TRIzol LS Reagent Research Articles

Endometriosis does not Alter Aromatase Gene Expression (CYP19A1) in Mural Lutein-granulosa Cells of Women Undergoing Assisted Reproduction Techniques – a Pilot Study

Purpose The aim of this study was to quantify aromatase gene expression in mural lutein-granulosa cells of women with endometriosis undergoing assisted reproduction techniques (ART, IVF or ICSI). Methods: a case-control study was performed on 11 women with endometriosis (all stages, ASRM criteria) and 11 women with male or tubal causes of infertility undergoing ART. There was no difference between the groups regarding age, amount of gonadotrophins used, days of induction, follicles, and eggs collected. Mural lutein-granulosa cells were harvested from pre-ovulatory follicles during oocyte retrieval and correctly isolated. After cells lyses and storage into Trizol LS Reagent®, RNA extraction and cDNA synthesis were performed. Quantification of relative gene expression for CYP19A1 (aromatase) was performed by real time PCR, using SYBR Green reagents. All experiments were performed in duplicates. Results there was no difference between the groups in the quantitative gene expression of CYP19A1 (aromatase) gene on mural lutein-granulosa cells (P>.05, Mann Whitney). Conclusions These results suggest that this enzyme, aromatase, may have a complex and refined control of its gene expression on this population of granulosa cells, and in spite of previous evidence showing its reduced activity on these cells in endometriosis, gene expression per se may not be affected by the disease.

First report of Pineapple mealybug wilt associated virus‐3 infecting pineapple in Cuba

Pineapple (Ananas comosus) is a common crop in tropical and subtropical areas of the world. Crop yields are seriously affected by mealybug wilt of pineapple (MWP), a viral disease with mealybugs (Dysmicoccus spp.) as vectors (Sether et al., 2005). Viruses associated with MWP are members of the genus Ampelovirus, family Closteroviridae. In Hawaii, Pineapple mealybug wilt-associated virus- (PMWaV-1) infection has been correlated with 5 to 15% of ratoon crop yield reduction and losses of up to 30% of production due to premature or asynchronous fruit ripeness. However, in that region, the most widespread virus species is PMWaV-2, which causes up to 100% fruit loss (Sether & Hu, 2002). Conversely, PMWaV-2 is uncommon in Australia, a country where MWP also causes major reduction of pineapple fruit yield. In Australia, MWP symptoms are strongly correlated with infections by PMWaV-3 alone or by both PMWaV-1 and -3 (Gambley et al., 2008). Due to the high nucleotide identity and the conserved genome organization between PMWaV-1 and PMWaV-3, it is considered that these two viruses have similar deleterious effects on either growth rate or pineapple fruit yield in Hawaii (Sether et al., 2005, 2009). Currently, there are 5,310 ha of pineapple orchards in Cuba, with annual fruit production that reached 28,908 tonnes in 2009. MWP disease is an economic problem for pineapple production in the island, causing up to 40% crop losses (Anonymous, 1989). PMWaV-2 was first detected in a diseased pineapple plant from Ciego de Avila in 1998, with molecular characterisation further provided by Borroto-Fernandez et al., (2007). During a survey for PMWaVs in 2009, thirty pineapple plants showing typical symptoms of MWP (foliar reddening, leaves with tips curved down and dieback) were collected in the Island of Youth, western region of the country (Fig. 1). Total RNA was extracted using the TRIzol LS Reagent kit (Invitrogen, Scotland, UK). RT-PCR assays for PMWaV-1, PMWaV-2 and PMWaV-3 detection were performed using the 225/226, 223/224 and 263/264 primer pairs, respectively; these amplify fragments corresponding to the HSP70h protein ORF of each viral species (Sether et al., 2005). Fragments of expected size for PMWaV-2 (c. 610 bp) and PMWaV-3 (c. 490 bp) were simultaneously amplified from seventeen plants with appropriate symptoms. Amplicons corresponding to PMWaV-1 were not obtained. DNA bands were purified, ligated to pGEM®-T Easy vector (Promega, Madison, USA) and two individual clones derived from each virus per infected plant were sequenced. PMWaV-3 derived amplicons shared ≥ 98% sequence identity with each other, and a representative sequence was deposited in GenBank (PMWaV-3 Cu, Accession No. GU563497). Sequence comparisons showed the closest nucleotide identity (98%) to PMWaV-3 from Hawaii (DQ399259). On the other hand, a representative sequence of PMWaV-2 Cu amplicons (FN825676) showed the closest nucleotide identity (99%) to PMWaV-2 from Taiwan (EU769115.1) and Thailand (EU016675.1). Phylogenetic analysis based on the PMWaV-3 amplicon sequence of Cuban and reference closteroviruses, grouped PMWaV-3 Cu and PMWaV-3 isolates from Hawaii and Thailand within the same phylogenetic cluster (Fig. 2). The phylogenetic tree also supported the sequence divergence of the cluster composed by PMWaV-3 and PMWaV-1 from that of PMWaV-2Hw, GLRaV-1 and GLRaV-3 (type member of the genus Ampelovirus) as previously observed (Sether et al., 2009). Gathering all these data, this is the first report of the presence of the PMWaV-3 in Cuban pineapple fields and in the Caribbean basin. Noteworthy is that pineapple plants affected by MWP were infected by both PMWaV-3 and PMWaV-2 which suggests that a complex of ampeloviruses may be widespread in Cuban pineapple fields. Results support the need to implement certification procedures for pineapple propagation materials to reduce the economic impact of MWP disease on pineapple crops in Cuba.

A Novel Protein Acts as a Negative Regulator of Prophenoloxidase Activation and Melanization in the Freshwater Crayfish Pacifastacus leniusculus

Melanization is an important immune component of the innate immune system of invertebrates and is vital for defense as well as for wound healing. In most invertebrates melanin synthesis is achieved by the prophenoloxidase-activating system, a proteolytic cascade similar to vertebrate complement. Even though melanin formation is necessary for host defense in crustaceans and insects, the process needs to be tightly regulated because of the hazard to the animal of unwanted production of quinone intermediates and melanization in places where it is not suitable. In the present study we have identified a new melanization inhibition protein (MIP) from the hemolymph of the crayfish, Pacifastacus leniusculus. Crayfish MIP has a similar function as the insect MIP molecule we recently discovered in the beetle Tenebrio molitor but interestingly has a completely different sequence. Crayfish MIP as well as Tenebrio MIP do not affect phenoloxidase activity in itself but instead interfere with the melanization reaction from quinone compounds to melanin. Importantly, crayfish MIP in contrast to Tenebrio MIP contains a fibrinogen-like domain, most similar to the substrate recognition domain of vertebrate l-ficolins. Surprisingly, an Asp-rich region similar to that found in ficolins that is likely to be involved in Ca2+ binding is present in crayfish MIP. However, crayfish MIP did not show any hemagglutinating activity as is common for the vertebrate ficolins. A mutant form of MIP with a deletion lacking four Asp amino acids from the Asp-rich region lost most of its activity, implicating that this part of the protein is involved in regulating the prophenoloxidase activating cascade. Overall, a new negative regulator of melanization was identified in freshwater crayfish that shows interesting parallels with proteins (i.e. ficolins) involved in vertebrate immune response.

IMP Dehydrogenase Type 1 Associates with Polyribosomes Translating Rhodopsin mRNA

IMP dehydrogenase (IMPDH) catalyzes the pivotal step in guanine nucleotide biosynthesis. Here we show that both IMPDH type 1 (IMPDH1) and IMPDH type 2 are associated with polyribosomes, suggesting that these housekeeping proteins have an unanticipated role in translation regulation. This interaction is mediated by the subdomain, a region of disputed function that is the site of mutations that cause retinal degeneration. The retinal isoforms of IMPDH1 also associate with polyribosomes. The most common disease-causing mutation, D226N, disrupts the polyribosome association of at least one retinal IMPDH1 isoform. Finally, we find that IMPDH1 is associated with polyribosomes containing rhodopsin mRNA. Because any perturbation of rhodopsin expression can trigger apoptosis in photoreceptor cells, these observations suggest a likely pathological mechanism for IMPDH1-mediated hereditary blindness. We propose that IMPDH coordinates the translation of a set of mRNAs, perhaps by modulating localization or degradation.

Rarity of Influenza A Virus in Spring Shorebirds, Southern Alaska

Rarity of Influenza A Virus in Spring Shorebirds, Southern Alaska

Characterization of a Novel Caenorhabditis elegans Prolyl 4-Hydroxylase with a Unique Substrate Specificity and Restricted Expression in the Pharynx and Excretory Duct

Collagen prolyl 4-hydroxylases (C-P4Hs) have a critical role in collagen synthesis, since 4-hydroxyproline residues are necessary for folding of the triple-helical molecules. Vertebrate C-P4Hs are alpha(2)beta(2) tetramers in which the beta subunit is identical to protein-disulfide isomerase (PDI). Three isoforms of the catalytic alpha subunit, PHY-1, PHY-2, and PHY-3, have been characterized from Caenorhabditis elegans, PHY-1 and PHY-2 being responsible for the hydroxylation of cuticle collagens, whereas PHY-3 is predicted to be involved in collagen synthesis in early embryos. We have characterized transcripts of two additional C. elegans alpha subunit-like genes, Y43F8B.4 and C14E2.4. Three transcripts were generated from Y43F8B.4, and a polypeptide encoded by one of them, named PHY-4.1, assembled into active (PHY-4.1)(2)/(PDI-2)(2) tetramers and PHY-4.1/PDI-2 dimers when coexpressed with C. elegans PDI-2 in insect cells. The C14E2.4 transcript was found to have a frameshift leading to the absence of codons for two residues critical for P4H catalytic activity. Thus, C. elegans has altogether four functional C-P4H alpha subunits, PHY-1, PHY-2, PHY-3, and PHY-4.1. The tetramers and dimers containing recombinant PHY-4.1 had a distinct substrate specificity from the other C-P4Hs in that they hydroxylated poly(l-proline) and certain other proline-rich peptides, including ones that are expressed in the pharynx, in addition to collagen-like peptides. These data and the observed restricted expression of the phy-4.1 transcript and PHY-4.1 polypeptide in the pharyngeal gland cells and the excretory duct suggest that in addition to collagens, PHY-4.1 may hydroxylate additional proline-rich proteins in vivo.

Optimization of an RNA isolation procedure from plasma samples

In this study, we aimed to optimize the isolation procedure of circulating RNA from large volumes of plasma. Simultaneously, the stability and integrity of RNA from plasma samples were examined. To investigate the isolation of circulating RNA, reverse transcription-PCR analysis in combination with capillary electrophoresis was used. The presence of amplifiable RNA in plasma from healthy volunteers and from breast cancer patients was analyzed. We found that circulating RNA in plasma was highly fragmented and degraded. Plasma RNA was most efficiently isolated from large volumes of samples after introducing the step of plasma concentration by evaporation and by using TRIzol LS reagent. A single freeze/thaw process had no significant effect on RNA integrity and quantity of plasma RNA. The average amount of RNA in plasma from breast cancer patients was lower than in plasma from healthy volunteers. The concentrating of large volumes of plasma facilitates isolation of plasma RNA and yields amplifiable RNA for at least fragments that are up to 310 bp long.

Neutrophil Elastase Converts Human Immature Dendritic Cells into Transforming Growth Factor-β1-Secreting Cells and Reduces Allostimulatory Ability

During microbial infection, neutrophils (polymorphonuclear leukocytes; PMNs) activate dendritic cells (DCs). However, early reports illustrated that neutrophil-derived mediators may suppress responses to mitogens. In the present study, we investigated the mechanism used by PMNs to modulate the immunostimulatory ability of DCs. Autologous syngeneic PMNs decreased T-cell proliferation induced by allogeneic DCs. Culture supernatant (CS) derived from PMNs also decreased allostimulation ability of immature DCs and increased the expression of transforming growth factor (TGF)-beta1 on DCs. A TGF-beta1 monoclonal antibody, a CD40 monoclonal antibody, or a serine protease inhibitor reversed the effect of PMN CS on DC allostimulatory ability. Furthermore, elastase reproduced the inhibitory effect of PMN CS on DC allostimulatory ability and the TGF-beta1 production. The role of elastase was confirmed by examining PMN CS from two patients with cyclic neutropenia, a disease due to mutations in the neutrophil elastase gene. These PMN CS samples had reduced elastase activity and were unable to increase DC TGF-beta1 production. Moreover, elastase and PMN CS induced IkappaBalpha degradation in DCs. We conclude that PMNs decrease DC allostimulatory ability via production of elastase leading to a switch of immature DCs into TGF-beta1-secreting cells.

Evidence of the co‐circulation of influenza h1n1, h1n2 and h3n2 viruses in the pig population of Korea

SWINE influenza is an acute, highly infectious respiratory disease of pigs, characterised by a sudden onset of coughing, high fever, nasal discharge, dyspnoea and weight loss throughout the whole herd (Easterday and Van Reeth 1999). Swine influenza virus causes large economic losses in the swine industry, and zoonotic transmission of swine influenza viruses from pigs to human beings has been reported (Dasco and others 1984, Wentworth and others 1994). Swine influenza viruses belong to the family Orthomyxoviridae, type A, and contain eight single-stranded RNA segments. Three subtypes, H1N1, H1N2 and H3N2 currently circulate in most pig populations throughout the world. The first virus isolated was an H1N1 subtype, and is known as the causative agent of classical swine influenza. However, the H1N1 subtype that is currently circulating is a variant of this (Roberts and others 1987). H1N2 influenza viruses were isolated from pigs in the USA in 1999 (Karasin and others 2000), in France in 1987 (Gourreau and others 1994), and in Japan in 1978 to 1980 and 1989 to 1992 (Ito and others 1998). H3N2 influenza viruses have caused respiratory disease and sometimes abortion among pigs in the USA since 1998 (Webby and others 2000). In the Republic of Korea, three subtypes, H1N1, H1N2 and H3N2, were identified from pigs’ lungs obtained from 2000 to 2002, in 2003, and in 1998, respectively (Song and others 2003, Choi and others 2004, Jung and Chae 2005). Since 2002, there have been no reports about the influenza virus subtypes circulating in the Korean pig population. This study aimed to examine the prevalence of type A influenza virus subtypes in pigs in the Republic of Korea from 2003 to 2004, and also to identify whether all three subtypes of influenza A viruses are co-circulating in the Korean pig population. A total of 159 pigs, ranging in age from four to 20 weeks, from 140 farms, between January 2003 and December 2004, were submitted from across Korea to the Department of Veterinary Pathology, Seoul National University, for diagnosis of swine influenza virus infection. All the herds had severe outbreaks of influenza-like respiratory disease, with pigs showing fever, anorexia, coughing, nasal and ocular discharge, and weight loss, especially during the winter season; 82 cases were submitted in 2003 and 77 cases were submitted in 2004. There were 49 cases from Kyounggi province, 37 from Chungcheung province, 38 from Cholla province and 35 from Kyoungsang province. Lung samples were taken from pigs showing signs of respiratory illness, and influenza virus was isolated from these using Madin-Darby canine kidney (MDCK) cells. Cytopathic viruses were mostly detected after three passages in MDCK cells from homogenates of lung tissue. RNA was extracted from the cells showing cytopathic effects with Trizol LS Reagent (Gibco BRL) according to the manufacturer’s instructions. The subtype of swine influenza virus was determined by reverse transcriptase-PCR (RT-PCR) as previously described by Jung and Chae (2005). For haemagglutinin (HA) subtyping, one set of primers (H1F:5'-GGGACATGTTACCCAGGAGAT-3', nucleotides 345 to 365; H1R: 5'-CTGCTTGACCTCTCACTTTGG-3', nucleotides 756 to 736) was designed to amplify 411 base pairs (bp) of the Veterinary Record (2007) 161, 104-105

An Efficient and Rapid Method for Recovery of Norovirus from Food Associated with Outbreaks of Gastroenteritis

Noroviruses have emerged as the most common cause of foodborne outbreaks of acute nonbacterial gastroenteritis. In this study, two methods for the extraction of viruses from deli ham were compared. Using both methods, as little as 1 to 10 reverse transcription (RT)–PCR units of inoculated norovirus and enterovirus could be detected by nested RT-PCR assays. The fastest and most efficient extraction method based on TRIzol LS Reagent was chosen to identify viruses in food items associated with three different outbreaks. Norovirus was detected using nested (real time) RT-PCR assays that target the genome region routinely used for diagnosis of human cases, thereby facilitating the comparison of sequences detected in food and clinical specimens. For one outbreak, a norovirus sequence (163/163 nucleotides) identical to those detected in clinical samples was found on salami sliced by a food handler with a recent history of gastroenteritis. For the other two outbreaks, norovirus was detected on leftovers of spareribs and ham, but fecal samples from affected persons were not available. The methods used in this study may be useful in future outbreak investigations because the extraction method is easy to perform and suitable for this particular type of food and the detection method facilitates direct comparison of patient and food data.

A Conserved Subtilisin Protease Identified in Babesia divergens Merozoites

Invasion of erythrocytes is an integral part of the Babesia divergens life cycle. Serine proteases have been shown to play an important role in invasion by related Apicomplexan parasites such as the malaria parasite Plasmodium falciparum. Here we demonstrate the presence of two dominant serine proteases in asexual B. divergens using a biotinylated fluorophosphonate probe. One of these active serine proteases (p48) and its precursors were recognized by anti-PfSUB1 antibodies. These antibodies were used to clone the gene encoding a serine protease using a B. divergens cDNA library. BdSub-1 is a single copy gene with no introns. The deduced gene product (BdSUB-1) clearly belongs to the subtilisin superfamily and shows significant homology to Plasmodium subtilisins, with the highest degree of sequence identity around the four catalytic residues. Like subtilisin proteases in other Apicomplexan parasites, BdSUB-1 undergoes two steps of processing during activation in the secretory pathway being finally converted to an active form (p48). The mature protease is concentrated in merozoite dense granules, apical secretory organelles involved in erythrocyte invasion. Anti-PfSUB1 antibodies have a potent inhibitory effect on erythrocyte invasion by B. divergens merozoites in vitro. This report demonstrates conservation of the molecular machinery involved in erythrocyte invasion by these two Apicomplexan parasites and paves the way for a comparative analysis of other molecules that participate in this process in the two parasites.

PPARβ/δ Regulates Paneth Cell Differentiation Via Controlling the Hedgehog Signaling Pathway

All 4 differentiated epithelial cell types found in the intestinal epithelium derive from the intestinal epithelial stem cells present in the crypt unit, in a process whose molecular clues are intensely scrutinized. Peroxisome proliferator-activated receptor beta (PPARbeta) is a nuclear hormone receptor activated by fatty acids and is highly expressed in the digestive tract. However, its function in intestinal epithelium homeostasis is understood poorly. To assess the role of PPARbeta in the small intestinal epithelium, we combined various cellular and molecular approaches in wild-type and PPARbeta-mutant mice. We show that the expression of PPARbeta is particularly remarkable at the bottom of the crypt of the small intestine where Paneth cells reside. These cells, which have an important role in the innate immunity, are strikingly affected in PPARbeta-null mice. We then show that Indian hedgehog (Ihh) is a signal sent by mature Paneth cells to their precursors, negatively regulating their differentiation. Importantly, PPARbeta acts on Paneth cell homeostasis by down-regulating the expression of Ihh, an effect that can be mimicked by cyclopamine, a known inhibitor of the hedgehog signaling pathway. We unraveled the Ihh-dependent regulatory loop that controls mature Paneth cell homeostasis and its modulation by PPARbeta. PPARbeta currently is being assessed as a drug target for metabolic diseases; these results reveal some important clues with respect to the signals controlling epithelial cell fate in the small intestine.

Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-Time and Conventional Reverse Transcription–PCR

Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system, NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription-PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding beta-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures. For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.

Characterization of the Condensin Component Cnap1 and Protein Kinase Melk as Novel E2F Target Genes Down-regulated by 1,25-Dihydroxyvitamin D3

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) has potent antiproliferative effects characterized by a hampered G(1)/S transition. cDNA microarrays were used to monitor expression of 21,492 genes in MC3T3-E1 mouse osteoblasts at 1, 6, 12, 24, and 36 h after treatment with 1,25(OH)(2)D(3). Statistical analysis revealed a cluster of genes that were strongly down-regulated by 1,25(OH)(2)D(3) and which not only function in cell cycle regulation and DNA replication but also mediate checkpoint control, DNA repair, chromosome modifications, and mitosis. Because many of these genes were shown earlier to be regulated by the transcriptional repressor E2F4, the intergenic regions of these 1,25(OH)(2)D(3)-down-regulated genes were searched for the presence of E2F binding sites. This led to the characterization of two novel E2F target genes, chromosome condensation-related SMC-associated protein 1 (Cnap1) and maternal embryonic leucine zipper kinase (Melk). Transfection studies and site-directed mutagenesis confirmed Cnap1 and Melk to be bona fide E2F targets. Repression of Cnap1 and Melk by 1,25(OH)(2)D(3) was confirmed not only in MC3T3-E1 cells but also in several other bone-unrelated cell types. This down-regulation as well as the antiproliferative effect of 1,25(OH)(2)D(3) depended on the pocket proteins p107 and p130 because 1,25(OH)(2)D(3) failed to repress these E2F target genes and lost its antiproliferative action in p107(-/-);p130(-/-) cells but not in pRb(-/-) cells.

PC26 COMPARISON OF TOLL LIKE RECEPTOR- AND TH1-/TH2-RELATED MRNA LEVELS IN SPANISH VERSUS GERMAN PREGNANT WOMEN AND NEONATES

Introduction: Epidemiological studies indicate that the exposure to microbes in early life is associated with a reduced risk to develop allergic disease. Toll-like receptors bind microbial compounds thereby triggering innate immune responses. We asked whether the mRNA expression levels of toll like receptors (TLR) and Th1-/Th2-related markers differ in placental cord blood of Spanish and German neonates. In a second step, cord blood mRNA expressions were correlated with mRNA levels in maternal blood. Methods: 47 pregnant women from Granada, Spain (n=23, mean age 29 yrs) and Munich, Germany (n=24, mean age 32 yrs) were participants of the NUHEAL study and were selected for this study. Cord blood and maternal peripheral blood were obtained immediately after delivery and placed in Trizol LS Reagent (In-vitrogen Lifesciences). After RNA-extraction and cDNA synthesis, IL-4, IL-13, CRTH2, CCR4 (Th2-associated), IFN-y, CXCR3, (Th1-associated) and IL-1, TLR2 and TLR4 (TLR) were quantified by real-time RT PCR (iCycler, Biorad, USA). SYBR Green was used as detection dye. The quantitation of cDNAs in low copy numbers was verified with fluorescing hydrolysis probes (IFN-y, IL-4, IL-13, CRTH2; TibMolBiol, Germany). Relative mRNA expressions were calculated as follows: 1/(Expression Gene-of-interest / Expression GAPDH). All reactions were run in duplicates and included untranscribed mRNA as negative controls. Amplification of the desired cDNAs was confirmed by sequencing. For comparisons between groups Mann-Whitney-U test was applied. Correlations r0.3 were considered positive (Spearman rho; SPSS Version 11). Results: Cord blood from Spanish neonates had significantly higher TLR2 (p<0.01) and TLR4 (p<0.001) mRNA-expression levels than cord blood from German neonates. In peripheral blood from Spanish mothers, TLR2 (p<0.01) and TLR4 mRNA (p<0.001) expression levels were also significantly higher as compared to German mothers. In contrast, Spanish women had lower IL-1 (p<0.05) and IL-4 mRNA (p<0.05) expression levels compared to German women. TLR2 and TLR4 mRNA expressions were highly correlated in mothers and theis neonates. mRNA expressions of the remaining markers did not differ between habitants of both countries. Conclusion: TLR2 and TLR4 mRNA expressions were clearly higher in Spanish women and neonates. We speculate that the differences in TLR expression is due to different microbial exposure in both cities. We further speculate that environmental factors may have an impact on the fetal immune system already in utero.

Cytomegalovirus and Epstein-Barr virus DNA transcription in endodontic symptomatic lesions.

Productive Herpesviridae infections are implicated in the etio-pathogenesis of aggressive periodontitis. However, virtually nothing is known about a possible role of herpesviruses in pulpal and periapical pathosis. This study employed a cDNA analysis to determine transcription of human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) and herpes simplex virus (HSV) in 14 recalcitrant periapical lesions and in 2 periapical healthy control sites. Periapical samples were collected in conjunction with periapical surgery and kept frozen until virologic examination. RNA was isolated from periapical tissue by using a guanidinium isothiocyanate-acid phenol procedure (TRIZOL LS Reagent, GIBCO BRL, Rockville, MD). cDNAs were amplified by means of oligonucleotides targeting highly conserved regions of the test viruses and the RT-PCR-100 amplification kit (Sigma-Aldrich, St Louis, MO). Standardization of PCR primer sensitivity and validation was carried out according to established methods. Amplification products were identified by agarose gel electrophoresis. HCMV transcript was detected in 12 of 13 symptomatic and in 1 asymptomatic periapical lesion. EBV transcript was demonstrated in 8 of the 13 symptomatic lesions but not in the asymptomatic periapical lesion. HCMV and EBV dual transcription occurred at higher frequency in periapical lesions showing radiographic bone destruction of 5 mm x 7 mm or larger than in smaller size lesions (P = 0.03; Chi-squared test). No HCMV or EBV transcription was identified in the 2 healthy control sites. HSV transcript was not detected in any study site. The present data suggest that HCMV or EBV infections participate in the pathogenesis of periapical symptomatic lesions. Herpesviruses may produce periapical pathosis as a direct result of viral infection and replication, or as a consequence of virally induced impairment of the host defense and subsequent increased virulence of resident bacterial pathogens.

Stabilization of Gene Expression Profiles in Blood after Phlebotomy

Emerging technologies such as quantitative, real-time reverse transcription-PCR (RT-PCR) and microarrays enable the accurate quantification of ribonucleic acids in clinical samples. Gene expression profiling of tumor samples with use of microarray technology has become the prototypic application of this new type of molecular diagnostics. DNA microarray analysis of different closely related tumor types revealed that this technology is able to distinguish different tumors (1). The concentrations of specific mRNAs, however, may change between specimen acquisition and the beginning of analysis, and these changes are not well understood. When blood is studied, specimen collection and sample preparation techniques by themselves may produce changes in gene expression ex vivo, e.g., it has been shown that anticoagulants cause ex vivo changes in cytokine production (2). If changes in gene expression occur after phlebotomy, the valid interpretation of basal mRNA expression data as sensitive markers in clinical studies requires the standardization of conditions for assaying patient blood samples. The current study was undertaken to analyze changes in gene expression in human peripheral blood stored ex vivo. We studied ex vivo changes in expression of several genes by use of quantitative, real-time RT-PCR and evaluated procedures for standardized blood sampling for molecular diagnostics. After receiving informed consent, we obtained blood from healthy donors (23–60 years of age). Whole blood was collected with use of the VACUTAINER® Safety-LokTM Blood Collection Set and either PAXgeneTM Blood RNA Tubes (2.5 mL draw volume; PreAnalytiX) or EDTA tubes (VACUTAINER PLUSTM 6-mL dipotassium EDTA tubes). This investigation was performed according to the International Declarations of Helsinki and Tokyo. After phlebotomy, PAXgene Blood RNA Tubes were stored at room temperature until processing. Alternatively, immediately after phlebotomy, 1 mL of water and 6 mL of Trizol LS reagent (Invitrogen) were added to 1 mL of whole …

Effect of Holding Conditions on the Detection of West Nile Viral RNA by Reverse Transcriptase-Polymerase Chain Reaction from Mosquito (Diptera: Culicidae) Pools: Table 1

We evaluated the effect of holding temperature and time between mosquito death and processing mosquito pools for virus detection on our ability to detect West Nile (WN) viral RNA from pools of mosquitoes by reverse transcriptase-polymerase chain reaction (RT-PCR). Pools of 24 uninfected Culex pipiens L. mosquitoes were "spiked" with either a single Cx. pipiens that had been inoculated previously with WN virus or with an uninfected mosquito. These pools were held dry at 20, 4, -20, or -70 degrees C for selected time intervals before all mosquito pools were triturated in TRIzol LS reagent and processed for detection of WN viral RNA. While infectious virus virtually disappeared from pools maintained at 20 degrees C by 48 h after mosquito death, neither holding temperature (20 to -70 degrees C) nor holding period (up to 2 wk) affected detection of WN viral RNA by real-time RT-PCR. These findings suggest that we need not keep mosquitoes chilled to be able to detect WN viral RNA effectively by RT-PCR. This should enhance the feasibility of field-based WN virus surveillance programs where only detection of WN viral RNA is the objective and maintenance of a cold chain may not be possible.

Presence of Fetal RNA in Maternal Plasma

The discovery of fetal DNA in maternal plasma (1) has opened up a new horizon on prenatal molecular diagnosis. Many groups have since shown that fetal genetic traits, such as RhD status and inherited genetic diseases, can be determined from fetal DNA in maternal plasma (2)(3)(4)(5). However, it is not known whether fetal RNA is also present in maternal plasma. Here, using a two-step reverse transcription (RT)-PCR assay, we demonstrate the presence of fetal-derived, male-specific mRNA in plasma of pregnant women carrying male fetuses. Pregnant women attending the Prenatal Diagnosis Unit at the Department of Obstetrics and Gynecology, Prince of Wales Hospital, Hong Kong were recruited with informed consent. The study was approved by the Clinical Research Ethics Committee. Women early and late in their pregnancies (n = 21 and 37, respectively) were recruited in this study. The mean gestational ages of the subjects in early and late pregnancies were 16 weeks (range, 11–19 weeks) and 33 weeks (range, 26–40 weeks), respectively. All early-pregnancy samples were obtained before any invasive procedure. On the other hand, late-pregnancy samples were collected either from women who had invasive procedures in early pregnancy (n = 21) or from women who did not have any prenatal invasive procedure (n = 16). All plasma samples were harvested within 30 min from EDTA-blood samples as described previously (1). Total RNA from plasma samples was isolated with the Trizol LS Reagent (Life Technologies) as instructed …

A rapid screening procedure for the identification of high-titer retrovirus packaging clones.

We have developed a viral RNA (vRNA) dot blot assay for rapid identification of high-titer retrovirus vector production by packaging cell clones. The procedure employs Trizol LS reagent to purify vRNA from packaging cell supernatants, a sensitive dot blot assay, and Phosphorlmager technology to quantify packaged viral genomes in 2 days. Experiments performed on viral supernatants of known biological titer demonstrated that the vRNA dot blot assay was extremely sensitive and that dot intensity correlated directly with viral titer. It is often necessary to analyze approximately 100 virus producing cell clones, making this method useful as a rapid screen to identify the highest virus producing clones. The vRNA dot blot assay consistently identified a subset of candidate high-titer producer cell clones. In three independent screens the supernatant with the highest biological titer was produced by one of the previously defined candidate high-titer producer clones. Our procedure greatly facilitates virus titration by: (1) rapidly eliminating the vast majority of low-titer producer cell clones; (2) accurately identifying the subset of candidate high-titer producer clones for further biological titration and assessment of the proviral genomic structure; and (3) reducing laborious tissue culture manipulations to a minimum. Furthermore, the reliance of this method on molecular detection makes it ideally suited for the isolation of high-titer clones lacking a drug selection marker.