Molecular Analysis Of Samples Research Articles

Erysiphe corylacearum as a New Pathogen of Hazelnut in the Czech Republic

In July 2021, leaves and shoot tops of the common hazel (Corylus avellana L.), with a whitish coating, were found in the Czech Republic (southern Moravia region). The infected hazel bushes were found along a road in a deciduous forest and in an urban garden. In most European countries, Phyllactinia guttata is found on the abaxial surface of the leaves in the form of a continuous whitish to light grey mycelium, possibly with large black chasmothecia. In our case, the mycelium was present on both sides of the leaves, but the symptoms and the incidence were much stronger on the adaxial side. The first symptoms usually appeared on the adaxial side of the leaves as small white radially expanding patches of mycelium. In the final stage, the spots merged and covered a substantial part of the leaf blade (50–85 % on the adaxial side, 5–25 % on the abaxial side). When the abaxial side of the leaves was infected, chlorotic spots were evident on the adaxial side. The spots of powdery mildew were small (3–15 mm), whitish, rounded to irregular, effuse eventually becoming confluent, and occurred primarily on the adaxial side of the leaves. Conidiophores (30–53×4–6 µm) grew on the amphigenic mycelium, were erect, consisted of 1–3 cells, i.e. cylindrical foot cell and followed 1–2 cells, from which hyaline ellipsoid to doliform-limoniform conidia (17–34 ×15–21) (n = 50) were individually detached. Single or in groups dark brown chasmothecia (77–116 µm in diameter) had up to hyaline 8–15 aseptate straight appendages (50–102 µm) with multiple (3–5×) dichotomously branched apexes and recurved tips. Chasmothecia contained 3–6 asci (42–62 × 34–55 µm) with 4–8 obovoid to broadly ellipsoidal hyaline ascospores (14–22 × 75 µm). Based on morphological characters, the powdery mildew was identified as Erysiphe corylacearum (2). Morphological identification was confirmed by molecular analysis of samples. DNA was extracted from symptomatic leaves tissue using the DNeasy Plant Mini Kit, (Quiagen, Hilden, Germany) and the rDNA internal transcribed spacer region of 2 isolates was amplified using primers PMITS1 and PMITS2 (Cunnington et al. 2003) and sequenced. BLAST analysis of our 720bp fragments (both identical and represented by GenBank accession no. OR432526) showed 100% sequence identity to ITS rDNA sequences of E. corylacearum other countries of Central Europe for example from Austria (MW031866), Italy (MW045428), Hungary (OQ411007), Germany (OP554268) or Slovakia (MT176105). Pathogenicity was verified on two-year-old plants of Corylus avellana. Healthy leaves were artificially infected by dusting conidia from infected leaves. Inoculated plants were incubated under controlled conditions (21–23 °C, 70–80 % relative humidity). Characteristic symptoms of powdery mildew appeared on the adaxial side of the leaves 9–12 days after inoculation. Control plants treated with distilled water remained symptomless. Powdery mildew isolated from inoculated leaves was morphologically identical to isolates from naturally infected leaves. The first record of E. corylacearum in Europe on cultivated hazelnut species was reported by Sezer et al. (2017) in Turkey in 2013. Within a few years, the E. corylacearum spread and was recorded on various species of Corylus in other European countries (for example Mezzalama et al., 2020; Rosati et al., 2021; Beenken et al., 2022; Boneva et al., 2023), East Asia (Arzanlou et al., 2018) and the USA (Meparishvili 2019). To our knowledge, this is the first report of Erysiphe corylacearum in the Czech Republic.

Leptospirose genital bovina: achados em touros mantidos em condições do bioma Caatinga

ABSTRACT: Leptospirosis is a disease that causes economic and social impact, as it affects wild and domestic animals and humans. There may be peculiarities in the epidemiology of this disease in the Caatinga biome, Brazil, where the environment is adverse and the etiologic agent, Leptospira spp., requires alternative transmission routes. Considering that in bovine leptospirosis the genital carrier is constantly neglected and the lack of reports on the role of bulls in the epidemiology of the bovine genital leptospirosis (BGL) syndrome, mainly in semiarid conditions such as Caatinga biome, this study aimed to investigate bulls maintained in Caatinga biome conditions as genital carriers of leptospires. Urinary tract (urine, bladder, and kidney) and reproductive tract (vas deferens, cauda epididymis, and vesicular gland) samples were collected from 42 slaughtered bulls. Microscopic agglutination test (MAT), polymerase chain reaction (PCR), and microbiological isolation were included as diagnostic methods. Anti-Leptospira spp. antibodies were found in 17 (40.48%) animals, while 26 animals (61.90%) had at least one organ or urine with leptospiral DNA, and 10 animals (23.81%) were positive at bacteriological culture. Sequenced samples targeting the LipL32 gene showed 99% similarity with Leptospira borgpetersenii. Molecular analysis of samples from the vas deferens and cauda epididymis is recommended for the diagnosis of genital leptospirosis in bulls and, if it is impossible to collect these tissues, semen can be used. In conclusion, this study provides important information relating to bulls from the Caatinga biome, Brazil, as carriers of genital leptospirosis. The results indicate that, even under adverse environmental conditions, leptospires may survive and propagate, mainly due to the characteristic of genital carriers for the sexually spreading of adapted Leptospira species without influence by external variables. Thus, prevention and control strategies for bovine leptospirosis need to include actions aimed at the genital carrier.

Comparison of microscopy and metabarcoding to identify pollen used by the critically endangered rusty patched bumble bee, Bombus affinis

Abstract Taxonomic analysis of pollen collected by bees can provide insights into their host plant use, providing information about the plant species selected for targeted conservation strategies. The two main identification approaches used are morphological analysis of pollen samples affixed to microscope slides (i.e., microscopic palynology) and molecular analysis of samples. Both methods are widely used for freshly collected materials and have been compared in multiple studies, yet their application to archived samples remains to be explored. Archived samples may be particularly useful for the study of rare or protected species, particularly when historical foraging patterns are uncertain. We used both methods to analyse pollen collected by the endangered rusty patched bumble bee species, Bombus affinis Cresson, applied to museum‐archived specimens. Pollen samples were removed from the corbiculae of bumble bees originally collected in Michigan between 1914 and 1974. Samples included 24 rusty patched bumble bees each with large pollen loads on both corbiculae, allowing for pollen from the same bee to be analysed using both methods. DNA metabarcoding detected more plant taxa than light microscopy, and DNA barcoding also had higher taxonomic resolution when compared to taxa determined using light microscopy. In many instances, pollen could only be confidently identified to tribe or family with light microscopy. Discrepancy between methods decreased when taxa identified via DNA metabarcoding were binned into ecologically relevant groups corresponding to those identified using light microscopy. Although binning demonstrated smaller within‐method variance, there was still minimal correspondence between the two methods. Results indicate there are benefits and biases unique to each method and highlight the utility of binning taxonomic results to morphological or ecological groupings.

Metformin impairs bile acid homeostasis in ethinylestradiol-induced cholestasis in mice

Metformin, an oral antidiabetic drug, recently demonstrated a reducing effect on bile acids (BA) plasma concentrations in one patient with intrahepatic cholestasis of pregnancy (ICP) by unknown mechanism. Therefore, the aim of the present study was to examine the effect of metformin on BA homeostasis and related molecular pathways in the liver and intestine using a mouse model of ICP. The cholestasis was induced in female C57BL/6 mice by repeated administration of ethinylestradiol (10 mg/kg BW s.c.) and/or metformin (150 mg/kg BW orally) over 5 consecutive days with subsequent bile collection and molecular analysis of samples. We demonstrated that metformin significantly increased the rate of bile secretion in control mice. This increase was BA dependent and was produced both by increased liver BA synthesis via induced cholesterol 7α-hydroxylase (Cyp7a1) and by increased BA reabsorption in the ileum via induction of the apical sodium-dependent BA transporter (Asbt). In contrast, metformin further worsened ethinylestradiol-induced impairment of bile secretion. This reduction was also BA dependent and corresponded with significant downregulation of Bsep, and Ntcp, major excretory and uptake transporters for BA in hepatocytes, respectively. The plasma concentrations of BA were consequently significantly increased in the metformin-treated mice. Altogether, our data indicate positive stimulation of bile secretion by metformin in the intact liver, but this drug also induces serious impairment of BA biliary secretion, with a marked increase in plasma concentrations in estrogen-induced cholestasis. Our results imply that metformin should be used with caution in situations with hormone-dependent cholestasis, such as ICP.

Colour variation of the intertidal hermit crabClibanarius virescensconsidering growth stage, geographic area in the Indo–West Pacific Ocean, and molecular phylogeny

AbstractMembers ofClibanarius virescensshow considerable intraspecific colour variation, including colouration of the second/third pereopods (green/white) and the dactyls of the second/third pereopods (with or without dark bands/patches). However, factors inducing these colour variations have not yet been elucidated. Here, we investigated the occurrence of colour variation in this species with particular emphasis on change of colouration associated with growth stage and region in specimens from tropical/subtropical to warm temperate areas in the Indo–West Pacific, including evidence from molecular phylogeny based on the cytochrome c oxidase subunit I (COI). We have, then, clarified that the colouration on the pereopod dactyls gradually changed from solid colour (yellow/white) to having dark-coloured area(s) or transverse band(s) as a result of the growth stage. The frequency of occurrence of the solid colour dactyls was higher than those of other colour types in tropical regions. Our results also indicated that the white ambulatory leg type was the colouration type that was frequently seen in juvenile stages. However, significant genetic differences were not detected between each colouration determined by molecular analysis of samples from 14 localities in the Indo-West Pacific region; in contrast, two genetically differentiated regional populations (North Australia; Phuket, Thailand; and Lombok, Indonesia) were detected. The present study, therefore, emphasizes the necessity for further study on the colour variation of marine animals focusing on growth stages and regional differences, with molecular data to facilitate the research on adaptation and/or speciation, especially in geographically widely distributed species.

Frequency and genotyping of alpha thalassemia in individuals undergoing premarital screening.

To evaluate the frequency of alpha thalassemia and detect mutations in the alpha genes in individuals undergoing premarital screening. The cross-sectional study was conducted at King Fahad Central Hospital, Jazan, Saudi Arabia, from January 2018 to May 2019, and comprised blood samples of individuals visiting the premarital screening clinic. The samples were analyzed for complete blood counts and haemoglobin electrophoresis. Molecular analysis of samples suspected for alpha thalassemia was done using alpha-globin StripAssay kit. Data was anlaysed using SPSS 20. Of the 3,970 samples analysed, 1,859(46.83%) were from males and 2,111(53.17%) from females. The overall frequency of suspected alpha thalassemia was 4.43% based upon haematological parameters including complete blood count and haemoglobin electrophoresis. Overall, 80 suspected samples were selected for genetic analyses, and, of them, 76 (95%) were positive for deletional and non-deletional mutations of alpha-globin genes, while 4 (5%) were negative for any of the 21 mutations tested. Alpha thalassemia was found to be highly prevalent in the study area.

Oleogelation of rapeseed oil with cellulose fibers as an innovative strategy for palm oil substitution in chocolate spreads

Abstract The texturing properties of mercerized cellulose dispersed in rapeseed oil without any prior thermal treatment were investigated as a function of botanical origin, cellulose content, mass fraction and fiber size through a molecular analysis of samples, macroscopic/microscopic observations and rheological experiments (continuous and oscillatory shear flows). A solid-like behavior of dispersions was demonstrated for a minimum cellulose content of about 60wt% in the vegetal powder. The oleogelation efficiency increases with the mass fraction of powder in oil and the maximum size of cellulose fibers as a result of weak attractive interactions and fiber entanglement giving rise to a network entrapping the liquid phase. An innovative strategy using a 30wt% dispersion of bamboo fibers in rapeseed oil was finally used to produce in a conch a chocolate spread with a high thermal stability at 38°C and healthier nutritional qualities compared to an equivalent product prepared with palm oil.

Impression Cytology Is a Non-invasive and Effective Method for Ocular Cell Retrieval of Zika Infected Babies: Perspectives in OMIC Studies.

ImportanceNon-invasive techniques for retrieving ocular surface cells from babies infected by zika virus (ZIKV) during the gestational period remain to be determined.ObjectivesThe aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from Zika infected babies with and without Congenital Zika Syndrome (CZS) in satisfactory amount and quality to enable easy adoption in the field and application in the context of genomic and molecular approaches.Design, Settings, and ParticipantsOcular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. After an authorized written informed consent from the parents, samples were collected from both eyes of 12 babies (4 babies with maternal ZIKV exposure during gestation and presence of clinical signs which included ocular abnormalities and microcephaly; 4 babies with maternal ZIKV exposure during gestation but no clinical signs; and 4 unaffected control babies with negative PCR for Zika virus and without clinical signs). Cells were used for microscopy analyses and evaluated for their suitability for downstream molecular applications in transcriptomic and proteomic experiments.ResultsOur optimized impression cytology protocol enabled the capture of a considerable number of viable cells. The microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed potential morphological alterations (cell keratinization, pyknosis, karyolysis, anucleation, and vacuolization). Molecular approaches were also performed in parallel. Genomic DNA and RNA were successfully isolated from all samples to enable the establishment of transcriptomic and proteomic studies.Conclusions and RelevanceOur method proved to be a suitable, fast, and non-invasive tool to obtain ocular cell preparations from babies with and without Zika infection. The method yielded sufficient cells for detailed morphological and molecular analyses of samples. We discuss perspectives for the application of impression cytology in the context of ZIKV studies in basic and clinical research.

Comparing the efficacy and cost-effectiveness of sampling methods for estimating population abundance and density of a recovering carnivore: the European pine marten (Martes martes)

Many methods are available to gather data on wildlife population parameters, such as population abundance and density, yet few have been compared or validated. We compared the efficacy of three survey methods (live trapping, hair tubes and scats) for estimating abundance and population density of the European pine marten (Martes martes) in Galloway Forest, Scotland. We evaluated these methods by, firstly, comparing the accuracy of the population estimate derived from each method, and, secondly, comparing the financial cost of each method. Molecular analysis of samples from all three methods was used to determine sex and individual genotype. Population abundance estimates were derived from capture-recapture programme Capwire. The non-invasive methods (hair tubes and scats combined) detected 81% of known individuals, although hair tubes and scats performed poorly alone, detecting 48% and 52% of individuals, respectively. Live trapping was the individual method that detected the most individuals (77%). Hair tubes were the most expensive method, both in financial cost and personnel hours, whilst scat sampling was the cheapest method. There was a highly significant association between the sex of the animal and the total number of detections by method. The population abundance estimate from all methods combined was 32 (95% CI 31–35) and the population density estimate was 0.27 martens/km2. This study indicates that a combined sampling approach comprising hair tubes and scats maximises the number of detections and provides a viable alternative to invasive live trapping.

Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa.

Background: Wolbachia, a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the Anopheles (An.) genera, but has recently been found in An. gambiae s.l. populations. As there are numerous Anopheles species that have the capacity to transmit malaria, we analysed a range of species to determine Wolbachia prevalence rates, characterise novel Wolbachia strains and determine any correlation between the presence of Plasmodium, Wolbachiaand the competing endosymbiotic bacterium Asaia. Methods: Anopheles adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC), Ghana, Uganda and Madagascar, between 2013 and 2017. Molecular analysis of samples was undertaken using quantitative PCR, Sanger sequencing, Wolbachia multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial 16S rRNA gene. Results: Novel Wolbachia strains were discovered in five species: An. coluzzii, An. gambiae s.s., An. arabiensis, An. moucheti and An. species 'A', increasing the number of Anopheles species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with Wolbachia supergroup B strains. We also provide evidence for resident strain variants within An. species 'A'. Wolbachia is the dominant member of the microbiome in An. moucheti and An. species 'A', but present at lower densities in An. coluzzii. Interestingly, no evidence of Wolbachia/Asaia co-infections was seen and Asaia infection densities were also shown to be variable and location dependent. Conclusions: The important discovery of novel Wolbachia strains in Anopheles provides greater insight into the prevalence of resident Wolbachia strains in diverse malaria vectors. Novel Wolbachia strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other Anopheles mosquito species, which could be used for population replacement or suppression control strategies.

Listeria, haurdunaldirako desafioa?

Azken urteotan, Gipuzkoan, listeriosiaren intzidentziaren igoera esanguratsua atzeman da. Arrisku-taldeen artean, haurdun dauden emakumeak aurkitzen dira. Transmisio-bide ohikoena helduetan, kutsatutako elikagaien bidezkoa da, eta fetuaren eta jaioberrien kasuan, berriz, transmisio bertikala (plazenta edo erditze-kanalean zehar). Kasuen erregistroa Ingurumen Ikerkuntza eta Osasun Publikoko teknikariek egin dute eta laginen azterketa molekularra Donostia Ospitaleko Mikrobiologia Zerbitzuan eta Mikrobiologia Zentro Nazionalean egin dira, PFGE teknika erabiliz andui ezberdinakisolatzeko. Aldi berean, Gipuzkoan izandako azken urteotako listeriosi kasuen bilaketa klinikoa eginez, zehaztu ahal izan da zein izan den haurdun eta jaioberrien kasuen ezaugarri eta bilakaera. 2008-2014 urte bitartean, Gipuzkoan 73 kasu berri jakinarazi ziren, eta horietatik 52, 2012-2014 urte bitartean. Urte horietako kasuen informazio kliniko eta mikrobiologikoak aztertu dira lan honetan. 2013. urtean, 22 kasurekin, azken 10 urteetako intzidentzia hirukoiztu zen. Urte horretan, haurdun eta jaioberriak arrisku-taldeen % 50 izan ziren (9 kasu).Haurdunaren infekzioa detektatzea zaila izan liteke, sintoma inespezifikoak eragiten ditu-eta (digestio-aparatuarekin lotutako sintomak eta sukarra), baina umekiarengan ondorio kaltegarriak izan ditzake (abortu septikoa, goiztiartasuna). Lan honetan aztertutako kasu gehienetan haurdunak sukarrarekin agertu ziren eta kasu gehienetan erditze goiztiarra gertatu zen. Kasuen banaketa eta ezaugarriak ezberdinak izan dira urte batetik bestera. 2012ko kasuek banaketa topografiko zehatz bat izan zuten eta 2014ko kasuak, ordea, elikagai konkretu bati loturikoak izan dira. Lortutako datuen arabera, eta behaketa horren ostean infekzio-bide nagusia elikagai kutsatuen bidezkoa dela ohartuta, prebentzio-neurriak areagotu behar direla atzeman dugu, eta, horretarako, elikagaien inguruko heziketa egokia eta lehen mailako prebentzio-neurriak azaltzen ditugu.

Rapid onset of cardiomyopathy in STZ-induced female diabetic mice involves the downregulation of pro-survival Pim-1

BackgroundDiabetic women are five times more likely to develop congestive heart failure compared with two fold for men. The underlying mechanism for this gender difference is not known. Here we investigate the molecular mechanisms responsible for this female disadvantage and attempt safeguarding cardiomyocytes viability and function through restoration of pro-survival Pim-1.Methods and ResultsDiabetes was induced by injection of streptozotocin in CD1 mice of both genders. Functional and dimensional parameters measurement using echocardiography revealed diastolic dysfunction in female diabetic mice within 8 weeks after STZ-induced diabetes. This was associated with significant downregulation of pro-survival Pim-1 and upregulation of pro-apoptotic Caspase-3, microRNA-1 and microRNA-208a. Male diabetic mice did not show any significant changes at this time point (P < 0.05 vs. female diabetic). Further, the onset of ventricular remodelling was quicker in female diabetic mice showing marked left ventricular dilation, reduced ejection fraction and poor contractility (P < 0.05 vs. male diabetic at 12 and 16 weeks of STZ-induced diabetes). Molecular analysis of samples from human diabetic hearts confirmed the results of pre-clinical studies, showing marked downregulation of Pim-1 in the female diabetic heart (P < 0.05 vs. male diabetic). Finally, in vitro restoration of Pim-1 reversed the female disadvantage in diabetic cardiomyocytes.ConclusionsWe provide novel insights into the molecular mechanisms behind the rapid onset of cardiomyopathy in female diabetics. These results suggest the requirement for the development of gender-specific treatments for diabetic cardiomyopathy.

Invasion Success in Cogongrass (Imperata cylindrica): A Population Genetic Approach Exploring Genetic Diversity and Historical Introductions

AbstractPropagule pressure significantly contributes to and limits the potential success of a biological invasion, especially during transport, introduction, and establishment. Events such as multiple introductions of foreign parent material and gene flow among them can increase genetic diversity in founding populations, often leading to greater invasion success. We applied the tools and theory of population genetics to better understand the dynamics of successful biological invasion. The focal species, cogongrass, is a perennial invasive grass species significantly affecting the Gulf Coast and southeastern region of the United States. The literature indicates separate, allopatric introductions of material from East Asia (Philippines and Japan) into the U.S. states of Mississippi and Alabama. Molecular analysis of samples from those two states utilized amplified fragment length polymorphism (AFLP) markers on 388 individuals from 21 localities. We hypothesized that previously isolated lineages of cogongrass are present and crossing in the Southeast. We observed genetic variation within localities (0.013 ≤ heterozygosity (He) ≤ 0.051, mean = 0.028 ± 0.001) with significant and substantial population structure (FST= 0.534, P &lt; 0.001). Population structure analyses detected two genetically defined and statistically supported populations, which appear to have experienced some admixture. The geographic distribution of those populations was consistent with the two-introduction scenario reported previously. These results are also consistent with contact in the invasive range of previously isolated lineages from the native range.

EBUS and EUS guided fine needle aspirations for molecular diagnostic analysis in lung cancer.

In daily clinical practice the diagnosis of lung cancer is often based on cytological specimens. These cytological samples are increasingly obtained by ultrasound-guided techniques with fine needle aspirations. Recent developments have shown that transesophageal ultrasound guided fine needle aspiration (EUS-FNA) and endobronchial ultrasound guided transbronchial fine needle aspiration (EBUS-TBNA) are minimally invasive diagnostic and staging procedures that have shown to be highly sensitive and accurate. Although several studies have shown that these cytological samples allow for reliable diagnosis and sub classification of non-small cell lung cancer, cytological samples for molecular analysis are not yet routinely used. In this paper we review the current literature regarding the results of molecular analysis of samples obtained by EUS-FNA and/or EBUS-TBNA, focusing on the targets for currently available treatments of non-small cell lung cancer like epidermal growth factor receptor (EGFR), Kirsten rat sarcoma oncogene (KRAS) and Echinoderm microtubule-associated protein-like 4 gene anaplastic lymphoma kinase gene translocation (EML4-ALK). We conclude that the cytological samples obtained by endosonography guided fine needle aspirations (EUS and EBUS) are highly accurate for molecular analysis. This analysis can be performed reliably in the vast majority of patients in daily practice.

Ocular Whipple's Disease: Therapeutic Strategy and Long-Term Follow-Up

To characterize the clinical features of ocular Whipple's disease (WD) and determine the long-term prognosis after antibiotic treatment. Retrospective case series. Medical records of patients referred between January 1993 and December 2010 were reviewed for chronic corticosteroid-resistant uveitis or neuro-ophthalmologic findings consistent with WD. Eleven patients (male/female = 9/2) were included in this study. Diagnosis was based on cytologic examination and molecular analysis of samples (cerebrospinal fluid, vitreous, duodenum, or any involved lymph node). It was based on cytology before the routine use of polymerase chain reaction (PCR) and on both cytology and molecular biology for more recent patients. Long-term antibiotic therapy included oral trimethoprim-sulfamethoxazole (TMP-SMX) and rifampin, TMP-SMX alone, rifampin alone, or tetracycline alone. (1) Demographic and clinical characteristics of patients with positive PCR for Tropheryma whipplei or periodic acid-Schiff-positive macrophages in the vitreous and (2) long-term prognosis after antibiotic treatment. Mean age at diagnosis was 63 years (range, 51-73 years). Average time between the onset of the disease and diagnosis was 2 years (range, 1 month to 11 years). Mean follow-up was 7.2 years (range, 0.25-18 years). Ophthalmologic findings consisted of chronic uveitis (9 patients), isolated bilateral optic disc swelling (1 patient), and Parinaud syndrome (1 patient). All patients had PAS-positive macrophages, and 6 patients had a positive PCR for T. whipplei. Nine patients were treated with TMP-SMX and rifampin. One patient treated with only tetracycline relapsed and was successfully treated with TMP-SMX. No major side effects were reported. Intraocular inflammation and neurologic manifestations were controlled in all cases. At the end of follow-up, 2 patients were off treatment, 2 patients had a neurologic relapse after treatment interruption, and 5 patients were still taking TMP-SMX. One patient was taking only rifampin. Two patients were lost to follow-up. Ocular WD seems to be a neurologic manifestation of WD. Trimethoprim-SMX with rifampin is an efficient treatment, and prolonging treatment for at least 1 year is recommended. Long-term low-dose antibiotic therapy may reduce the rate of relapse, neurologic involvement, and death.

Molecular characterisation of the intestinal microbiota of farmed Atlantic salmon (Salmo salar L.)

In this study, the intestinal microbiota of farmed Atlantic salmon (Salmo salar L.) was examined with traditional culture-based techniques, and by molecular analysis of the 16S rDNA. The aim of the study was to evaluate the use of cultivation-based and molecular-based techniques, and compare the results obtained by the two methods. Samples were collected from the fore-, mid- and hind-gut, respectively. PCR and denaturing gradient gel electrophoresis (DGGE) analyses were performed on DNA extracted directly from the gut content and from bacteria cultivated on Tryptic Soy Agar (TSA). Population fingerprints of the predominant microbiota were generated by DGGE analysis of universal V3 16S rDNA PCR amplicons, and distinct bands from DGGE were sequenced. Results show that the salmon intestine was dominated by Lactobacillus spp., Lactococcus sp., Bacillus sp., Photobacterium phosphoreum, Acinetobacter sp., Pseudomonas sp. and Vibrio sp. Molecular analyses of samples from direct DNA extraction enabled detection of lactic acid bacteria. Prior cultivation detected Vibrio sp., Acinetobacter sp. and Pseudomonas sp., which were not detected by the direct analyses. In addition to the PCR–DGGE approach, 50 TSA isolates from the hind-gut were pure cultured and identified by partial sequencing of the 16S rDNA. Vibrio spp. and P. phosphoreum dominated among these isolates. The data provided demonstrate the use of PCR–DGGE as an alternative method for studying the intestinal microbiota of fish.

P53 mutation profiling of multiple esophageal carcinoma using laser capture microdissection to demonstrate field carcinogenesis.

Inactivation of the p53 tumor suppressor gene is one of the most frequent genetic alterations observed in human esophageal carcinomas. In patients with esophageal carcinoma, one of the significant pathological features of the tumor is the presence of multiple lesions within the esophagus. However, the molecular mechanisms involved in the occurrence of multiple lesions have remained elusive. To characterize p53 alterations in multiple esophageal carcinomas and to study their roles in carcinogenesis, we performed p53 immunohistochemical and p53 mutation analyses using laser capture microdissection on surgically resected human esophageal carcinomas from 11 patients: 9 patients with multiple esophageal carcinomas, 1 with an intramural metastasis lesion within the esophagus and 1 with an intraepithelial carcinoma lesion contiguous to the main lesion. In each of the patients with multiple esophageal carcinomas, we examined samples from 1 main lesion and 1 representative concomitant lesion. Molecular analyses of samples from fresh-frozen normal tissues and tumor tissues of the main lesion (whole tumor) were also performed by the same method. p53 protein accumulation was observed in 16 (72.7%) of 22 lesions from the 11 cases. No p53 mutation was found in normal esophageal tissues. In the 9 cases of multiple esophageal carcinomas, point mutations were detected in the whole tumor in 1 (11.1%) case, in the microdissected tumor samples of main lesions in 3 (33.3%) cases and in the microdissected tumor samples of concomitant lesions in 3 (33.3%) cases. For the microdissected tumor samples, there was a 54.5% (12/22) concordance rate between the results of immunostaining and molecular analysis. In the 8 cases of whole tumors in which a p53 mutation was not observed, 2 cases revealed p53 mutation in the microdissected tumor samples of the main lesion. All 6 cases of multiple esophageal carcinomas that showed a p53 mutation in the microdissected tumor sample had a discordant p53 mutational status between the main and concomitant lesions. In contrast, both the intramural metastasis lesion and the intraepithelial carcinoma contiguous to the main lesion showed p53 mutational patterns identical to those of the main lesions. In conclusion, the analysis of microdissected DNA by laser capture microdissection is useful for characterizing the heterogeneity of the p53 gene mutation in multiple carcinoma lesions that cannot be accurately analyzed in whole esophageal tumor DNA. The finding of different p53 gene mutations among multiple esophageal carcinoma lesions suggest further evidence of multicentric or field carcinogenesis of the human esophagus.

Molecular ecology of a Neolithic meadow: The DNA of the grass remains from the archaeological site of the Tyrolean Iceman

The paper reports on the molecular analysis of samples of approximately 5,300-year-old grass found at the alpine archaeological site where the so-called Tyrolean Iceman was discovered. The grass comes from a ‘cloak’ made of long grass blades and/or the stuffing of the ‘snow footwear’ worn by the Iceman. The results show that while the largest fraction of the DNA extractable from the grass is of ‘foreign’ origin, a much smaller part belongs to the original genetic material of the grass itself, and can be used as a valuable taxonomic clue to the plant species utilized by neolithic men to manufacture their equipment. On the other hand, the ‘foreign’ DNA, or at least a portion of it, comes from microorganisms-mainly filamentous fungi and unicellular algae-which seem to have been associated with the grass since the time the grass was harvested.