Annotation and characterization of the Plasmodium vivax rhoptry neck protein 4 (PvRON4)

Background: The tight junction (TJ) is one of the most important structures established during merozoite invasion of host cells and a large amount of proteins stored in Toxoplasma and Plasmodium parasites’ apical organelles are involved in forming the TJ. Plasmodium falciparum and Toxoplasma gondii apical membrane antigen 1 (AMA-1) and rhoptry neck proteins (RONs) are the two main TJ components. It has been shown that RON4 plays an essential role during merozoite and sporozoite invasion to target cells. This study has focused on characterizing a novel Plasmodium vivax rhoptry protein, RON4, which is homologous to PfRON4 and PkRON4. Methods: The ron4 gene was re-annotated in the P. vivax genome using various bioinformatics tools and taking PfRON4 and PkRON4 amino acid sequences as templates. Gene synteny, as well as identity and similarity values between open reading frames (ORFs) belonging to the three species were assessed. The gene transcription of pvron4, and the expression and localization of the encoded protein were also determined in the VCG-1 strain by molecular and immunological studies. Nucleotide and amino acid sequences obtained for pvron4 in VCG-1 were compared to those from strains coming from different geographical areas. Results: PvRON4 is a 733 amino acid long protein, which is encoded by three exons, having similar transcription and translation patterns to those reported for its homologue, PfRON4. Sequencing PvRON4 from the VCG-1 strain and comparing it to P. vivax strains from different geographical locations has shown two conserved regions separated by a low complexity variable region, possibly acting as a “smokescreen”. PvRON4 contains a predicted signal sequence, a coiled-coil α-helical motif, two tandem repeats and six conserved cysteines towards the carboxyterminus and is a soluble protein lacking predicted transmembranal domains or a GPI anchor. Indirect immunofluorescence assays have shown that PvRON4 is expressed at the apical end of schizonts and co-localizes at the rhoptry neck with PvRON2.

PvRON2, a new Plasmodium vivax rhoptry neck antigen

Background: Rhoptries are specialized organelles from parasites belonging to the phylum Apicomplexa; they secrete their protein content during invasion of host target cells and are sorted into discrete subcompartments within rhoptry neck or bulb. This distribution is associated with these proteins’ role in tight junction (TJ) and parasitophorous vacuole (PV) formation, respectively. Methods: Plasmodium falciparum RON2 amino acid sequence was used as bait for screening the codifying gene for the homologous protein in the Plasmodium vivax genome. Gene synteny, as well as identity and similarity values, were determined for ron2 and its flanking genes among P. falciparum, P. vivax and other malarial parasite genomes available at PlasmoDB and Sanger Institute databases. Pvron2 gene transcription was determined by RT-PCR of cDNA obtained from the P. vivax VCG-1 strain. Protein expression and localization were assessed by Western blot and immunofluorescence using polyclonal anti-PvRON2 antibodies. Co-localization was confirmed using antibodies directed towards specific microneme and rhoptry neck proteins. Results and discussion: The first P. vivax rhoptry neck protein (named here PvRON2) has been identified in this study. PvRON2 is a 2,204 residue-long protein encoded by a single 6,615 bp exon containing a hydrophobic signal sequence towards the amino-terminus, a transmembrane domain towards the carboxy-terminus and two coiled coil a-helical motifs; these are characteristic features of several previously described vaccine candidates against malaria. This protein also contains two tandem repeats within the interspecies variable sequence possibly involved in evading a host’s immune system. PvRON2 is expressed in late schizonts and localized in rhoptry necks similar to what has been reported for PfRON2, which suggests its participation during target cell invasion. Conclusions: The identification and partial characterization of the first P. vivax rhoptry neck protein are described in the present study. This protein is homologous to PfRON2 which has previously been shown to be associated with PfAMA-1, suggesting a similar role for PvRON2.

Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (PvRON1)

Background: Plasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as PfRONs, Pf92, Pf38, Pf12 and Pf34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (PvRON1) and examine its antigenicity in natural P. vivax infections. Methods: The PvRON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I PvRON1 sequence were synthesized and inoculated in rabbits for obtaining anti-PvRON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the PvRON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA. Results: In the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that PvRON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The PvRON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax. Conclusions: This study shows the identification and characterization of the P. vivax rhoptry neck protein 1 in the VCG-1 strain. Taking into account that PvRON1 shares several important characteristics with other Plasmodium antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the Aotus monkey model are thus recommended.

Influence of the lateral pterygoid muscle on the growth of the mandibular condilar cartilage

At the end of the last century, a model to explain clinical observations related to the mandibular growth was developed. According to it, the lateral pterigoid muscle (LPM) was one of the main modulators of the differentiation of mesenquimal cells inside the condyle to condroblasts or osteoblasts, and therefore of the growth of the mandibular condilar cartilage (CCM). The main components of the model were the humoral and the mechanical. Nowadays, the humoral would include growth factors such as IGF-I, FGF-2 and VEGF, which seem to be involved in mandibular growth. Since skeletal muscle can secrete these growth factors, there is a possibility that LPM modulates the growth of CCM by a paracrine or endocrine mechanism. The mechanical component derived from the observations that both the blood flow inside the temporomandibular joint (ATM) and the action of the retrodiscal pad on the growth of the CCM, depend, in part, on the contractile activity of the LPM. Despite the fact that there are some results suggesting  hat LPM is activated under conditions of mandibular protrusion, there is no full agreement on whether this can stimulate the growth of CCM. In this review, the contributions and limitations of the works related to mandibular growth are discussed and a model which integrates the available information to explain the role of the LPM in the growth of the CCM is proposed.

Prevalencia de síntomas osteomusculares en miembros superiores y los probables factores de riesgo asociados en los estudiantes de musica en una institución universitaria de Bogotá,Colombia, 2013

Los músicos por su práctica instrumental tienen una alta demanda de desempeño físico, especialmente de los miembros superiores y están expuestos a varios factores de riesgo biomecánico que pueden resultar en problemas de salud. Objetivo: determinar la prevalencia de sintomatología osteomuscular de miembros superiores y los probables factores de riesgo asociados, en los estudiantes expuestos a la actividad musical durante el segundo semestre del año 2013 en una institución universitaria de Bogotá, Colombia. Método: se realizó un estudio descriptivo de corte trasversal en 134 estudiantes de todos los semestres de música en una institución universitaria. Se aplicó el Cuestionario nórdico estandarizado para análisis de síntomas músculo esqueléticos y una encuesta ad hoc que contemplaba aspectos sociodemográficos y antecedentes académicos, patológicos, factores de exposición y hábitos. Resultados: Las prevalencias generales encontradas en el estudio, son similares a las que refieren algunos estudios revisados que contemplan ciertas variables afines a las que se estudiaron. La prevalencia de síntomas osteomusculares cervico-braquial fue de 77.9%. La prevalencia de molestias en cuello fue mayor en las mujeres (64.3%) que en los hombres (37.4%) (OR=3.02, IC 95%=1.26, 7.18). La prevalencia de síntomas en manos/muñecas que le impidió hacer su trabajo en los últimos 12 meses fue mayor en los estudiantes que refirieron alguna enfermedad (29.4%) que en los que no la manifestaron (10.2%), (OR=3.69, IC 95%=1.34, 10.19). La prevalencia de molestias en cuello que les impidió hacer su trabajo en los últimos 12 meses fue mayor en los estudiantes que practicaron algún pasatiempo con sus brazos (10.4%) versus los que no lo practicaron, cuya frecuencia fue 0.0%. Los instrumentos musicales de mayor práctica fueron cuerda y percusión y se asociaron a prevalencia de síntomas osteomusculares cérvico-braquiales con una distribución por segmentos similar. Los tiempos de práctica semanales y la antigüedad en la práctica, conduce a síntomas cervico-braquiales. Conclusiones: Este estudio coincide con la distribución de las prevalencias encontradas en poblaciones de estudiantes de música revisadas, con respecto a la sintomatología, a los segmentos cervico-braquiales de mayor afectación, a la significancia del género femenino con respecto al masculino, al tipo de instrumentos y a los tiempos de práctica entre otros. Esto plantea la necesidad de educar a nuestros músicos en la detección temprana de síntomas desde su formación de pregrado o quizás mucho antes.

Aportes de la evidencia epidemiológica para el estudio de las condiciones de riesgo laboral, asociada a la presentación del síndrome del túnel del carpo (stc)

Las formas de adquirir una enfermedad son diversas. Los seres humanos están expuestos a diferentes factores de riesgo ambientales, físicos y ocupacionales entre otros, estos últimos se presentan con mayor o menor frecuencia dependiendo de la actividad laboral que realiza un trabajador, del tiempo que emplea para cumplir la tarea o la manera en que ejecuta ésta. Estos factores se ven asociados con aspectos propios del individuo que de alguna manera los aumentan o los disminuyen. Este proyecto investigativo pretende realizar una aproximación a la evidencia epidemiológica que se contempla en el estudio de las condiciones de riesgo laboral asociadas a la presentación del Síndrome del Túnel del Carpo (STC), con el fin de dejar elementos que contribuyan a la elaboración de diferentes instrumentos de evaluación de las condiciones de riesgo, que pueden estar vinculadas a la presentación de este problema.

Factores socio-demográficos, laborales y factores de riesgo ergonómicos relacionados con la aparición de síntomas osteomusculares de la actividad del bicitaxismo en una localidad de la ciudad de Bogotá (2014)

Resumen Objetivo: Determinar los factores socio-demográficos, laborales y los factores de riesgo ergonómico, relacionados con la aparición de síntomas osteomusculares de la actividad del Bicitaxismo en el barrio Toberín de la Localidad de Usaquén de la Ciudad Bogotá, para recomendar estrategias de prevención. Materiales y métodos: Estudio observacional descriptivo de corte transversal en 150 personas que trabajan en el Bicitaxismo. Se realizó un muestreo simple y aleatorio. La identificación de los factores asociados con síntomas osteomusculares se hizo mediante la aplicación de la metodología Ergopar (Ergonomía Participativa para la prevención de trastornos musculoesqueléticos de origen laboral), en el grupo de personas que trabajan en el Bicitaxismo. Se estimó la proporción de personas con trastornos músculo esqueléticos en este grupo de trabajadores y se exploró asociación mediante la prueba Chi cuadrado de Pearson o prueba exacta de Fisher, usando nivel de significación del 5%, con el programa SPSS 20. Resultados: Los resultados sugirieron que en el bicitaxismo hay asociación estadísticamente significativa con los factores de edad y aparición de dolor en espalda lumbar valor p (0,009), manos/muñecas valor p (0,001), piernas con un valor p (0,022), rodillas valor p (0,001), tiempo de trabajo con la aparición de dolor en codos valor p (0,013), horas de trabajo con la aparición de molestias en codos valor p (0,000), y en piernas valor p (0,002), Días de trabajo, con la aparición de molestias en manos/muñecas valor p (0,003), dolor en piernas valor p (0), y dolor en rodillas valor p (0,014), e igualmente exigencias físicas con la aparición de molestias en manos/muñecas valor p (0,002) y dolor en piernas con un valor p (0,044) Conclusión: Se evidenció asociación entre la actividad del bicitaxismo y la aparición de síntomas osteomusculares de molestia/dolor en los diferentes segmentos musculares, tales como espalda lumbar, codos, manos/muñecas, piernas y rodillas

Prevalencia de los síntomas osteomusculares y los factores de riesgo asociados, en trabajadores de una empresa de geomática, Colombia 2014

Objetivo Identificar la prevalencia de síntomas osteomusculares, por segmentos y los factores de riesgo asociados, en los trabajadores de una empresa de Geomática, en Colombia en el año 2014. Metodología Se llevó a cabo un estudio descriptivo de corte transversal con una población de 169 trabajadores, distribuidos en 2 grupos, el grupo de campo que desarrolla actividades de topografía y el grupo de oficina donde se realizan procesamiento de datos en Geomática y actividades administrativas. A cada trabajador se le aplicó el cuestionario ERGOPAR que interroga la exposición o factores de riesgo y la presencia de síntomas osteomusculares. Resultados: El personal de oficina presenta mayor frecuencia de síntomas osteomusculares en el cuello 72%, la región lumbar 55%, los codos 17,7%, las manos y muñecas 57.3%. Presentándose con mayor frecuencia en las mujeres los síntomas en cuello 80% y manos 64%, mientras que los mayores porcentajes en personal de campo se presentan en las piernas 21%, las rodillas 26% y pies 11,5%. Se encontró asociación significativa entre la sedestación durante más de cuatro horas, con dolor en cuello (p=0.02) y dolor en región lumbar (p=0.03); inclinar el cuello hacia delante durante más de cuatro horas, con dolor en el cuello (p=0.006); repetir cada pocos segundos la flexión de muñecas (p=001) y utilizar los dedos de manera intensiva por más de 4 horas (p=0.01) con dolor en manos y las variables jornada laboral y puesto de trabajo con dolor en pies. Conclusiones La prevalencia de síntomas osteomusculares en los trabajadores de la empresa estudiada es alta. Dado que se encontró asociación significativa con las variables sociodemográficas y laborales. La alta prevalencia de sintomatología puede ser explicada por la exposición a carga física laboral, por posturas de trabajo, por movimientos repetitivos y características propias de género.

Effect of combined heat and high-pressure treatments on the texture of chicken breast muscle (Pectoralis fundus)

Commercially supplied chicken breast muscle was subjected to simultaneous heat and pressure treatments. Treatment conditions ranged from ambient temperature to 70 °C and from 0.1 to 800 MPa, respectively, in various combinations. Texture profile analysis (TPA) of the treated samples was performed to determine changes in muscle hardness. At treatment temperatures up to and including 50 °C, heat and pressure acted synergistically to increase muscle hardness. However, at 60 and 70 °C, hardness decreased following treatments in excess of 200 MPa. TPA was performed on extracted myofibrillar protein gels that after treatment under similar conditions revealed similar effects of heat and pressure. Differential scanning calorimetry analysis of whole muscle samples revealed that at ambient pressure the unfolding of myosin was completed at 60 °C, unlike actin, which completely denatured only above 70 °C. With simultaneous pressure treatment at >200 MPa, myosin and actin unfolded at 20 °C. Unfolding of myosin and actin could be induced in extracted myofibrillar protein with simultaneous treatment at 200 MPa and 40 °C. Electrophoretic analysis indicated high pressure/temperature regimens induced disulfide bonding between myosin chains.

Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

Effect of plasma insulin and branched-chain amino acids on skeletal muscle protein synthesis in fasted lambs

The increase in fractional rate of protein synthesis (K-s) in the skeletal muscle of growing rats during the transition from fasted to fed state has been explained by the synergistic action of a rise in plasma insulin and branched-chain amino acids (BCAA). Since growing lambs Also exhibit an increase in K-s with level of feed intake, the objective of the present study was to determine if this synergistic relationship between insulin and BCAA also occurs in ruminant animals. Six 30 kg fasted (72 h) lambs (8 months of age) received each of four treatments, which were based on continuous infusion into the jugular vein for 6 h of: (1) saline (155 mmol NaCl/l); (2) a mixture of BCAA (0.778 mumol leucine, 0.640 mumol isoleucine and 0.693 mumol valine/min.kg); (3) 18.7 mumol glucose/min.kg (to induce endogenous insulin secretion): (4) co-infusion of BCAA and glucose. Within each period all animals received the same isotope of phenylalanine, (Phe) as follows: (1) L-[1-C-13]Phe; (2) L-phenyl-[ring H-2(5)]-alanine; (3) L-[N-15]Phe; (4) L-[ring 2,6-H-3]Phe. Blood was sampled serially during infusions to measure plasma concentrations of insulin, glucose and amino acids, and plasma free Phe isotopic activity; biopsies were taken 6 h after the beginning of infusions to determine K-s in in. longissimus dorsi and vastus muscle. Compared with control (saline-infused) lambs, K-s was increased by an average of 40% at the end of glucose infusion, but this effect was not statistically significant in either of the muscles sampled. BCAA infusion, alone or in combination with glucose, also had no significant effect on K-s compared with control sheep. K-s was approximately 60% greater for vastus muscle than for m. longissimus dorsi (P<0.01), regardless of treatment. It is concluded that there are signals other than insulin and BCAA that are responsible for the feed-induced increase in K-s in muscle of growing ruminant animals.