Digoxigenin-labeled Probes Research Articles

Restoration of E-cadherin expression in pancreatic ductal adenocarcinoma treated with microRNA-101

To investigate the possibility of inhibiting the progression of pancreatic ductal adenocarcinoma (PDAC) by facilitating the expression of E-cadherin through the enforced expression of microRNA-101 (miR-101). In situ hybridization was conducted with archival tissue using a double digoxigenin-labeled probe. Chromatin immunoprecipitation (ChIP) assay was conducted with EZ-Magna ChIPTM A. Gene profile analysis, Western blot, and immunoprecipitation assays were performed using standard protocols. We found that decreased miR-101 expression observed in archival patient tissues was significantly associated with poor prognosis indicated by low-intensity staining in high-grade tumors. ChIP assays using anti-enhancer of zeste homolog 2 (EZH2) antibodies indicated not only the interaction of EZH2 to the CDH1 (E-cadherin) promoter, but also that this interaction was significantly diminished in cells transfected with pre-miR-101. We observed a global downregulation of trimethylated lysine 27 of H3 histone (H3K27me3) along with upregulation of the enzymes histone deacetylase -1 and -2 with the re-expression of miR-101. Further, we observed lesser levels of transcriptional factors that inhibit the CDH1 promoter with pre-miR-101 treatment. Western blot analysis confirmed the enhanced E-cadherin expression. PANC-1 cells transduced with pre-miR-101 displayed markedly attenuated growth in SCID mice. These results suggest the potential therapeutic use of miR-101-enforced expression for inhibition of PDAC.

A gene-protein assay for human epidermal growth factor receptor 2 (HER2): brightfield tricolor visualization of HER2 protein, the HER2 gene, and chromosome 17 centromere (CEN17) in formalin-fixed, paraffin-embedded breast cancer tissue sections

BackgroundThe eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH) or immunohistochemistry (IHC), respectively. Our objective was to combine the US Food and Drug Administration (FDA)-approved HER2 & chromosome 17 centromere (CEN17) brightfield ISH (BISH) and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section.MethodsThe HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE) samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative)] and Calu-3 [HER2 positive (amplified gene, protein positive)]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5) and a conventional 3,3'-diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigenin-labeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays.ResultsHER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after) the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene-protein assay demonstrated high concordance with those obtained using the separate HER2 IHC and HER2 & CEN17 BISH assays, respectively.ConclusionsWe have developed a protocol that allows simultaneous visualization of the HER2 IHC and HER2 & CEN17 BISH targets. This automated protocol facilitated the determination of HER2 protein and HER2 gene status in randomly selected breast cancer samples, particularly in cases that were equivocal or exhibited tumor heterogeneity. The HER2 gene-protein assay produced results virtually equivalent to those of the single FDA-approved HER2 IHC and HER2 & CEN17 BISH assays.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041964038705297

Molecular characterization of IMP-type metallo- -lactamases among multidrug-resistant Achromobacter xylosoxidans

To analyse the metallo-β-lactamase (MBL) genes and their genetic environments in clinical isolates of Achromobacter xylosoxidans. From January 2004 to December 2010, four MBL-producing, multidrug-resistant (MDR) A. xylosoxidans strains were isolated and analysed at a tertiary care university hospital in Japan. Species-level identification was confirmed by 16S ribosomal RNA gene sequencing. Molecular typing was performed using PFGE, the presence of MBL genes was detected using PCR, and integron gene cassettes were examined by cloning and sequence analysis of integron PCR products. The plasmids obtained from individual isolates were analysed based on EcoRI restriction patterns, Southern hybridizations using digoxigenin-labelled probes for bla(IMP-1) and bla(IMP-19) as well as conjugation and transformation experiments. No clonal relationship was found among the four A. xylosoxidans isolates. Three isolates harboured bla(IMP-1) and one isolate harboured bla(IMP-19). These MBL genes were carried on class 1 integrons. Four different class 1 integron gene cassette arrays were found, including orf1-bla(IMP-1)-aacA4, bla(IMP-1)-bla(IMP-1)-nit1/nit2-catB3-bla(IMP-1)-bla(IMP-1), aacA4-bla(IMP-1) and bla(IMP-19). The restriction pattern of the plasmid DNA obtained from each isolate was unique and the hybridization analyses revealed the presence of each MBL gene within a plasmid. Moreover, all of the plasmids carrying an MBL gene could be transformed into Escherichia coli HST08. This study provides genetic evidence for the existence of IMP-type MBL genes in MDR A. xylosoxidans isolates. Moreover, the present findings provide evidence that A. xylosoxidans can receive IMP-type MBL genes via plasmid-mediated transfer, which contributes to their carbapenem resistance.

Oligo-DNA custom macroarray for monitoring major pathogenic and non-pathogenic fungi and bacteria in the phyllosphere of apple trees.

BackgroundTo monitor the richness in microbial inhabitants in the phyllosphere of apple trees cultivated under various cultural and environmental conditions, we developed an oligo-DNA macroarray for major pathogenic and non-pathogenic fungi and bacteria inhabiting the phyllosphere of apple trees.Methods and FindingsFirst, we isolated culturable fungi and bacteria from apple orchards by an agar-plate culture method, and detected 32 fungal and 34 bacterial species. Alternaria, Aureobasidium, Cladosporium, Rhodotorula, Cystofilobasidium, and Epicoccum genera were predominant among the fungi, and Bacillus, Pseudomonas, Sphingomonas, Methylobacterium, and Pantoea genera were predominant among the bacteria. Based on the data, we selected 29 major non-pathogenic and 12 phytopathogenic fungi and bacteria as the targets of macroarray. Forty-one species-specific 40-base pair long oligo-DNA sequences were selected from the nucleotide sequences of rDNA-internal transcribed spacer region for fungi and 16S rDNA for bacteria. The oligo-DNAs were fixed on nylon membrane and hybridized with digoxigenin-labeled cRNA probes prepared for each species. All arrays except those for Alternaria, Bacillus, and their related species, were specifically hybridized. The array was sensitive enough to detect 103 CFU for Aureobasidium pullulans and Bacillus cereus. Nucleotide sequencing of 100 each of independent fungal rDNA-ITS and bacterial 16S-rDNA sequences from apple tree was in agreement with the macroarray data obtained using the same sample. Finally, we analyzed the richness in the microbial inhabitants in the samples collected from apple trees in four orchards. Major apple pathogens that cause scab, Alternaria blotch, and Marssonina blotch were detected along with several non-phytopathogenic fungal and bacterial inhabitants.ConclusionsThe macroarray technique presented here is a strong tool to monitor the major microbial species and the community structures in the phyllosphere of apple trees and identify key species antagonistic, supportive or co-operative to specific pathogens in the orchard managed under different environmental conditions.

First Report of China Rose (Hibiscus rosa-sinensis) as a Host of Alfalfa mosaic virus in Spain.

China rose (Hibiscus rosa-sinensis L.) is an ornamental plant grown throughout the tropics and subtropics. In June 2011, a China rose plant (sample CV-1) showing bright yellow "aucuba"-type mosaic, mainly at the center of the leaves, was found in a public garden in Caleta de Vélez (Málaga Province, southern Spain). Electron microscope examination of negatively stained preparations from the symptomatic plant revealed the presence of semispherical to bacilliform virus-like particles of 30 to 56 × 16 nm. Sap extracts also reacted positively in double-antibody sandwich (DAS)-ELISA to antiserum against Alfalfa mosaic virus (AMV) (Bioreba AG, Reinach, Switzerland). RNA of this sample was extracted with the RNeasy Kit (Qiagen, Valencia, CA) and tested by reverse-transcription (RT)-PCR with AMV specific primers (2), using AMV and GoTaq Master Mixes (Promega, Madison, WI) for cDNA synthesis and amplification, respectively. After cloning and sequencing, the ~750-bp DNA fragment was confirmed as the coat protein (CP) gene of AMV (GenBank Accession No. HE591387) with the highest nucleotide identity of 96% to AMV isolates belonging to subgroup IIA (e.g., GenBank Accession No. AJ130707). Sap from affected leaves of sample CV-1 was mechanically inoculated onto herbaceous indicator plants (Chenopodium amaranticolor, C. quinoa, and Ocimum basilicum). Both Chenopodium species developed chlorotic local lesions followed by mosaic within 3 days after inoculation, and O. basilicum showed bright yellow mosaic of calico type 2 weeks postinoculation. These symptoms are consistent with those reported for AMV in these hosts (1). Virus infection in the inoculated plants was confirmed by DAS-ELISA and RT-PCR. To gain insight on the prevalence and genetic variability of AMV in China rose, a survey was carried out in nearby locations in the provinces of Málaga (14 samples from Torre del Mar and 5 samples from Rincón de la Victoria) and Granada (12 samples from La Herradura). Leaf samples were analyzed by tissue blot hybridization with an AMV-specific digoxigenin-labeled RNA probe obtained from the RNA 1 of the Spanish isolate Tec1 (3), and only two samples from Torre del Mar tested positive. One of these samples (TM-2) was used to amplify by RT-PCR the AMV CP gene that was cloned and sequenced (GenBank Accession No. HE591386). The highest nucleotide identity of the TM-2 CP gene (98%) was with the subgroup IIB Spanish isolate Tec1, whereas identity with the CV-1 isolate was 95%. Nevertheless, phylogenetic analysis (neighbor-joining method) showed that both CV-1 and TM-2 isolates belong to the recently proposed AMV subgroup IIB (3), which includes the Tec1 isolate and two other isolates from ornamental plants, Phlox paniculata from the United States (GenBank Accession No. DQ124429) and Viburnum lucidum from Spain (GenBank Accession No. EF427449). These results show that AMV subgroup IIB is emerging as a complex cluster of virus isolates that currently are reported to infect only ornamentals. To our knowledge, this is the first report of AMV naturally occurring in China rose.

Comparative evaluation of specific methods for labeling of Encephalitozoon cuniculi in paraffin wax–embedded tissue samples

Detection of the microsporidian Encephalitozoon cuniculi in tissue samples is considered difficult. The aim of the current study was to determine whether immunohistochemistry (IHC) and in situ hybridization (ISH) represent reliable methods for the detection of E. cuniculi in postmortem tissue samples of rabbits. Paraffin-embedded tissue sections of brain and kidneys of 48 naturally infected pet rabbits, 10 negative controls, and the eyes of 3 further rabbits were used for all investigations. By IHC in 19 animals (37.3%), spores could be clearly detected and were all equally stained. By ISH using a digoxigenin-labeled oligonucleotide probe, only 6 animals (11.8%) proved undoubtedly positive. In these cases, many parasite-like objects revealed strong typical purple-black positive signals. However, several of the examined samples showed only partial staining of the pathogen or unclear results. Thus, in order to find an explanation for these inconsistent ISH results and to take a more detailed look at the different developmental stages of the organism, electron microscopy was applied. Empty spores, which had already discharged their polar filaments, prevailed in total number. Taken together, both techniques are rather insensitive, but under the condition that sufficient numbers of microsporidia are present, IHC can be recommended for specific identification of E. cuniculi in tissue samples. In contrast, ISH failed to detect some developmental stages of the organism, and, as such, ISH is therefore considered an inappropriate diagnostic method.

CDNA cloning and primary structure analysis of transglutaminase from bluefin tuna Thunnus orientalis

A cDNA clone encoding a tissue-type transglutaminase (TGase) was isolated from the fast muscle of bluefin tuna Thunnus orientalis using a method that combines reverse transcription-PCR and rapid amplification of cDNA ends. The full-length cDNA was 2,872 bp with an open reading frame coding for a protein of 678 amino acid residues. The deduced amino acid sequence had a calculated molecular mass of 74,193 Da and a theoretical isoelectric point of 7.39. The bluefin tuna TGase showed a high amino acid identity of 75% to that of Atlantic salmon Salmo salar, and a low identity of 38% to that of red seabream Pagrus major. Three residues, namely, Cys269, His326, and Asp349, were conserved in a catalytic site, as has also been observed in TGases from other fish and mammals. In phylogenetic analysis, the bluefin tuna TGase cloned in this study was classified into tissue-type TGase2. Southern blot analysis of bluefin tuna genomic DNA digested with restriction enzymes PstI and KpnI using a digoxigenin-labeled probe revealed two bands of different sizes, suggesting the presence of multiple TGase genes in the bluefin tuna.

Identification of programmed cell death related genes in bamboo

The event of bamboo flowering and subsequent death of bamboo cells, a rare phenomenon is an interesting model to study gene expression/function in the context of the programmed cell death (PCD) in plant. To identify genes involved in autolytic cell death in bamboo (Bambusa arundinacea/Bambusa bambos Voss), a suppressive subtractive cDNA hybridization (SSH) was performed between cDNA isolated from control (healthy), as driver and test internodal tissue (45days after setting of seeds), as tester. In-silico data revealed that 82% of total ESTs (231) were non-significant (unidentified proteins) while remaining ESTs were classified as protein with known/predicted function/s. Among these, net distribution and differential expression patterns of 11 important B. arundinacea PCD specific ESTs were studied using RNA slot-blot, qRT-PCR and semi-quantitative RT. In-situ localization of mRNA-transcripts for selected bamboo PCD-specific ESTs namely V2Ba48 (Aldehyde dehydrogenase 2) and V2Ba19 (Glycogen phosphorylase) were detected using digoxigenin-labeled corresponding anti-sense RNA probes employing Confocal Laser Scanning Microscope (CLSM). Differential expression-kinetics of the aforementioned genes were confirmed during the progress of PCD after setting of seeds. Global appearance of V2Ba48, V2Ba19, V2Ba95 (Ubiquitin thioesterase) and V2Ba89 (Nebulin isoform 2) genes were identified in monocot (Oryza sativa) and dicots (Arabidopsis thaliana and Nicotiana tabacum). This is the first report on systematic analysis of genes involved in death of bamboo cells that may provide critical information regarding key metabolic/regulatory genes involved in plant PCD.

Altered expression of microRNA miR-146a correlates with the development of chronic renal inflammation

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that act as post-transcriptional regulators of target mRNA. In this study, we sought to identify the microRNA underlying local inflammation in a murine model of chronic kidney disease (CKD). In microarray analysis of kidneys, the expression of miR-146a/b was elevated in B6.MRLc1 CKD mice that spontaneously develop renal inflammation with age. Primary-microRNA analysis found that elevated miR-146a/b expression in the kidneys of B6.MRLc1 mice was mainly derived from miR-146a rather than miR-146b, and this expression increased with the development of CKD. Histopathological scores for glomerular and interstitial lesions, mRNA expression of inflammatory mediators, and macrophage infiltration were significantly higher in B6.MRLc1 than C57BL/6 mice and were positively correlated with miR-146a expression. In situ hybridization and laser microdissection-RT-PCR showed that miR-146a expression in interstitial lesions containing inflammatory cells was higher than in the glomerulus. The increased expression of the inflammatory-associated genes RELA, IRAK1, IL1B, IL10, and CXCLs was noted in miR-146a/b-silenced human monocytes. The amount of miR-146a was higher in urine sediments of B6.MRLc1 than of C57BL/6 mice. Thus, miR-146a expression in the kidneys and its urinary excretion was specifically associated with the development of interstitial lesions and correlated with inflammatory cell infiltration.

Schmallenberg Virus in Central Nervous System of Ruminants

Schmallenberg Virus in Central Nervous System of Ruminants

Incidence and genetic stability of Potato spindle tuber pospiviroid in potato in Turkey

The prevalence of Potato spindle tuber pospiviroid (PSTVd) infection in randomly selected potato tubers was determined via nonisotopic dot blot hybridization and reverse transcriptase polymerase chain reaction (RT-PCR). In January and February of 2010, 168 seedling tubers of Solanum tuberosum, representing 27 cultivars, were received from different seedling potato producers in Turkey. Digoxigenin-labeled RNA probes of PSTVd were synthesized from the cloned PCR products and applied in dot blot hybridization to detect viroids in RNA extracts isolated from dormant potato tubers. PSTVd was found in 7 samples and 6 cultivars (Innovator, Lady Jo, Russet Burbank, Agria, Provento and Konsül). Some RNA samples, although positive by RT-PCR test, did not give a hybridization signal by dot blot assay. The complete genome of a selected isolate (PSTVd-TR) was cloned and sequenced. Primary and secondary structure analysis showed that the RNA genome of the PSTVd-TR isolate (GenBank Accession No. HQ456944) differed from other compared isolates by only a few nucleotides. The highest identity (99%) was in a PSTVd isolate that was identified in Solanum spp. in Italy (Accession No. EF459700). The infectivity of PSTVd-TR isolate was shown 2 weeks after the mechanical inoculation of sap extracts onto seedlings of tomato cultivar Joker. The PSTVd infections were proven by RT-PCR assay in symptomless tomato seedlings.

INTERPHASE CYTOGENETIC ANALYSIS OF LUNG ADENOCARCINOMAS WITH BRONCHIOLOALVEOLAR PATTERN

Aneuploidy has been suggested as a marker for stratification of many neoplasms but its potential usefulness in adenocarcinoma (ADC) with bronchioloalveolar (BAC) pattern has not been well defined. We examined paraffin-embedded tissue sections from 28 cases of ADC with BAC pattern as well as 7 benign lung lesions and 9 normal lung tissue samples for chromosomal aneuploidy by in situ hybridization using digoxigenin-labelled probes for chromosomes 1 and X. Of the 28 ADC with BAC pattern, 17 (61%) were diploid and 11 (39%) were aneuploid. Of the 17 diploid cases, 7 (41%) were male and 10 (59%) were female and of the 11 aneuploid cases, 2 (18%) were male, 9 (82%) were female. Regarding the cell type, 24 (86%) were adenocarcinomas in situ (AIS) so called BAC and minimally invasive ADC (MIA), and 4 cases (14%) were invasive ADC. Of the 12 cases each of AIS and MIA, 9 (75%) and 8 (67%) had diploid pattern respectively. Of the 4 invasive ADC cases, all had aneuploid pattern. Seventeen cases (71%) with T1 tumor size (> 0 mm ≤30 mm), had diploid and 4 cases (100%) with T2 tumor size (> 30 mm ≤70 mm) had aneuploid pattern. Statistical analyses showed that nuclear diploidy was significantly correlated with AIS and MIA tumor types while aneuploidy correlated with invasive ADC type (P=0.025). Also a significant correlation was found between ploidy and tumor size (P=0.033). In conclusion, these findings suggest that DNA ploidy analysis provides useful information for the assessment of cellular kinetics and reflect histopathological subtypes in ADC with BAC pattern that are destined to behave aggressively.

First Report of Tomato chlorosis virus Infecting Tomato in Sudan.

In March 2011, interveinal yellowing and necrosis symptoms on middle and lower leaves were observed in tomato (Solanum lycopersicum L., cv. Castle Rock) plants grown in three adjacent greenhouses of the Agricultural Research Corporation at Wad Medani (Gezira State, Sudan). These symptoms resembled those caused by Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) (4) (genus Crinivirus, family Closteroviridae). Whitefly (Bemisia tabaci) infestation was also observed in these greenhouses. Total RNA was extracted by TRIzol Reagent (Invitrogen, Carlsbad, CA) from symptomatic leaves and analyzed by dot-blot hybridization with digoxigenin-labelled RNA probes to the coat protein (CP) gene of ToCV and to the minor coat protein (CPm) gene of TICV. Positive signal was obtained only with the ToCV probe. Reverse transcription (RT)-PCR reactions were performed with two pairs of primers specific for the detection of ToCV, MA380(+) (5'-GTGAGACCCCGATGACAGAT-3') and MA381(-) (5'-TACAGTTCCTTGCCCTCGTT-3'), specific to the CP gene (ToCV RNA 2) (3), and MA396(+) (5'-TGGTCGAACAGTTTGAGAGC-3') and MA397(-) (5'-TGAACTCGAATTGGGACAGA-3'), specific to the RNA-dependent RNA polymerase (RdRp) gene (ToCV RNA 1) (1). DNA fragments of the expected sizes (436 and 763 bp, respectively) were obtained, thus supporting the presence of ToCV in the symptomatic samples. Amplified DNA fragments were cloned in pGEM-T Easy vector (Promega, Madison, WI) and one clone per amplicon was sequenced (Macrogen Inc., Seoul, South Korea). The highest nucleotide sequence identity of the CP gene fragment obtained (GenBank Accession No. JN411685) was 99.2% related with North American ToCV isolates from Florida (DQ234674), Colorado (DQ234675), and Georgia (HQ879842), while the RdRp gene fragment (JN411686) was more closely related (99.0%) to the Spanish AT80/99 isolate (DQ983480). Although yellowing symptoms similar to those reported here have been observed sporadically during the last few years in open-field tomato crops in the state of Gezira, additional studies are needed to determine the prevalence and economic impact of ToCV infections in tomato cultivation in Sudan. To our knowledge, ToCV has been found in continental Africa only in Morocco and South Africa, in the Mediterranean climate areas in the northern and southern edges of the continent, respectively (2). The finding of ToCV infecting tomato in Sudan raises the question of whether this virus is emerging also in other tropical areas of the continent and illustrates the need to monitor whitefly-infested areas within Africa for the presence of ToCV.

The spatio-temporal patterning of Hoxa9 and Hoxa13 in the developing zebrafish enteric nervous system

Hirschsprung's disease is characterised by the absence of ganglion cells in the distal bowel, a process which is controlled by complex genetic pathways. Homeobox genes have a major role in gut development and this is depicted by the enteric Hox code which describes the different spatial and temporal expression of Hox genes. Hoxa9 and Hoxa13 mutations have been discovered in patients with Hirschsprung's disease (HD). The aim of this study was to determine the spatio-temporal pattern of Hoxa9 and Hoxa13 in enteric nervous system (ENS) development using the zebrafish model. Purified plasmids that contained the gene of interest were obtained and inoculated into culture medium to exponentially increase the number of bacteria containing the plasmid. Cells were then harvested by centrifugation and plasmid DNA was extracted, which was then linearised and precipitated. RNA digoxigenin-labelled probes were made by in vitro transcription reaction. In situ hybridisation was carried out using these probes on zebrafish embryos which were collected from 24 to 120 h post fertilisation (hpf), by which time the zebrafish intestine is fully developed. Embryos were then mounted in glycerol and imaged using an Olympus B40 microscope and images were taken using an Olympus Super F1.8 digital camera. At 24 hpf, Hoxa9 expression is seen in the forebrain and hindbrain and also in the very distal myotome whereas Hoxa13 expression, however, is seen only at the forebrain and hindbrain. At 48 hpf, Hoxa9- and Hoxa13-labelled cells are seen migrating distally from the forebrain into the notochord and spinal cord. At 72 hpf, Hoxa9-labelled cells can be seen throughout the spinal cord whereas Hoxa13 positive cells are seen migrating down from the spinal cord and in the proximal gut. By 96 hpf, Hoxa9- and Hoxa13-labelled cells have migrated down the full length of the spinal cord and along the proximal and mid intestine. By 120 hpf, Hoxa9 and Hoxa13 positive cells can be seen along the entire length of the zebrafish intestine. These results show further evidence that Hoxa9 and Hoxa13 are involved in the early and organised patterning of ENS development in the zebrafish model.

In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe

The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these...

Identification of the Cluster Control Region for the Protocadherin-β Genes Located beyond the Protocadherin-γ Cluster

The clustered protocadherins (Pcdhs), Pcdh-α, -β, and -γ, are transmembrane proteins constituting a subgroup of the cadherin superfamily. Each Pcdh cluster is arranged in tandem on the same chromosome. Each of the three Pcdh clusters shows stochastic and combinatorial expression in individual neurons, thus generating a hugely diverse set of possible cell surface molecules. Therefore, the clustered Pcdhs are candidates for determining neuronal molecular diversity. Here, we showed that the targeted deletion of DNase I hypersensitive (HS) site HS5-1, previously identified as a Pcdh-α regulatory element in vitro, affects especially the expression of specific Pcdh-α isoforms in vivo. We also identified a Pcdh-β cluster control region (CCR) containing six HS sites (HS16, 17, 17', 18, 19, and 20) downstream of the Pcdh-γ cluster. This CCR comprehensively activates the expression of the Pcdh-β gene cluster in cis, and its deletion dramatically decreases their expression levels. Deleting the CCR nonuniformly down-regulates some Pcdh-γ isoforms and does not affect Pcdh-α expression. Thus, the CCR effect extends beyond the 320-kb region containing the Pcdh-γ cluster to activate the upstream Pcdh-β genes. Thus, we concluded that the CCR is a highly specific regulatory unit for Pcdh-β expression on the clustered Pcdh genomic locus. These findings suggest that each Pcdh cluster is controlled by distinct regulatory elements that activate their expression and that the stochastic gene regulation of the clustered Pcdhs is controlled by the complex chromatin architecture of the clustered Pcdh locus.

TRPA1 contributes to specific mechanically activated currents and sensory neuron mechanical hypersensitivity

The mechanosensory role of TRPA1 and its contribution to mechanical hypersensitivity in sensory neurons remains enigmatic. We elucidated this role by recording mechanically activated currents in conjunction with TRPA1 over- and under-expression and selective pharmacology. First, we established that TRPA1 transcript, protein and functional expression are more abundant in smaller-diameter neurons than larger-diameter neurons, allowing comparison of two different neuronal populations. Utilising whole cell patch clamping, we applied calibrated displacements to neurites of dorsal root ganglion (DRG) neurons in short-term culture and recorded mechanically activated currents termed intermediately (IAMCs), rapidly (RAMCs) or slowly adapting (SAMCs). Trpa1 deletion (–/–) significantly reduced maximum IAMC amplitude by 43% in small-diameter neurons compared with wild-type (+/+) neurons. All other mechanically activated currents in small- and large-diameter Trpa1−/− neurons were unaltered. Seventy-three per cent of Trpa1+/+ small-diameter neurons responding to the TRPA1 agonist allyl-isothiocyanate (AITC) displayed IAMCs to neurite displacement, which were significantly enhanced after AITC addition. The TRPA1 antagonist HC-030031 significantly decreased Trpa1+/+ IAMC amplitudes, but only in AITC responsive neurons. Using a transfection system we also showed TRPA1 over-expression in Trpa1+/+ small-diameter neurons increases IAMC amplitude, an effect reversed by HC-030031. Furthermore, TRPA1 introduction into Trpa1−/− small-diameter neurons restored IAMC amplitudes to Trpa1+/+ levels, which was subsequently reversed by HC-030031. In summary our data demonstrate TRPA1 makes a contribution to normal mechanosensation in a specific subset of DRG neurons. Furthermore, they also provide new evidence illustrating mechanisms by which sensitisation or over-expression of TRPA1 enhances nociceptor mechanosensitivity. Overall, these findings suggest TRPA1 has the capacity to tune neuronal mechanosensitivity depending on its degree of activation or expression.

Application of in-situ hybridization for the detection and identification of avian malaria parasites in paraffin wax-embedded tissues from captive penguins

In captive penguins, avian malaria due to Plasmodium parasites is a well-recognized disease problem as these protozoa may cause severe losses among valuable collections of zoo birds. In blood films from naturally infected birds, identification and differentiation of malaria parasites based on morphological criteria are difficult because parasitaemia is frequently light and blood stages, which are necessary for identification of parasites, are often absent. Post-mortem diagnosis by histological examination of tissue samples is sometimes inconclusive due to the difficulties in differentiating protozoal tissue stages from fragmented nuclei in necrotic tissue. The diagnosis of avian malaria would be facilitated by a technique with the ability to specifically identify developmental stages of Plasmodium in tissue samples. Thus, a chromogenic in-situ hybridization (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 18S rRNA, was developed for the detection of Plasmodium parasites in paraffin wax-embedded tissues. This method was validated in comparison with traditional techniques (histology, polymerase chain reaction), on various tissues from 48 captive penguins that died at the zoological garden Schönbrunn, Vienna, Austria. Meronts of Plasmodium gave clear signals and were easily identified using ISH. Potential cross-reactivity of the probe was ruled out by the negative outcome of the ISH against a number of protozoa and fungi. Thus, ISH proved to be a powerful, specific and sensitive tool for unambiguous detection of Plasmodium parasites in paraffin wax-embedded tissue samples.

The balance between the expressions of hASH1 and HES1 differs between large cell neuroendocrine carcinoma and small cell carcinoma of the lung

To clarify the biological differences between small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC), we investigated the expression of two bHLH type transcription factors, human achaete-scute homolog 1 (hASH1) and hairy/enhancer of split 1 (HES1), which positively and negatively regulate the neuroendocrine differentiation of respiratory epithelial cells, respectively. Eighty-eight formalin-fixed and paraffin-embedded pulmonary carcinomas (32 SCLC, 32 LCNEC, 14 adenocarcinomas, and 10 squamous cell carcinomas) and 14 SCLC and 1 LCNEC derived cell lines were used. hASH1 and HES1 mRNA were detected using a highly sensitive in situ hybridization method with digoxigenin-labeled cRNA probes and biotinylated tyramide. The staining results were scored from 0 to 12 by multiplying the staining intensity by the percentage of positive tumor cells. The mean staining score of hASH1 mRNA was significantly higher in SCLC than in LCNEC (p<0.01); conversely, that of HES1 mRNA was lower in SCLC than in LCNEC (p<0.01). These findings reveal that SCLC more strongly expresses the neuroendocrine phenotype, while LCNEC shows characteristics more similar to the ciliated epithelium phenotype, suggesting that the biological characteristics of these two tumors are different.

First Report of Grapevine yellow speckle viroid 1 and Hop stunt viroid in Grapevine (Vitis vinifera) in New Zealand.

In February 2009, grapevines (Vitis vinifera) in a commercial vineyard in Auckland were showing shortened, spindly canes with tiny leaves. Approximately 10% of the vines were affected. An RNeasy Plant Mini Kit (Qiagen, Valencia, CA) was used to isolate total RNA from leaves collected from six symptomatic (cvs. BAC0022A and Syrah) and eight symptomless vines (cvs. BAC0022A, Syrah, and Chardonnay). RNA was tested by reverse transcription-PCR for the presence of Australian grapevine viroid, Citrus exocortis viroid, Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2, and Hop stunt viroid (HSVd). Four of the six symptomatic and all the symptomless vines tested positive for GYSVd-1 using primers 5'-TGTGGTTCCTGTGGTTTCAC-3' and 5'-ACCACAAGCAAGAAGATCCG-3', which amplify the complete genome (368 bp), and published primers PBCVd100C/194H (3), which amplify a 220-bp region of the genome. Amplicons from each PCR were transformed into a pCR 4-TOPO vector (Invitrogen, Carlsbad, CA), cloned, and sequenced. Sequence from both PCRs aligned identically to generate a consensus sequence (GenBank Accession No. HQ447056), which showed 99% nt identity to GYSVd-1 (GenBank No. X87906) by BLASTN analysis. All symptomatic and symptomless vines also tested positive for HSVd using primers C/H-HSVd (4) and HSVd-C60/H79 (1), which amplify the complete genome (298 bp). Amplicons from each isolate were cloned and sequenced. Sequence from both PCRs were aligned. Clones from all isolates, with the exception of one, aligned identically to create a consensus sequence (GenBank No. HQ447057) that showed 99% nt identity to Chinese HSVd isolates from grapevine (GenBank Nos. DQ371436-59) by BLASTN analysis. Sequence from the remaining isolate (GenBank No. HQ447056) was identical to a German Riesling grape isolate of HSVd (GenBank No. X06873). The presence of each viroid was further confirmed in PCR-positive plants by dot-blot hybridization with digoxigenin-labeled synthetic ssRNA probes specific to the full-length genomes of GYSVd-1 and HSVd (S. Harper and L. Ward, unpublished data). To our knowledge, this is the first report of GYSVd-1 and HSVd in V. vinifera in New Zealand. Since both viroids were detected in symptomatic and symptomless plants, the symptoms observed in the vineyard cannot be attributed to viroid infection. Symptoms described for GYSVd-1 include leaf spots and flecks, but no disease symptoms have been reported in grapes as a result of HSVd (2). Viruses found in the vines include Grapevine leaf roll virus-3, Grapevine viruses A and B, and Rupestris stem pitting associated virus, but these are not thought to be the cause of the symptoms. Two sequence types of HSVd were found, suggesting at least two separate introductions of HSVd into the vineyard. The vineyard is more than 40 years old so both viroids may have been present for some years. Export of wine from New Zealand was worth 1 billion dollars in 2009, so there is potential for these viroids to have an economic impact if symptoms are expressed. HSVd has been reported from China, Europe, Japan, Middle East, Pakistan, and the United States. GYSVd-1 has been reported from Australia, China, East Mediterranean, Europe, Japan, and the United States.