Recombinant Fasciola hepatica Fatty Acid Binding Protein as a Novel Anti-Inflammatory Biotherapeutic Drug in an Acute Gram-Negative Nonhuman Primate Sepsis Model.

Due to their phylogenetic proximity to humans, nonhuman primates (NHPs) are considered an adequate choice for a basic and preclinical model of sepsis. Gram-negative bacteria are the primary causative of sepsis. During infection, bacteria continuously release the potent toxin lipopolysaccharide (LPS) into the bloodstream, which triggers an uncontrolled systemic inflammatory response leading to death. Our previous research has demonstrated in vitro and in vivo using a mouse model of septic shock that Fh15, a recombinant variant of the Fasciola hepatica fatty acid binding protein, acts as an antagonist of Toll-like receptor 4 (TLR4) suppressing the LPS-induced proinflammatory cytokine storm. The present communication is a proof-of concept study aimed to demonstrate that a low-dose of Fh15 suppresses the cytokine storm and other inflammatory markers during the early phase of sepsis induced in rhesus macaques by intravenous (i.v.) infusion with lethal doses of live Escherichia coli. Fh15 was administered as an isotonic infusion 30 min prior to the bacterial infusion. Among the novel findings reported in this communication, Fh15 (i) significantly prevented bacteremia, suppressed LPS levels in plasma, and the production of C-reactive protein and procalcitonin, which are key signatures of inflammation and bacterial infection, respectively; (ii) reduced the production of proinflammatory cytokines; and (iii) increased innate immune cell populations in blood, which suggests a role in promoting a prolonged steady state in rhesus macaques even in the presence of inflammatory stimuli. This report is the first to demonstrate that a F. hepatica-derived molecule possesses potential as an anti-inflammatory drug against sepsis in an NHP model. IMPORTANCE Sepsis caused by Gram-negative bacteria affects 1.7 million adults annually in the United States and is one of the most important causes of death at intensive care units. Although the effective use of antibiotics has resulted in improved prognosis of sepsis, the pathological and deathly effects have been attributed to the persistent inflammatory cascade. There is a present need to develop anti-inflammatory agents that can suppress or neutralize the inflammatory responses and prevent the lethal consequences of sepsis. We demonstrated here that a small molecule of 14.5 kDa can suppress the bacteremia, endotoxemia, and many other inflammatory markers in an acute Gram-negative sepsis rhesus macaque model. These results reinforce the notion that Fh15 constitutes an excellent candidate for drug development against sepsis.

ArsH protects Pseudomonas putida from oxidative damage caused by exposure to arsenic.

The two As resistance arsRBC operons of Pseudomonas putida KT2440 are followed by a downstream gene called arsH that encodes an NADPH-dependent flavin mononucleotide reductase. In this work, we show that the arsH1 and (to a lesser extent) arsH2 genes of P. putida KT2440 strengthened its tolerance to both inorganic As(V) and As(III) and relieved the oxidative stress undergone by cells exposed to either oxyanion. Furthermore, overexpression of arsH1 and arsH2 endowed P. putida with a high tolerance to the oxidative stress caused by diamide (a drainer of metabolic NADPH) in the absence of any arsenic. To examine whether the activity of ArsH was linked to a direct action on the arsenic compounds tested, arsH1 and arsH2 genes were expressed in Escherichia coli, which has an endogenous arsRBC operon but lacks an arsH ortholog. The resulting clones both deployed a lower production of reactive oxygen species (ROS) when exposed to As salts and had a superior endurance to physiological redox insults. These results suggest that besides the claimed direct action on organoarsenicals, ArsH contributes to relieve toxicity of As species by mediating reduction of ROS produced in vivo upon exposure to the oxyanion, e.g. by generating FMNH2 to fuel ROS-quenching activities.

Intraperitoneal administration of the anti-IL-23 antibody prevents the establishment of intestinal nematodes in mice.

Previous studies have established that an increased Th-9 response creates a hostile environment for nematode parasites. Given that IL-23, a cytokine required for maintenance of the IL-17-secreting phenotype, has inhibitory effects on IL-9 production, we hypothesized that reducing circulating IL-23 by treatment with anti-IL-23 antibodies would reduce the establishment and development of parasitic intestinal nematodes. In this study, we show that animals treated with anti-IL-23 monoclonal antibodies showed a drastic reduction in the number of mouse pinworms (Aspiculuris tetraptera) recovered from the intestine (p < 0.001) at 23 days post-infection compared to the untreated animals. The cytokine levels in Peyer's patches (PP) in treated and infected animals increase the expression of interleukins such as IL-25, IL-21, and IL-9, augmenting mucus production in the crypts, and boosting chemokines, such as OX40 and CCL20 in the mucosa. Our results suggest that the Th17/Th2 regulatory mechanism provoked by the administration of the anti-IL-23 antibody prevents the implantation of the intestinal nematode in mice. The diminished inflammatory IL-17 levels alter the Th9 environment perhaps as a consequence of IL-17 inhibiting IL-9 expression. These Th9 conditions may explain the successful treatment against Inflammatory Bowel Disease (IBD) both with antibodies against IL-23 or through parasitization with nematodes.

Concordancia entre la PET cerebral con 18F-FDG y los biomarcadores en líquido cefalorraquídeo en el diagnóstico de enfermedad de Alzheimer / Concordance between brain 18F-FDG PET and cerebrospinal fluid biomarkers in diagnosing Alzheimer's disease

Objetivos. El hipometabolismo cortical posterior por PET con 18F-FDG (PET-FDG) y la alteración de los niveles del péptido Aß1-42 y las proteínas Tau total (tTau) y Tau fosforilada (pTau) en líquido cefalorraquídeo (LCR) son biomarcadores establecidos para el diagnóstico de la enfermedad de Alzheimer (EA). Evaluamos la concordancia y la relación entre los resultados de la PET-FDG y los biomarcadores en LCR en pacientes sintomáticos con sospecha de EA. Material y métodos. Revisión retrospectiva de 120 pacientes con deterioro cognitivo admitidos en la Unidad de Neurología Cognitiva a los que se les ha realizado punción lumbar para la determinación de biomarcadores en LCR y una PET-FDG cerebral. Para el análisis de concordancia (coeficiente Kappa), el resultado de la PET-FDG y del conjunto de los biomarcadores-LCR se clasificó en cada paciente como normal, no-concluyente, o compatible-EA. Se efectuó además una regresión logística incluyendo las variables cuantitativas Aß1-42, tTau y pTau como predictores y la PET-FDG como variable dependiente. Resultados. El coeficiente Kappa ponderado entre PET-FDG y biomarcadores-LCR fue de 0,46 (IC 95%: 0,35-0,57). En el análisis por regresión logística, la Aß1-42 y la tTau fueron en conjunto capaces de discriminar un resultado PET metabólicamente sugestivo de EA de uno no sugestivo de EA, con una sensibilidad del 91% y una especificidad del 93% aplicando la recta de corte Aß1-42=44+1,3×tTau. Conclusiones. La concordancia entre la PET-FDG cerebral y los biomarcadores-LCR es moderada, lo cual indica su valor complementario en el diagnóstico de EA. Los niveles de Aß1-42 y tTau en LCR son buenos predictores del estatus metabólico característico de EA por PET-FDG cerebral (AU)

Objectives. Cortical posterior hypometabolism on PET imaging with 18F-FDG (FDG-PET), and altered levels of Aß1-42 peptide, total Tau (tTau) and phosphorylated Tau (pTau) proteins in cerebrospinal fluid (CSF) are established diagnostic biomarkers in Alzheimer's disease (AD). An evaluation has been made of the concordance and relationship between the results of FDG-PET and CSF biomarkers in symptomatic patients with suspected AD. Material and methods. A retrospective review was carried out on 120 patients with cognitive impairment referred to our Cognitive Neurology Unit, and who were evaluated by brain FDG-PET and a lumbar puncture for CSF biomarkers. In order to calculate their Kappa coefficient of concordance, the result of the FDG-PET and the set of the three CSF biomarkers in each patient was classified as normal, inconclusive, or AD-compatible. The relationship between the results of both methods was further assessed using logistic regression analysis, including the Aß1-42, tTau and pTau levels as quantitative predictors, and the FDG-PET result as the dependent variable. Results. The weighted Kappa coefficient between FDG-PET and CSF biomarkers was 0.46 (95% CI: 0.35-0.57). Logistic regression analysis showed that the Aß1-42 and tTau values together were capable of discriminating an FDG-PET result metabolically suggestive of AD from one non-suggestive of AD, with a 91% sensitivity and 93% specificity at the cut-off line Aß1-42=44+1.3×tTau. Conclusions. The level of concordance between FDG-PET and CSF biomarkers was moderate, indicating their complementary value in diagnosing AD. The Aß1-42 and tTau levels in CSF help to predict the patient FDG-PET cortical metabolic status (AU)

Concordance between brain 18F-FDG PET and cerebrospinal fluid biomarkers in diagnosing Alzheimer's disease. / Concordancia entre la PET cerebral con 18F-FDG y los biomarcadores en líquido cefalorraquídeo en el diagnóstico de enfermedad de Alzheimer.

OBJECTIVES: Cortical posterior hypometabolism on PET imaging with 18F-FDG (FDG-PET), and altered levels of Aß1-42 peptide, total Tau (tTau) and phosphorylated Tau (pTau) proteins in cerebrospinal fluid (CSF) are established diagnostic biomarkers in Alzheimer's disease (AD). An evaluation has been made of the concordance and relationship between the results of FDG-PET and CSF biomarkers in symptomatic patients with suspected AD. MATERIAL AND METHODS: A retrospective review was carried out on 120 patients with cognitive impairment referred to our Cognitive Neurology Unit, and who were evaluated by brain FDG-PET and a lumbar puncture for CSF biomarkers. In order to calculate their Kappa coefficient of concordance, the result of the FDG-PET and the set of the three CSF biomarkers in each patient was classified as normal, inconclusive, or AD-compatible. The relationship between the results of both methods was further assessed using logistic regression analysis, including the Aß1-42, tTau and pTau levels as quantitative predictors, and the FDG-PET result as the dependent variable. RESULTS: The weighted Kappa coefficient between FDG-PET and CSF biomarkers was 0.46 (95% CI: 0.35-0.57). Logistic regression analysis showed that the Aß1-42 and tTau values together were capable of discriminating an FDG-PET result metabolically suggestive of AD from one non-suggestive of AD, with a 91% sensitivity and 93% specificity at the cut-off line Aß1-42=44+1.3×tTau. CONCLUSIONS: The level of concordance between FDG-PET and CSF biomarkers was moderate, indicating their complementary value in diagnosing AD. The Aß1-42 and tTau levels in CSF help to predict the patient FDG-PET cortical metabolic status.

Self-adjuvanting C18 lipid vinil sulfone-PP2A vaccine: study of the induced immunomodulation against Trichuris muris infection.

Despite the importance of the adjuvant in the immunization process, very few adjuvants merge with the antigens in vaccines. A synthetic self-adjuvant oleic-vinyl sulfone (OVS) linked to the catalytic region of recombinant serine/threonine phosphatase 2A from the nematode Angiostrongylus costaricensis (rPP2A) was used for intranasal immunization in mice previously infected with Trichuris muris The animal intranasal immunization with rPP2A-OVS showed a reduction of 99.01% in the number of the nematode eggs and 97.90% in adult. The immunohistochemical analysis of the intestinal sections showed that in immunized animals with lipopeptide the mucus was significantly higher than in the other experimental groups. Also, these animals presented significantly different chemokine, CCL20 and CCL11, levels. However, although the number and size of Tuft cells did not vary between groups, the intensity of fluorescence per cell was significant in the group immunized with the rPP2A-OVS. The results of the present study suggest that mice immunized with the lipopeptide are capable of activating a combined Th17/Th9 response. This strategy of immunization may be of great applicability not only in immunotherapy and immunoprophylaxis to control diseases caused by nematodes but also in pathologies necessitating action at the level of the Th9 response in the intestinal mucosa.

Meningoencefalitis por Cryptococcus neoformans en adolescente con desnutrición / Cryptococcus neoformans meningoencephalitis in a malnourished teenager

La criptococosis es una micosis causada por dos hongos levaduriformes encapsulados del género Cryptoccoccus, ingresa al organismo por vía inhalatoria con diseminación al sistema nervioso central, su prevalencia es mayor en inmunodeprimidos por VIH SIDA. Presentamos el caso de un paciente masculino de 13 años de edad, VIH negativo, de la etnia Lenca, procedente de zona rural de Honduras, con historia de contacto prolongado con heces de paloma (Columba livia), quien se presentó con síndrome de hipertensión endocraneana. Tinta china de líquido cefalorraquídeo reportó levaduras encapsuladas compatibles con Cryptococcus neoformans spp confirmado por cultivo. Su único antecedente de inmunosupresión fue la desnutrición. Tuvo evolución favorable con la terapia combinada anfotericina b y fluconazol

Cryptococcosis is a fungal infection caused by two encapsulated yeasts of the genus Cryptoccoccus. The microorganism enters the body through inhalation and it disseminates to the central nervous system. Its prevalence is higher in immunocompromised patients, especially those with HIV-AIDS. We report the case of a 13-year old male patient, negative for HIV infection, who belongs to the Lenca ethnic group, from a rural area in Honduras and a positive history of contact with pigeon feces (Columba livia). The patient presented with intracranial hypertension syndrome. An India-ink examination of his cerebrospinal fluid revealed encapsulated yeasts compatible with Cryptococcus neoformans spp., confirmed by culture. The presentation of this case is important because of being a pediatric HIV negative patient, whose only previous immunosuppression was malnutrition. Additionally, he had a history of contact with pigeon feces, resulting in risk factors for developing the disease. We should also take into account the imp ortance of the epidemiological correlation in order to achieve an early diagnosis and an adequate response to therapy. In this case, the combined therapy with fluconazole and amphotericin, led to a favorable outcome

Reproductive outcomes of Alpine goats primed with progesterone and treated with human chorionic gonadotropin during the anestrus-to-estrus transition season.

This study aimed to determine the possible effects of a single injection of human chorionic gonadotropin (hCG) as a means for estrus induction in acyclic French-Alpine goats during the reproductive transition period at 25°N, 103°W. The potential effects of hCG upon ovarian function and reproductive performance of goats were also assessed. Multiparous acyclic French-Alpine goats (n = 39; 37.4 ± 8 .5 kg) were primed with 20mg progesterone (P4) 1 day prior to hCG administration. Thereafter, does were treated either with saline (hCG-0; n = 10), 50 (hCG-50; n = 9), 100 (hCG-100; n = 10), or 300 IU of hCG (hCG-300; n = 10). Ovarian structures and pregnancy were monitored by transrectal ultrasonography. In addition, after hCG application, goats were monitored twice daily (0800 and 1800 h) to detect estrus signs, with the use of aproned, sexually active bucks treated with testosterone. Goats were bred 12h after the onset of estrus. Two days after hCG administration, the number of large follicles was higher (P < 0.05) in the hCG-50 and hCG-300 groups (1.7 ± 0.1 and 1.8 ± 0.2, respectively) compared with the hCG-100 and hCG-0 groups (1.4 ± 0.2 and 1.1 ± 0.1, respectively). Although none of the hCG-0-goats depicted estrus, the estrus response from the hCG-50, hCG-100, and hCG-300 groups over the 7-d breeding period was 67%, 100%, and 90%, respectively (P > 0.05), being always accompanied by ovulation. Pregnancy rate (67, 100, and 70%), kidding rate (55%, 80%, and 70%), and litter size (1.6 ± 0.5, 1.5 ± 0.5, and 1.5 ± 0.5) for hCG-50, hCG-100, and hCG-300, respectively, did not differ among the hCG-treated does. Therefore, the combined use of P4-priming plus a 100-IU hCG injection is an effective protocol for inducing estrus in non-cycling Alpine goats during the anestrus-to-estrus transition period, which is of key importance for both goat producers and industrializers.

Metagenomic investigation of the geologically unique Hellenic Volcanic Arc reveals a distinctive ecosystem with unexpected physiology.

Hydrothermal vents represent a deep, hot, aphotic biosphere where chemosynthetic primary producers, fuelled by chemicals from Earth's subsurface, form the basis of life. In this study, we examined microbial mats from two distinct volcanic sites within the Hellenic Volcanic Arc (HVA). The HVA is geologically and ecologically unique, with reported emissions of CO2 -saturated fluids at temperatures up to 220°C and a notable absence of macrofauna. Metagenomic data reveals highly complex prokaryotic communities composed of chemolithoautotrophs, some methanotrophs, and to our surprise, heterotrophs capable of anaerobic degradation of aromatic hydrocarbons. Our data suggest that aromatic hydrocarbons may indeed be a significant source of carbon in these sites, and instigate additional research into the nature and origin of these compounds in the HVA. Novel physiology was assigned to several uncultured prokaryotic lineages; most notably, a SAR406 representative is attributed with a role in anaerobic hydrocarbon degradation. This dataset, the largest to date from submarine volcanic ecosystems, constitutes a significant resource of novel genes and pathways with potential biotechnological applications.

Oclusión de arteria ciliorretiniana en la hemocromatosis / Cilioretinal artery occlusion in hemochromatosis

CASO CLÍNICO: Se presenta el caso de una mujer de 31 años con pérdida brusca de visión de un ojo debido a una oclusión de arteria ciliorretiniana. En la exploración presentaba hepatomegalia y en la analítica los niveles séricos de hierro, saturación de transferrina y ferritina estaban elevados. Los perfiles de autoinmunidad y de hipercoagulabilidad fueron normales. El estudio doppler-ultrasónico de los troncos supraaórticos fue anodino, pero la ecografía cardíaca evidenció una miocardiopatía con calcificación subendocárdica. El estudio genético para la hemocromatosis fue positivo. DISCUSIÓN: La calcificación subendocárdica secundaria a hemocromatosis puede ser la causa de la oclusión embólica de la arteria ciliorretiniana. El cuadro embólico ocular fue la forma de presentación de la hemocromatosis en nuestra paciente

CLINICAL CASE: We report a case of a 31 year-old woman with a sudden visual loss due to a cilioretinal artery occlusion. The physical examinination showed hepatomegaly. Serum iron and ferritin and transferrin saturation were unusually high. The doppler scan of carotid arteries showed no relevant signs of atheromatous disease. Dilated cardiomiopaty was revealed in the B-scan with subendocardial calcium deposits. Genetic tests were positive for hemochromatosis. DISCUSSION: Subendocardial calcification due to hemochromatosis could be the embolic source in our patient. This embolic ocular disease is the first presentation of hemochromatosis in this patient

The two paralogue phoN (phosphinothricin acetyl transferase) genes of Pseudomonas putida encode functionally different proteins.

Phosphinothricin (PPT) is a non-specific inhibitor of glutamine synthetase that has been employed as herbicide for selection of transgenic plants expressing cognate resistance genes. While the soil bacterium Pseudomonas putida KT2440 has been generally considered PPT-sensitive, inspection of its genome sequence reveals the presence of two highly similar open reading frames (PP_1924 and PP_4846) encoding acetylases with a potential to cause tolerance to the herbicide. To explore this possibility, each of these genes (named phoN1 and phoN2) was separately cloned and their activities examined in vivo and in vitro. Genetic and biochemical evidence indicated that phoN1 encodes a bona fide PPT-acetyl transferase, the expression of which suffices to make P. putida tolerant to high concentrations of the herbicide. In contrast, PhoN2 does not act on PPT but displays instead activity against methionine sulfoximine (MetSox), another glutamine synthetase inhibitor. When the geometry of the substrate-binding site of PhoN1 was grafted with the equivalent residues of the predicted PhoN2 structure, the resulting protein increased significantly MetSox resistance of the expression host concomitantly with the loss of activity on PPT. These observations uncover intricate biochemical and genetic interactions among soil microorganisms and how they can be perturbed by exposure to generic herbicides in soil.

[Cilioretinal artery occlusion in hemochromatosis]. / Oclusión de arteria ciliorretiniana en la hemocromatosis.

CLINICAL CASE: We report a case of a 31 year-old woman with a sudden visual loss due to a cilioretinal artery occlusion. The physical examinination showed hepatomegaly. Serum iron and ferritin and transferrin saturation were unusually high. The doppler scan of carotid arteries showed no relevant signs of atheromatous disease. Dilated cardiomiopaty was revealed in the B-scan with subendocardial calcium deposits. Genetic tests were positive for hemochromatosis. DISCUSSION: Subendocardial calcification due to hemochromatosis could be the embolic source in our patient. This embolic ocular disease is the first presentation of hemochromatosis in this patient.

Functional coexistence of twin arsenic resistance systems in Pseudomonas putida†KT2440.

The genome of the soil bacterium Pseudomonas putida KT2440 bears two virtually identical arsRBCH operons putatively encoding resistance to inorganic arsenic species. Single and double chromosomal deletions in each of these ars clusters of this bacterium were tested for arsenic sensitivity and found that the contribution of each operon to the resistance to the metalloid was not additive, as either cluster sufficed to endow cells with high-level resistance. However, otherwise identical traits linked to each of the ars sites diverged when temperature was decreased. Growth of the various mutants at 15°C (instead of the standard 30°C for P. putida) uncovered that ars2 affords a much higher resistance to As (III) than the ars1 counterpart. Reverse transcription polymerase chain reaction of arsB1 and arsB2 genes as well as lacZ fusions to the Pars1 and Pars2 promoters traced the difference to variations in transcription of the corresponding gene sets at each temperature. Functional redundancy may thus be selected as a stable condition - rather than just as transient state - if it affords one key activity to be expressed under a wider range of physicochemical settings. This seems to provide a straightforward solution to regulatory problems in environmental bacteria that thrive under changing scenarios.

Premarket evaluations of the IMDx C. difficile for Abbott m2000 Assay and the BD Max Cdiff Assay.

Clostridium difficile-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing C. difficile has been difficult, as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAATs) are quickly becoming the standard of care. We compared the performance of two automated investigational/research use only (IUO/RUO) NAATs for the detection of C. difficile toxin genes, the IMDx C. difficile for Abbott m2000 Assay (IMDx) and the BD Max Cdiff Assay (Max). A prospective analysis of 111 stool specimens received in the laboratory for C. difficile testing by the laboratory's test of record (TOR), the BD GeneOhm Cdiff Assay, and a retrospective analysis of 88 specimens previously determined to be positive for C. difficile were included in the study. One prospective specimen was excluded due to loss to follow-up discrepancy analysis. Of the remaining 198 specimens, 90 were positive by all three methods, 9 were positive by TOR and Max, and 3 were positive by TOR only. One negative specimen was initially inhibitory by Max. The remaining 95 specimens were negative by all methods. Toxigenic C. difficile culture was performed on the 12 discrepant samples. True C. difficile-positive status was defined as either positive by all three amplification assays or positive by toxigenic culture. Based on this definition, the sensitivity and specificity were 96.9% and 95% for Max and 92.8% and 100% for IMDx. In summary, both highly automated systems demonstrated excellent performance, and each has individual benefits, which will ensure that they will both have a niche in clinical laboratories.

Tobacco: A New Natural Host of Tomato chlorosis virus in Spain.

In March 2013, symptoms of mild leaf curling, mosaic, and interveinal yellowing were observed in tobacco (Nicotiana tabacum) plants grown in a row surrounding the exterior of a greenhouse containing a tomato crop in Guía de Isora, Tenerife (Canary Islands, Spain). The tobacco plants were found lightly infested by the whitefly (Hemiptera: Aleyrodidae) Bemisia tabaci. The greenhouses in this area are devoted to the commercial production of tomato. The farmers grow some tobacco plants inside and outside of them as a reservoir of parasitoids and depredators of B. tabaci. This insect is the natural vector of the main viruses severely affecting tomato in the Canary Islands, the begomovirus Tomato yellow leaf curl virus and the crinivirus Tomato chlorosis virus (ToCV). ToCV was detected in Spain in 1997 (2) and has become established in most of the coastal provinces of eastern and southern continental Spain and in the Canary Islands. Approximately 50% of the tomato plants grown inside the greenhouse close to the tobacco plants showed typical ToCV symptoms, and infection by this virus was confirmed in the seven plants tested by reverse transcription (RT)-PCR using specific coat protein gene (CP) primers (see below). Total RNA was extracted with TRIzol Reagent (Invitrogen) from leaves of five tobacco plants showing the symptoms mentioned above and analyzed by dot-blot hybridization using digoxigenin-labeled RNA probes to the CP gene of ToCV. Positive signal was obtained for all five plants. RT-PCR reactions were performed with specific primers for the detection of ToCV, MA380(+) (5'-GTGAGACCCCGATGACAGAT-3') and MA381(-) (5'-TACAGTTCCTTGCCCTCGTT-3'), specific to the CP gene (ToCV RNA 2) (3), and MA396(+) (5'-TGGTCGAACAGTTTGAGAGC-3') and MA397(-) (5'-TGAACTCGAATTGGGACAGA-3'), specific to the RNA-dependent RNA polymerase (RdRp) gene (ToCV RNA 1) (1). DNA fragments of the expected size (436 and 763 bp, respectively) were obtained, thus supporting the presence of ToCV in the symptomatic samples. The amplified product of the RdRp gene fragment from one sample was directly sequenced (Macrogen Inc., South Korea) and resulted closely related to ToCV isolates from Sudan (GenBank Accession No. JN411686, 99.6% nt identity) and Spain (DQ983480, 99.4% nt identity), thereby confirming the infection by this virus. Partial sequence of the ToCV isolate from tobacco was deposited in GenBank under accession no. KJ175084. In addition, all five plants resulted positive when analyzed by ELISA for Tomato spotted wilt virus and Potato virus Y and by PCR for Tomato yellow leaf curl virus (data not shown), all three viruses reported to infect naturally tobacco. Although tobacco has been reported as an experimental host of ToCV (4), to our knowledge, this is the first report of this species as a natural host of this virus. The finding of ToCV infecting tobacco raises the question of whether this virus could emerge as a pathogen of this crop and questions the use that farmers make of tobacco as reservoirs of natural enemies for whitefly control in tomato. References: (1) G. Lozano et al. J. Virol. 83:12973, 2009. (2) J. Navas-Castillo et al. Plant Dis. 84:835, 2000. (3) H. P. Trenado et al. Eur. J. Plant Pathol. 118:193, 2007 (4) W. M. Wintermantel and G. C. Wisler. Plant Dis. 90:814, 2006.

Comparison of three real-time PCR for the quantification of polyomavirus BK.

BACKGROUND: Reactivation of latent polyomavirus BK is associated with nephropathy (PVAN) after renal transplantation. BK viral load determinations are a highly sensitive and specific method for predicting risk for PVAN. OBJECTIVES AND STUDY DESIGN: The performance of three real-time PCR for BKV DNA quantification (MultiCode(®)-RTx BK virus ASR [MC-RTx], MGB-Alert BKV ASR [MGB] and a laboratory developed assay [LDA]) were evaluated against a conventional PCR (test of record, TOR) in terms of linearity, dynamic range, and accuracy. RESULTS: The LOD (log(10) copies/ml) were 2.0, 2.0 and 3.0 for MC-RTx, MGB and LDA, respectively with a commercial plasma panel and 2.0, 2.6 and 3.5 with a urine panel. These assays demonstrated excellent linearity (r(2) = 1.0) and reproducibility (CV range = 0.7-20.4%, 0.9-13.2%, and 0.5-13%, respectively). In an analysis of 100 clinical specimens, all 76 samples defined as true positive for BKV DNA (positive by two or more methods or a recent history of positivity) were detected with MC-RTx, while only 64 were detected with MGB and 55 were detected with LDA. BKV DNA was not detected by any method in the true negative specimens. Based on these results, the sensitivities were 100% for MC-RTx, 84% for MGB and 72% for LDA. The greatest linear correlation with the mean concentration was observed with MC-RTx (r(2) = 0.96) with two samples (3%) with greater than 0.5 log(10) variance in quantification versus seven (11%) with MGB and ten (18%) with LDA. CONCLUSIONS: These real-time assays for BKV load demonstrated excellent performance characteristics, with the MC-RTx demonstrating the greatest sensitivity.

The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes.

The 'Standard European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.

Monitoring biodegradative enzymes with nanobodies raised in Camelus dromedarius with mixtures of catabolic proteins.

Functional studies of biodegradative activities in environmental microorganisms require molecular tools for monitoring catabolic enzymes in the members of the native microbiota. To this end, we have generated repertories of single-domain V(HH) fragments of camel immunoglobulins (nanobodies) able to interact with multiple proteins that are descriptors of environmentally relevant processes. For this, we immunized Camelus dromedarius with a cocktail of up to 12 purified enzymes that are representative of major types of detoxifying activities found in aerobic and anaerobic microorganisms. Following the capture of the antigen-binding modules from the mRNA of the camel lymphocytes and the selection of sub-libraries for each of the enzymes in a phage display system we found a large number of V(HH) modules that interacted with each of the antigens. Those associated to the enzyme 2,3 dihydroxybiphenyl dioxygenase of Burkholderia xenovorans LB400 (BphC) and the arsenate reductase of Staphylococcus aureus (ArsC) were examined in detail and found to hold different qualities that were optimal for distinct protein recognition procedures. The repertory of anti-BphC V(HH) s included variants with a strong affinity and specificity for linear epitopes of the enzyme. When the anti-BphC V(HH) library was recloned in a prokaryotic intracellular expression system, some nanobodies were found to inhibit the dioxygenase activity in vivo. Furthermore, anti-ArsC V(HH) s were able to discriminate between proteins stemming from different enzyme families. The easiness of generating large collections of binders with different properties widens considerably the molecular toolbox for analysis of biodegradative bacteria and opens fresh possibilities of monitoring protein markers and activities in the environment.

Effectiveness of intranasal vaccination against Angiostrongylus costaricensis using a serine/threonine phosphatase 2 A synthetic peptide and recombinant antigens.

Intranasal immunization was assayed in C57BL/6 mice against Angiostrongylus costaricensis using a synthetic and a recombinant peptide belonging to the catalytic region of the serine/threonine phosphatase 2 A (PP2A) of the parasite. Immunization was carried out with the synthetic peptide (SP) polymerized either with itself or with the beta fraction of the cholera toxin (CTB) and then enclosed in nanocapsules of phosphatidyl choline, cholesterol and Quil A (ISCOM). Another group of mice was immunized with recombinant peptide. Immunization consisted of two intranasal inoculations at two-week intervals, and the challenge with L3 larvae was made one month after the last vaccination. The effectiveness of immunization was evaluated 30 days after infection by analysis of the number of parasites in the arteries of the immunized mice, as well as by measuring spleen sizes in the experimental groups. The response induced was determined by identifying the isotypes of IgG as well as the IgE and IgA specific antigen response. The interleukins produced by the splenocyte culture of the different groups were assessed after exposing them to the peptide used in the immunization. From our results, 60%, 80%, and 100% protection against the A. costaricensis challenge was achieved in mice immunized with polymerized synthetic peptide in ISCOM, synthetic peptide polymerized with the CTB in ISCOM and inclusion bodies respectively. Splenomegaly was found to be less evident in the immunized mice than in the controls. A significant increase in IFN gamma and IL-17 levels was observed in the group with 100% protection. The results showed that vaccination through the nasal mucosa may constitute a useful method of immunization and result in a protective immune response against A. costaricensis.

Análisis coste-efectividad del diagnóstico del síndrome del túnel carpiano mediante estudio electrofisiológico / Cost effectiveness analysis of the diagnosis of carpal tunnel syndrome using electrophysiological studies

Introducción. La elevada demanda de estudios electrofisiológicos(EEF) en pacientes paucisintomáticos con sospechade síndrome del túnel carpiano (STC) crea una sobrecargaen gabinetes de exploraciones. En el sector de referenciade nuestro hospital estas solicitudes provienen en su mayoríade atención primaria, pero también de otras especialidades.Muchos EEF en STC son normales o con alteraciones levesy por ello su resultado no conlleva una modificación dela actitud terapéutica. Por tanto, ante una sospecha de STCsin evidencia clínica de degeneración axonal no está claro siel EEF resulta coste-efectivo.Métodos. Realizamos un modelo de decisión y evaluacióneconómica que compara tres estrategias: opción A (EEFa todos los pacientes remitidos con sospecha de STC), opciónB (cribado previo por neurólogo y EEF sólo si sospechade STC axonal) y opción C (cribado previo por neurólogo yEEF sólo si sospecha de STC, axonal o no). Estudiamos durante2 meses a pacientes remitidos al gabinete de neurologíapor sospecha de STC. El EEF se consideró como la pruebaque establecía el diagnóstico final. De cada estrategia se determinaronlas consecuencias en términos de diagnósticoscorrectos, costes y relación coste/efectividad (C/E). Se realizóademás un análisis de sensibilidad. El total de estudiosrealizados fue 188, siendo la opción C la mejor relación C/E,pero también la más costosa.Conclusiones. Si hay «cribado neurológico» la relaciónC/E es mejor, pero resulta más cara. Sería recomendable potenciarla opción A, con un coste intermedio y una relaciónC/E aceptable. Sin embargo, ello genera una gran presiónasistencial y presupuestaria en los centros del sistema públicode salud, por lo que es necesario mejorar también eldiagnóstico y los criterios de derivación del STC (AU)

Introduction. The high demand for electrophysiologicalstudies (EP studies) in paucisymptomatic patientswith suspected carpal tunnel syndrome (CTS) createsoverloads in neurological examination departments.Most of these requests in the referenced section of ourhospital come from primary health care, however it alsocomes from other specialists. Many EP Studies for CTSare normal or have minimal alterations so that no changein the therapeutic attitude is required. Thus, it is notclear whether EP studies are cost-effective for suspectedCTS without clinical evidence of axonal degeneration.Methods. A decision-making and economic evaluationmodel was made to compare three strategies: option A(EP Studies for all patients with suspected CTS), option B(prior selection by neurologist and EP studies only ifaxonal CTS was suspected) and option C (prior selectionby neurologist and EP studies only if CTS, axonal orotherwise was suspected). The study was conducted overa two month period on patients referred to the neurologydepartment with suspected CTS. EP studies were consideredto be the proof that established the final diagnosis.The consequences were determined in terms of correctdiagnoses, cost and cost/effectiveness ratio for each strategy.A total number of 188 studies were performed,option C being the most cost-effective, but also the mostexpensive.Conclusions. When there is neurological screening,the cost-effectiveness ratio is better but it is also moreexpensive. It is recommended to favor option A with intermediatecost and an acceptable cost-effectiveness ratio.However, this also generates great pressure on bud-45 gets and care facilities which means that CTS diagnosis and criteria for referring patients to the neurologicalexamination departments must be improved (AU)