Embryonic gene expression of Coregonus palaea (whitefish) under pathogen stress as analyzed by high-throughput RNA-sequencing.

Most fishes produce free-living embryos that are exposed to environmental stressors immediately following fertilization, including pathogenic microorganisms. Initial immune protection of embryos involves the chorion, as a protective barrier, and maternally-allocated antimicrobial compounds. At later developmental stages, host-genetic effects influence susceptibility and tolerance, suggesting a direct interaction between embryo genes and pathogens. So far, only a few host genes could be identified that correlate with embryonic survival under pathogen stress in salmonids. Here, we utilized high-throughput RNA-sequencing in order to describe the transcriptional response of a non-model fish, the Alpine whitefish Coregonus palaea, to infection, both in terms of host genes that are likely manipulated by the pathogen, and those involved in an early putative immune response. Embryos were produced in vitro, raised individually, and exposed at the late-eyed stage to a virulent strain of the opportunistic fish pathogen Pseudomonas fluorescens. The pseudomonad increased embryonic mortality and affected gene expression substantially. For example, essential, upregulated metabolic pathways in embryos under pathogen stress included ion binding pathways, aminoacyl-tRNA-biosynthesis, and the production of arginine and proline, most probably mediated by the pathogen for its proliferation. Most prominently downregulated transcripts comprised the biosynthesis of unsaturated fatty acids, the citrate cycle, and various isoforms of b-cell transcription factors. These factors have been shown to play a significant role in host blood cell differentiation and renewal. With regard to specific immune functions, differentially expressed transcripts mapped to the complement cascade, MHC class I and II, TNF-alpha, and T-cell differentiation proteins. The results of this study reveal insights into how P. fluorescens impairs the development of whitefish embryos and set a foundation for future studies investigating host pathogen interactions in fish embryos.

Sanitary quality of edible bivalve mollusks in Southeastern Brazil using an UV based depuration system

The increase in seafood production, especially in mariculture worldwide, has brought out the need of continued monitoring of shellfish production areas in order to ensure safety to human consumption. The purpose of this research was to evaluate pathogenic protozoa, viruses and bacteria contamination in oysters before and after UV depuration procedure, in brackish waters at all stages of cultivation and treatment steps and to enumerate microbiological indicators of fecal contamination from production site up to depuration site in an oyster cooperative located at the Southeastern estuarine area of Brazil. Oysters and brackish water were collected monthly from September 2009 to November 2010. Four sampling sites were selected for enteropathogens analysis: site 1- oyster growth, site 2- catchment water (before UV depuration procedure), site 3 - filtration stage of water treatment (only for protozoa analysis) and site 4- oyster's depuration tank. Three microbiological indicators ! were examined at sites 1, 2 and 4. The following pathogenic microorganisms were searched: Giardia cysts, Cryptosporidium oocysts, Human Adenovirus (HAdV), Hepatitis A virus (HAV), Human Norovirus (HnoV) (genogroups I and II), JC strain Polyomavirus (JCPyV) and Salmonella sp. Analysis consisted of molecular detection (qPCR) for viruses (oysters and water samples); immunomagnetic separation followed by direct immunofluorescence assay for Cryptosporidium oocysts and Giardia cysts and also molecular detection (PCR) for the latter (oysters and water samples); commercial kit (Reveal-Neogee (R)) for Salmonella analysis (oysters). Giardia was the most prevalent pathogen in all sites where it was detected: 36.3%, 18.1%, 36.3% and 27.2% of water from sites 1, 2, 3 and 4 respectively; 36.3% of oysters from site 1 and 54.5% of depurated oysters were harboring Giardia cysts. The huge majority of contaminated samples were classified as Giardia duodenalis. HAdv was detected in water and o! ysters from growth site and HnoV GI in two batches of oysters ! (site 1) in huge concentrations (2.11 x 10(13), 3.10 x 10(12) gc/g). In depuration tank site, Salmonella sp., HAV (4.84 x 10(3)) and HnoV GII (7.97 x 10(14)) were detected once in different batches of oysters. Cryptosporidium spp. oocysts were present in 9.0% of water samples from site four. These results reflect the contamination of oysters even when UV depuration procedures are employed in this shellfish treatment plant. Moreover, the molecular comprehension of the sources of contamination is necessary to develop an efficient management strategy allied to shellfish treatment improvement to prevent foodborne illnesses. (C) 2011 Elsevier Ltd. All rights reserved.

Aetiologies of pulseless electrical activity in out-of-hospital cardiac arrests:A retrospective study and analysis of specific causes

Background: Pulseless electrical activity (PEA) cardiac arrest is defined as a cardiac arrest (CA) presenting with a residual organized electrical activity on the electrocardiogram. In the last decades, the incidence of PEA has regularly increased, compared to other types of CA like ventricular fibrillation or pulseless ventricular tachycardia. PEA is frequently induced by reversible conditions. The "4 (or 5) H" & "4 (or 5) T" are proposed as a mnemonic to asses for Hypoxia, Hypovolemia, Hypo- /Hyperkalaemia, Hypothermia, Thrombosis (cardiac or pulmonary), cardiac Tamponade, Toxins, and Tension pneumothorax. Other pathologies (intracranial haemorrhage, severe sepsis, myocardial contraction dysfunction) have been identified as potential causes for PEA, but their respective probability and frequencies are unclear and they are not yet included into the resuscitation guidelines. The aim of this study was to analyse the aetiologies of PEA out-of-hospital CA, in order to evaluate the relative frequencies of each cause and therefore to improve the management of patients suffering a PEA cardiac arrest. Method: This retrospective study was based on data routinely and prospectively collected for each PEMS intervention. All adult patients treated from January 1st 2002 to December 2012 31st by the PEMS for out-of-hospital cardiac arrest, with PEA as the first recorded rhythm, and admitted to the emergency department (ED) of the Lausanne University Hospital were included. The aetiologies of PEA cardiac arrest were classified into subgroups, based on the classical H&T's classification, supplemented by four other subgroups analysis: trauma, intra-cranial haemorrhage (ICH), non-ischemic cardiomyopathy (NIC) and undetermined cause. Results: 1866 OHCA were treated by the PEMS. PEA was the first recorded rhythm in 240 adult patients (13.8 %). After exclusion of 96 patients, 144 patients with a PEA cardiac arrest admitted to the ED were included in the analysis. The mean age was 63.8 ± 20.0 years, 58.3% were men and the survival rate at 48 hours was 29%. 32 different causes of OHCA PEA were established for 119 patients. For 25 patients (17.4 %), we were unable to attribute a specific cause for the PEA cardiac arrest. Hypoxia (23.6 %), acute coronary syndrome (12.5%) and trauma (12.5 %) were the three most frequent causes. Pulmonary embolism, Hypovolemia, Intoxication and Hyperkaliemia occurs in less than 10% of the cases (7.6 %, 5.6 %, 3.5%, respectively 2.1 %). Non ischemic cardiomyopathy and intra-cranial haemorrhage occur in 8.3 % and 6.9 %, respectively. Conclusions: According to our results, intra-cranial haemorrhage and non-ischemic cardiomyopathy represent noticeable causes of PEA in OHCA, with a prevalence equalling or exceeding the frequency of classical 4 H's and 4 T's aetiologies. These two pathologies are potentially accessible to simple diagnostic procedures (native CT-scan or echocardiography) and should be included into the 4 H's and 4 T's mnemonic.

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

Role of Membrane Transporters, P-Glycoprotein and Mrp1, in The Placental Transfer of Drugs With Special Reference to Saquinavir and Quetiapine

Drug transporting membrane proteins are expressed in various human tissues and blood-tissue barriers, regulating the transfer of drugs, toxins and endogenous compounds into or out of the cells. Various in vitro and animal experiments suggest that P-glycoprotein (P-gp) forms a functional barrier between maternal and fetal blood circulation in the placenta thereby protecting the fetus from exposure to xenobiotics during pregnancy. The multidrug resistance-associated protein 1 (MRP1) is a relatively less studied transporter protein in the human placenta. The aim of this study series was to study the role of placental transporters, apical P-gp and basal MRP1, using saquinavir as a probe drug, and to study transfer of quetiapine and the role of P-gp in its transfer in the dually perfused human placenta/cotyledon. Furthermore, two ABCB1 (encoding P-gp) polymorphisms (c.3435C>T, p.Ile1145Ile and c.2677G>T/A, p.Ala893Ser/Thr) were studied to determine their impact on P-gp protein expression level and on the transfer of the study drugs. Also, the influence of the P-gp protein expression level on the transfer of the study drugs was addressed. Because P-gp and MRP1 are ATP-dependent drug-efflux pumps, it was studied whether exogenous ATP is needed for the function of ATP-dependent transporter in the present experimental model. The present results indicated that the addition of exogenous ATP was not necessary for transporter function in the perfused human placental cotyledon. Saquinavir and quetiapine were both found to cross the human placenta; transplacental transfer (TPTAUC %) for saquinavir was <0.5% and for quetiapine 3.7%. Pharmacologic blocking of P-gp led to disruption of the blood-placental barrier (BPB) and increased the placental transfer of P-gp substrate, saquinavir, into the fetal circulation by 6- to 8-fold. In reversed perfusions P-gp, MRP1 and possibly OATP2B1 had a negligible role in the fetal-to-maternal transfer of saquinavir. The TPTAUC % of saquinavir was about 100-fold greater from the fetal side to the maternal side compared with the maternal-to-fetal transfer. P-gp activity is not likely to modify the placental transfer of quetiapine. Higher P-gp protein expression levels were associated with the variant allele 3435T, but no correlation was found between the TPTAUC % of saquinavir and placental P-gp protein expression. The present results indicate that P-gp activity drastically affects the fetal exposure to saquinavir, and suggest that pharmacological blockade of the P-gp activity during pregnancy may pose an increased risk for adverse fetal outcome. The blockade of P-gp activity could be used in purpose to obtain higher drug concentration to the fetal side, for example, in prevention (to decrease virus transfer to fetal side) or in treating sick fetus.

Metodologias analíticas para determinação das fumonisinas em milho e alimentos à base de milho

Fumonisins are mycotoxins occurring worldwide, mainly in maize and maize-based food products, which could affect animal and human health. This paper reviews analytical methodologies for the determination of these fungal toxins in foods. It includes extraction, cleanup, derivatization procedures, detection, quantification, and confirmation procedures. Initial attempts at gas chromatographic methods and thin layer chromatography were supplanted by liquid chromatographic methods, mainly performed with fluorometric detection, or mass spectrometry detection, enabling the analysis of polar and thermolabile chemicals without chemical derivatization, which results in lower limits of detection. Alternative methods, such as enzyme linked immunosorbent assay or zone capillary zone electrophoresis, are also described.

Natural Science in the Bothnian Sea During 1975-2008: A Review

The Baltic Sea is one of the most studied areas in the world. However, parts of its northernmost reach, the Bothnian Sea, seem to be under represented in the natural scientific literature compared to other parts of the Baltic. The Bothnian Sea represents a unique inland sea environment for the scientific community to study due to its shallowness and low salinity. The natural sciences research carried out on the Bothnian Sea has been reviewed between 1975 and 2008. This time period was chosen to continue on from an earlier review paper ending in 1974. Along with the number of papers published the goal was also to review the content of the papers, indentifying dominating themes to evaluate gaps in the current knowledge on the Bothnian Sea and provide recommendations for topics of future research focus. In a classification into specific research topics biodiversity was the leading research focus followed by chemical and physical oceanography, pollution and toxins, and fish biology and fisheries. The current good condition of the Bothnian Sea is highly valued with its historically less eutrophic and clearer waters when compared to the Baltic. However, today the Bothnian Sea is facing eutrophication resulting from nutrient-rich water transported by the many rivers draining into it from Sweden and Finland making it an area in need of protection and preservation. More human activity will also concentrate on the Bothnian Sea in the future. Therefore the use of the sea and its coastal areas must be planned carefully to minimize the harmful effects of this increasing human activity. To achieve this more information is needed for the basis of Integrated Coastal Zone Management (ICZM) and maritime spatial planning (MSP). For example, for the Bothnian Sea the information on the underwater nature which is essential for ICZM is so far missing to a large extent. Specific biological, chemical and physical oceanographic information is needed to combine with economic analyses and environmental policies regarding this region. More research of a multidisciplinary nature is required on the unique Bothnian Sea environment and this we feel is best achieved through a joint Finnish-Swedish research strategy.

Double-bilayer: a new phase formed by lysophospholipids and the corresponding fatty acid

The product of catalytic activity of the enzyme phospholipase A2, which resembles the core unit of animal toxins, on phospholipids is a 1:1 mixture of lysolipid and fatty acid. This mixture was studied by time-resolved simultaneous small- and wide angle x-ray diffraction over the temperature range from 23 to 53.5ºC. An unusually large lamellar structure was observed, with d = 11 nm, contradicting the complex functional dimer model between lysolipid and fatty acid. It can be explained by formation of a "double-bilayer", a new phase consisting of two different bilayers, one formed by lysophospholipid and other by fatty acid, bound together by head group interactions. Its strucutre was confirmed by simulations of the X-ray scattering pattern.

Lannoitetehtaan prosessivesien käsittelyvaihtoehtojen soveltuvuuden arviointi

Phosphorus and nitrogen cause eutrophication of water bodies, causing severe damage to the ecosystem. Eutrophication of the waters causes oxygen depletion, which in turn increases fish mortality, releasing toxins in waters. The released toxins can cause damage to animals and humans, which is the reason in many countries to set emission limits for waste water. Nutrients exist naturally, but due to human activities there is high nutrient leaching to water bodies. Human activity is one of the main reasons to the eutrophication. The aim of this thesis was to estimate the suitability of different water treatment options for Yara Finland’s fertilizer plant’s process waters in Siilinjärvi. The fertilizer plant process waters are high concentrate and especially nitrogen concentrations are high, which bring challenge to the treatment. At the theoretical part was investigated conventional and as well advanced wastewater treatment methods like reverse osmosis, adsorption and ion exchange. Beside different treatment methods corporate environmental requirements, responsibility and strategies were researched. At the empirical part of the thesis the goal was to find out possibil-ities to intensify the efficiency of purification at lamella clarifier with chemical precipitation. In addition possibility to use already existing chemical purifying plant for process waters was estimated. As a result of the research Yara has a possibility to intensify lamella clarifier’s action by addi-tion of calcium hydroxide and thus to obtain the phosphorus and fluorine to precipitate out of the water. But in practice this would be too expensive. It is possible to eliminate nitrogen compounds by adsorption or ammonia stripping, both methods requires additional testing. It is possible to process waters in chemical purifying plant, if ammonium nitrogen has been reduced before. Reverse osmosis is possible to exploit for the phosphoric acid plant’s waters.

Hérnia encarcerada em orifício de trocarte lateral diagnosticada por tomografia computadorizada

Incisional hernia is an uncommon complication in laparoscopic surgery. The majority of the hernias are located in the umbilical site. Nevertheless, they can occur in the lateral trocar site, although they are rarely diagnosed. We report a case of a 55 year-old patient who underwent a videolaparoscopic hysterectomy and developed small bowel obstruction on the third postoperative day. This initially gave rise to the diagnosis of paralytic ileum. The definitive diagnosis of incarcerated hernia in the lateral trocar site was established after an abdominal computed tomography was performed.

A new concept in prophylaxis and therapy: paramunization by poxvirus inducers

The so-called primitive, innate or paraspecific immune system is the phylogenetically older part of the complex immune system. It enables the organism to immediately attack various foreign substances, infectious pathogens, toxins and transformed cells of the organism itself. ,,Paramunity" is defined as an optimal regulated and activated, antigen-nonspecific defence, acquired through continuous active and succesful confrontation with endogenous and exogenous noxes or by means of ,,paramunization" with so called ,,paramunity inducers". Paramunity inducers based on different pox virus species (e.g. Baypamun®, Duphapind®, Conpind) have turned out to be effective and safe when applied with human beings as well as with animals. Pox virus inducers activate phagocytosis and NK-cells in addition to regulation of various cytokines, notably interferon a and g, IL 1, 2, CSF and TNF which comprise the network of the complex paraspecific immune system. The results of experimental work as well as practical use in veterinary medicine have shown that paramunization by pox inducers goes far beyond the common understanding of so-called ,,immuno-therapy". They are ,,bioregulators", because they have 1. a regulatory effect on a disturbed immune system in the sense of an optimal homoeostasis, and 2. simultaneously a regulatory effect between the immune, nervous, circulatory and hormone system. Therefore, the use of paramunization by pox inducers opens a new way of prophylaxis and therapy, not only with regard to infections, but also with regard to different other indications.

Staphylococcal toxin genes in strains isolated from cows with subclinical mastitis

The present study was carried out in 11 dairy herds in four municipal districts of the rural area of the State of Pernambuco, Brazil. Out of 984 quarter milk (246 cows), 10 (1.0%) were positive for clinical mastitis, 562 (57.1%) for subclinical mastitis and 412 (41.9%) were negative. A total of 81 Staphylococcus spp. isolates were obtained from milk samples from the cows diagnosed with subclinical mastitis. From these, 53 (65.0%) were S. aureus, 16 (20.0%) coagulase-positive staphylococci (CPS) and 12 (15.0%) coagulase-negative staphylococci (CNS). The isolates were further investigated for the presence of toxin genes by multiplex and uniplex PCR. The main gene observed was seg followed by seh, sei and sej. The distribution of these observed genes among the isolates obtained from different areas showed a regional pattern for the SEs. The presence of toxin genes in the strains isolated from bovine milk demonstrates a potential problem for public health.

Serogroups and virulence genes of Escherichia coli isolated from psittacine birds

Escherichia coli isolates from 24 sick psittacine birds were serogrouped and investigated for the presence of genes encoding the following virulence factors: attaching and effacing (eae), enteropathogenic E. coli EAF plasmid (EAF), pili associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin (afa), capsule K1 (neu), curli (crl, csgA), temperature-sensitive hemagglutinin (tsh), enteroaggregative heat-stable enterotoxin-1 (astA), heat-stable enterotoxin -1 heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga-like toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf1), haemolysin (hly), aerobactin production (iuc) and serum resistance (iss). The results showed that the isolates belonged to 12 serogroups: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 and O166. The virulence genes found were: crl in all isolates, pap in 10 isolates, iss in seven isolates, csgA in five isolates, iuc and tsh in three isolates and eae in two isolates. The combination of virulence genes revealed 11 different genotypic patterns. All strains were negative for genes encoding for EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 and stx2. Our findings showed that some E. coli isolated from psittacine birds present the same virulence factors as avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC) and Enteropathogenic E. coli (EPEC) pathotypes.

Perreyia flavipes larvae toxicity

Fresh or thawed Perreyia flavipes larvae were ground and mixed with water and orally ad ministered to sheep. At 5mg/kg, neither clinical nor enzymatic changes were observed. Unique do ses of 7.5 and 10mg/kg induced characteristic clinical signs of Perreyia sp. larvae poisoning, increased GGT and AST values, and decreased glycemic curves. However, doses of 5, 10, and 15mg/kg repeated at 30 or 15 days intervals caused no disease and mild disease followed by death, respectively. These fin dings indicate that these animals probably developed some degree of tolerance to the toxins in P. flavipes larvae. Ultrastru ctural examination of liver revealed proliferation of the smooth endoplasmic reticulum in the hepatocytes, which may be associated with an increased ability to metabolize toxins and could consequently lead to the tolerance observed in the present study. Further investigations may elucidate whether such tolerance effects could be applied as a control measure for P. flavipes poioning or other hepatotoxic diseases. In addition, clinicopathological findings were discussed.

A surveillance of enteropathogens in piglets from birth to seven days of age in Brazil

The purpose of the study was to evaluate the real importance of anaerobic enteropathogens and rotavirus in contrast to more common agents as cause of diarrhea in piglets within the first week of life. Sixty 1- to 7-day-old piglets, 30 diarrheic and 30 non-diarrheic (control), from 15 different herds were selected, euthanized and necropsied. Samples of the jejunum, ileum, colon, cecum and feces were collected from the piglets and analyzed to determine the presence of the following enteropathogens: enterotoxigenic Escherichia coli (ETEC), Clostridium perfringens types A and C, Clostridium difficile, rotavirus and Isospora suis. Among diarrheic piglets, 23.3% were positive for C. difficile, 70% for C. perfringens type A cpb2+, 14.3% for rotavirus and 10% for ETEC. Among non-diarrheic control piglets, 10% were positive for C. difficile, 76.7% for C. perfringens type A cpb2+, 0% for rotavirus, 3.3% for ETEC and 3.3% for I. suis. C. perfringens type C was not detected in any of the animals. Histological lesions characteristic of C. difficile, E. coli and rotavirus were observed. However, no C. perfringens type A suggestive lesions were detected. There was a positive correlation between mesocolon edema and the presence of C. difficile toxins. Although C. perfringens type A cpb2+ was the most frequently detected enteropathogen, there was no association between its presence and diarrhea or macro or microscopic changes. C. difficile and Rotavirus were the most relevant pathogens involved with neonatal diarrhea in this study, and histopathology associated with microbiological test proved to be the key to reach a final diagnosis.