Recently a novel DNA virus (Torque Teno Virus (TTV) has been identified in Japan and shown to be associated with elevated aminotransferase s levels after transfusion. However the exact role of TTV in pathogenesis of liver disease is yet to be established. The purpose of this study was to determine the prevalence of TTV in thalassemic patients and its relationship with elevated alanine-aminotransfrase (ALT) and aspartateaminotransfrase (AST). This cross-sectional analysis study was conducted on 452 thallasemic patients. Serums were collected from all of the patients, first ALT and AST levels were determined. Then, after DNA extraction, TTV DNA was amplified and detected using semi-nested PCR, followed by gel electrophoresis. Demographic characteristics and clinical data were collected from each participant for statistical analysis. The findings showed that 160 of 452 (35.4%) samples had TTV DNA detected by PCR. From 160 TTV DNA positive, 98 (61.20%) were female and 62 (38.80%) of them were male (P=0.549). The mean ALT and AST values in TTV positive group were higher than in TTV negative group, and the difference was statistically significant (p<0.0001). The result showed that the prevalence of TTV in thalassemic patients in Jahrom is less than other studies in Iran and the mean ALT and AST values in TTV positive individuals were about 2 times more than in TTV negative individuals.

/pdf/molecular-prevalence-and-clinical-importance-of-torque-teno-2fi9z1ixeg.pdf

Molecular Prevalence and Clinical Importance of Torque Teno Virus Infection in Thalassemia Patients as High Risk Individuals

This paper aims to discuss various aspects of the use of reference genes in qPCR technique used in the thousands of present studies. Most frequently, these are housekeeping genes and they must meet several criteria so that they can lay claim to the name. Lots of papers report that in different conditions, for different organisms and even tissues the basic assumption—the constant level of the expression is not maintained for many genes that seem to be perfect candidates. Moreover, their transcription can not be affected by experimental factors. Sounds simple and clear but a great number of designed protocols and lack of consistency among them brings confusion on how to perform experiment properly. Since during selection of the most stable normalizing gene we can not use any reference gene, different ways and algorithms for their selection were developed. Such methods, including examples of best normalizing genes in some specific cases and possible mistakes are presented based on available sources. Numerous examples of reference genes applications, which are usually in too few numbers in relevant articles not allowing to make a solid fundament for a reader, will be shown along with instructive compilations to make an evidence for presented statements and an arrangement of future qPCR experiments. To include all the pitfalls and problems associated with the normalization methods there is no way not to begin from sample preparation and its storage going through candidate gene selection, primer design and statistical analysis. This is important because numerous short reviews available cover the topic only in lesser extent at the same time giving the reader false conviction of complete topic recognition.

/pdf/reference-genes-in-real-time-pcr-589l6vcqns.pdf

Reference genes in real-time PCR.

Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies in Arabidopsis have established that JA also performs a critical role in anther and pollen development but is not essential for other developmental aspects of the plant's life cycle. Here, we describe the phenotypic and molecular characterization of a sterile mutant of tomato (jasmonic acid-insensitive1 [jai1]) that is defective in JA signaling. Although the mutant exhibited reduced pollen viability, sterility was caused by a defect in the maternal control of seed maturation, which was associated with the loss of accumulation of JA-regulated proteinase inhibitor proteins in reproductive tissues. jai1 plants exhibited several defense-related phenotypes, including the inability to express JA-responsive genes, severely compromised resistance to two-spotted spider mites, and abnormal development of glandular trichomes. We demonstrate that these defects are caused by the loss of function of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. These findings indicate that the JA/COI1 signaling pathway regulates distinct developmental processes in different plants and suggest a role for JA in the promotion of glandular trichome-based defenses.

The Tomato Homolog of CORONATINE-INSENSITIVE1 Is Required for the Maternal Control of Seed Maturation, Jasmonate-Signaled Defense Responses, and Glandular Trichome Development

Endophytes are micro-organisms including bacteria and fungi that survive within healthy plant tissues and promote plant growth under stress. This review focuses on the potential of endophytic microbes that induce abiotic stress tolerance in plants. How endophytes promote plant growth under stressful conditions, like drought and heat, high salinity and poor nutrient availability will be discussed. The molecular mechanisms for increasing stress tolerance in plants by endophytes include induction of plant stress genes as well as biomolecules like reactive oxygen species scavengers. This review may help in the development of biotechnological applications of endophytic microbes in plant growth promotion and crop improvement under abiotic stress conditions. Significance and impact of the study Increasing human populations demand more crop yield for food security while crop production is adversely affected by abiotic stresses like drought, salinity and high temperature. Development of stress tolerance in plants is a strategy to cope with the negative effects of adverse environmental conditions. Endophytes are well recognized for plant growth promotion and production of natural compounds. The property of endophytes to induce stress tolerance in plants can be applied to increase crop yields. With this review, we intend to promote application of endophytes in biotechnology and genetic engineering for the development of stress-tolerant plants.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/lam.12855

Induction of abiotic stress tolerance in plants by endophytic microbes.

Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes.

/pdf/identification-and-evaluation-of-new-reference-genes-in-25lo3duqzr.pdf

Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data

Plants respond to various stresses by expressing distinct sets of genes. The effects of multiple stresses on plants and their interactions are not well understood. We have discovered that salt stress causes the accumulation of proteinase inhibitors and the activation of other wound-related genes in tomato (Lycopersicon esculentum) plants. Salt stress was also found to enhance the plant's response to wounding locally and systemically. The tomato mutant (def-1), which has an impairment in the octadecanoid pathway, displayed a severe reduction in the accumulation of proteinase inhibitors under salt stress, indicating that salt stress-induced accumulation of proteinase inhibitors was jasmonic acid dependent. The analysis of salt stress in another tomato mutant, spr-1, which carries a mutation in a systemin-specific signaling component, and transgenic tomato plants that express an antisense-prosystemin cDNA, showed that prosystemin activity was not required for the salt-induced accumulation of proteinase inhibitors, but was necessary to achieve maximal levels. These results suggest that a prosystemin independent- but jasmonic acid-dependent pathway is utilized for proteinase inhibitor accumulation in response to salt stress.

http://www.plantphysiol.org/content/plantphysiol/132/4/2098.full.pdf

Salt Stress Activation of Wound-Related Genes in Tomato Plants

Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress

Methanol is a byproduct of cell wall modification, released through the action of pectin methylesterases (PMEs), which demethylesterify cell wall pectins. Plant PMEs play not only a role in developmental processes but also in responses to herbivory and infection by fungal or bacterial pathogens. Molecular mechanisms that explain how methanol affects plant defenses are poorly understood. Here we show that exogenously supplied methanol alone has weak effects on defense signaling in three dicot species, however it profoundly alters signaling responses to danger- and microbe-associated molecular patterns (DAMPs, MAMPs) such as the alarm hormone systemin, the bacterial flagellum-derived flg22 peptide, and the fungal cell wall-derived oligosaccharide chitosan. In the presence of methanol the kinetics and amplitudes of DAMP/MAMP-induced MAP kinase (MAPK) activity and oxidative burst are altered in tobacco and tomato suspension-cultured cells, in Arabidopsis seedlings and tomato leaf tissue. As a possible consequence of altered DAMP/MAMP signaling, methanol suppressed the expression of the defense genes PR-1 and PI-1 in tomato. In cell cultures of the grass tall fescue (Festuca arundinacea, Poaceae, Monocots), methanol alone activates MAPKs and increases chitosan-induced MAPK activity, and in the darnel grass Lolium temulentum (Poaceae), it alters wound-induced MAPK signaling. We propose that methanol can be recognized by plants as a sign of the damaged self. In dicots, methanol functions as a DAMP-like alarm signal with little elicitor activity on its own, whereas it appears to function as an elicitor-active DAMP in monocot grasses. Ethanol had been implicated in plant stress responses, although the source of ethanol in plants is not well established. We found that it has a similar effect as methanol on responses to MAMPs and DAMPs.

/pdf/methanol-and-ethanol-modulate-responses-to-danger-and-511zt5ydod.pdf

Methanol and ethanol modulate responses to danger- and microbe-associated molecular patterns.

Quantitative real-time reverse transcriptionpoly merase chain reaction (RT-PCR) provides an important tool for analyzing gene expression if proper internal standards are used The aim of this study was to identify and evaluate reference genes for use in real-time quantitative RT-PCR in perennial ryegrass (Lolium perenne L) during plant development Partial sequences of nine L perenne housekeeping genes were obtained by RT-PCR using degenerate primers designed from the corresponding genes in closely related species Primers for quantitative RT-PCR were designed based on partial sequences The housekeeping genes were evaluated for their expression stability in different tissues at various stages of development The analysis found that eEF-1α and eIF-4a were the most stable and β-TUB was the least stable of the genes tested when all tissues were analyzed together Analysis by geNorm indicated that the four most stably expressed housekeeping genes (eEF-1α, eIF-4a, 25S rRNA, and GAPDH) should be utilized when normalizing gene expression during plant developmental studies For root crown tissues at different stages of development, eIF-4a and 25S rRNA were the most stably expressed of the housekeeping genes tested In leaf tissues, eEF-1α and UBQ5 were the most stably expressed of the housekeeping genes tested We found that using two housekeeping genes as reference genes is suffi cient during RT-PCR gene expression studies when analyzing either root crown or leaf tissues during different stages of development

Evaluation of Reference Genes for Quantitative RT-PCR in Lolium perenne

A polymerase chain reaction (PCR)-based method for detection of Neotyphodium endophytes in seed and plant tissue from tall fescue (Festuca arundinacea Schreb.), Italian (Lolium multiflorum Lam.), and perennial (Lolium perenne L.) ryegrasses was developed. The primers were designed to amplify products from a conserved region of the Neotyphodium spp. tubulin 2 gene which is present in N. coenophialum (Morgan-Jones and W. Gams) Glenn, C.W. Bacon, and Hanlin (endophyte of tall fescue), N. lolii (Latch, M.J. Chr., and Samuels) Glenn, C.W. Bacon, and Hanlin (endophyte of perennial ryegrass), and N. occultans C.D. Moon, B. Scott, and M.J. Chr. (endophyte of Italian ryegrass). PCR yielded the expected amplification products from infected seed lots for tall fescue (358 base pairs [bp]), Italian ryegrass (364 bp), and perennial ryegrass (370 bp). Based on DNA mixture tests and bulk seed analysis, the PCR assay was sensitive enough to detect as little as one infected seed per 50 seeds tested. In addition, the primer set detected the Neotyphodium spp. endophyte in all plant tissues except roots. Comparison of the PCRbased assay to microscopic examination and immunoblot detection of endophytes in seed lots showed that all three methods compared favorably to one another. However, none of the three methods could distinguish between viable and nonviable endophyte in seed. This PCR method provides an accurate, sensitive approach for detecting the presence of the endophyte in these grasses, while maintaining enough specificity to discriminate Neotyphodium spp. from related fungal contaminants such as ergot [Claviceps purpurea (Fr.:Fr.) Tul.].

A Sensitive PCR-Based Assay to Detect Neotyphodium Fungi in Seed and Plant Tissue of Tall Fescue and Ryegrass Species

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