Real-time RT-PCR Research Papers - Academia.edu (original) (raw)

Neonatal sepsis is one of the most prevalent infections today, claiming the lives of almost one million newborns in developing countries. Our objective is to determine the bacteria that cause sepsis in newborns in Egyptian hospitals, as... more

Neonatal sepsis is one of the most prevalent infections today, claiming the lives of almost one million newborns in developing countries. Our objective is to determine the bacteria that cause sepsis in newborns in Egyptian hospitals, as well as to discover their resistance profiles, allowing for the selection of appropriate drug combinations against multidrug resistant (MDR) bacteria. Nine hundred and eighty-nine blood samples were collected from neonates in Al Demerdash and Ain Shams University Specialized Hospital from March 2019 to March 20121 according to the standard protocols. Bacterial identification and antimicrobial susceptibility test AST were performed using VITEK® 2 system. The results revealed that 51.4 % of patients had positive blood cultures, with 60.3 % females and 39.7 %. Males. Sepsis with late onset was detected in 95% of positive cases, while sepsis with early onset was observed in only 5% of the total positive cases. According to the Original Research Article

This report describes the generation of novel encapsidated RNA particles and their evaluation as in-tube internal controls in diagnostic real-time reverse-transcription PCR (rRT-PCR) assays for the detection of RNA viruses. A cassette... more

This report describes the generation of novel encapsidated RNA particles and their evaluation as in-tube internal controls in diagnostic real-time reverse-transcription PCR (rRT-PCR) assays for the detection of RNA viruses. A cassette containing sequences of 2 diagnostic primer sets for foot-and-mouth disease virus (FMDV) and a set for swine vesicular disease virus (SVDV) was engineered into a full-length cDNA clone containing the RNA-2 segment of Cowpea Mosaic Virus (CPMV). After co-inoculation with a plasmid that expressed CPMV RNA-1, recombinant virus particles were rescued from cowpea plants (Vigna unguiculata). RNA contained in these particles was amplified in diagnostic rRT-PCR assays used for detection of FMDV and SVDV. Amplification of these internal controls was used to confirm that rRT-PCR inhibitors were absent from clinical samples, thereby verifying negative assay results. The recombinant CPMVs did not reduce the analytical sensitivity of the rRT-PCRs when amplification of the insert was performed in the same tube as the diagnostic target. This system provides an attractive solution to the production of internal controls for rRT-PCR assays since CPMV grows to high yields in plants, the particles are thermostable, RNase resistant and simple purification of RNA-2 containing capsids yields a preparation which is non-infectious.

The chronic inflammation of arterial walls is associated with the development of atherosclerosis. Earlier we reported that avenanthramide (Avn) s-enriched extract of oats (AvnsO) significantly suppressed interleukin (IL)-1β-stimulated... more

The chronic inflammation of arterial walls is associated with the development of atherosclerosis. Earlier we reported that avenanthramide (Avn) s-enriched extract of oats (AvnsO) significantly suppressed interleukin (IL)-1β-stimulated secretion of proinflammatory cytokines, such as IL-6, IL-8, and MCP-1, by human aortic endothelial cells (HAEC). The main objective of the current study was to determine if the mechanism of inhibitory effect of these polyphenols from oats on the expression of proinflammatory cytokines is mediated through modulation of nuclear factor κB (NF-κB)-dependent transcription. Confluent HAEC monolayers were treated for 24 h with AvnsO, and synthetically prepared Avn-c suppressed IL-β-stimulated activation of NF-κB in a concentration-dependent manner. CH3-Avn-c, a synthetically prepared methyl ester derivative of Avn-c with a high biological potency, significantly and dose dependently decreased mRNA expression and secretion of IL-6, IL-8, and MCP-1 by HAEC as determined by real-time RT-PCR and ELISA, and it inhibited IL-1β-and TNFα-stimulated NF-κB activation as determined by a NF-κB DNA binding assay and a NF-κB luciferase reporter assay. AvnsO and Avn-c as well as CH3-Avn-c also inhibited the NF-κB-dependent reporter gene expression activated by TNFR-associated factor 2 and 6 (TRAF2, TRAF6) and NFκB-inducing kinase (NIK). CH3-Avn-c also significantly and dose dependently decreased the phosphorylation level of IκB kinase (IKK) and IκB, and prevented IκB degradation as measured by Western blotting. In addition, CH3-Avn-c markedly increased the overall levels of high mass ubiquitin-conjugated protein levels while it mildly inhibited proteasome activity. These observations suggest that Avns, unique polyphenols from oats, decrease the expression of endothelial proinflammatory cytokines at least in part through inhibition of NF-κB activation by inhibiting the phosphorylation of IKK and IκB, and by suppressing proteasome activity.

Melanin is the pigment that determines skin color. Melanin synthesis is catalysed by the enzyme tyrosinase and is controlled by TYR, TYRP1 and TYRP2 genes. The objective of this study was to evaluate the anti pigmentation property of palm... more

Melanin is the pigment that determines skin color. Melanin synthesis is catalysed by the enzyme tyrosinase and is controlled by TYR, TYRP1 and TYRP2 genes. The objective of this study was to evaluate the anti pigmentation property of palm tocotrienol rich fraction by determining melanin synthesis and expression of genes involved in its regulation in skin melanocytes. Palm tocotrienol rich fraction (TRF) which contains 75% α α α α-tocotrienol and 25% tocopherol was used to inhibit melanin synthesis which was determined by determining melanin level and tyrosinase enzyme activity. Expression of TYR, TYRP1 and TYRP2 genes was determined by quantitative real time reverse transcriptase polymerase chain reaction (real time RT-PCR). Primary culture of skin melanocytes was divided into two groups; untreated control and cells that were treated with 500 µg/ml tocotrienol rich fraction for 24 h. Our results showed that there was a reduction in tyrosinase activity and melanin content in melanocytes treated with tocotrienol rich fraction compared to control (p < 0.05). Expression of TYRP2 gene in melanocytes treated with tocotrienol rich fraction was also decreased (p < 0.05) compared to control. In conclusion, palm tocotrienol rich fraction has an anti pigmentation property that inhibit melanin synthesis by inhibiting tyrosinase activity and down regulating TYRP2 gene expression.

Type 2 diabetes patients exhibit subclinical inflammation but the regulatory mechanisms are poorly understood. We sought to evaluate the role of miR-146a expression along with its downstream proinflammatory signals in relation to glycemic... more

Type 2 diabetes patients exhibit subclinical inflammation but the regulatory mechanisms are poorly understood. We sought to evaluate the role of miR-146a expression along with its downstream proinflammatory signals in relation to glycemic control and insulin resistance. Study subjects (n = 20 each) comprised of clinically well characterized Type 2 diabetes patients and control non-diabetic subjects. miRNA and mRNA expression levels were probed in peripheral blood mononuclear cells (PBMC) by Real-time RT-PCR and plasma levels of TNFa and IL-6 were measured by ELISA.

Context: Excess production of aldosterone or cortisol has profound effects on cardiovascular function and impacts other major organ systems. The mechanisms leading to the autonomous hypersecretion of aldosterone or cortisol in... more

Context: Excess production of aldosterone or cortisol has profound effects on cardiovascular function and impacts other major organ systems. The mechanisms leading to the autonomous hypersecretion of aldosterone or cortisol in aldosterone-producing adenoma (APA) or cortisol-producing adenoma (CPA) are unknown.

A real time RT-PCR, using the LightCycler, was developed and compared with rapid antigen enzyme immunoassay (AgEIA) and enhanced virus culture for rapid detection of influenza A viruses in stored and prospectively collected respiratory... more

A real time RT-PCR, using the LightCycler, was developed and compared with rapid antigen enzyme immunoassay (AgEIA) and enhanced virus culture for rapid detection of influenza A viruses in stored and prospectively collected respiratory specimens. Specific hybridization probes were used for simultaneous detection and differentiation between H1N1 and H3N2 subtypes. The sensitivity of the RT-PCR for influenza A H1N1 was 120 copies and H3N2 350 copies of in vitro transcribed RNA. A specimen was considered positive for influenza A when it was culture positive or at least two methods yielded a positive test result. Using these criteria, with stored samples, the RT-PCR sensitivity, specificity, positive and negative predictive values were 82.9, 95.5, 98.9 and 52.5%, respectively. In specimens collected prospectively the RT-PCR sensitivity, specificity, positive and negative predictive values were 100, 87.9, 82.8 and 100%, respectively. There was complete concordance with subtype differentiation by hybridization probe melting temperature analysis and haemagglutination inhibition assay.

Herpes simplex virus type1 VP22 (HSV-1 VP22) Green fluorescent protein (GFP) a b s t r a c t An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of... more

Herpes simplex virus type1 VP22 (HSV-1 VP22) Green fluorescent protein (GFP) a b s t r a c t An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.

Recently, a novel approach to a highly sensitive and quantitative detection of rare earth element (REE) ions including La3+, Eu3+ and Tb3+, by the polymerase chain reaction (PCR) technique, has been reported. The detection of REE ions is... more

Recently, a novel approach to a highly sensitive and quantitative detection of rare earth element (REE) ions including La3+, Eu3+ and Tb3+, by the polymerase chain reaction (PCR) technique, has been reported. The detection of REE ions is based on the catalytic nature of REE ions targeting the deoxyribonucleic acid (DNA), thus monitoring of the ions can be achieved by reading the level of intact DNA by PCR. Despite of its high sensitivity (at ppb to ppt levels), the conventional PCR-based REE detection protocol requires certain length of time (1-2 hours). In the present study, we modified the PCR-based REE detection protocols by employing the high-speed PCR, and performed the automated and rapid detection of La3+ in small-sized aqueous samples within 5min.

Quantitative reverse transcription PCR (qRT-PCR) is one of the most precise and widely used methods of gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the... more

Quantitative reverse transcription PCR (qRT-PCR) is one of the most precise and widely used methods of gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the expression stability of potential reference genes in common buckwheat (Fagopyrum esculentum) in order to find the optimal reference for gene expression analysis in this economically important crop. Recently sequenced buckwheat floral transcriptome was used as source of sequence information. Expression stability of eight candidate reference genes was assessed in different plant structures (leaves and inflorescences at two stages of development and fruits). These genes are the orthologs of Arabidopsis genes identified as stable in a genome-wide survey gene of expression stability and a traditionally used housekeeping gene GAPDH. Three software applications -geNorm, NormFinder and BestKeeper -were used to estimate expression stability and provided congruent results. The orthologs of AT4G33380 (expressed protein of unknown function, Expressed1), AT2G28390 (SAND family protein, SAND) and AT5G46630 (clathrin adapter complex subunit family protein, CACS) are revealed as the most stable. We recommend using the combination of Expressed1, SAND and CACS for the normalization of gene expression data in studies on buckwheat using qRT-PCR. These genes are listed among five the most stably expressed in Arabidopsis that emphasizes utility of the studies on model plants as a framework for other species.

Three-dimensional porous scaffolds prepared from regenerated silk fibroin using either an all-aqueous process or a process involving an organic solvent, hexafluoroisopropanol (HFIP), have shown promise in cell culture and tissue... more

Three-dimensional porous scaffolds prepared from regenerated silk fibroin using either an all-aqueous process or a process involving an organic solvent, hexafluoroisopropanol (HFIP), have shown promise in cell culture and tissue engineering applications. However, their biocompatibility and in vivo degradation have not been fully established. The present study was conducted to systematically investigate how processing method (aqueous vs. organic solvent) and processing variables (silk fibroin concentration and pore size) affect the short-term (up to 2 months) and long-term (up to 1 year) in vivo behavior of the protein scaffolds in both nude and Lewis rats. The samples were analyzed by histology for scaffold morphological changes and tissue ingrowth, and by real-time RT-PCR and immunohistochemistry for immune responses. Throughout the period of implantation, all scaffolds were well tolerated by the host animals and immune responses to the implants were mild. Most scaffolds prepared from the all-aqueous process degraded to completion between 2 and 6 months, while those prepared from organic solvent (hexafluoroisopropanol (HFIP)) process persisted beyond 1 year. Due to widespread cellular invasion throughout the scaffold, the degradation of aqueous-derived scaffolds appears to be more homogeneous than that of HFIP-derived scaffolds. In general and especially for the HFIP-derived scaffolds, a higher original silk fibroin concentration (e.g. 17%) and smaller pore size (e.g. 100-200 mm) resulted in lower levels of tissue ingrowth and slower degradation. These results demonstrate that the in vivo behavior of the three-dimensional silk fibroin scaffolds is related to the morphological and structural features that resulted from different scaffold preparation processes. The insights gained in this study can serve as a guide for processing scenarios to match desired morphological and structural features and degradation time with tissue-specific applications.

Avian influenza virus (AIV) causes great economic losses for the poultry industry worldwide and threatens the human population with a pandemic. The conventional detection method for AIV involves sample preparation of viral RNA extraction... more

Avian influenza virus (AIV) causes great economic losses for the poultry industry worldwide and threatens the human population with a pandemic. The conventional detection method for AIV involves sample preparation of viral RNA extraction and purification from raw sample such as bird droppings. In this study, magnetic beads were applied for immunoseparation and purification of AIV from spiked chicken fecal sample. The beads were conjugated with monoclonal antibodies against the AIV nucleoprotein, which is conserved in all the AIV. The bead-captured virus was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) without RNA extraction because of effective removal of RT-PCR inhibitors. The developed bead-based assay showed a similar detection limit comparable to the RNA extraction and the classic virus isolation method. Using ready-to-use antibody-conjugated bead, the method requires less than 5 h. Furthermore, the method has potential to integrate into a Lab-on-a-chip system for rapid detection and identification of AIV. Crown

The illicit use of supraphysiological doses of androgenic steroids (AAS) has been suggested as a cause of arrhythmia in athletes. The objectives of the present study were to investigate the time-course and the cellular, ionic and... more

The illicit use of supraphysiological doses of androgenic steroids (AAS) has been suggested as a cause of arrhythmia in athletes. The objectives of the present study were to investigate the time-course and the cellular, ionic and molecular processes underlying ventricular repolarization in rats chronically treated with AAS. Male Wistar rats were treated weekly for 8 weeks with 10 mg/kg of nandrolone decanoate (DECA n = 21) or vehicle (control n = 20). ECG was recorded weekly. Action potential (AP) and transient outward potassium current (I to ) were recorded in rat hearts. Expression of KChIP2, Kv1.4, Kv4.2, and Kv4.3 was assessed by real-time PCR. Hematoxylin/eosin and Picrosirius red staining were used for histological analysis. QTc was greater in the DECA group. After DECA treatment the left, but not right, ventricle showed a longer AP duration than did the control. I to current densities were 47.5% lower in the left but not in the right ventricle after DECA. In the right ventricle the I to inactivation time-course was slower than in the control group. After DECA the left ventricle showed lower KChIP2 (∼ 26%), Kv1.4 (∼ 23%) and 4.3 (∼ 70%) expression while the Kv 4.2 increased in 4 (∼ 250%) and diminished in 3 (∼ 30%) animals of this group. In the right ventricle the expression of I to subunits was similar between the treatment and control groups. DECA-treated hearts had 25% fewer nuclei and greater nuclei diameters in both ventricles. Our results strongly suggest that supraphysiological doses of AAS induce morphological remodeling in both ventricles. However, the electrical remodeling was mainly observed in the left ventricle.

TaqMan real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) using purified RNA targets or coupled to tissue-print and squash procedures was developed to detect and quantify Citrus tristeza virus (CTV) RNA-targets in plant... more

TaqMan real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) using purified RNA targets or coupled to tissue-print and squash procedures was developed to detect and quantify Citrus tristeza virus (CTV) RNA-targets in plant tissues and in single aphids. With this method all CTV isolates tested from different hosts and origins were detected. The sensitivity of conventional realtime RT-PCR was 1,000 times higher than immunocapture (IC)-RT-nested PCR and 10 6 times higher than enzyme linked immunosorbent assay (ELISA). The quantitation limit ranged from 1.7×10 2 to 1.7× 10 9 transcript copies. The estimated number of CTV RNA-targets detected in different organs of a CTVinfected tree ranged from 4.5×10 5 to 6.5×10 8 copies when purified RNA was used as template and from 1.9×10 4 to 3.7×10 6 when tissue-printed material was used. In single squashed aphids the number of copies ranged from 4.73×10 3 to 1.23×10 5 . Reliable quantitation of CTV targets present in infected plant material or acquired by single aphid species, was achieved with tissue-print and squash procedures combined with real-time RT-PCR, both of which do not require extraction procedures or nucleic acid purification.

Abiotic stress, such as extreme temperature, drought, or excessive salinity, is one of the leading causes of crop loss worldwide. Microspore-derived (MD) cell suspension cultures of Brassica napus L. cv. Jet Neuf have been shown to be a... more

Abiotic stress, such as extreme temperature, drought, or excessive salinity, is one of the leading causes of crop loss worldwide. Microspore-derived (MD) cell suspension cultures of Brassica napus L. cv. Jet Neuf have been shown to be a useful system for studying the biochemistry of developing oilseeds. In the present study, we describe the application of MD cell suspension cultures of B. napus as a system for studying gene expression in response to abiotic stress, and demonstrate emybryogenic competence in cultures that have been continuously subcultured for more than 20 years. MD cell suspension cultures of B. napus L. cv Jet Neuf were exposed to low temperature or osmotic stress and the expression profile of known stress responsive genes was evaluated. The gene expression profile of BN115, a known cold-responsive gene in B. napus, was similar to that described for intact cold-acclimated plants. Likewise, two late embryogenesis abundant (Lea) genes were shown to be upregulated in response to low temperature or osmotic stress. The results demonstrate that B. napus MD cell suspension cultures are a useful system for the investigation of changes in gene expression in plants brought about by abiotic stress.

The expression of neuroglobin (Ngb) and cytoglobin (Cygb), two recently discovered globins with a potential neuroprotective activity against hypoxia and oxidative stress, was investigated in the cerebellum of young rats (postnatal day 20)... more

The expression of neuroglobin (Ngb) and cytoglobin (Cygb), two recently discovered globins with a potential neuroprotective activity against hypoxia and oxidative stress, was investigated in the cerebellum of young rats (postnatal day 20) after being exposed to chronic mild carbon monoxide (CO) at 25 ppm during prenatal (group A), prenatal and postnatal (group B), the postnatal period only (group C), and air (group D). The expression of genes associated with hypoxia signaling pathways was also investigated in the rat cerebella by real-time RT-PCR after CO exposure. Ngb and Cygb mRNAs did not change in any CO-exposed group. Quantitative immunohistochemistry showed no significant change in Ngb protein; however, there was a significant increase of Cygb protein in rats from groups A, B, and C when compared with group D. In group B, genes related to the generation of reactive oxygen species (Nos2) and lipid metabolism (Apat2) were upregulated. In contrast, no changes were found in the expression of 8 genes typically upregulated by hypoxic conditions (Angptl4, Arnt2, Casp1, Crebbp, Hif1a, Hif3a, Mt3, or Vegfa) in any CO-exposed group, suggesting that hypoxia-related gene expression is not altered by this mild CO exposure. Cygb but not Ngb may protect cerebellar cells from the chronic presence of CO exposure during prenatal and postnatal development.

Ancylostoma braziliense belongs to the family Ancylostomatidae and infects cats and dogs in various parts of the tropical world. It is also a zoonotic parasite causing cutaneous larva migrants in humans. There are very few, either... more

Ancylostoma braziliense belongs to the family Ancylostomatidae and infects cats and dogs in various parts of the tropical world. It is also a zoonotic parasite causing cutaneous larva migrants in humans. There are very few, either biological or molecular, studies of this species. In this study, differential display was used to identify differentially expressed genes in male and female A. braziliense. Nineteen new sequences were identified and examined by real-time RT-PCR to confirm male-female specificity. Ten were more expressed in males, while two were more expressed in females. Molecules shown to be important in other host-parasite relationships were also found in this study. #

The fact sheet b elow explores the roles commonly found in radio. Some are unique to SYN Radio, while others are exclusive to commercial stations. Radio Programming Manager This role holds responsibility for all content broadcast by the... more

The fact sheet b elow explores the roles commonly found in radio. Some are unique to SYN Radio, while others are exclusive to commercial stations. Radio Programming Manager This role holds responsibility for all content broadcast by the station. The position changes annually at SYN due to its policy of providing opportunities to as many young people as possible. In most cases at other community and commercial stations, this is a permanent position. Their responsibility is to oversee all programs and presenters, ensuring the smooth operating of the station. Within SYN, this position also liaises with the Radio Programming Committee at the turn of each radio season to select new programs from the collection of applicants, supporting the programs throughout the season. In commercial stations, the duties of this role fall within the title of Content Coordinator or Program Director-making decisions on programming, music, and reviewing demos of new talent. SYN Radio Programming Committee Members of the Committee (usually numbered between 4-7) work alongside the Radio Programming Manager in programming new radio seasons from the applications submitted, as well as receiving a group of programs to support more closely. Music Director This prominent role at SYN is also found at particular stations with a strong music focus e.g. Triple J. The role is the main interface between radio stations and the music industry, overseeing the musical direction the station takes, as well as providing programs access to the music they require. The recognisable "sound" of a station's music can be attributed to the Music Director and their team.

The cytokine-dependent, CD34+ human acute myeloid leukaemia cell line MUTZ-3 was used to generate immature dendritic-like cells and their validity as an alternative to primary CD34+ progenitor-derived DC (CD34-DC) for testing... more

The cytokine-dependent, CD34+ human acute myeloid leukaemia cell line MUTZ-3 was used to generate immature dendritic-like cells and their validity as an alternative to primary CD34+ progenitor-derived DC (CD34-DC) for testing nickel-induced sensitization was assessed. Expression levels of the DC maturation markers HLA-DR, CD86, CD83 and CD11c were studied using flow cytometry after 24 and 48 hours exposure to the model compound nickel sulphate (NiSO4.6H20) (100 and 300 µM). No maturation of MUTZ-3 DC was observed, whereas significantly upregulated expression levels of CD83 and CD86 were noticed in CD34-DC after 24 hours treatment with 300 µM nickel sulphate compared to control cells. Differential expression of the cytokine genes IL1 , IL6, IL8, CCL2, CCL3, CCL3L1, CCL4 was analysed using real-time RT-PCR after 6, 10 and 24 hours of nickel exposure. In response to 100 µM nickel sulphate MUTZ-3 DC revealed slightly upregulated mRNA levels only after 24 hours, whereas 300 µM induced transcription of CCL3, CCL3L1 and IL8 significantly already after 6 or 10 hours. These cytokine data correspond to earlier observations in CD34-DC, in which effects were already obtained at 100 µM nickel sulphate.

Entamoeba histolytica calreticulin (EhCRT) is remarkably immunogenic in humans (90-100% of invasive amoebiasis patients). Nevertheless, the study of calreticulin in this protozoan is still in its early stages. The exact location,... more

Entamoeba histolytica calreticulin (EhCRT) is remarkably immunogenic in humans (90-100% of invasive amoebiasis patients). Nevertheless, the study of calreticulin in this protozoan is still in its early stages. The exact location, biological functions, and its role in pathogenesis are yet to be fully understood. The aim of the present work is to determine the location of EhCRT in virulent trophozoites in vivo and the expression of the Ehcrt gene during the development of experimentally induced amoebic liver abscesses (ALA) in hamsters. Antibodies against recombinant EhCRT were used for the immunolocalization of EhCRT in trophozoites through confocal microscopy; immunohistochemical assays were also performed on tissue sections of ALAs at different times after intrahepatic inoculation. The expression of the Ehcrt gene during the development of ALA was estimated through both in situ RT-PCR and real-time RT-PCR. Confocal assays of virulent trophozoites showed a distribution of EhCRT in the cytoplasmic vesicles of different sizes. Apparently, EhCRT is not exported into the hepatic tissue. Real-time RT-PCR demonstrated an over-expression of the Ehcrt gene at 30 min after trophozoite inoculation, reaching a peak at 1-2 h; thereafter, the expression fell sharply to its original levels. These results demonstrate for the first time in an in vivo model of ALA, the expression of Ehcrt gene in E. histolytica trophozoites and add evidence that support CRT as a resident protein of the ER in E. histolytica species. The in vivo experiments suggest that CRT may play an important role during the early stages of the host-parasite relationship, when the parasite is adapting to a new environment, although the protein seems to be constitutively synthesized. Moreover, trophozoites apparently do not export EhCRT into the hepatic tissue in ALA.

Fusarium proliferatum is together with Fusarium verticillioides the main source of fumonisins, a health risk mycotoxin, contaminating agro-products. Contrary to F. verticillioides, it colonizes a wide range of host plants besides maize,... more

Fusarium proliferatum is together with Fusarium verticillioides the main source of fumonisins, a health risk mycotoxin, contaminating agro-products. Contrary to F. verticillioides, it colonizes a wide range of host plants besides maize, such as wheat or barley among others, in particular in certain regions (Southern Europe). The phylogenetic study performed in this work using a wide sample of isolates from diverse hosts and origins revealed a high variability, while no host preferences could be sustained. A real time RT-PCR assay was also developed specific for F. proliferatum on the basis on fumonisin biosynthetic gene, FUM1, which allowed discrimination from F. verticillioides. FUM1 gene expression showed a high and significant correlation (0.77) with fumonisin production, representing a valuable tool for specific and sensitive diagnosis of metabolically active fumonisin-producing F. proliferatum isolates and for evaluating the influence on environmental conditions on FUM1 gene regulation. The ability to produce fumonisins was also widely distributed indicating that F. proliferatum can represent a risk for health similarly to F. verticillioides. Moreover, the wide range of plants susceptible to colonization by F. proliferatum suggests that the impact of fumonisin risk in a number of commodities might need a revision.

The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of... more

The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a ''gold'' standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. As a first step towards standardization, we describe a series of RT-qPCR protocols that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible. We would like to emphasize, however, that RT-qPCR data constitute only a snapshot of information regarding the quantity of a given transcript in a cell or tissue. Any assessment of the biological consequences of variable mRNA levels must include additional information regarding regulatory RNAs, protein levels and protein activity. The entire protocol described here, encompassing all stages from initial assay design to reliable qPCR data analysis, requires approximately 15 h.

The world is suffering from a massive delusion based on the belief that a test for RNA is a test for a deadly new virus, a virus that has emerged from wild bats or other animals in China, supported by the western assumption that Chinese... more

The world is suffering from a massive delusion based on the belief that a test for RNA is a test for a deadly new virus, a virus that has emerged from wild bats or other animals in China, supported by the western assumption that Chinese people will eat anything that moves.
If the virus exists, then it should be possible to purify viral particles. From these particles RNA can be extracted and should match the RNA used in this test. Until this is done it is possible that the RNA comes from another source, which could be the cells of the patient, bacteria, fungi etc. There might be an association with elevated levels of this RNA and illness, but that is not proof that the RNA is from a virus. Without purification and characterization of virus particles, it cannot be accepted that an RNA test is proof that a virus is present.
Definitions of important diseases are surprisingly loose, perhaps embarrassingly so. A couple of symptoms, maybe contact with a previous patient, and a test of unknown accuracy, is all you often need. While the definition of SARS, an earlier coronavirus panic, was self-limiting, the definition of the new coronavirus disease is open-ended, allowing the imaginary epidemic to grow. Putting aside the existence of the virus, if the coronavirus test has a problem with false positives (as all biological tests do) then testing an uninfected population will produce only false-positive tests, and the definition of the disease will allow the epidemic to go on forever.
This strange new disease, officially named COVID-19, has none of its own symptoms. Fever and cough, previously blamed on uncountable viruses and bacteria, as well as environmental contaminants, are most common, as well as abnormal lung images, despite those being found in healthy people. Yet, despite the fact that only a minority of people tested will test positive (often less than 5%), it is assumed that this disease is easily recognized. If that was truly the case, the majority of people selected for testing by doctors should be positive.
The coronavirus test is based on PCR, a DNA manufacturing technique. When used as a test it does not produce a positive/negative result, but simply the number of cycles required to detect sufficient material to beat the arbitrary cutoff between positive and negative. If positive means infected and negative means uninfected, then there are cases of people going from infected to uninfected and back to infected again in a couple of days.
A lot of people say it is better to be safe than sorry. Better that some people are quarantined who are uninfected than risk a pandemic. But once people test positive, they are likely to be treated, with treatments similar to SARS. Doctors faced with what they believe is a deadly virus treat for the future, for anticipated symptoms, not for what they see today. This leads to the use of invasive oxygenation, high dose corticosteroids, antiviral drugs and more. In this case, some populations of those diagnosed (e.g. in China) are older and sicker than the general population and much less able to withstand aggressive treatment. After the SARS panic had subsided doctors reviewed the evidence, and it showed that these treatments were often ineffective, and all had serious side effects, such as persistent neurologic deficit, joint replacements, scarring, pain and liver disease. As well as higher mortality.

Advanced molecular technology has become a crucial tool for identifying new genes with importance in medicine, agriculture, animal production, health, environment, industry other related areas. Among the applications of molecular... more

Advanced molecular technology has become a crucial tool for identifying new genes with importance in medicine, agriculture, animal production, health, environment, industry other related areas. Among the applications of molecular techniques is important to highlight the use of the Polymerase Chain Reaction (PCR) in the identification and characterization of viral, bacterial, parasitic and fungal agents. PCR is a process used in molecular biology to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Mechanisms involved in this methodology are similar to those occurring in vivo during DNA replication. Through this paper we will review procedure, advantages, types & applications of PCR.

Histamine plays an important role in the regulation of various immunological functions. To evaluate the role of histamine in contact hypersensitivity, contact dermatitis was induced with dinitrofluorobenzene (DNFB) in histidine... more

Histamine plays an important role in the regulation of various immunological functions. To evaluate the role of histamine in contact hypersensitivity, contact dermatitis was induced with dinitrofluorobenzene (DNFB) in histidine decarboxylase knockout (HDC2/2) histamine-deficient and wild-type mice. The DNFB-induced increase of the ear thickness was significantly higher in HDC2/2 mice than in wild-type mice. Using flow cytometry, significantly lower percentages of CD4 1 Th and CD8 1 Tc cells, and significantly higher percentages of CD45R 1 B cells were observed in the regional lymph nodes in HDC2/2 mice than in wild-type mice. In the ear specimens of both groups, the majority of the infiltrating cells were neutrophils and macrophages at 24 and 48 h after challenge. Using immunohistochemistry, we observed significantly more CD45 1 leukocytes in HDC2/2 mice than in wild-type mice. The expression of Th1 (IL-2, IFN-c, TNF-a) and Th2 (IL-4) mRNAs was examined by quantitative real time RT-PCR in the ear samples. The levels of Th1 cytokine mRNAs both at 24 and 48 h after challenge and IL-4 mRNA at 48 h showed a significantly higher increase in HDC2/2 mice than in wild-type mice. These results suggest that histamine plays a negative immunoregulatory role in DNFB-induced contact hypersensitivity.

A closed tube isothermal Invader ® assay (Third Wave Technologies Inc., Madison, Wisconsin, USA) was adapted for the detection of African swine fever virus (ASFV) DNA. Several ASFV Invader ® assays were designed successfully and tested on... more

A closed tube isothermal Invader ® assay (Third Wave Technologies Inc., Madison, Wisconsin, USA) was adapted for the detection of African swine fever virus (ASFV) DNA. Several ASFV Invader ® assays were designed successfully and tested on a real-time PCR instrument (iCycler TM , BioRad). The assay exhibiting the lowest signal/noise ratio (VP73 ASFV Invader ® Assay) was analysed further using serial 10-fold dilutions of Lisbon 60 ASFV viral genome. The assay sensitivity was determined to be in the order of 2500 copies of ASFV DNA and showed a dynamic range of 4 logs, from 2.5 × 10 6 to 2500 copies. The high specificity of the test was demonstrated by the lack of cross-reactivity to the clinically similar but heterologous virus, classical swine fever virus. The sensitivity of the Invader ® assay is sufficient for the testing of acutely infected viremic animals in which the viral load will be high. The robustness and ease of use of the ASFV Invader ® assay, combined with the possibility to run and read the assay using simple and relatively inexpensive equipment, makes it suitable for laboratories lacking containment facilities and/or real-time PCR instrumentation or on a regional basis for on-site diagnosis close to putative sites of ASFV outbreaks. Agüero, M., Fernàndez, J., Romero, L., Sànchez-Masqaraque, C., Arias, M., Sànchez-Viscaíno, J.M., 2003. Highly sensitive PCR assay for routine diagnosis of African swine fever virus in clinical samples. A multi-site study for detection of the factor V (Leiden) mutation from genomic DNA using a homogenous invader microtiter plate fluorescence resonance energy transfer (FRET) assay.

The main autocrine/paracrine role of the active metabolite of vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) (1,25-D(3)), is inhibition of cell growth and induction of cell differentiation and/or apoptosis. Synthesis and degradation of the... more

The main autocrine/paracrine role of the active metabolite of vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) (1,25-D(3)), is inhibition of cell growth and induction of cell differentiation and/or apoptosis. Synthesis and degradation of the secosteroid occurs not only in the kidney but also in normal tissue or malignant extrarenal tissues such as the colon. Because 25-hydroxyvitamin D(3) 24-hydroxylase (CYP24A1) is considered to be the main enzyme determining the biological half-life of 1,25-D(3), we have examined expression of the CYP24A1 mRNA (by real-time RT-PCR) and protein (by immunohistochemistry) in normal human colon mucosa, colorectal adenomas, and adenocarcinomas in 111 patients. Although 76% of the normal and benign colonic tissue was either completely devoid of or expressed very low levels of CYP24A1, in the majority of the adenocarcinomas (69%), the enzyme was present at high concentrations. A parallel increased expression of the proliferation marker Ki-67 in the same sam...

Downregulation of the transcription factor AtMYB103 using transgenic technology results in early tapetal degeneration and pollen aberration during anther development in Arabidopsis thaliana. This paper describes the functional analysis of... more

Downregulation of the transcription factor AtMYB103 using transgenic technology results in early tapetal degeneration and pollen aberration during anther development in Arabidopsis thaliana. This paper describes the functional analysis of the AtMYB103 gene in three knock-out mutants. Two male sterile mutants, ms188-1 and ms188-2, were generated by ethyl-methane sulfonate (EMS) mutagenesis. A map-based cloning approach was used, and ms188 was mapped to a 95.8-kb region on chromosome 5 containing an AtMYB103 transcription factor. Sequence analysis revealed that ms188-1 had a pre-mature stop codon in the AtMYB103 coding region, whereas ms188-2 had a CCT fi CTT base-pair change in the first exon of AtMYB103, which resulted in the replacement of a proline by a leucine residue in the R2R3 domain. The third mutant, an AtMYB103 transposon-tagging line, also showed a male sterile phenotype. Allelism tests indicated that MS188 and AtMYB103 belong to the same locus. Cytological observation revealed defective tapetum development and altered callose dissolution in ms188 plants. Additionally, most of the microspores in mature anthers were degraded and surviving microspores lacked exine. AtMYB103 encoded an R2R3 MYB protein that is predominantly located in the nucleus. Real-time RT-PCR analysis indicated that the callase-related gene A6 was regulated by AtMYB103. Expression of the exine formation gene MS2 was not detected in mutant anthers. These results implicate that AtMYB103 plays an important role in tapetum development, callose dissolution and exine formation in A. thaliana anthers.

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The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. This... more

The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.

Decidualization of the human endometrium is critical for implantation, but the mechanisms involved are largely unknown. Activin subunits are expressed in endometrium during decidualization. From its known actions in cell differentiation... more

Decidualization of the human endometrium is critical for implantation, but the mechanisms involved are largely unknown. Activin subunits are expressed in endometrium during decidualization. From its known actions in cell differentiation and tissue remodelling, we hypothesized that activin A is involved in the paracrine regulation of decidualization. We examined the expression of activin receptors (ActRs) by semi-quantitative and real-time RT-PCR. mRNA for all ActR subtypes (Ia, Ib, IIa and IIb) was detected in endometrium, with maximal expression in the early secretory phase and in early pregnancy. ActR protein was localized exclusively to stromal and endothelial cells. This expression pattern was confirmed by in-situ hybridization. Activin bioavailability is locally regulated by its binding protein, follistatin, and also by the antagonist, inhibin. Inhibin competition for ActRII binding is enhanced by the binding protein, betaglycan. Follistatin and betaglycan were also detected in the endometrium, localized to stromal and epithelial cells. This co-expression of activin subunits, receptors and binding proteins indicates that stromal cells are capable of responding to activin, and that there is tight local regulation of activin action within the endometrium. As activin production is up-regulated in decidual cells, this provides further evidence for an involvement of activins during stromal cell decidualization.

Winter rye (Secale cereale L.) is known to be recalcitrant to tissue culture response (TCR). Moreover, the mechanisms controlling TCR are poorly recognized. In the present study, a Genetically Directed Differential Subtraction Chain... more

Winter rye (Secale cereale L.) is known to be recalcitrant to tissue culture response (TCR). Moreover, the mechanisms controlling TCR are poorly recognized. In the present study, a Genetically Directed Differential Subtraction Chain (GDDSC) strategy was used to isolate genomic regions associated with TCR. Two pairs of bulks, R-NR and E > 90–E < 25, were prepared, and for each pair, the bulk was used both as a tester and as a driver. After eight rounds of subtraction, 45 unique GDDSC products were obtained. To verify the connection between GDDSCs and TCR two approaches were applied: Real-Time RT-PCR analysis and genetic mapping. The expression profiles of four out of six investigated products agrees with the phenotype and subtraction direction. Two from the developed GDDSC-SCAR markers showed polymorphism in lines L9 and L318, the parental components of a mapping population. The polymorphic SCAR GDDSC markers were mapped on the rye chromosome 4R (SCAR-GDDSC NR 440BamHI) and 6R (SCAR-GDDSC E < 25 340BamHI). The marker E < 25_340B9 was localized in the border region of QTL for embryogenic callus production.

A quantitative two-step multiplex real-time reverse transcriptase (RT-) PCR assay for the simultaneous detection of genogroup I (GI) and genogroup II (GII) noroviruses (NoVs) is described below. A murine norovirus 1 (MNV-1) real-time PCR... more

A quantitative two-step multiplex real-time reverse transcriptase (RT-) PCR assay for the simultaneous detection of genogroup I (GI) and genogroup II (GII) noroviruses (NoVs) is described below. A murine norovirus 1 (MNV-1) real-time PCR detection assay described recently was integrated successfully into the multiplex assay, making it possible to detect GI and GII NoVs and MNV-1 in one reaction tube with MNV-1 plasmid DNA as real-time PCR internal amplification control (IAC).

Dopamine, a neurotransmitter present in all vertebrates, is involved in processes such as motor function, learning and behavior, sensory activities, and neuroendocrine control of pituitary hormone release. In the female eel, we have... more

Dopamine, a neurotransmitter present in all vertebrates, is involved in processes such as motor function, learning and behavior, sensory activities, and neuroendocrine control of pituitary hormone release. In the female eel, we have analyzed how gonadal steroids regulate brain expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine. TH mRNA levels were assayed by quantitative real-time RT-PCR. THpositive nuclei were further localized by in situ hybridization (ISH) and immunohistochemistry, and the location of TH nuclei that project to the pituitary was determined using DiI retrograde tracing. Chronic in vivo treatment with testosterone increased TH mRNA specifically in the periglomerular area of the olfactory bulbs, and in the nucleus preopticus anteroventralis (NPOav). NPOav was DiI-labeled showing that this nucleus is hypophysiotropic in the eel. The non-aromatizable 5 -dihydrotestosterone gave identical results in both areas, whereas estradiol-17 had no stimulatory effect, showing that the observed stimulatory effects of testosterone were androgendependent. In teleosts, dopamine neurons originating from the NPOav directly inhibit gonadotropic function, and our results indicate an androgen-dependent positive feedback on this neuroendocrine control in the eel. In mammals, dopamine interneurons in the olfactory bulbs are involved in the enhancement of olfactory sensitivity and discrimination. Our results in the European eel suggest an androgen-dependent stimulation of olfactory processing, a sensory function believed to be important in eel navigation during its reproductive migration towards the oceanic spawning grounds. To our knowledge, this is the first evidence from any vertebrate of an androgendependent effect on dopaminergic activity in the olfactory bulbs, providing a new basis for understanding the regulation by gonadal steroids of central dopaminergic systems in vertebrates.

The real-time reverse transcription polymerase chain reaction (RT-PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. This combines the nucleic acid... more

The real-time reverse transcription polymerase chain reaction (RT-PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. This combines the nucleic acid amplification and detection steps into one homogeneous assay and obviates the need for gel electrophoresis to detect amplification products. Use of appropriate chemistries and data analysis eliminates the need for Southern blotting or DNA sequencing for amplicon identification. Its simplicity, specificity and sensitivity, together with its potential for high throughput and the ongoing introduction of new chemistries, more reliable instrumentation and improved protocols, has made real-time RT-PCR the benchmark technology for the detection and/or comparison of RNA levels.

In this study different instruments and methods used for tissue homogenization, RNA extraction and quantitative PCR (qPCR) based detection of grapevine RNA viruses were evaluated. Semi-automated and automated homogenization techniques... more

In this study different instruments and methods used for tissue homogenization, RNA extraction and quantitative PCR (qPCR) based detection of grapevine RNA viruses were evaluated. Semi-automated and automated homogenization techniques were compared to process samples from grapevine petioles and cambial tissue. Four different high throughput automated nucleic acid extraction platforms were compared with the RNeasy plant extraction kit for their capacity and efficiency of extracting viral RNA from grapevine infected tissues. The RNA prepared from each extraction platform was then used as template for a comparative analysis of qPCR by One Step RT-qPCR, Two Step RT-qPCR and low density array (LDA) detection. This study showed that a thorough homogenization of grapevine tissues using the Tissue Lyser as well as DNase digestion of the purified RNA prior to cDNA synthesis improved the virus detection and yielded the lowest quantitation cycle (Cq) values in RT-qPCR. Comparison of different RNA extraction methods showed that methods implementing the magnetic bead-based technology were superior to other methods used. Comparing different qPCR detection methods, One

To eradicate the Hepatitis C Virus from the bodies of the infected individuals Interferon and Ribavirin based therapy is used. HCV is highly prevalent in District Mardan Khyber Pakhtunkhwa that is why it is important to determine the... more

To eradicate the Hepatitis C Virus from the bodies of the infected individuals Interferon and Ribavirin based therapy is used. HCV is highly prevalent in District Mardan Khyber Pakhtunkhwa that is why it is important to determine the response of standard interferon based therapy in Chronic HCV patients of this region. A total of 215 patients were selected for interferon based therapy. The patients were selected from three different Tehsil of District Mardan. After confirmation of active HCV infection by q-PCR, standard interferon with Ribavirin was given to patients for 6 months. After completion of therapy, end of treatment virologic response (ETR) was calculated. After completion of the 6 months long therapy, the results obtained were as. Out of total 215 patients, 168 (78.13%) were negative for HCV RNA and showing end of treatment response (ETR) while 47 (21.86%) were positive for HCV RNA and did not show ETR. In Tehsil Mardan, out of 102 patients who had completed therapy, 76 patients (74.51%) showed ETR and 26 (25.49%) did not show the ETR. In Tehsil Kattlang, we found that out of total 51 patients who had taken 6 months therapy, 41 (80.39%) were negative for HCV RNA and 10 (19.61%) were resistant to therapy while in Tehsil Tkhatbhai, out of 62, 50 (80.64%) were negative and 12 (19.35%) were positive. The above discussion shows that antiviral therapy against HCV infection in chronic HCV patients of District Mardan KPK province is 78.13%. The high response rate may be due to the prevalence of genotypes 2 and 3.

In order to determine the suitability of reference or housekeeping genes as internal controls in real-time reverse transcriptase PCR (RT-PCR) assays for quantification of target mRNAs, we studied the levels of expression of four candidate... more

In order to determine the suitability of reference or housekeeping genes as internal controls in real-time reverse transcriptase PCR (RT-PCR) assays for quantification of target mRNAs, we studied the levels of expression of four candidate reference genes in maritime pine by real-time RT-PCR. The expression levels obtained for glyceraldehyde-3-phosphate-dehydrogenase, 18S ribosomal RNA, eukaryotic translation initiation factor eIF4AII and ubiquitin in nine stages of embryo development revealed that none of the genes tested proved to be suitable as an internal control. Copy number quantification of the four transcripts showed an average relative variation of seven fold. We propose that the combination of a precise method for RNA quantification, internal controls for monitoring RT reaction and PCR efficiency and a robust external standard curve can guarantee a reliable absolute quantification of mRNA transcripts in real time RT-PCR. This approach may avoid the controversy in the use of housekeeping genes and may assume special significance in tissues undergoing developmental changes.

The chondrogenic differentiation of bone marrow-derived human mesenchymal stem cells (MSCs) in a collagen type I hydrogel, which is in clinical use for matrix-based autologous chondrocyte transplantation (ACT), was investigated. Collagen... more

The chondrogenic differentiation of bone marrow-derived human mesenchymal stem cells (MSCs) in a collagen type I hydrogel, which is in clinical use for matrix-based autologous chondrocyte transplantation (ACT), was investigated. Collagen hydrogels with 2.5 3 10 5 MSCs/mL were fabricated and cultured for 3 weeks in a serum-free, defined, chondrogenic differentiation medium containing 10 ng/mL TGF-b1 or 100 ng/mL BMP-2. Histochemistry revealed morphologically distinct, chondrocytelike cells, surrounded by a sulfated proteoglycan-rich extracellular matrix in the TGF-b1 and BMP-2 treated group, with more elongated cells seen in the BMP-2 treated group. Immunohistochemistry detected collagen type II (Col II) in the TGF-b1 and BMP-2 treated group. Collagen type X (Col X) staining was positive in the TGF-b1 but only very weak in the BMP-2 treated group. RT-PCR analyses revealed a specific chondrogenic differentiation with the expression of the cartilage specific marker genes Col II, Col X, and aggrecan (AGN) in the TGF-b1 and the BMP-2 treated group, with earlier expression of these marker genes in the TGF-b1 treated group. Interestingly, MSC-gels cultured in DMEM with 10% FBS (control) indicated few isolated chondrocyte-like cells but no expression of Col II or Col X could be detected. The results show, that MSCs cultured in a collagen type I hydrogel are able to undergo a distinct chondrogenic differentiation pathway, similar to that described for MSCs cultured in high-density pellet cultures. These findings are valuable in terms of ex vivo predifferentiation or in situ differentiation of MSCs in collagen hydrogels for articular cartilage repair. 2007 Wiley Periodicals, Inc. J Biomed Mater Res 83A: [626][627][628][629][630][631][632][633][634][635] 2007

Caveolae, a specialized form of lipid rafts, are cholesterol-and sphingolipid-rich membrane microdomains implicated in potocytosis, endocytosis, transcytosis, and as platforms for signal transduction. One of the major constituents of... more

Caveolae, a specialized form of lipid rafts, are cholesterol-and sphingolipid-rich membrane microdomains implicated in potocytosis, endocytosis, transcytosis, and as platforms for signal transduction. One of the major constituents of caveolae are three highly homologous caveolin isoforms (caveolin-1, caveolin-2, and caveolin-3). The present study expands the analysis of caveolin isoform expression in C6 glioma cells. Three complementary approaches were used to assess their differential expression during the dibutyryl-cyclic AMP-induced differentiation of C6 cells into an astrocyte-like phenotype. Immunoblotting, conventional RT-PCR, and real-time RT-PCR analysis established the expression of the caveolin-3 isoform in C6 cells, in addition to caveolin-1 and caveolin-2. Similar to the other isoforms, caveolin-3 was associated with light-density, detergent-insoluble caveolae membrane fractions obtained using sucrose-density gradient centrifugation. The three caveolin isoforms display different temporal patterns of mRNA/protein expression during the differentiation of C6 cells. Western blot and real-time RT-PCR analysis demonstrate that caveolin-1 and caveolin-2 are up-regulated during the late stages of the differentiation of C6 cells. Meanwhile, caveolin-3 is gradually down-regulated during the differentiation process. Indirect immunofluorescence analysis via laser-scanning confocal microscopy reveals that the three caveolin isoforms display similar subcellular distribution patterns. In addition, co-localization of caveolin-1/caveolin-2 and caveolin-1/caveolin-3 was detected in both C6 glioma phenotypes. The findings reveal a differential temporal pattern of caveolin gene expression during phenotypic differentiation of C6 glioma cells, with potential implications to developmental and degenerative events in the brain.

Background: Norovirus (NoV) is recognised as one of the most common causes of foodborne infections. Contaminated shellfish, food, water and hospitals are well documented sources of the virus. Objective: NoV in diarrheic children has not... more

Background: Norovirus (NoV) is recognised as one of the most common causes of foodborne infections. Contaminated shellfish, food, water and hospitals are well documented sources of the virus. Objective: NoV in diarrheic children has not previously been investigated in Istanbul, Turkey, hence the aim of this study was to detect and investigate the frequency and phylogeny of human NoV genogroups I and II in children with acute gastroenteritis. Study design: 238 stool samples were collected from diarrheic children from 2 hospitals (Cerrahpasa Medical School and Haseki) in Istanbul and analysed by ELISA, RT-PCR and real-time RT-PCR using both SYBR Green and probe-based assays for human NoV. Primers targeting the RNA-polymerase gene were used for RT-PCR to allow DNA sequencing of Turkish NoV strains and phylogenetic analysis to be performed. Results: NoV GII was detected in 36 (15.1%) of 238 samples by SYBR Green real-time RT-PCR, 10.9% by a probe-based real-time RT-PCR and 10.5% by ELISA (Ridascreen). Genogroup II (GII) the Turkish NoVs clustered with including GII4 (72.2%), GII16 (5.5%), GIIb (16.7%) and GIIe (5.5%). Two variants of GII4 (GII4-2006b and GII4-2008), GII16 and recombinant noroviruses (GIIb and GIIe) were identified. Conclusion: This study shows a high frequency and genetic diversity of NoV GII infections in children with acute gastroenteritis in Istanbul, Turkey. .tr (H. Yilmaz). and water. Norovirus is the most common cause of food-borne illness accounting for 54% of food-borne disease outbreaks in 2006 in the USA. 9 However, the frequency and phylogeny of NoV in diarrheic children have not previously been investigated in Istanbul, Turkey.

This study was conducted to characterize the immunological parameters of chickens vaccinated with two formulated inactivated vaccines, water in oil (WO) and water in oil in water (WOW), prepared from velogenic Newcastle disease virus... more

This study was conducted to characterize the immunological parameters of chickens vaccinated with two formulated inactivated vaccines, water in oil (WO) and water in oil in water (WOW), prepared from velogenic Newcastle disease virus (vNDV) genotype VIIj isolated from outbreak among vaccinated chickens. Six groups (G1–G6) of commercial broiler chickens were established (n = 20). The G1–G3 were received homologous (WO and WOW) and heterologous (LaSota) inactivated vaccines, respectively. The G4 was vaccinated with live heterologous (LaSota) vaccine, while G5 and G6 were kept as control positive and control negative non-vaccinated groups. The antibody titers were measured against vNDV and LaSota antigens using hemagglutination inhibition (HI) test, the cytokine gene expressions of IFNγ, IL1β, IL4, IL6, IL8, and IL18 were quantified using real-time RT-PCR, and the virus shedding was titrated on chicken embryo fibroblast cells after challenging by vNDV. The classical clinical signs and 100% mortality were observed only in G5 after vNDV challenging. The highest HI titers were detected in G1, G2, and G3 using NDV/168 antigen with no significant differences among them. These groups showed higher HI titer than G4 (2-4log2). Cytokine gene expression of IFNγ, IL1, IL6, IL8, and IL18 were significantly downregulated in vaccinated chickens with upregulation of IL4 than non-vaccinated challenge group. Viral shedding titers were significantly (0.0001, p ≤ 0.001) reduced in all samples form vaccinated chickens. In conclusion, the prepared vaccines produced highly efficient immunological responses and could be used for controlling the NDV infection.