in breast cancer seems to be associated with hypermethylation of LATS2 promoter regions. Further LATS2 as a tumor suppressor can be recognized as a promising Biomarker in Breast cancer pathogenesis. Though, further studies, targeting larger sets of breast cancer population are required to establish LATS2 as a promising biomarker. Highlights • Expression and Methylation status of LATS2 gene was studied in 140 Breast cancer cases. • The promoter was hypermethylated and expression was downregulated at mRNA and Protein level. • Multiparametric analysis showed high association between methylation, expression and clinical parameters of patients. • LATS2 may serve as potential biomarker, at least in Breast cancer. [ABSTRACT FROM AUTHOR]

Methamphetamine (METH) abuse is one the most worldwide problems with wide-ranging effects on the central nervous system (CNS). Chronic METH abuse can associate with cognitive abnormalities and neurodegenerative changes in the brain. Agmatine, a cationic polyamine, has been proposed as a neuromodulator that modulates many effects of abused drugs. The aim of this study was to determine if agmatine can decrease the impairment effect of METH on memory and hippocampal CaMKII-α gene expression, a gene that plays a major role in memory. Male wistar rats (200–220 g) were allocated into 7 groups, including 5 groups of saline, METH (1, 2 mg/kg), Agmatine (5, 10 mg/kg) and 2 groups of agmatine (5, 10 mg/kg) with higher doses of METH (2 mg/kg) for 5 consecutive days ( n  = 8 in each group). All injections were done intraperitoneally and agmatine was administrated 10 min before METH treatment. Furthermore, Passive avoidance learning (PAL) test was assessed on the 5th day. Retention test was done 24 h after training and the rats were sacrificed immediately. Hippocampi were removed and stored at −80 °C. Finally, hippocampal CaMKII-α gene expression was measured using Quantitative Real-time PCR. Our data showed that chronic METH dose-dependently impaired PAL retrieval, as it decreased step-through latency (STL) and increased time spent in the dark compartment (TDC). While Agmatine with a higher dose (10 mg/kg) significantly decreased impairment effect of METH (2 mg/kg) on PAL and memory. Also, molecular results revealed that METH (2 mg/kg) markedly decreased hippocampal CaMKII-α gene expression while agmatine (10 mg/kg) co-adminstration prevented it. Taken together, the results propose that agmatine may provide a potential therapy for learning and memory deficits induced by METH. [ABSTRACT FROM AUTHOR]

Pimpinella haussknechtii is an annual native plant grows in west of Iran. Phytochemical study of the fruits of P. haussknechtii led to the isolation of a novel irregular sesquiterpene lactone with a new skeletone and oxidation pattern named: pimpinelol. The structure of the isolated compound was elucidated by extensive spectroscopic methods. Cytotoxic activity of Pimpinelol was evaluated using standard MTT assay against breast cancer cells. Induced endoplasmic reticulum stress and related gene expressions were evaluated with fluorescence microscopy analysis and real time PCR, respectively. These findings confirmed that Pimpinelol concentrations dependently increased protein aggregation and the mRNA expression of ATF-4, CHOP, GADD34 and TRIB3 in MCF-7 breast cancer cell line. According to the obtain results, we demonstrated for the first time that Pimpinelol decreased breast cancer cell viability by inducing ER stress. [ABSTRACT FROM AUTHOR]

Rice tungro is the most important viral disease affecting rice in South and Southeast Asia, caused by two viruses rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV). Transgenic resistance using RNA-interference (RNAi) has been reported individually against RTBV and RTSV earlier. Here we report the development of transgenic rice plants expressing RNAi against both RTBV and RTSV simultaneously. A DNA construct carrying 300 bp of RTBV DNA and 300 bp of RTSV cDNA were cloned as the two arms in hairpin orientation in a binary plasmid background to generate RNAi against both viruses simultaneously. Transgenic rice plants were raised using the above construct and their resistance against RTBV and RTSV was quantified at the T 1 plants. Levels of both the viral nucleic acids showed a fall of 100- to 500-fold in the above plants, compared with the non-transgenic controls, coupled with the amelioration of stunting. The transgenic plants also retained higher chlorophyll levels than the control non-transgenic plants after infection with RTBV and RTSV. Small RNA analysis of virus inoculated transgenic plants indicated the presence of 21 nt and 22 nt siRNAs specific to RTBV and RTSV. The evidence points towards an active RNAi mechanism leading to resistance against the tungro viruses in the plants analysed. [ABSTRACT FROM AUTHOR]

Begomoviruses (family Geminiviridae ) are frequently associated with alphasatellites and betasatellites in the Old World. Tomato yellow leaf curl virus , one of the most damaging begomovirus species worldwide, was recently found associated with betasatellites in the eastern coast of the Mediterranean Sea, and in the Middle East region. Tomato yellow leaf curl virus (TYLCV)/betasatellite associations were shown to increase TYLCV virulence in experimental conditions. The sustainability of TYLCV/satellite associations in tomato was assessed here by estimating accumulation levels of satellites in comparison to TYLCV, vector transmission efficiency, and by testing how far the popular Ty-1 resistance gene used in most TYLCV-resistant tomato cultivars in the Mediterranean Basin is effective against betasatellites. Three satellites previously isolated from okra in Burkina Faso—of the species Cotton leaf curl Gezira betasatellite , Cotton leaf curl Gezira alphasatellite and Okra leaf curl Burkina Faso alphasatellite —were shown to accumulate at levels similar to, or higher than, the helper virus TYLCV-Mld in tomato plants from 32 to 150 days post inoculation (dpi). Cotton leaf curl Gezira betasatellite (CLCuGB) reduced TYLCV-Mld accumulation whereas alphasatellites did not. Transmission tests were performed with B. tabaci from plants infected with TYLCV-Mld/CLCuGB- or TYLCV-Mld/Okra leaf curl Burkina Faso alphasatellite. At 32 dpi, both satellites were transmitted to more than 50% of TYLCV-infected test plants. Betasatellite transmission, tested further with 150 dpi source plants was successful in more than 30% of TYLCV-infected test plants. Ty-1 resistant tomato plants co-infected with TYLCV (–Mld or –IL) and CLCuGB exhibited mild leaf curling and mosaic symptoms at the early stage of infection associated with a positive effect on TYLCV-IL accumulation, while resistant plants infected with TYLCV only, were asymptomatic. Together with previous experimental studies, these results further emphasize the potential risk of betasatellites to tomato cultivation, including with Ty-1 resistant cultivars. [ABSTRACT FROM AUTHOR]

A 60-day feeding trial was conducted to optimize the dietary protein (30, 28 & 26%) and lipid (6,7 & 8%) levels for optimum inclusion of 33% de-oiled rice bran based (DORB) diets on the growth performance, feed utilization, expression of insulin like growth factor (IGF-I and IGF-II) and RNA/DNA ratio in the Labeo rohita . The formulated diets were different combination of protein and lipid levels while fixing the level of DORB at 33% viz., P30L6, P30L7, P30L8, P28L6, P28L7, P28L8, P26L6, P26L7 and P26L8 (P-Protein, L-Lipid), respectively.Two hundred seventy fingerlings (average wt. 5.56 g ± 0.2) were equally distributed in nine treatments in triplicates with 10 fish per tank. The weight gain % (WG) and specific growth rate (SGR) increased with the decreasing protein level and increasing lipid level in diets. Highest weight gain % and specific growth rate was along with lowest FCR were recorded in the group fed with 26% protein and 7% lipid. The protein efficiency ratio (PER) was significantly ( p  < 0.05) higher in P26L7 and P26L8 groups. The expression of IGF-I mRNA was found to be higher in P26L7 group as compared to other groups. Though the expression of IGF-II was significantly ( p  < 0.05) higher in P26L7 group, it was similar to the P26L8 group. The RNA concentration and RNA/DNA ratio was found significantly ( p  < 0.05) higher in the P26L7 group. Dietary protein level did not affect significantly ( p  > 0.05) on whole body protein and lipid content. Significantly ( p  < 0.05) higher body lipid level and lower protein level was found at 26% protein and 8% dietary lipid fed group. Hence, diet containing 33% DORB with 26% crude protein and 7% lipid level is optimal for supporting maximum growth and feed utilization of Labeo rohita. [ABSTRACT FROM AUTHOR]

Cashew ( Anacardium occidentale ) nut can trigger serious reactions in allergic patients, including anaphylaxis and death. Labelling the presence of cashew nuts in food products is mandatory and consequently, sensitive and specific analytical methods must be developed. In this study, Ana o allergen coding sequences have been sequenced in several cashew varieties. Two hydrolysis probes, locked nucleic acid (LNA) and TaqMan, have been designed and their efficiency, sensitivity, limit of detection and specificity for Ana o 1 coding-sequence detection have been compared. Reliable Real Time PCR assays to detect and quantify up to 10 ppm of cashew nuts in complex mixtures have been developed. Moreover, the influence of boiling and autoclave treatment on cashew nut detectability has been analysed by qPCR, showing both probes similar performance. This analytical method was able to detect up to 1000 ppm with good functionality in autoclave treated samples. Boiling did not affect cashew nut detectability. Both hydrolysis probes are suitable for Ana o 1 coding sequence detection. Applicability of the assay has been studied by analysing several food products, and comparing the results with those of a commercial ELISA kit. [ABSTRACT FROM AUTHOR]

The cytosine embedded copper based metal–organic framework (Bio-MOF) was synthesized by facile one-step sonochemical method by simply mixing of 4–4, biphenyldicarboxylic, cytosine and copper nitrate (Bio-Cu-H 2 bpdc-Cy). The prepared bio-MOF was characterized by XRD, FTIR and FE-SEM techniques. The effect of Cu-H 2 bpdc-Cy on the expression of the rsbA gene was evaluated in the clinical and standard Proteus mirabilis and study of MIC of Cu-H 2 bpdc-Cy by microdilution against them that have the rsbA gene. According to different concentrations of MIC, MBC concentrations was cultured on blood agar culture medium. Regarding to the concentration of MIC, gene expression changes were obtained by real-time PCR. MIC for standard and clinical strains of Proteus mirabilis was 1.6 and 1.8 mg/ml, and also MBC was obtained to be 1.8 and 2.0 mg/ml, respectively. Finally, in the real time PCR method, expression of the rsbA gene in presences of bio-Cu-H 2 bpdc-Cy was reduced, but has no effect on the gene expression of the Housekeeping DNA Gyrase-B gene. Considering the effect of Cu-H 2 bpdc-Cy on the rsbA gene in Proteus mirabilis bacteria, it is possible to use of Cu-H 2 bpdc-Cy agent as a therapeutic supplement against this bacterium. [ABSTRACT FROM AUTHOR]

Résumé En 2017, au laboratoire de l’hôpital de Beauvais, un appareil de PCR en temps réel est installé. Ce travail décrit la vérification de méthode, en portée A de la trousse de PCR triplexe en temps réel, Urethritis basic ® , FTD, pour la détection qualitative de Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) et Mycoplasma genitalium (MG). Pour répondre à la norme NF 15189, une analyse des risques ainsi que la vérification de certains critères de performance sont réalisées sur site. La fidélité intermédiaire pour NG est évaluée sur 2 échantillons donnant des résultats douteux au premier passage. Lors des passages suivants les résultats sont négatifs. La comparaison avec l’ancienne méthode en point final (GenoQuick ® CT, Hain Lifescience) pour la recherche de CT est réalisée sur 46 échantillons, 4 résultats sont discordants (tous positifs avec l’ancienne méthode et négatifs avec la nouvelle). L’exactitude est vérifiée par deux EEQ pour CT et NG : les résultats sont conformes. Les essais de contamination inter- échantillons montrent l’absence de contamination. Enfin, une vérification continue à l’aide de graphiques de Levey-Jennings construites à partir des « Cycles threshold » (Ct) des contrôles est mise en place. Ce travail a permis de conclure à l’aptitude de la méthode tout en confirmant l’intérêt de contrôler les échantillons trouvés douteux pour le gonocoque. Il propose également un outil de surveillance continue des performances. Method verification, in range A, of a triplex real time PCR for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium In 2017, at the Beauvais hospital laboratory, a real time PCR system has been acquired. This work describes the method verification of a real time PCR kit, Urethritis basic ® , FTD, in range A, for the qualitative detection of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Mycoplasma genitalium (MG). According to the ISO 15189 standard, a risk analysis and a verification of some performance criteria are performed on site. Intermediate precision for NG is evaluated on two samples giving equivocal results. Following runs gives negative results. Comparison between the new and the previous end-point PCR method (GenoQuick ® CT, Hain Lifescience) for the detection of CT is performed on 46 samples. Four samples show discordant results (all positive with the previous method and negative with the new one). Accuracy is assessed by two EQA (External Quality Assessment) for CT and NG and gave expected results. Our tests of carry-over contamination, by alternating positive and negative samples, did not identify any contamination. Finally, we implemented continuous verification by Levey-Jennings charts established using threshold cycles of controls. This work let to conclude on the ability of the method while confirming the interest of controlling equivocal NG results. In addition, it provides a tool to continuously monitor the performance. [ABSTRACT FROM AUTHOR]

The Vibrio splendidus species is ubiquitous in the marine environment but have also been recognized as pathogenic for several aquatic animals. This species belongs to the Splendidus clade which is composed of 16 genetically related species. To our knowledge, there are no available tools able to discriminate the V. splendidus species from the other members of the clade. Thus, we developed a Real Time PCR based on the toxR gene specific to V. splendidus species. Specificity tests were performed on 74 reference strains and 116 field strains and gave 96.05% inclusivity and 100% exclusivity results. The limit of detection of the PCR was determined according to the XP-U47-600-2 AFNOR norm and was estimated at 18 copies genome per reaction. This work describes a reliable, sensible and specific tool to discriminate V. splendidus species from other members of the Splendidus clade and thus could be clearly suitable to perform taxonomic investigations. [ABSTRACT FROM AUTHOR]

Aim To find out the association between Human Papilloma Virus (HPV) genotypes 16/18 in Pakistani patients with oral squamous cell carcinoma (OSCC). Methods DNA from oral rinse of 300 subjects was taken. The subjects included 100 cases with OSCC and 200 controls. Samples were analyzed by both conventional and real time PCR using “HPV consensus Gp5+/Gp6+ and HPV 16, 18 specific primers”. Results Out of 300 persons, 74/300 (25%) were found to be infected with HPV: “46/100(46%) from cases and 74/200(14%) from controls”. The distribution was: HPV16, 6/300 (8%): 4/100 (9%) from OSCC group and 2/200 (8%) from controls while HPV 18 was 9/300(12%): 5/100(11%) from cases and 4/200(16%) from controls. Out of 300 subjects, 26(35%) were infected by “both HPV 16/18 (23(50%) from cases and 3(12%) from controls”. Persons who were infected with HPV 16&18 had higher chances to develop OSCC as compared to those who didn’t have HPV 16/18 (AOR: 21.4, 95% CI: 5.73 – 80.8). Conclusion The exposure to high risk strains of Human papilloma virus (16/18) in combination can be fabricotor of trouble (p < 0.001, Adjusted odds ratio; 21.42) in OSCC. [ABSTRACT FROM AUTHOR]

Objective The aim of this study was to analyze the etiology and clinical consequences of viral respiratory infections in 18 elderly care centers (ECC) in Lisbon, which housed a total of 1022 residents. Methods Nasopharyngeal swabs were collected whenever an elderly had symptoms of acute respiratory infections (ARI). PCR and RT-PCR were performed for influenza A/B, human parainfluenza virus 1–4, adenovirus, human metapneumovirus (HMPV), respiratory syncytial virus (RSV), rhinovirus, enterovirus, human coronavirus and human Bocavirus (HBoV). Array cards for atypical bacteria were also used in severe cases. Results In total, 188 episodes of ARI were reported, being rhinovirus the most frequently detected (n = 53), followed by influenza A(H3) (n = 19) and HBoV (n = 14). Severe infections were reported in 19 patients, 11 of which were fatal, Legionela pneumophila , rhinovirus, HMPV and RSV associated with these fatalities. Nine influenza strains were analyzed, all antigenically dissimilar from vaccine strain 2013/14. “Age”, “HMPV” and “Respiratory disease” showed an association with severe infection. Conclusions In this study an etiologic agent could be found in 60% of the acute respiratory episodes. These data provides information about the circulating viruses in ECC and highlights the importance of searching both viruses and atypical bacteria in severe ARI. [ABSTRACT FROM AUTHOR]

Objective The detection of BRAF V600E mutation in papillary thyroid carcinoma (PTC) may be helpful to offer diagnostic confirmation. Additionally, such detection may provide a targeted therapeutic approach for the radioactive iodine resistant patients to predict adverse outcomes. To compare the results of immunohistochemistry (IHC) method using the anti-BRAF V600E (VE1) antibody with the Quantitative real-time polymerase chain reaction (qPCR) approach in examining BRAF V600E mutation in PTC, we investigated the sensitivity and specificity of BRAF V600E (clone VE1) mouse monoclonal antibody in detecting the BRAF V600E mutation and correlated BRAF V600E mutation with clinicopathologic features in PTC. Methods IHC and qPCR were performed in 40 cases of paraffin-embedded PTCs tissues. The association between BRAFV600E mutation and clinicopathologic features of PTC was assessed with the χ 2 test. Results The concordance rate between IHC and qPCR analyses was 95% (38/40). The BRAF V600E (VE1) antibody has a sensitivity of 100% (34/34) and specificity of 66.67% (4/6) for detecting the mutation. Our study showed that there was no significant association of BRAF V600E mutation with the gender, age, tumor size and lymph node metastasis in PTCs. Conclusion We may draw the conclusion that detection of BRAF V600E mutation by immunohistochemistry is highly sensitive and specific. Immunohistochemical detection of the mutated BRAF V600E protein in PTC may facilitate mutational analysis in the clinical setting. [ABSTRACT FROM AUTHOR]

The aim of this work was to study the transcriptional effects of glucocorticoids on corticosteroid hormone receptors, prereceptors (11β-hydroxysteroid dehydrogenase 1 and 2, 11β-HSD1 and 2), and chaperones molecules regulating intracellular trafficking of the receptors (FKBP51 and FKBP52) in thymus of veal calves. Moreover, the expression of FKBP51 and FKBP52 gene were investigated in beef cattle thymus. In the cervical thymus of veal calves, dexamethasone administration in combination with estradiol decreased FKBP51 expression ( P < 0.01). The same treatment increased mineralocorticoid receptor (MR) ( P < 0.01) and 11β-HSD1 expression ( P < 0.05) compared to control group in the cervical thymus of veal calves. The thoracic thymus of veal calves treated with dexamethasone and estradiol showed a decrease of FKBP51 ( P < 0.05), FKBP52 ( P < 0.05), glucocorticoid receptor ( P < 0.05), and MR expression ( P < 0.05) compared to control group in the thoracic thymus of veal calves. The gene expression of FKBP51 decreased both in cervical ( P < 0.01) and thoracic thymus ( P < 0.01) of beef cattle treated with dexamethasone and estradiol. In addition, also prednisolone administration reduced FKBP51 expression in the cervical thymus ( P < 0.01) and in the thoracic thymus of beef cattle ( P < 0.01). The gene expression of FKBP52 increased only in the cervical thymus following dexamethasone administration ( P < 0.01). The decrease of FKBP51 gene expression in thymus could be a possible biomarker of illicit dexamethasone administration in bovine husbandry. Moreover, so far, an effective biomarker of prednisolone administration is not identified. In this context, the decrease of FKBP51 gene expression in thymus of beef cattle following prednisolone administration could play an important role in the indirect identification of animals illegally treated with prednisolone. [ABSTRACT FROM AUTHOR]

Summary Conventional methods for diagnosis of invasive aspergillosis (IA) lack sensitivity and specificity. Serological methods still have many cases of cross-reactivity. However, molecular techniques seem to arise as a rapid approach, specific and direct that could be used in the diagnosis of IA. In this study, we analyzed 88 serum samples from patients of having IA using GM-ELISA test, nested PCR with primers for the rRNA 18S of Aspergillus genus and real time PCR specific for A. fumigatus . Among the 88 samples, 64 of them had positive GM titers and 23 had positive nested PCRs; 18 of the 23 PCR-positive samples were also GM-positive. On the other hand, 18 samples were detected positive by reel time PCR; 13 positive samples were also detecting positive by nested PCR. QPCR revealed 26 % of the patients with IA, while nested PCR and galactomannan ELISA revealed respectively 34 % and 94 % of the patients with IA. Probable IA was diagnosed in 18 and possible IA was diagnosed in 6 episodes. Forty-four episodes were defined as not having IA. The positive and negative predictive values were respectively 100 %, and 88 % for QPCR, 100 % and 97 % for nested PCR, and 28 % and 60 % for GM test. These results suggesting that combined use of methods might improve the diagnosis of IA. [ABSTRACT FROM AUTHOR]

In recent years, researchers have focused on viral and plant immunostimulants which could have beneficial effects in disease prevention and control in shrimp culture. At present, the application of the recombinant VP28 protein (r-VP28) and herbal immunostimulant has been considered as a more effective approach to prevent white spot syndrome (WSS) by enhancing the immune response in shrimp. In the present study, expression of selected immune related genes in response to r-VP28 and herbal immunostimulant mix (HIM) were separately studied qualitatively and quantitatively by RT-PCR and real time PCR, respectively during ontogenetic development from nauplius to juvenile stage in Litopenaeus vannamei . The mRNA expression level of immune related genes such as anti-lipopolysaccharides (ALF), Lysozyme, cMnSOD, Crustin, Prophenoloxidase, Tumor necrosis factor receptor-associated factor 6 (TRAF6) and Haemocyanin were found to be up-regulated significantly in different ontogenetic development stages of shrimp fed with r-VP28 and HIM formulated diets. Relative percent survival (RPS) was determined in shrimp fed with immunostimulants formulated diets after oral challenge with WSSV. The survival of WSSV challenged shrimp was found to be higher in immunostimulants treated groups when compared to untreated group. The results of PCR, ELISA and real time PCR revealed the absence of WSSV in WSSV-challenged shrimp after 20 days of treatment with immunostimulants. Among these immunostimulants, HIM was found to be more effective when compared to r-VP28. After a survey of literature, we are of the opinion that this might be the first report on the expression of immune genes during ontogenetic development of L. vannamei in response to immunostimulants. [ABSTRACT FROM AUTHOR]

The success of Somatic cell nuclear transfer (SCNT) primarily depends on the extent of reprogramming of donor cells genome. The error of reprogramming may lead to inappropriate expression of embryonic genes at any stage of development. Under the present study the relative expression of different genes related to pluripotency (Oct-4 and Nanog), growth factors (IGF-2 and IGF-2R) and DNA methyltransferase gene (Dnmt-1) was evaluated in SCNT embryos at 8–16 cells, morula and blastocyst stages as compared to IVF group. In SCNT, significantly higher degree of relative expression was observed for Oct-4 in morula (1.41) and blastocysts (1.14) as compared to 8–16 cells (referral stage) whereas in IVF, a lower expression was observed at morula (0.82) stage. The expression of Nanog in SCNT embryos was increased significantly in morula (2.23) and decreased subsequently in blastocyst (0.56), whereas it was increased significantly from 8 to 16 cells to morula (1.62) and blastocyst (4.5) of IVF group. The IGF-2 and IGF-2R showed significantly higher expression rates in morula and blastocysts of SCNT (6.56, 5.90 and 1.11, 1.4) and IVF (8.69, 8.25 and 2.96, 3.91) embryos, respectively as compared to referral stage. The expression of Dnmt-1 was significantly higher in SCNT morula (1.29) and blastocyst (1.15) however in IVF, it was similar in 8–16 cells stage and morula but, higher in blastocyst (1.58). The dissimilar pattern of gene expression of SCNT might be a consequence of incomplete reprogramming of donor nucleus which resulted into lower blastocyst rate of SCNT as compared to IVF embryos. [ABSTRACT FROM AUTHOR]

Neospora caninum is a major cause of abortion in cattle and represents an important veterinary health problem of great economic significance. The Medicines for Malaria Venture (MMV) Pathogen Box, an open-source collection of 400 compounds with proven anti-infective properties against a wide range of pathogens, was screened against a N. caninum beta-galactosidase reporter strain grown in human foreskin fibroblasts. A primary screening carried out at 1 µM yielded 40 compounds that were effective against N. caninum tachyzoites. However, 30 of these compounds also affected the viability of the host cells. The 10 remaining compounds exhibited IC 50 values between 4 and 43 nM. Three compounds with IC 50 values below 10 nM, namely MMV676602, MMV688762 and MMV671636, were further characterized in vitro in more detail with respect to inhibition of invasion versus intracellular proliferation, and only MMV671636 had an impact on intracellular proliferation of tachyzoites. This was confirmed by transmission electron microscopy, showing that the primary target of MMV671636 was the mitochondrion. MMV671636 treatment of experimentally infected mice significantly reduced the number of animals with lung and brain infection, and these mice also exhibited a significantly reduced titer of antibodies directed against N. caninum antigens. Thus, MMV671636 is a promising starting point for the development of a future neosporosis therapy. [ABSTRACT FROM AUTHOR]

Since 2003, India has had a well-established influenza surveillance network, though Influenza C virus was not the focus of study. We therefore retrospectively analyzed clinical samples from Pune, western India collected during January 2009 to August 2015, by real-time RT-PCR. Three of 2530 samples of patients with influenza-like illness (ILI) or severe acute respiratory illness (SARI) showed positivity for Influenza C virus infection, while 105 and 31 samples were positive for Influenza A and B viruses respectively. Influenza C viruses were successfully isolated using the embryonated egg system and whole genomes were sequenced and analyzed phylogenetically. HE gene-based phylogeny showed that two viruses C/India/P119564/2011 and C/India P121719/2012 clustered with the C/Sao Paulo/378/82 (SP82) lineage, whereas C/India/P135047/2013 clustered with the C/Kanagawa/1/76 (KA76) lineage. The internal gene of these viruses grouped in two lineages. The PB1, PB2, M and NS genes of the study viruses grouped with C/Yamagata/26/81 (YA81), while the P3 (PA) and NP genes grouped with C/Mississippi/80 (MS80). Bayesian clock studies conclude that the Indian strains may have emerged through multiple reassortment events. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small (19–25 base long), non-coding RNAs that regulate post-transcriptional gene expression by cleaving targeted mRNAs in several eukaryotes. The miRNAs play vital roles in multiple biological and metabolic processes, including developmental timing, signal transduction, cell maintenance and differentiation, diseases and cancers. Experimental identification of microRNAs is expensive and lab-intensive. Alternatively, computational approaches for predicting putative miRNAs from genomic or exomic sequences rely on features of miRNAs viz. secondary structures, sequence conservation, minimum free energy index (MFEI) etc . To date, not a single miRNA has been identified in bubaline ( Bubalus bubalis ), which is an economically important livestock. The present study aims at predicting the putative miRNAs of buffalo using comparative computational approach from buffalo whole genome shotgun sequencing data (INSDC: AWWX00000000.1). The sequences were blasted against the known mammalian miRNA. The obtained miRNAs were then passed through a series of filtration criteria to obtain the set of predicted (putative and novel) bubaline miRNA. Eight miRNAs were selected based on lowest E-value and validated by real time PCR (SYBR green chemistry) using RNU6 as endogenous control. The results from different trails of real time PCR shows that out of selected 8 miRNAs, only 2 (hsa-miR-1277-5p; bta-miR-2285b) are not expressed in bubaline PBMCs. The potential target genes based on their sequence complementarities were then predicted using miRanda. This work is the first report on prediction of bubaline miRNA from whole genome sequencing data followed by experimental validation. The finding could pave the way to future studies in economically important traits in buffalo. [ABSTRACT FROM AUTHOR]

Analysis of crustacean species plays a role in authenticity issues as well as allergen detection. A real-time PCR-based screening assay was developed for the detection of crustaceans in food. In order to cover most relevant species in one analytical step, PCR systems were newly developed for the detection of shrimps (Penaeidae), lobster ( Homarus sp.), Common shrimp ( Crangon crangon ), river prawns ( Macrobrachium sp.) and Chinese mitten crab ( Eriocheir sinensis ). In addition a published system targeting Northern prawn ( Pandalus borealis ) was selected. All PCR-systems are based on mitochondrial 16S rRNA gene sequences and were optimized to be run with a standard program at a universal annealing temperature of 60 °C. Validation experiments confirmed a sensitivity of the PCR systems of 0.01–0.1 genome copies or 0.04–2.5 pg DNA, respectively. Specificity was demonstrated with 25,000 copies of pure genomic DNA from about thirty plant and animal species relevant in food and the performance in food matrix was evaluated. Finally primer and probes were pre-spotted steadily on 96-well PCR plates and the practical applicability of the assay was proven with selected food products. The assay enables detection of multiple species of market relevance. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the longitudinal pattern of M. hyopneumoniae detection in self-replacement gilts at various farms and to characterize the genetic diversity among samples. A total of 298 gilts from three M. hyopneumoniae positive farms were selected at 150 days of age (doa). Gilts were tested for M. hyopneumoniae antibodies by ELISA, once in serum at 150 doa and for M. hyopneumoniae detection in laryngeal swabs by real time PCR two or three times. Also, 425 piglets were tested for M. hyopneumoniae detection in laryngeal swabs. A total of 103 samples were characterized by Multiple Locus Variable-number tandem repeats Analysis. Multiple comparison tests were performed and adjusted using Bonferroni correction to compare prevalences of positive gilts by ELISA and real time PCR. Moderate to high prevalence of M. hyopneumoniae in gilts was detected at 150 doa, which decreased over time, and different detection patterns were observed among farms. Dam-to-piglet transmission of M. hyopneumoniae was not detected. The characterization of M. hyopneumoniae showed 17 different variants in all farms, with two identical variants detected in two of the farms. ELISA testing showed high prevalence of seropositive gilts at 150 doa in all farms. Results of this study showed that circulation of M. hyopneumoniae in self-replacement gilts varied among farms, even under similar production and management conditions. In addition, the molecular variability of M. hyopneumoniae detected within farms suggests that in cases of minimal replacement gilt introduction bacterial diversity maybe farm specific. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is considered one of the most important food-borne parasitic zoonoses globally and sheep are important intermediate hosts of the parasite. Meat and milk from infected sheep are considered an important source of infection for humans. Here, the authors evaluated T. gondii infection in the Italian Cornigliese sheep breed using meat juice ELISA, and in vitro assay for followed by Real Time-PCR and PCR-RFLP. Twenty-one hearts were collected at slaughter. Meat juice serology was carried out on all samples, while eleven hearts with the highest antibody titres were subjected to acid-peptic digestion and seeding onto Vero cells. DNA was extracted at three different time points following seeding. PCR-positive samples were then genotyped by PCR-RFLP. All the meat juice samples were positive for IgG antibodies against p30 protein of T. gondii . Five of the 11 samples, seeded onto Vero cells, were positive in PCR made on DNA extracted after 21 days of culture and the PCR-RFLP revealed a Type-II or Type II variant profile at 9/10 loci. Two out of five samples showed an increase in terms of parasite growth by comparing the Cq values at three different time points. To the authors’ knowledge, this is the first report of in vitro cultivation of T. gondii from muscle tissue of naturally-infected sheep. In vitro assays may be a promising alternative to bioassays and further studies are necessary in order to improve assay performance and to identify possible early markers of parasite proliferation. [ABSTRACT FROM AUTHOR]

Caspase proteases play an essential role in cell apoptosis and inflammation, thus matter greatly in animal development and other biological processes. As a ubiquitous environmental pollutant, pentachlorophenol (PCP) is considered to have adverse effects on animal apoptosis during embryonic development, yet the evidence that PCP interfere with caspase genes was seldom reported. To uncover the effects of PCP on caspases expression in early embryos of zebrafish, two concentrations of PCP (5 μg/L and 200 μg/L) were chosen and 14 types of caspase genes at two different developmental stages, 8 h post-fertilization (hpf) and 24 hpf were analyzed. Lower survival and hatching rates, distinct developmental delay and morphological deformities of head and tail were observed. PCP, especially in the high concentration, significantly altered the expressions of most caspase genes. At 8 hpf, PCP had the most significant inductive effects on gene casp8l2 with fold changes (FCs) of 6.87 at 5 μg/L and 4.48 at 200 μg/L, and casp6l1 (with FCs of 3.15/3.69), and inhibitory effects on caspa (with FCs of 0.93/0.53) and caspb (with FCs of 0.99/0.57). At 24 hpf, PCP had the most significant effects on casp6l2 , casp9 , and caspc . PCP exposure possibly disrupted intrinsic apoptosis pathway considering its effects on casp9 expression. In addition, most caspase genes exhibited higher levels at 24 hpf than 8 hpf except caspc . Our results suggested that PCP had different effects on varied caspase genes, which probably resulting in a profound impact on caspase proteins and apoptosis processes and, ultimately, developmental abnormality. [ABSTRACT FROM AUTHOR]

Objective (i) To validate a simple and efficient method for extracting high quality RNA from small samples of bone tissue, (ii) to test its application on limited amounts of grafted oral bone and (iii) to analyze the gene expression of the OPG/RANK/RANKL system and IL-6 in “spontaneous healing” and grafted tissue. Design 26 patients in need of extraction of one lower molar tooth were divided in 3 groups. In group A (8 patients) the alveolar socket was left for spontaneous healing, in group B (8 patients) it was filled with a hydroxyapatite scaffold while in group C (10 patients) it was filled with hydroxyapatite granules. A small amount of bone was scraped from the alveolar site and sent for analysis. Four months later a new bone specimen was harvested during implant bed preparation. Results IL-6 increased over time in all groups and in particular to the grafted groups. RANK, RANKL and OPG increased over time in all groups, except for RANK in group B. The RANKL/OPG ratio showed a negative value in group A and even more in group B, while it was positive in group C. Conclusions The alveolar site grafted with a granular biomaterial behaved similar to the physiological healing group but displayed a slow remodeling process. RANK, RANKL, OPG and the RANKL/OPG ratio might be able to distinguish among different biomaterials and represent different healing patterns according to different clinical conditions. [ABSTRACT FROM AUTHOR]

Background & aims We evaluated the performance of a rapid method to quantify HCV-RNA in the hepatic and extrahepatic compartments, by using for the first time the Abbott Real Time HCV-assay. Methods Non-tumoral (NT), tumoral (TT) liver samples, lymph nodes and ascitic fluid from patients undergoing orthotopic-liver-transplantation (N = 18) or liver resection (N = 4) were used for the HCV-RNA quantification; 5/22 patients were tested after or during direct acting antivirals (DAA) treatment. Total RNA and DNA quantification from tissue-biopsies allowed normalization of HCV-RNA concentrations in IU/μg of total RNA and IU/10 6 liver-cells, respectively. Results HCV-RNA was successfully quantified with high reliability in liver biopsies, lymph nodes and ascitic fluid samples. Among the 17 untreated patients, a positive and significant HCV-RNA correlation between serum and NT liver-samples was observed (Pearson: rho = 0.544, p = 0.024). Three DAA-treated patients were HCV-RNA “undetectable” in serum, but still “detectable” in all tested liver-tissues. Differently, only one DAA-treated patient, tested after sustained-virological-response, showed HCV-RNA “undetectability” in liver-tissue. Conclusions HCV-RNA was successfully quantified with high reliability in liver bioptic samples and extrahepatic compartments, even when HCV-RNA was “undetectable” in serum. Abbott Real Time HCV-assay is a good diagnostic tool for HCV quantification in intra- and extra-hepatic compartments, whenever a bioptic sample is available [ABSTRACT FROM AUTHOR]

The labelled of pistachio on food products is mandatory and, as a consequence, the development of suitable analytical methodologies to detect this nut in processed foods is advisable. In this work, two different qPCR assays to detect pistachio, SYBR ® Green and locked nucleic acid (LNA) probes, are tested and compared. Pis v allergen coding sequences have been amplified and cloned in different pistachio varieties, and specific primers and probes for each allergen have been designed. According to our results, LNA probe-real time PCR appears to be the most sensitive and specific method, reaching 10 mg/kg of pistachio. The effect of temperature and/or pressure on pistachio DNA detection was also analysed by LNA probe-based qPCR. Data showed a reduced amplificability of pistachio after thermal treatment under pressure, nevertheless, this effect was not observed after boiling. The applicability of this method has been studied by analysing 14 food products and by comparison with a commercial ELISA kit. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in pigs, and has tremendous negative economic impact on the swine industry worldwide. PRRSV is classified into the two distinct genotypes: type 1 and type 2, and most of the described PRRSV isolates in China are type 2. Rapid and sensitive detection of PRRSV is of great importance for the disease control and regional eradication programs. Recombinase polymerase amplification (RPA) has emerged as a novel isothermal amplification technology for the molecular diagnosis of infectious diseases. In this study, a fluorescence reverse transcription RPA (RT-RPA) assay was developed to detect the type 2 PRRSV using primers and exo probe specific for the viral nucleocapsid gene. The reaction was performed at 40 °C within 20 min. The RT-RPA assay could detect both the classical (C-PRRSV) and highly pathogenic PRRSV (HP-PRRSV), but there was no cross-reaction to other pathogens. Using the in vitro transcribed PRRSV RNA as template, the analytical sensitivity of RT-RPA was 690 copies. The assay performance was evaluated by testing 60 field samples and compared to real-time RT-PCR. The detection rate of RT-RPA was 86.6% (52/60), while the detection rate of real-time RT-PCR was 83.3% (50/60). This simple, rapid and reliable method could be potentially applied for rapid detection of PRRSV in point-of-care and rural areas. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) is a chronic autoimmune disease, and the most common cause of nontraumatic disability in young people. The etiology of this disease is not well defined yet. Cytokines play an important role in differentiation, maturation and survival of a wide range of cells, including cells of the immune system. Suppressor of cytokine signaling (SOCS) proteins are the most important regulators of this cytokine signaling pathway. The aim of present study was to compare the expression levels of SOCS1, SOCS2, SOCS3 and SOCS5 genes in the blood of 50 relapsing-remitting MS (RR-MS) patients and 50 healthy controls by Taqman Quantitative Real-Time PCR in patients and healthy control group. We observed that SOCS1 and SOCS5 expression was significantly down-regulated (P = 0.045 and P = 0.044, respectively); whereas, no significant difference was observed between MS patients and controls for SOCS2 and SOCS3 gene expression (P = 0.747 and P = 0.439, respectively). In addition, there was no significant correlation between the expression of SOCS1, SOCS2, SOCS3 and SOCS5 genes and clinical findings, such as the level of physical disability in the MS patients according to the Kurtzke Expanded Disability Status Scale (EDSS) criterion and disease duration. However, a significant positive correlation was observed between expression levels of SOCS genes. This study shows that loss of balance among various members of the SOCS family proteins may contribute to pathophysiology of multiple sclerosis. [ABSTRACT FROM AUTHOR]

A resistant strain of Aphis craccivora Koch was selected with pirimicarb for 12 generations. The selected strain exhibited 47-fold resistance compared to the susceptible laboratory strain. The resistant strain also showed cross-resistance to carbosulfan, malathion, chlorpyrifos methyl and thiamethoxam. Low resistance to fenitrothion and acetamiprid was also observed. Bioassays in combination with biochemical synergist studies revealed that the higher inhibition of carboxylesterase using the synergist tributyl phosphorotrithioate (DEF) was associated with increased toxicity of pirimicarb in the resistant strain. On the other hand, piperonyl butoxide (PBO) and diethyl maleate (DEM) had less inhibitory effect on mixed function oxidases (MFO) and glutathione-S-transferase (GST) in the resistant strain. The activity of carboxylesterase was 29-fold greater in the resistant strain, whereas the activity of MFO and GST in the resistant strain was only 5.5 and 1.7- fold greater, respectively. The activity of acetylcholine esterase (AChE) in the resistant strain was 2 fold higher than in the susceptible strain. The I 50 (the concentration of pirimicarb that inhibit 50% of the enzyme activity) ratio of R-strain to S-strain was 12.6. Molecular studies using real-time quantitative PCR showed that the transcription level of Ace 2 gene in the resistant strain was 3.4-fold higher than that in the susceptible strain. In conclusion, mechanism of resistance in the pirimicarb resistant strain of A. craccivora may include overexpression of Ace2 gene and higher activity of the detoxification enzymes esterases and partly MFO. [ABSTRACT FROM AUTHOR]

Pork DNA was detected in meat mixtures using both conventional PCR and real-time PCR (RT-PCR). Thirty meat mixtures containing beef, chicken, camel, rabbit, goat and sheep with varying percentage of pork (0%, 1%, 5%, 10%, and 20%) and 75 commercial food products, were analyzed using conventional and RT-PCR to determine the presence of pork DNA. Pork DNA standard curves and cycle threshold (Ct) values were used for quantification. The detection limits for pork DNA in the mixtures were 0.22, 0.047, 0.048, 0.0000037, 0.015 ng/μl respectively. Unlike conventional PCR, RT-PCR detected pork DNA in nine processed food samples [chicken sausages (2), chicken luncheon (2), turkey meat loaf, milk chocolate with soft nougat, jelly, cake, and candies] at pork DNA concentrations of 0.0001 ng/μl or less. [ABSTRACT FROM AUTHOR]

A cross-sectional study was carried out to determine the prevalence and diagnostic performance of microscopy and real time PCR (RT-PCR) for 14 intestinal parasites in a Venezuelan rural community with a long history of persistent intestinal parasitic infections despite the implementation of regular anthelminthic treatments. A total of 228 participants were included in this study. A multiplex RT-PCR was used for the detection of Dientamoeba fragilis , Giardia intestinalis , Cryptosporidium sp. and a monoplex RT-PCR for Entamoeba histolytica . Furthermore, a multiplex PCR was performed for detection of Ascaris lumbricoides, Strongyloides stercoralis, Necator americanus and Ancylostoma duodenale. Combined microscopy-PCR revealed prevalences of 49.3% for A. lumbricoides , 10.1% for N. americanus (no A. duodenale was detected), 2.0% for S. stercoralis , 40.4% for D. fragilis , 35.1% for G. intestinalis , and 7.9% for E. histolytica/dispar . Significant increases in prevalence at PCR vs . microscopy were found for A. lumbricoides, G. intestinalis and D. fragilis . Other parasites detected by microscopy alone were Trichuris trichiura (25.7%), Enterobius vermicularis (3.4%), Blastocystis sp. (65.8%), and the non-pathogenic Entamoeba coli (28.9%), Entamoeba hartmanni (12.3%), Endolimax nana (19.7%) and Iodamoeba bütschlii (7.5%). Age- but no gender-related differences in prevalences were found for A. lumbricoides, T. trichiura, G. intestinalis, and E. histolytica/dispar . The persistently high prevalences of intestinal helminths are probably related to the high faecal pollution as also evidenced by the high prevalences of non-pathogenic intestinal protozoans. These results highlight the importance of using sensitive diagnostic techniques in combination with microscopy to better estimate the prevalence of intestinal parasites, especially in the case of D. fragilis trophozoites, which deteriorate very rapidly and would be missed by microscopy. In addition, the differentiation between the pathogenic E. histolytica and the non-pathogenic E. dispar can be attained. However, microscopy remains an important diagnostic tool since it can detect other intestinal parasites for which no PCR is available. [ABSTRACT FROM AUTHOR]

Paratuberculosis is a disease affecting mainly ruminants, and it is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Control of the disease relies primarily on test-and-removal that is not easy to apply effectively for reasons associated with its pathogenesis. Therefore the aim of this preliminary study was to investigate whether positivity of sheep to MAP-specific antibody and DNA, detected by ELISA and faecal real time polymerase chain reaction (PCR) respectively, varies between specific stages of breeding i.e. before and after parturition, and before the mating season. Rectal faeces (n = 120) and whole blood (n = 120) samples were collected from a flock of sheep with no record of clinical paratuberculosis. The sheep under study are maintained throughout the year, in isolated confinement with no access to pasture or other animals, and a continuously monitored diet. The proportion of positive animals to real time PCR before parturition (13.9%) was found to be significantly lower (χ 2 = 10.67, P = 0.0015 ) than the proportion of positive animals after parturition (59.5%), and before the mating season (47.6%). The proportion of positive animals after parturition and before the mating season did not differ significantly. PCR analysis of one sample collected from each animal after parturition, allowed detection of more than 71% of the reactors. In conclusion, faecal shedding of MAP detected by real time PCR seems to be decreased before parturition, which probably makes the specific period less suitable for detection of shedders in sheep with no clinical signs of paratuberculosis. [ABSTRACT FROM AUTHOR]

The effect of the extract of Zataria multiflora ( Z. multiflora ) on IFN-γ, FOXP3, IL-4, TGF-β, and IL-17 gene expression was evaluated in cultured splenocytes obtained from control, nontreated asthma or sensitized mice (group S), Sensetized animals treated with dexamethasone or three concentrations of Z. multiflora extract (200, 400 and 800 μg/ml) (n = 6, for each group). IFN-γ and FOXP3 gene expressions were significantly decreased ( P < 0.001 for both cases) but IL-4 ( P < 0.001) and IL-17 ( P < 0.05) were increased in group S compared to control group. Z. Multiflora extract 800 μg/ml, significantly upregulated IFN-γ gene expression ( P < 0.01) and its 400 and 800 μg/ml concentrations increased FOXP3 gene expression ( P < 0.05 and P < 0.001, respectively) compared to group S. Z. multiflora extract at all concentrations (200, 400 and 800 μg/ml) decreased TGF-β gene expression and its lowest concentration significantly reduced IL-17 gene expression compared to group S ( P < 0.001 for all cases). Only IL-4 and TGF-β gene expression was significantly decreased following treatment with dexamethasone ( P < 0.001 for both cases). The results indicated an increase in IFN-γ and FOXP3 but decrease in TGF-β and IL-17 gene expression profile in sensitized splenocytes treated with the extract, which might be partially due to the presence of one of its constituent, carvacrol. [ABSTRACT FROM AUTHOR]