Whole genome sequence phylogenetic analysis of four Mexican rabies viruses isolated from cattle.

Phylogenetic analysis of the rabies virus in molecular epidemiology has been traditionally performed on partial sequences of the genome, such as the N, G, and P genes; however, that approach raises concerns about the discriminatory power compared to whole genome sequencing. In this study we characterized four strains of the rabies virus isolated from cattle in Querétaro, Mexico by comparing the whole genome sequence to that of strains from the American, European and Asian continents. Four cattle brain samples positive to rabies and characterized as AgV11, genotype 1, were used in the study. A cDNA sequence was generated by reverse transcription PCR (RT-PCR) using oligo dT. cDNA samples were sequenced in an Illumina NextSeq 500 platform. The phylogenetic analysis was performed with MEGA 6.0. Minimum evolution phylogenetic trees were constructed with the Neighbor-Joining method and bootstrapped with 1000 replicates. Three large and seven small clusters were formed with the 26 sequences used. The largest cluster grouped strains from different species in South America: Brazil, and the French Guyana. The second cluster grouped five strains from Mexico. A Mexican strain reported in a different study was highly related to our four strains, suggesting common source of infection. The phylogenetic analysis shows that the type of host is different for the different regions in the American Continent; rabies is more related to bats. It was concluded that the rabies virus in central Mexico is genetically stable and that it is transmitted by the vampire bat Desmodus rotundus.

Exploring the potential of Burkholderia sacchari to produce polyhydroxyalkanoates.

AIM: Evaluation of the capability of Burkholderia sacchari to incorporate different monomers into polyhydroxyalkanoates (PHA). METHODS AND RESULTS: Thirty different carbon sources were evaluated as cosubstrates for B. sacchari growing on glucose with the intention to promote the incorporation of different monomers into the PHA produced by this species. With odd-numbered fatty acids, incorporation of the 3HV monomer was achieved, up to 65 mol% in the case of valerate. With 4-hydroxybutyrate, incorporation of 4HB was obtained, representing 9·1 mol%. With hexanoic acid, the production of P3HB-co-3HHx was achieved, containing up to 1·6 mol% of 3HHx. The molar fraction of 3HHx was found to be dependent on the ratio of glucose to hexanoic acid supplied. Metabolic flux analysis revealed a high efficiency of B. sacchari in converting carbon sources into P3HB-co-3HHx. Nevertheless, hexanoic acid was only poorly converted to 3HHx. CONCLUSIONS: Burkholderia sacchari is able to incorporate 3HV, 4HB and 3HHx in PHA containing mainly 3HB. The 3HHx content of P3HB-co-3HHx can be controlled by varying the glucose to hexanoic acid ratio. Burkholderia sacchari is highly efficient in converting carbon sources into PHA; however, only 2% of the hexanoic acid supplied could be converted to 3HHx. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing an approach to modulate the composition of P3HB-co-3HHx produced by bacteria using mixtures of carbohydrate and hexanoic acid as carbon source.

[Implementation of "fast-track" treatment in paediatric complicated appendicitis]. / Aplicación de un modelo terapéuticofast-track en la apendicitis aguda complicada del paciente pediátrico.

OBJECTIVE: Acute appendicitis is the most common emergency surgical pathology in childhood and there is no consensus on its management. Fast-track treatment, based on optimizing perioperative care has reduced morbidity and mortality of surgical pathologies, including simple acute appendicitis. The aim of our study was to assess the effects of a fast-track protocol in complicated acute appendicitis. METHODS: Ambispective cohort study. Historical unexposed cohort: children with complicated appendicitis and appendectomy in our hospital during 2008-2009. Exposed cohort: children operated in 2010-2011 and who performed protocol. The protocol treatment was done after a literature review, adapting the principles of fast-track to a potentially severe urgent disease: early mobilization, limited drainage-tubes and short antibiotic regimens Taking hospital stay as the resulting variable, the calculated sample size for alpha = 0.05 and power = 90% was 54, being X1 = 7 +/- 3DS and X2=5. RESULTS: We included 151 patients, historical cohort 81 and current cohort 70, which excluded 31 children who did not meet protocol because of surgeon choice. Both groups showed homogeneity due to the absence of differences in sex, age, weight, type of appendicitis (gangrenous, perforated, generalized peritonitis) or surgical approach. The average stay decreased 2.71 days (p <0.001) due to the protocol, without any complication increase (abscess, postoperative ileus, readmission). CONCLUSIONS: Complicated appendicitis in children is common and potentially serious, and optimization of treatment should be a primary goal of our practice. Application of a fast-track protocol can provide clinical and economic benefits, although this requires an appropriate multidisciplinary management.

Fungal endophytes: diversity and functional roles.

All plants in natural ecosystems appear to be symbiotic with fungal endophytes. This highly diverse group of fungi can have profound impacts on plant communities through increasing fitness by conferring abiotic and biotic stress tolerance, increasing biomass and decreasing water consumption, or decreasing fitness by altering resource allocation. Despite more than 100 yr of research resulting in thousands of journal articles, the ecological significance of these fungi remains poorly characterized. Historically, two endophytic groups (clavicipitaceous (C) and nonclavicipitaceous (NC)) have been discriminated based on phylogeny and life history traits. Here, we show that NC-endophytes represent three distinct functional groups based on host colonization and transmission, in planta biodiversity and fitness benefits conferred to hosts. Using this framework, we contrast the life histories, interactions with hosts and potential roles in plant ecophysiology of C- and NC-endophytes, and highlight several key questions for future work in endophyte biology.

Patient profiles and their relationship with the CES-D scale at the Diabetes Center for Puerto Rico

PURPOSE: The prevalence of diabetes mellitus in Puerto Ricans has been identified and reported as being disproportionately higher as compared to other metabolic pathologies. Recently, diabetes has been identified as the third cause of mortality in Puerto Rico (Puerto Rico Health Department, Vital Statistics Annual Report, 1999-2001). The Research Center, Education and Medical Services for Diabetes in Puerto Rico (also known as the [quot ]Centro de Diabetes para Puerto Rico[quot ] [CDPR]) is a public corporation in the island created by the government to reduce diabetes prevalence, mortality and morbidity. METHOD: The CDPR offers Diabetes Self Management Educational Training Program Schools for patients (DSMETPS) island wide. The research design was an ex-post facto. As part of the process, patients are administered an extensive sociodemographic and health information questionnaire, which also includes the CES-D (a symptomatology depressive scale). This study pretends to describe the diabetic patient profiles (n=27) using information from the DSMETPS of the CDPR and explore the association with the CES-D. Variables such as patients' needs, knowledge and understanding of the condition (i.e., pathology management, type and medications utilized and exercise and nutritional patterns), patient attitudes to diabetes and their relations with the CES-D were explored. RESULTS: Results show a negative association, controlling for age and gender, between patients diabetic education/knowledge and CES-D score. CONCLUSIONS: Diabetes educators in Puerto Rico need to identify depressive symptomatology in order to prevent mental health complications in their patients since this may affect their future treatment and prognosis. An interdisciplinary team is recommended to improve the effectivity of the intervention.

Biomecánica de las prótesis de rodilla / Biomechanics of the knee prosthesis

Las prótesis totales de rodilla constituyen uno de los implantesmás colocados en el ámbito de la ortopedia. El númerode modelos existentes en el mercado es muy elevadoy todos tienen sus características. A pesar de ello podemosagrupar las artroplastias de rodilla por estas caracterísricas,siendo las prótesis de platillo móvil el grupo más novedoso.Revisamos la bibliogrfía más reciente sobre la biomecánicade las prótesis totales de rodilla, englobando aspectos comolos materiales, el polietileno, la fijación, la geometría ola presencia del ligamento cruzado posterior para entenderen profundidad el funcionemiento de una PTR

Total knee prostheses (TKAs) are one of the most frequentimplants we use like orthpaedists. There are a largenumber of models and all of them have their characteristics.Despite of it we can group TKAs in avery few ones inwhich mobile-bearing arthroplasty in the newness. We reviewthe literature about biomechanics of TKAs includingmaterials, UHMWPE, fixation of the prostheses, geometryor presence of the cruciate posterior ligament to really understandarthroplasty working

Biomecánica de la rodilla / Biomechanics of the knee

La biomecánica de la rodilla todavía no se conoce exactamente.Diversos patrones se han establecido, pero ningunoha alcanzado el funcionamiento real de la articulación.Revisamos la bibliografía y presentamos la evolucióndel entendimiento del movimiento de la rodilla hasta llegaral modelo de seis grados de libertad en la zona patelofemoraly en la tibiofemoral. Este modelo parece ser el másaproximado a la realidad en cuanto a la cinemática de larodilla

Knee´s biomechanics is not well known yet. A largenumber of patterns has been stablished, but neither ofthem has reached the right articular movement. We reviewthe literature and present the understanding about kneemovement until six grades of freedom model in patello-femoraland tibio-femoral zones. This model seems to be themost approximate to reality in knee kinematics

Fungal biology and agriculture: revisiting the field.

Plant pathology has made significant progress over the years, a process that involved overcoming a variety of conceptual and technological hurdles. Descriptive mycology and the advent of chemical plant-disease management have been followed by biochemical and physiological studies of fungi and their hosts. The later establishment of biochemical genetics along with the introduction of DNA-mediated transformation have set the stage for dissection of gene function and advances in our understanding of fungal cell biology and plant-fungus interactions. Currently, with the advent of high-throughput technologies, we have the capacity to acquire vast data sets that have direct relevance to the numerous subdisciplines within fungal biology and pathology. These data provide unique opportunities for basic research and for engineering solutions to important agricultural problems. However, we also are faced with the challenge of data organization and mining to analyze the relationships between fungal and plant genomes and to elucidate the physiological function of pertinent DNA sequences. We present our perspective of fungal biology and agriculture, including administrative and political challenges to plant protection research.

Hepatotoxicity of ketoconazole in Sprague-Dawley rats: glutathione depletion, flavin-containing monooxygenases-mediated bioactivation and hepatic covalent binding.

1. This study has examined ketoconazole (KT)-induced hepatotoxicity in vivo and in vitro, using male Sprague-Dawley rats with [(3)H]KT (1.5 micro Ci mg(-1)) at 40 and 90 mg KT kg(-1) doses. Blood and liver samples were collected from 0 to 24 h for alanine aminotransaminase (ALT), glutathione (GSH) and covalent binding analyses. 2. Covalent binding occurred as early as 0.5 h, peaked at 2 h (0.026 +/- 0.01 nmol KT mg(-1) protein) and 8 h (0.088 +/- 0.04 nmol KT mg(-1) protein) for 40 and 90 mg KT kg(-1) doses, respectively. ALT levels increased at 0.5 h for the 40 and 90 mg KT kg(-1) doses (44.3 and 56.4 U ml(-1), respectively) relative to control, 22.7 U ml(-1). At 24 h, the 90 mg KT kg(-1) dose reduced hepatic GSH levels from 9.92 +/- 1.1 to 4.76 +/- 0.3 nmol GSH mg(-1) protein. 3. The role of the flavin-containing monooxygenases (FMO) utilized Sprague-Dawley microsomes with 1, 10 and 100 micro M [(3)H]KT. Maximum covalent binding occurring at 100 micro M KT. Heat inactivation of microsomal FMO significantly decreased covalent binding by 75%, whereas 1 mM GSH significantly reduced covalent binding by 65%. 4. Thus, KT-induced hepatotoxicity is dose- and time-dependent and appears to be FMO mediated, in part, to metabolites that may react with protein and, possibly, GSH.

Determination of the optimal conditions for laryngeal pacing with the Itrel II implantable stimulator.

OBJECTIVE: To determine the optimal stimulus paradigm, electrode orientation, and configuration of an implantable stimulator used to reanimate the posterior-cricoarytenoid (PCA) muscle in case of bilateral vocal fold paralysis (BVFP). STUDY DESIGN: Acute studies were conducted on 13 canines implanted with Itrel II systems with or without PCA innervation. PCA stimulus-response characteristics were obtained by measuring stimulated vocal fold displacement endoscopically. RESULTS: The denervated PCA was only 10% to 25% as responsive to stimulation as the innervated PCA. However, the response could be increased to 38% and 61% if the Itrel was modified to deliver 1 and 2 msec pulses, respectively. Stimuli delivered centrally to the muscle 5 mm from the median raphe improved performance. CONCLUSION AND SIGNIFICANCE: The optimal stimulus paradigm identified in this study (1 msec pulses delivered at 30 to 40 Hz and 2 to 8.5 mA) has been applied to implanted BVFP patients and improved outcome. Information regarding optimal electrode orientation could also be important to future clinical trials.

The biocompatibility, integrity, and positional stability of an injectable microstimulator for reanimation of the paralyzed larynx.

The biocompatibility, integrity, positional stability, and potential use of hermetically sealed injectable wireless micromachined microstimulators were investigated for reanimation of the paralyzed larynx. The device, consisting of silicon and glass, has been tested and proven to be biocompatible with no evidence of pathological tissue reaction or rejection up to one-year implantation in the rat dorsum and canine larynx. By one month, each unit was encapsulated by a thin membrane, which thickened to form a fibrous layer of less than 500 microm at 6-12 months. The microstimulators demonstrated long-term in vivo durability: the hermetic seal of only one in ten devices was breached. Once implanted, migration of the device varied from 0 to 3 cm, depending upon the extent of surgical dissection. No discernable migration was noted when the tissue dissection was minimal. Studies utilizing a modified device equipped with electrodes indicated that migration was nominal with sufficient positional stability to ensure activation of target muscles for glottis opening. This study supported the feasibility of using a microstimulator for reanimation of paralyzed laryngeal muscles that open the airway during breathing. This innovative approach to treatment would alleviate the need for a tracheotomy or surgical resection of the vocal fold.

Influence of prenylated and non-prenylated flavonoids on liver microsomal lipid peroxidation and oxidative injury in rat hepatocytes.

Prenylated chalcones from hops and beer were compared with non-prenylated flavonoids [chalconaringenin (CN), naringenin (NG), genistein (GS) and quercetin (QC)] for their ability to inhibit lipid peroxidation in rat liver microsomes. Chalcones with prenyl- or geranyl-groups (5 and 25 microM) were more effective inhibitors of microsomal lipid peroxidation than CN, NG or GS induced by Fe(2+)/ascorbate. Prenylated chalcones were effective inhibitors of microsomal lipid peroxidation induced by Fe(3+)-ADP/NADPH and by tert-butyl hydroperoxide (TBH) but to a lesser extent compared to the Fe(2+)/ascorbate system. An increase of prenyl substituents decreased antioxidant activity in the lipid peroxidation systems. Certain flavonoids behaved as prooxidants in the iron-dependent lipid peroxidation systems. For example, at 5 microM, NG enhanced iron/ascorbate-induced lipid peroxidation whereas CN, diprenylxanthohumol and tetrahydroxanthohumol enhanced Fe(3+)-ADP/NADPH-induced lipid peroxidation. None of the flavonoids (25 microM), except QC, inhibited NADPH cytochrome P450-reductase activity of rat liver microsomes, suggesting that the mechanism of inhibition of lipid peroxidation induced by Fe(3+)-ADP/NADPH is not due to inhibition of the reductase enzyme. Chalcones exhibiting antioxidant activity against TBH-induced lipid peroxidation such as xanthohumol and 5'-prenylxanthohumol, and NG, with no antioxidant property at 5 microM concentration protected cultured rat hepatocytes from TBH toxicity. Other antioxidants (desmethylxanthohumol and CN) in the TBH system were not cytoprotective. These results demonstrate the importance of prenyl groups in the antioxidant activity of hop chalcones in the various in vitro systems of lipid peroxidation. Furthermore, the antioxidant activity of the flavonoids has little or no bearing on their ability to protect rat hepatocytes from the toxic effects of TBH.

Failed cementless total knee arthroplasty presenting as osteolysis of the fibular head.

Large osteolytic lesions can occur adjacent to cementless total knee arthroplasty components. This occurrence frequently is related to suboptimal design features leading to the generation of metal or polyethylene wear debris. Occasionally, such lesions can mimic a bone tumor. A case of a lytic lesion of the fibular head adjacent to a failed cementless knee arthroplasty is described along with a description of the damaged retrieved component that led to the failure.

Isoform specificity of N-deacetyl ketoconazole by human and rabbit flavin-containing monooxygenases.

N-Deacetyl ketoconazole (DAK) is the major metabolite of orally administered ketoconazole. This major metabolite has been demonstrated to be further metabolized predominately by the flavin-containing monooxygenases (FMOs) to the secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole (N-hydroxy-DAK) by adult and postnatal rat hepatic microsomes. Our current investigation evaluated the FMO isoform specificity of DAK in a pyrophosphate buffer (pH 8.8) containing the glucose 6-phosphate NADPH-generating system. cDNA-expressed human FMOs (FMO1, FMO3, and FMO5) and cDNA-expressed rabbit FMOs (FMO1, FMO2, FMO3, and FMO5) were used to assess the metabolism of DAK to its subsequent FMO-mediated metabolites by HPLC analysis. Human and rabbit cDNA-expressed FMO3 resulted in extensive metabolism of DAK in 1 h (71.2 and 64.5%, respectively) to N-hydroxy-DAK (48.2 and 47.7%, respectively) and two other metabolites, metabolite 1 (11.7 and 7.8%, respectively) and metabolite 3 (10.5 and 10.0%, respectively). Previous studies suggest that metabolite 1 is the nitrone formed after successive FMO-mediated metabolism of N-hydroxy-DAK. Moreover, these studies display similar metabolic profiles seen with adult and postnatal rat hepatic microsomes. The human and rabbit FMO1 metabolized DAK predominately to the N-hydroxy-DAK in 1 h (36.2 and 25.3%, respectively) with minimal metabolism to the other metabolites (

Exogenous surfactant therapy in newborns.

The introduction of surfactant replacement therapy for the management of respiratory distress syndrome (RDS) in the premature infant is one of the major advances in neonatal intensive care. After almost 50 years of intense research, the concerted efforts of basic scientists and clinicians have been rewarded. Despite the remarkable effects of surfactant therapy of RDS, the impact on the incidence of chronic lung disease has been unclear. With the recognition of the role of surfactant inactivation in the pathogenesis of other respiratory disorders of newborns (i.e., meconium aspiration syndrome and pneumonia) newer applications as well as new challenges have emerged. This article provides an overview of the physiology of the surfactant system and the current uses of this therapy in newborns.

Flavin-containing monooxygenase-mediated metabolism of N-deacetyl ketoconazole by rat hepatic microsomes.

Although ketoconazole is extensively metabolized by hepatic microsomal enzymes, the route of formation and toxicity of suspected metabolites are largely unknown. Reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice. DAK may be susceptible to successive oxidative attacks on the N-1 position by flavin-containing monooxygenases (FMO) producing potentially toxic metabolites. Previous laboratory findings have demonstrated that postnatal rat hepatic microsomes metabolize DAK by NADPH-dependent monooxygenases to two metabolites as determined by HPLC. Our current investigation evaluated DAK's metabolism in adult male and female rats and identified metabolites that may be responsible for ketoconazole's hepatotoxicity. DAK was extensively metabolized by rat liver microsomal monooxygenases at pH 8.8 in pyrophosphate buffer containing the glucose 6-phosphate NADPH-generating system to three metabolites as determined by HPLC. The initial metabolite of DAK was a secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole, which was confirmed by liquid chromatography/mass spectrometry and NMR spectroscopy. Extensive metabolism of DAK occurred at pH 8.8 in pyrophosphate buffer (female 29% and male 53% at 0.25 h; female 55% and male 57% at 0.5 h; and female 62% and male 66% at 1.0 h). Significantly less metabolism of DAK occurred at pH 7.4 in phosphate buffer (female 11%, male 17% at 0.25 h; female 20%, male 31% at 0.5 h; and female 27%, male 37% at 1 h). Heat inactivation of microsomal-FMO abolished the formation of these metabolites from DAK. SKF-525A did not inhibit this reaction. These results suggest that DAK appears to be extensively metabolized by adult FMO-mediated monooxygenation.

Antiproliferative and cytotoxic effects of prenylated flavonoids from hops (Humulus lupulus) in human cancer cell lines.

Six flavonoids [xanthohumol (XN), 2',4',6',4-tetrahydroxy-3'-prenylchalcone (TP); 2',4',6',4-tetrahydroxy-3'-geranylchalcone (TG); dehydrocycloxanthohumol (DX); dehydrocycloxanthohumol hydrate (DH); and isoxanthohumol (IX)] from hops (Humulus lupulus) were tested for their antiproliferative activity in human breast cancer (MCF-7), colon cancer (HT-29) and ovarian cancer (A-2780) cells in vitro. XN, DX and IX caused a dose-dependent (0.1 to 100 microM) decrease in growth of all cancer cells. After a 2-day treatment, the concentrations at which the growth of MCF-7 cells was inhibited by 50% (IC50) were 13.3, 15.7 and 15.3 microM for XN, DX and IX, respectively. After a 4-day treatment, the IC50 for XN, DX and IX were 3.47, 6.87 and 4.69 microM, respectively. HT-29 cells were more resistant than MCF-7 cells to these flavonoids. In A-2780 cells, XN was highly antiproliferative with IC50 values of 0.52 and 5.2 microM after 2 and 4 days of exposure, respectively. At 100 microM, all the hop flavonoids were cytotoxic in the three cell lines. Growth inhibition of XN- and IX-treated MCF-7 cells was confirmed by cell counting. XN and IX inhibited DNA synthesis in MCF-7 cells. As antiproliferative agents, XN (chalcone) and IX (flavanone isomer of XN) may have potential chemopreventive activity against breast and ovarian cancer in humans.

Predictive value of in vitro model systems in toxicology.

The application of in vitro model systems to evaluate the toxicity of xenobiotics has significantly enhanced our understanding of drug- and chemical-induced target toxicity. From a scientific perspective, there are several reasons for the popularity of in vitro model systems. From the public perspective, in vitro model systems enjoy increasing popularity because their application may allow a reduction in the number of live animals employed in toxicity testing. In this review, we present an overview of the use of in vitro model systems to investigate target organ toxicity of drugs and chemicals, and provide selective examples of these model systems to better understand cutaneous and ocular toxicity and the role of drug metabolism in the hepatotoxicity of selected agents. We conclude by examining the value and use of in vitro model systems in industrial development of new pharmaceutical agents.

Characterization of a linear DNA plasmid from the filamentous fungal plant pathogen Glomerella musae [Anamorph: Colltotrichum musae (Berk. & Curt.) Arx.].

A 7.4-kilobase (kb) DNA plasmid was isolated from Glomerella musae isolate 927 and designated pGML1. Exonuclease treatments indicated that pGML1 was a linear plasmid with blocked 5' termini. Cell-fractionation experiments combined with sequence-specific PCR amplification revealed that pGML1 resided in mitochondria. The pGML1 plasmid hybridized to cesium chloride-fractionated nuclear DNA but not to A + T-rich mitochondrial DNA. An internal 7.0-kb section of pGML1 was cloned and did not hybridize with either nuclear or mitochondrial DNA from G. musae. Sequence analysis revealed identical terminal inverted repeats (TIR) of 520 bp at the ends of the cloned 7.0-kb section of pGML1. The occurrence of pGML1 did not correspond with the pathogenicity of G. musae on banana fruit. Four additional isolates of G. musae possessed extrachromosomal DNA fragments similar in size and sequence to pGML1.