Weak Positive Signals Research Articles

Is Artemia susceptible to the microsporidium Ecytonucleospora hepatopenaei (EHP) infection?

Following the three-stage approach of the WOAH (World Organisation for Animal Health) standard on the confirmation of susceptible species to a specific pathogen, Artemia franciscana juveniles were challenged with immersion exposure (IM) in a homogenate suspension of Penaeus vannamei hepatopancreas infected with Ecytonucleospora hepatopenaei (EHP) and per os administration (PO) with chlorella soaked in EHP-infected hepatopancreatic homogenate suspension. Using TaqMan probe-based quantitative PCR, we detected that loads of EHP in A. franciscana juveniles from both the IM and PO groups were about 101 copies/ng-DNA on the 3rd day post-exposure (3 dpe). However, no more EHP was detectable in the challenged A. franciscana juveniles on 7 dpe. Nested PCR targeting the EHP spore wall protein gene did not produce a target band in A. franciscana juveniles on either 3 dpe or 7 dpe. The confirmative evidence of the absence of sustained infection with EHP in the exposed Artemia was substantiated using in situ DIG-labeling-loop-mediated DNA amplification (ISDL) on A. franciscana juvenile sections, which revealed a few weak grey-blue positive signals appeared on 3 dpe but disappeared on 7 dpe. The results indicated that exposure to EHP resulted in the invasion of Artemia in intestinal epithelial cells, but the infection was unable to persist, confirming Artemia is not susceptible to EHP. Therefore, the research countered the assertion of Artemia as a potential EHP reservoir.

Development of an image processing software for quantification of histological calcification staining images.

Quantification of the histological staining images gives important insights in biomedical research. In wet lab, it is common to have some stains off the target to become unwanted noisy stains during the generation of histological staining images. The current tools designed for quantification of histological staining images do not consider such situations; instead, the stained region is identified based on assumptions that the background is pure and clean. The goal of this study is to develop a light software named Staining Quantification (SQ) tool which could handle the image quantification job with features for removing a large amount of unwanted stains blended or overlaid with Region of Interest (ROI) in complex scenarios. The core algorithm was based on the method of higher order statistics transformation, and local density filtering. Compared with two state-of-art thresholding methods (i.e. Otsu's method and Triclass thresholding method), the SQ tool outperformed in situations such as (1) images with weak positive signals and experimental caused dirty stains; (2) images with experimental counterstaining by multiple colors; (3) complicated histological structure of target tissues. The algorithm was developed in R4.0.2 with over a thousand in-house histological images containing Alizarin Red (AR) and Von Kossa (VK) staining, and was validated using external images. For the measurements of area and intensity in total and stained region, the average mean of difference in percentage between SQ and ImageJ were all less than 0.05. Using this as a criterion of successful image recognition, the success rate for all measurements in AR, VK and external validation batch were above 0.8. The test of Pearson's coefficient, difference between SQ and ImageJ, and difference of proportions between SQ and ImageJ were all significant at level of 0.05. Our results indicated that the SQ tool is well established for automatic histological staining image quantification.

Deep Learning on chromatographic data for Segmentation and Sensitive Analysis

Lateral flow immunoassay (LFIA) is one of the most common methods in point-of-care testing, which is widely applied in some conditions for various applications. Image segmentation is an increasingly popular experimental paradigm to efficiently test the target area in LFIA. However, due to process pollution, and problems related to the experimental operation and irregular structure of the background of the reaction, currently available tools cannot be used to extract correct signals from these images, which affects the accuracy of detection. Machine learning has significantly improved modern biochemical analysis by pushing the limits of traditional techniques for the recognition and processing of images. In this paper, the U-Net, a variant of the convolutional neural network (CNN) is used for the quantitative analysis of LFIA images for the accurate quantification of single- and multi-target images. By graying, binarizing, and labeling different concentrations of test strips, the target area of LFIA images containing the T-/C-lines is extracted and obtained. Then it provides updated trends and directions for the development of LFIA technology. Several indicators are introduced to evaluate the proposed U-Net structure to verify the feasibility and effectiveness of its image processing capability. When the trained U-Net model was used to process images, the peak signal-to-noise ratio was 22.4 dB, significantly higher than prevalent methods in the area that have reported only a 4 dB improvement in the quality of the graphics. The intersection-over-union between samples also increased to above 93%. Our results show that the proposed method has significant potential for detecting a segmented target in an LFIA area, especially weak positive signals and multichannel detection. With other modifications, this deep learning method can be applied as a powerful tool to study rapid detection devices, systems, and biological images.

Pharmacological Treatment of Methamphetamine/Amphetamine Dependence: A Systematic Review.

BackgroundStimulant drugs are second only to cannabis as the most widely used class of illicit drug globally, accounting for 68 million past-year consumers. Dependence on amphetamines (AMPH) or methamphetamine (MA) is a growing global concern. Yet, there is no established pharmacotherapy for AMPH/MA dependence. A comprehensive assessment of the research literature on pharmacotherapy for AMPH/MA dependence may inform treatment guidelines and future research directions.MethodsWe systematically reviewed the peer-reviewed literature via the electronic databases PubMed, EMBASE, CINAHL and SCOPUS for randomised controlled trials reported in the English language examining a pharmacological treatment for AMPH/MA dependence or use disorder. We included all studies published to 19 June 2019. The selected studies were evaluated for design; methodology; inclusion and exclusion criteria; sample size; pharmacological and (if included) psychosocial interventions; length of follow-up and follow-up schedules; outcome variables and measures; results; overall conclusions and risk of bias. Outcome measures were any reported impact of treatment related to AMPH/MA use.ResultsOur search returned 43 studies that met our criteria, collectively enrolling 4065 participants and reporting on 23 individual pharmacotherapies, alone or in combination. Disparate outcomes and measures (n = 55 for the primary outcomes) across studies did not allow for meta-analyses. Some studies demonstrated mixed or weak positive signals (often in defined populations, e.g. men who have sex with men), with some variation in efficacy signals dependent on baseline frequency of AMPH/MA use. The most consistent positive findings have been demonstrated with stimulant agonist treatment (dexamphetamine and methylphenidate), naltrexone and topiramate. Less consistent benefits have been shown with the antidepressants bupropion and mirtazapine, the glutamatergic agent riluzole and the corticotropin releasing factor (CRF-1) antagonist pexacerfont; whilst in general, antidepressant medications (e.g. selective serotonin reuptake inhibitors [SSRIs], tricyclic antidepressants [TCAs]) have not been effective in reducing AMPH/MA use.ConclusionsNo pharmacotherapy yielded convincing results for the treatment of AMPH/MA dependence; mostly studies were underpowered and had low treatment completion rates. However, there were positive signals from several agents that warrant further investigation in larger scale studies; agonist therapies show promise. Common outcome measures should include change in use days. Future research must address the heterogeneity of AMPH/MA dependence (e.g. coexisting conditions, severity of disorder, differences between MA and AMPH dependence) and the role of psychosocial intervention.Electronic supplementary materialThe online version of this article (10.1007/s40263-020-00711-x) contains supplementary material, which is available to authorized users.

Face mask sampling reveals antimicrobial resistance genes in exhaled aerosols from patients with chronic obstructive pulmonary disease and healthy volunteers

IntroductionThe degree to which bacteria in the human respiratory tract are aerosolised by individuals is not established. Building on our experience sampling bacteria exhaled by individuals with pulmonary tuberculosis using...

Detection of Herpesvirus anguillae (AngHV-1) in European eel Anguilla anguilla (L.) originating from northern Poland-assessment of suitability of selected diagnostic methods.

The Community Action Plan requests EU member states to implement measures that ensure the recovery of the severely depleted European eel stocks. One of the main threats is posed by Anguillid herpesvirus 1 (AngHV-1) leading to increased mortality in both wild and farmed eels. Following recommendations of the OIE to minimize the risk of obtaining false-negative results, the main aim of the study was to optimize diagnostic methods for AngHV-1 detection using conventional PCR, nested PCR and insitu hybridization assay. While 53.3% of the individual organ samples were tested positive for AngHV-1 by PCR, the additional virus analysis via nested PCR revealed that the actual prevalence was 93.3%. In the cell cultivation passages, a cytopathic effect was hardly found in the first two rounds. In the third passage onto cell cultures, a lytic CPE was detected. The identification and confirmation of the viruses obtained from cell cultures as well as directly from the organ tissues were proceeded by PCR, nested PCR and sequencing of the PCR products. While no positive signal was detectable in the first round by PCR using samples from the third cell culture passages, the nested PCR provided weak but visible positive signals.

Flavanol-Rich Lychee Fruit Extract Prevents Diabetes-induced Muscle Loss And Palmitate-induced Myotube Loss

Diabetes-induced muscle loss is associated with stimulated the muscle RING-finger protein 1 (MuRF1), a muscle specific E3 ubiquitin ligase. Elevated lipid metabolites impair myogenesis. Flavanol-rich lychee fruit extract (FRLFE) exhibited anti-diabetic and -obesity properties which would be an alternative approach for maintaining muscle homeostasis. PURPOSE: The underlying mechanisms of FRLFE on maintaining muscle homeostasis was studied in vivo and in vitro. METHODS: Dietary (10 wk) FRLFE supplementation (200 mg/kg diet) on the skeletal muscle size and transcription factors such as NF-κB and Foxo3a involved in regulation of MuRF1 were investigated in diabetic db/db mice (n=10/per group). The roles of FRLFE on cell cycle and senescent phenotype were investigated in palmitate-induced insulin resistance in C2C12 muscle cells. The statistical significance of the differences among the groups (P < 0.05) was determined by one-way ANOVA and following post hoc assessment by Student-Newman-Keuls Method. RESULTS: The average cross-sectional area was significantly increased by 1.6-fold after administration of FRLFE compared with non-treated db/db mice (420μm2). Prevention of muscle loss by FRLFE was associated with down regulation of MuRF1 mRNA expression (410% vs. 100%, p<0.05). Decreased NF-κB expression in the nuclear fraction was observed in FRLFE treated db/db mice compared with untreated mice (260% vs. 60%, p<0.05). At transcriptional level, FRLFE abated ceramide-induced MuRF1 promoter activity (200% vs. 100%, p<0.05). Restoration of SIRT1 expression (30% vs. 150%, p<0.05) prevented Foxo3a nuclear localization in FRLFE treated db/db mice. A strong β-galactosidase staining positive signal was observed in the palmitate-treated myoblasts. For cell cycle analysis, significantly increased proportion of cells in G2-phase by palmitate was observed compared with group without palmitate treatment (28.9% vs. 18.3%). Weak β-galactosidase staining positive signals in accordance with decreased proportion of cells in G2-phase (21.7%) were observed in FRLFE-treated myoblasts. CONCLUSION: The efficacy of FRLFE on preserving skeletal muscle mass and myogenic process indicates that FRLFE acts as an effective supplement for diabetes and/or obesity-induced muscle atrophy.

Real-Time PCR Detection of Paenibacillus spp. in Raw Milk To Predict Shelf Life Performance of Pasteurized Fluid Milk Products

Psychrotolerant sporeformers, specifically Paenibacillus spp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. While Paenibacillus spp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number of Paenibacillus spp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detect Paenibacillus spp. in fluid milk and to discriminate between Paenibacillus and other closely related spore-forming bacteria. Specificity was confirmed using 16 Paenibacillus and 17 Bacillus isolates. All 16 Paenibacillus isolates were detected with a mean cycle threshold (C(T)) of 19.14 ± 0.54. While 14/17 Bacillus isolates showed no signal (C(T) > 40), 3 Bacillus isolates showed very weak positive signals (C(T) = 38.66 ± 0.65). The assay provided a detection limit of approximately 3.25 × 10(1) CFU/ml using total genomic DNA extracted from raw milk samples inoculated with Paenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection of Paenibacillus. Heat-treated milk samples where Paenibacillus (≥1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection of Paenibacillus that has the potential to identify raw milk with microbial spoilage potential as a pasteurized product.

Mangrove microniches determine the structural and functional diversity of enriched petroleum hydrocarbon-degrading consortia

In this study, the combination of culture enrichments and molecular tools was used to identify bacterial guilds, plasmids and functional genes potentially important in the process of petroleum hydrocarbon (PH) decontamination in mangrove microniches (rhizospheres and bulk sediment). In addition, we aimed to recover PH-degrading consortia (PHDC) for future use in remediation strategies. The PHDC were enriched with petroleum from rhizosphere and bulk sediment samples taken from a mangrove chronically polluted with oil hydrocarbons. Southern blot hybridization (SBH) assays of PCR amplicons from environmental DNA before enrichments resulted in weak positive signals for the functional gene types targeted, suggesting that PH-degrading genotypes and plasmids were in low abundance in the rhizosphere and bulk sediments. However, after enrichment, these genes were detected and strong microniche-dependent differences in the abundance and composition of hydrocarbonoclastic bacterial populations, plasmids (IncP-1α, IncP-1β, IncP-7 and IncP-9) and functional genes (naphthalene, extradiol and intradiol dioxygenases) were revealed by in-depth molecular analyses [PCR-denaturing gradient gel electrophoresis and hybridization (SBH and microarray)]. Our results suggest that, despite the low abundance of PH-degrading genes and plasmids in the environmental samples, the original bacterial composition of the mangrove microniches determined the structural and functional diversity of the PHDC enriched.

Production, characterization and application of monoclonal antibody to spherulocytes: A subpopulation of coelomocytes of Apostichopus japonicus

One monoclonal antibody (mAb 3F6) against coelomocytes of sea cucumber Apostichopus japonicus was developed by immunization of Balb/C mice. Analyzed by indirect immunofluorescence assay test (IIFAT), immunocytochemical assay (ICA), Western blotting and fluorescenceactivated cell sorter (FACS), mAb 3F6 showed specific for spherulocytes of A. japonicus. The mAb 3F6 recognized an antigen of molecular weight 136 kDa in Western blotting. Isotype analysis revealed mAb 3F6 as IgG type. The flow cytometry assay confirmed the microscopy observations and showed coelomocytes positive to mAb 3F6. The antigencity of haemocytes or coelomocytes of Hemicentrotus pulcherrimus, Scapharca subcrenata, Asterina pectinifera, Asterias rollestoni, Ruditapes philipinarum, Patinopecten yessoensis and Mytilus edulis was compared and the result showed that none of them was positive with mAb 3F6. Most of cells free in polian vesicle were positive with mAb 3F6. The positive cells are in spherical shape, 5–7 μm in diameter, smaller than coelomic spherulocytes of A. japonicus. The result of immunofluorescent staining with cells in hemal vessel showed that there were strong positive signals on cytoplasm of some spherical cells with diameter of 7–8 μm. Some other cells with higher nucelo-plasmic ratio, about 5–6 μm in diameter showed weak positive signals on membrane. Immunohistochemistry assay revealed that positive signals were mostly observed in the lumen structure of rete mirabile and haemocoel of the respiratory tree. In addition, the outer epidermis of body wall and tentacle also showed positive.

Immunogenicity Profile in Mice of a Pegylated Recombinant Erwinia Chrysanthemi-Derived L-Asparaginase.

Abstract 2034Poster Board II-11 Background:Hypersensitivity is the most common dose-limiting toxicity observed with any of the currently available L-asparaginases, vital components of multi-agent chemotherapy for childhood acute lymphoblastic leukemia (ALL). In a significant proportion of patients, administration of L-asparaginase preparations also results in the development of high titers of serum IgG antibodies which in the majority of cases interfere with the therapeutic effect of the enzyme. L-asparaginases are extracted either from Escherichia coli or Erwinia chrysanthemi and when signs of hypersensitivity occur with the Escherichia coliproducts, used as front line therapy, patients are switched to the Erwinia chrysanthemi preparations. Despite the availability of second line products, hypersensitivity reactions and neutralizing antibodies remained frequently observed, providing the rationale for developing new L-asparaginases. Using recombinant and state-of-the-art PEGylation technologies, Asparec®, a new Erwinia chrysanthemi-derived L-asparaginase (crisantaspase), has been designed with the objective of developing a compound with improved pharmacokinetic (PK) and pharmacodynamic (PD) properties and reduced immunogenicity as compared to the native enzyme. Materials & Methods:A recombinant crisantaspase has been engineered and further modified by covalent conjugation to methoxy-polyethylene glycol (PEG) molecules of 2, 5 or 10 kDa using a stable PEG-protein linkage, yielding PEG conjugates of high specific activity (range: 483 to 543 U/mg). Studies performed in mice have shown that the 5 kDa PEG r-crisantaspase conjugate showed improved PK/PD properties as compared to other tested PEG-r-crisantaspase conjugates and Erwinase®, the commercially available crisantaspase. In order to characterize the immunogenicity profile of PEG-r-crisantaspase conjugates and select a candidate for further development, groups of 8 female mice received intravenous administration of PEG-r-crisantaspase conjugates (5 U/kg) versus Erwinase® (250 U/kg) twice a week at Week 1, 2, 3, 4 and 8. Levels of specific antibodies against r-crisantaspase or PEG-r-crisantaspase conjugates were measured using indirect Elisa method with absorbance read at 450 nm. Results:High absorbance values reflective of high titers of anti-crisantaspase antibodies were observed for Erwinase® starting at Week 2 of treatment (mean±SD: 1.816±0.57) and were maintained for the whole study period (2.946±0.283 at Week 8). In contrast, no significant anti-crisantaspase antibody levels were noted following treatment with the 2, 5 or 10 kDa PEG-r-crisantaspase conjugates (at Week 8: 0.026±0.008, 0.019±0.010, and 0.021±0.004, respectively). Treatment with the 2 kDa PEG-r-crisantaspase conjugate did not induce significant levels of specific anti-conjugate antibodies within the first 4 weeks of treatment. Mean absorbance values slightly increased with time resulting in weak positive signals at the end of the treatment period (0.183±0.422). Mean values were slightly greater following treatment with the 5 kDa and 10 kDa PEG- r-crisantaspase conjugates starting week 1 and increased with time. At the end of the treatment period, values were markedly higher with the 10 kDa (1.094±0.578) as compared to the 5 kDa PEG-r-crisantaspase conjugate (0.371±0.392). Similarly, the number of animals with significant mean absorbance values (>0.5) was higher with 10 kDa PEG-r-crisantaspase conjugate as compared to the 2 and 5 kDa PEG-r-crisantaspase conjugates. Conclusions:These data clearly indicate that PEGylation significantly reduced immunogenicity of r-crisantaspase. Data are in agreement with PK/PD results, providing additional support for the development of the 5 kDa PEG-r-crisantaspase conjugate as a promising second line treatment for ALL. Disclosures:Allas:Alize Pharma: Employment. Sahakian:Alize Pharma: Employment. Abribat:Alize Pharma: Employment.

PCR-based identification ofPythiumspp. causing cavity spot in carrots and sensitive detection in soil samples

On the basis of ITS sequences PCR primers were designed for the identification of the five Pythium species found to be most important for the development of carrot cavity spot in Norway: P. intermedium, P. sulcatum, P. sylvaticum, P. violae and P. ‘vipa’. The P. ‘vipa’ isolates had a unique ITS sequence, differed morphologically from all other Pythium isolates, and thus probably represent a new species. The PCR primers were species-specific with no cross-reaction to other Pythium species or to fungal isolates from carrot tested. The detection limits varied for the different primer pairs. The two most sensitive assays allowed detection of as little as 5 fg DNA. All five Pythium species could be detected in lesions from diseased carrots. Weak positive signals were obtained from some carrot samples without symptoms. PCR assays allowed detection of pathogens in soil. In samples of soil known to produce cavity spots on cropped carrots, strong signals were obtained. In several soil samples more than one of the five Pythium species could be detected. The utilization of this diagnostic PCR assay in analysis of field soil and carrot tissue might in the future be exploited to reduce the incidence of this serious carrot disease.

The suitability of different DHB isomers as matrices for the MALDI-TOF MS analysis of phospholipids: which isomer for what purpose?

Although the analysis of large biomolecules is the prime application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), there is also increasing interest in lipid analysis. Since lipids possess relatively small molecular weights, matrix signals should be as small as possible to avoid overlap with lipid peaks. Although 2,5-dihydroxybenzoic acid (DHB) is an established MALDI matrix, the question whether just this isomer is ideal for lipid analysis was not yet addressed. UV absorptions of all six DHB isomers were determined and their laser desorption spectra recorded. In addition, all isomers were used as matrices to record positive and negative ion mass spectra of selected phospholipids (phosphatidylcholine and -serine): In the order 2,5-, 2,6-, 2,3- and 2,4-DHB, the quality of the positive ion lipid spectra decreases. This correlates well with the decreasing acidity of the applied DHB isomers. The 3,4- and 3,5- isomers give only very weak positive ion signals especially of acidic lipids. In contrast, the most suitable matrices in the negative ion mode are 2,5-, 2,4- and 3,5-DHB. 2,6-DHB does not provide any signal in the negative ion mode due to its marked acidity. Finally, differences in the crystallization behavior of the pure matrix and the matrix/lipid co-crystals were also monitored by atomic force microscopy (AFM): 2,5-DHB gave the smallest crystals and the skinniest layer. It is concluded that basically all DHB isomers can be used as MALDI matrices but the 2,5-isomer represents the most versatile compound.

Development and evaluation of a one-step real-time reverse transcription polymerase chain reaction assay for the detection of salmonid alphaviruses in serum and tissues

We designed 4 primer pairs to amplify conserved regions of the E1 or nsP4 genes of salmonid alphavirus (SAV) and evaluated their performance in optimized 1-step SYBR green real-time RT-PCR (RRT-PCR) assays. A single primer pair, amplifying a 227 bp segment of E1 was then chosen for further study. This RRT-PCR was shown to be highly repeatable and reproducible over a wide range of RNA dilutions, with a linear relationship between cycle threshold (Ct) value and RNA concentration over a 10(7) dilution range. The limit of detection was calculated to be < or = 1.5 TCID50 ml(-1). When applied to sera previously screened by virus isolation for SAV viraemia, the RRT-PCR correctly identified all 13 culture-positive samples, as well as finding an additional 28 sera positive. Relative semi-quantitation of sera showed a very highly significant relationship between copy number and TCID50 (p < 0.001, R2 = 0.9563). Following experimental infection of salmon, heart samples were consistently positive until 21 d post infection (dpi), with (weak) positive signals still detectable in 50% of fish 70 dpi.

Genomic approach of AIDS pathogenesis: exhaustive genotyping of the TNFR1 gene in a French AIDS cohort

Large-scale genomic studies in cohorts have been made possible for the last few years thanks to the progress of molecular biology and bioinformatics. This systematic approach allows a better understanding of the molecular mechanisms of disease development and as a consequence can contribute to the rational design of new diagnostic and therapeutic tools. We present here the exhaustive genotyping of a candidate gene, tumor necrosis factor receptor 1 ( TNFR1), in the genetic of resistance to immunodeficiency virus (GRIV) AIDS cohort. This gene was chosen because it is likely to be involved in the apoptosis pathways of CD4 + and CD8 + T-cells during human immunodeficiency virus 1 (HIV-1) infection. Seven frequent polymorphisms were characterized in 319 HIV-1 seropositive patients from the GRIV cohort with extreme disease progression phenotypes, slow progression or rapid progression, and in 427 healthy controls. The TNFR1 gene locus does not appear to be part of any haploblock and contains only a small haploblock of two successive SNPs. One promoter SNP (TNFR1_17444594, position –581) and one intronic SNP (TNFR1_27223241, position +11511) gave weak positive signals of association (resp. P = 0.03 and P = 0.04) as well as two haplotypes. To our knowledge, this is the first genetic association study dealing with the TNFR1 gene in AIDS and the putative associations identified will need to be validated through other AIDS cohort analyses or by further biological experimentation.

The phylogeny and evolution of the genus Claviceps

Phylogenetic trees of 16 Claviceps species were constructed based on alignments of 5.8S rDNA and the adjacent ITS1 and ITS2 spacers. Two highly supported clades were found: (1) C. paspali, C. zizaniae, C. grohii, C. sulcata, C. fusiformis, and C. purpurea; and (2) C. citrina, C. phalaridis, two unidentified Claviceps spp. (isolates PM and SG), C. sorghicola, C. gigantea, C. sorghi, C. africana, C. viridis, and C. pusilla. No relationship was found between the species placement and its morphological markers. The probe from C. purpurea gene cpd1 for dimethylallyl tryptophan synthase, the first enzyme of alkaloid biosynthesis, was hybridized to Pst 1 digested genomes of the above species under non-stringent conditions. Hybridizing DNA was present in all species of clade 1, although the signal of the C. paspali gene was weaker. In clade 2, only C. africana, C. gigantea, and C. pusilla gave weak positive signals. Colorimetric detection found small amounts of alkaloids in cultures of Claviceps sp. SG and PM but despite that, no cpd1 hybridizing bands were found. The occurrence of two major clades of Claviceps and their biogeography suggests, that the genus originates from South America and that the evolution of its species was influenced by comigration with their hosts and with the global climatic changes that influenced spreading of grass subfamilies.

Tissue distribution and developmental expression of type XVI collagen in the mouse

The expression of a recently identified collagen, α1(XVI), in adult mouse tissue and developing mouse embryo was examined by immunohistochemistry and in situ hybridization. A polyclonal antiserum was raised against a recombinant fusion protein, which contained a segment of 161 amino acids in the N-terminal noncollagenous domain of the human α1(XVI) collagen. Immunoprecipitation of metabolically labelled human or mouse fibroblast cell lysates with this antibody revealed a major, bacterial collagenase sensitive polypeptide of ∼210 kDa. The size agrees with the prediction from the full-length cDNA. Immunofluorescence examination of adult mouse tissues using the affinity purified antibody revealed a rather broad distribution of the protein. The heart, kidney, intestine, ovary, testis, eye, arterial walls and smooth muscles all exhibited significant levels of expression, while the skeletal muscle, lung and brain showed very restricted and low signals. During development, no significant expression of the mRNA or protein was observed in embryo of day 8 of gestation, but strong signals was detected in placental trophoblasts. Expression in embryos was detectable first after day 11 of gestation with weak positive signals appearing in the heart. In later stages of development, stronger RNA hybridizations were observed in a variety of tissues, particularly in atrial and ventricular walls of the developing heart, spinal root neural fibers and skin. These data demonstrate that type XVI collagen represents another collagenous component widely distributed in the extracellular matrix and may contribute to the structural integrity of various tissues.

DETECTION OF ERWINIA AMYLOVORA BY NESTED PCR AND PCR-DOT-BLOT AND REVERSE BLOT HYBRIDIZATIONS

The sensitivity and specificity of four methods based on the polymerase chain reaction (PCR) for detection of Erwinia amylovora were compared. A previously developed single-round PCR assay was used to amplify a specific 1-kb DNA fragment of plasmid pEA29, known to be unique to and conserved in E. amylovora. The ends of the 1-kb single-round PCR product were sequenced, and two new oligonucleotide primers were designed from sequences internal to the original primers and used in nested PCR. These primers directed amplification of a 844-bp fragment from the 1-kb first-round PCR product directly from E. amylovora cells but not from other bacteria associated with apple. Less than one cell of E. amylovora from pure culture was detected with nested PCR-an increase in sensitivity of 1,000-fold compared to single-round PCR. The lower limit of detection of PCR-dot-blot and reverse-blot hybridization methods was approximately 20 cells. Weak positive signals were sometimes produced by bacteria other than E. amylovora in hybridization experiments but only if nonspecific PCR products were visible on ethidium bromide-stained agarose gels. When asymptomatic apple tissue was screened, E. amylovora was detected in 61, 84, and 100 % of leaf samples; 0, 66, and 80 % of axillary bud samples; and 4, 27, and 75 % of mature fruit calyx samples by first-round PCR, PCR-dot-blot hybridization, and nested PCR, respectively. The potential applications of each method are discussed

Detection ofErwinia amylovoraby Nested PCR and PCR-Dot-Blot and Reverse-Blot Hybridizations

The sensitivity and specificity of four methods based on the polymerase chain reaction (PCR) for detection of Erwinia amylovora were compared. A previously developed single-round PCR assay was used to amplify a specific 1-kb DNA fragment of plasmid pEA29, known to be unique to and conserved in E. amylovora. The ends of the 1-kb single-round PCR product were sequenced, and two new oligonucleotide primers were designed from sequences internal to the original primers and used in nested PCR. These primers directed amplification of a 844-bp fragment from the 1-kb first-round PCR product directly from E. amylovora cells but not from other bacteria associated with apple. Less than one cell of E. amylovora from pure culture was detected with nested PCR-an increase in sensitivity of 1,000-fold compared to single-round PCR. The lower limit of detection of PCR-dot-blot and reverse-blot hybridization methods was approximately 20 cells. Weak positive signals were sometimes produced by bacteria other than E. amylovora in hybridization experiments but only if nonspecific PCR products were visible on ethidium bromide-stained agarose gels. When asymptomatic apple tissue was screened, E. amylovora was detected in 61, 84, and 100 % of leaf samples; 0, 66, and 80 % of axillary bud samples; and 4, 27, and 75 % of mature fruit calyx samples by first-round PCR, PCR-dot-blot hybridization, and nested PCR, respectively. The potential applications of each method are discussed

Tissue distribution and developmental expression of type XVI collagen in the mouse

Abstract The expression of a recently identified collagen, α1(XVI), in adult mouse tissue and developing mouse embryo was examined by immunohistochemistry and in situ hybridization. A polyclonal antiserum was raised against a recombinant fusion protein, which contained a segment of 161 amino acids in the N-terminal noncollagenous domain of the human α1(XVI) collagen. Immunoprecipitation of metabolically labelled human or mouse fibroblast cell lysates with this antibody revealed a major, bacterial collagenase sensitive polypeptide of ∼210 kDa. The size agrees with the prediction from the full-length cDNA. Immunofluorescence examination of adult mouse tissues using the affinity purified antibody revealed a rather broad distribution of the protein. The heart, kidney, intestine, ovary, testis, eye, arterial walls and smooth muscles all exhibited significant levels of expression, while the skeletal muscle, lung and brain showed very restricted and low signals. During development, no significant expression of the mRNA or protein was observed in embryo of day 8 of gestation, but strong signals was detected in placental trophoblasts. Expression in embryos was detectable first after day 11 of gestation with weak positive signals appearing in the heart. In later stages of development, stronger RNA hybridizations were observed in a variety of tissues, particularly in atrial and ventricular walls of the developing heart, spinal root neural fibers and skin. These data demonstrate that type XVI collagen represents another collagenous component widely distributed in the extracellular matrix and may contribute to the structural integrity of various tissues.