Efectos de la imitación en la interacción social recíproca en un niño con Trastorno del Espectro Autista Grave / Effects of imitation on mutual social interaction in children with severe autism spectrum disorder / Efectes de la imitació en la interacción social recíproca en un nen amb trastorns de l'espectre autista greu

Los niños con trastorno del espectro autista (TEA) presentan dificultades de empatía y relación social. Estudios previos sugieren un déficit primario en el sistema de neuronas espejo. En este estudio de caso único se ha aplicado la metodología observacional para investigar el uso de la imitación en un proceso de una psicoterapia de orientación psicoanalítica en un niño con TEA grave. Los resultados sugieren que el uso de la imitación en este tipo de psicoterapia es una opción recomendable, ya que favorece los procesos de diferenciación y mejora las capacidades de interacción

Children with autism spectrum disorders (ASD) show difficulties in empathy and social interaction. Previous studies suggest primary deficits in the mirror system brain network. In this single case study observational methodology has been used to investigate the process of a psychoanalytic psychotherapy with imitation in a child with severe ASD. Results suggest that the use of imitation within this psychotherapy approach promote the differentiation processes and stimulates social interaction

Efectes de la imitació en la interacció social recíproca en un nen amb trastorn de l’espectre autista greu. Els nens amb trastorn de l’espectre autista (TEA) presenten dificultats d’empatia i relació social. Estudis previs suggereixen un dèficit primari en el sistema de neurones mirall. En aquest estudi de cas únic s’ha aplicat la metodologia observacional per investigar l’ús de la imitació en un procés d’una psicoteràpia d’orientació psicoanalítica en un nen amb TEA greu. Els resultats suggereixen que l’ús de la imitació en aquest tipus de psicoteràpia és una opció recomanable, ja que afavoreix els processos de diferenciació i millora les capacitats d'interacció

Catéteres tunelizados. Complicaciones en su inserción / No disponible

Introducción: La creación de accesos vasculares para hemodiálisis en pacientes afectos de insuficiencia renal terminal, se presenta periódicamente como un problema. Por ello, la disponibilidad de alternativas a la fístula arteriovenosa es algo necesario; distintos tipos de catéteres tunelizados se vienen desarrollando, entre los cuales el catéter de Tesio es el mejor conocido y utilizado. Frecuentemente es implantado por cirujanos vasculares o radiólogos, lo que conlleva una dependencia para los servicios de nefrología que puede retrasar la obtención del nuevo acceso vascular y el inicio o reinicio de tratamiento. Material y métodos: Se realizó un estudio descriptivo con el objetivo de analizar las complicaciones inmediatas durante la inserción de catéteres centrales tunelizados para hemodiálisis y evaluar la posibilidad de su consecución por nefrólogos y personal especialista en diálisis. Entre enero de 2003 y diciembre de 2005 se implantaron 175 catéteres centrales tunelizados de Tesio en nuestra unidad de hemodiálisis. Se registró edad y sexo del paciente, tiempo en diálisis, diagnóstico de hipertensión, diabetes mellitus o cardiopatía previa, antecedentes de tratamiento anticoagulante o antiagregante, dificultad percibida por el nefrólogo para el desarrollo de la técnica, sangrado, presión arterial sistólica y diastólica, frecuencia cardíaca, saturación de oxígeno y cambios en la monitorización de ECG, al inicio de la intervención, durante la canalización de la vena, inserción del dilatador y catéter, y al final de la misma. Resultados: Los pacientes fueron 88 hombres y 82 mujeres, con media de edad de 64 años. El 21% de los pacientes llevaban habitualmente tratamiento con antiagregante o anticoagulante, que había sido retirado en los días previos. El tamaño ecográfico de la vena era mayor de 1 cm en el 79% de los casos. De 175 inserciones sólo 3 pacientes (2%) presentaron signos de inestabilidad hemodinámica, dos de ellos por descenso de tensión arterial y uno por descenso de saturación de oxígeno; ninguno de ellos requirió soporte asistido, no apareció hemorragia ni otras complicaciones. Conclusiones: La implantación de catéteres tunelizados, bajo anestesia local, presenta mínimas complicaciones e incidencias durante su inserción. Puede llevarse a cabo por un nefrólogo y personal especializado, en hospitales con el respaldo de unidades que permitan abordar las posibles complicaciones (AU)

Introduction: The creation of vascular accesses for hemodialysis in patients affected with terminal kidney failure affections, is presented periodically as a problem. Because of it, the availability of alternatives to the arteriovein fistula is something necessary; different types of tunnelled dialysis catheters are being developed, among which the Tesio catheter is the most well known and most widely utilized. Often it is implanted by vascular surgeons or radiologists; this creates dependence for the nephrology services that can delay obtaining the new vascular access and initiating or reinitiating the treatment. Material and methods: A descriptive study with the objective of analyzing the immediate complications during the insertion of tunnelled central catheters for hemodialysis and to evaluate the possibility of its attainment was carried out by nephrologists and personnel specialized in dialysis. Between january of 2003 and december of 2005, 175 Tesio tunnelled central catheters were implanted in our hemodialysis unit. Age and sex of the patient was registered, time in dialysis, diagnosis of hypertension, diabetes mellitus or prior heart disease, previous anticoagulant or platelet antiaggregate treatment, difficulty perceived by the nephrologist for the development of the technique, bleeding, systolic and diastolic arterial pressure, cardiac frequency, oxygen saturation and changes in the monitoring of electrocardiogram, at the beginning of the intervention, during the channelling of the vein, insertion of the dilatators and catheter, and at the end of it. Results: The patients were 88 men and 82 women, with an average of age of 64. 21% of the patients habitually followed a treatment with platelet antiaggregate or anticoagulant, which had been withdrawn in the prior days. The ultrasound size of the vein was greater than one cm in 79% of the cases. Of 175 insertions only three patients (2%) presented signs of hemodynamic instability, two of them due to a descent of arterial tension and one by oxygen saturation descent; none of them required assisted backup, neither hemorrhage nor other complications appeared. Conclusions: The implant of tunnelled catheters, under local anesthesia, presents minimal complications and incidents during its insertion. It can be carried out by a nephrologist and specialized personnel, in hospitals with backup units that can undertake any possible complications (AU)

[Tunneled catheters. Complications during insertion]. / Catéteres tunelizados. Complicaciones en su inserción.

INTRODUCTION: The creation of vascular accesses for hemodialysis in patients affected with terminal kidney failure affections, is presented periodically as a problem. Because of it, the availability of alternatives to the arteriovein fistula is something necessary; different types of tunnelled dialysis catheters are being developed, among which the Tesio catheter is the most well known and most widely utilized. Often it is implanted by vascular surgeons or radiologists; this creates dependence for the nephrology services that can delay obtaining the new vascular access and initiating or reinitiating the treatment. MATERIAL AND METHODS: A descriptive study with the objective of analyzing the immediate complications during the insertion of tunnelled central catheters for hemodialysis and to evaluate the possibility of its attainment was carried out by nephrologists and personnel specialized in dialysis. Between January of 2003 and December of 2005, 175 Tesio tunnelled central catheters were implanted in our hemodialysis unit. Age and sex of the patient was registered, time in dialysis, diagnosis of hypertension, diabetes mellitus or prior heart disease, previous anticoagulant or platelet antiaggregate treatment, difficulty perceived by the nephrologist for the development of the technique, bleeding, systolic and diastolic arterial pressure, cardiac frequency, oxygen saturation and changes in the monitoring of electrocardiogram, at the beginning of the intervention, during the channelling of the vein, insertion of the dilatators and catheter, and at the end of it. RESULTS: The patients were 88 men and 82 women, with an average of age of 64. 21% of the patients habitually followed a treatment with platelet antiaggregate or anticoagulant, which had been withdrawn in the prior days. The ultrasound size of the vein was greater than one cm in 79% of the cases. Of 175 insertions only three patients (2%) presented signs of hemodynamic instability, two of them due to a descent of arterial tension and one by oxygen saturation descent; none of them required assisted backup, neither hemorrhage nor other complications appeared. CONCLUSIONS: The implant of tunnelled catheters, under local anesthesia, presents minimal complications and incidents during its insertion. It can be carried out by a nephrologist and specialized personnel, in hospitals with backup units that can undertake any possible complications.

A novel alcohol oxidase/RNA-binding protein with affinity for mycovirus double-stranded RNA from the filamentous fungus Helminthosporium (Cochliobolus) victoriae: molecular and functional characterization.

We have cloned and sequenced a novel alcohol oxidase (Hv-p68) from the filamentous fungus Helminthosporium (Cochliobolus) victoriae that copurifies with mycoviral double-stranded RNAs. Sequence analysis revealed that Hv-p68 belongs to the large family of FAD-dependent glucose methanol choline oxidoreductases and that it shares significant sequence identity (>67%) with the alcohol oxidases of the methylotrophic yeasts. Unlike the intronless alcohol oxidases from methylotrophic yeasts, a genomic fragment of the Hv-p68 gene was found to contain four introns. Hv-p68, purified from fungal extracts, showed only limited methanol oxidizing activity, and its expression was not induced in cultures supplemented with methanol as the sole carbon source. Northern hybridization analysis indicated that overexpression of Hv-p68 is associated with virus infection, because significantly higher Hv-p68 mRNA levels (10- to 20-fold) were detected in virus-infected isolates compared with virus-free ones. We confirmed by Northwestern blot analysis that Hv-p68 exhibits RNA binding activity and demonstrated that the RNA-binding domain is localized within the N-terminal region that contains a typical ADP-binding beta-alpha-beta fold motif. The Hv-p68 gene, or closely similar genes, was present in all species of the genus Cochliobolus but absent in the filamentous fungus, Penicillium chrysogenum, as well as in two nonmethylotrophic yeasts examined. This study represents the first reported case that a member of the FAD-dependent glucose methanol choline oxidoreductase family, Hv-p68, may function as an RNA-binding protein.

A cellular protein with an RNA-binding activity co-purifies with viral dsRNA from mycovirus-infected Helminthosporium victoriae.

A cellular protein that co-purifies with mycoviral dsRNA was isolated from the plant pathogenic fungus Helminthosporium victoriae (telomorph: Cochliobolus victoriae) infected with two viruses, the totivirus Helminthosporium victoriae 190S virus and the chrysovirus-like Helminthosporium victoriae 145S virus (Hv145SV). The cellular protein, which was, designated Hv-p68, accumulated to higher levels in virus-infected isolates compared to virus-free ones. The majority of the Hv145S dsRNAs were found in association with Hv-p68 and not packaged in virions. Hv-p68 could also be detected as a minor component of the virus capsid. Evidence is presented that Hv-p68 occurs in vivo as an octamer and that it possesses RNA-binding activities. Based on partial amino acid sequence analysis, Hv-p68 was shown to share significant sequence identity with alcohol oxidases from methylotrophic yeasts. Hv-p68 is proposed to play a role in viral RNA packaging/replication and in regulating viral pathogenesis.

Expression of the Totivirus Helminthosporium victoriae 190S virus RNA-dependent RNA polymerase from its downstream open reading frame in dicistronic constructs.

The undivided double-stranded RNA (dsRNA) genome of Helminthosporium victoriae 190S virus (Hv190SV) (genus Totivirus) consists of two large overlapping open reading frames (ORFs). The 5'-proximal ORF encodes a capsid protein (CP), and the downstream, 3'-proximal ORF encodes an RNA-dependent RNA polymerase (RDRP). Unlike the RDRPs of some other totiviruses, which are expressed as a CP-RDRP (Gag-Pol-like) fusion protein, the Hv190SV RDRP is detected only as a separate, nonfused polypeptide. In this study, we examined the expression of the RDRP ORF fused in frame to the coding sequence of the green fluorescent protein (GFP) in bacteria and Schizosaccharomyces pombe cells. The GFP fusions were readily detected in bacteria transformed with the monocistronic construct RDRP:GFP; expression of the downstream RDRP:GFP from the dicistronic construct CP-RDRP:GFP could not be detected. However, fluorescence microscopy and Western blot analysis indicated that RDRP:GFP was expressed at low levels from its downstream ORF in the dicistronic construct in S. pombe cells. No evidence that the RDRP ORF was expressed from a transcript shorter than the full-length dicistronic mRNA was found. A coupled termination-reinitiation mechanism that requires host or eukaryotic cell factors is proposed for the expression of Hv190SV RDRP.

Assembly of the Hv190S totivirus capsid is independent of posttranslational modification of the capsid protein.

The genome of Helminthosporium victoriae 190S totivirus (Hv190SV) consists of two large overlapping open reading frames (ORFs), encoding a capsid protein (CP) and an RNA-dependent RNA polymerase. The capsid of Hv190SV, even though encoded by a single gene, contains three closely related capsid polypeptides: p88, p83, and p78. p88 and p83 are phosphorylated, whereas p78, which is derived from p88 via proteolytic processing at the C terminus, is nonphosphorylated. In this study we expressed the CP ORF in bacteria and determined that a single product comigrating with virion p88 was generated. Evidence from in vivo phosphorylation studies indicated that the bacterially expressed p88 was unmodified, and thus autophosphorylation was ruled out. Enzymatic-dephosphorylation experiments using 32P-labeled p88 as a substrate demonstrated that the phosphorylated and nonphosphorylated forms of p88 could not be differentiated based on their mobilities in SDS gels and suggested that the two forms occur in purified virions. We also showed that the unmodified p88 is competent for assembly into virus-like particles, indicating that neither phosphorylation nor proteolytic processing of CP is required for capsid assembly. Posttranslational modification of CP, however, is proposed to play an important role in the life cycle of Hv190SV, including regulation of transcription/replication and/or packaging/release from virions of the viral (+) strand RNA transcript.

Expression, assembly, and proteolytic processing of Helminthosporium victoriae 190S totivirus capsid protein in insect cells.

The dsRNA genome (5.2 kbp) of Helminthosporium victoriae 190S totivirus (Hv190SV) consists of two large overlapping open reading frames (ORFs). The 5' proximal ORF codes for the capsid protein (CP) and the 3' ORF codes for an RNA-dependent RNA polymerase. Although the capsid of Hv190SV is encoded by a single gene, it is composed of two major closely related polypeptides, either p88 and p83 or p88 and p78. Whereas p88 and p83 are phosphoproteins, p78 is nonphosphorylated. Expression of the CP ORF in insect cells generated both p78 and p88 which assembled into virus-like particles. The finding that p78, p83, and p88 share a common N-terminal amino acid sequence is consistent with the determination that N-terminal, but not C-terminal, CP deletions were incompetent for assembly. Evidence was obtained that p78 is derived from p88 via proteolytic cleavage at the C-terminus. Proteolytic processing may play a regulatory role in the virus life cycle since it leads to dephosphorylation of CP and a subsequent decrease in virion transcriptional activity.

Purification and analysis of a polydnavirus gene product expressed using a poly-histidine baculovirus vector.

The VHv1.1 polydnavirus gene has been implicated in suppressing the encapsulation response in parasitized insects [Li and Webb (1994) J. Virol. 68, 7482-7489]. In order to characterize this gene product and to further our analysis of its immunosuppressive function, we expressed the VHv1.1 using a custom-designed C-terminal poly-histidine baculovirus vector which allows for high expression and single-step purification of the protein. The 34 kDa VHv1.1 protein was expressed in baculovirus-infected cell cultures and in H. virescens larvae. Highly enriched preparations of the secreted VHv1.1 protein were obtained after affinity chromatography using a NTA-(Ni2+) resin. Characterization with purified preparations of the VHv1.1 protein established that the protein is N-glycosylated, containing glycogroups which are PNGase F-sensitive but Endo H-resistant. The recombinant VHv1.1 protein bound to hemocytes in vitro and in vivo and was endocytosed in a manner similar to the native protein produced in CsPDV-infected larvae.

Expression and hemocyte-targeting of a Campoletis sonorensis polydnavirus cysteine-rich gene in Heliothis virescens larvae.

The polydnavirus associated with the parasitic wasp Campoletis sonorensis is injected into the lepidopteran insect, Heliothis virescens, during parasitization, after which viral gene products suppress the cellular immune system of the hosts. Four related cysteine-rich polydnavirus gene have been identified in parasitized H. virescens larvae and grouped into a family. In this study, we investigated the expression and hemocyte targeting of the cysteine-rich VHv1.4 protein. Full-length and truncated VHv1.4 proteins were produced in a bacterial expression system, and the purified proteins were used to raise polyclonal antisera. In immunoblots the VHv1.4 protein was detected in parasitized insects as early as 6 h and throughout the entire course of parasitism. The VHv1.4 protein appeared predominantly in the plasma fraction of hemolymph from parasitized larvae, suggesting that this protein is secreted. The VHv1.4 protein expressed from a recombinant baculovirus was secreted in two lepidopteran cell lines and in larvae injected with the recombinant virus. Digestion with endoglycosidases suggests that the VHv1.4 protein is glycosylated at multiple N-glycosylation sites. Immunofluorescence assays showed that the VHv1.4 protein binds to the hemocytes, most notably the granulocytes, in H. virescens larvae. After binding, the VHv1.4 protein was internalized, probably by endocytosis. Specific binding of the VHv1.4 to granulocytes implies an important function in the suppression of host cellular encapsulation response.

Expression of polydnavirus genes under polydnavirus promoter regulation in insect larvae infected with baculovirus recombinants.

We have evaluated the use of baculoviruses to deliver Campoletis sonorensis polydnavirus (CsPDV) genomic DNA into lepidopteran larvae to facilitate the identification of functional CsPDV genes. Genomic fragments consisting of regulatory (promoter) and coding sequences for two CsPDV genes (VHv1.1 and WHv1.6) were used to generate CsPDV-baculovirus recombinants and evaluate the expression of genes under the regulation of the CsPDV promoters. Northern blot and primer extension studies established that CsPDV genes were expressed under the control of their own promoters in these CsPDV-baculovirus recombinants. Transcripts were detected as early as 4 h post-infection indicating that temporal activity of CsPDV promoters was retained. The VHv1.1 gene product as expressed from CsPDV-baculovirus recombinants was identical in size and in functional properties to that produced in CsPDV-infected insects. CsPDV-baculovirus recombinants may be useful for the screening and characterization of polydnavirus genes with functional activities that can only be evaluated in insect larvae.

Characterization of a novel protein associated with the parasitization of lepidopteran hosts by an endoparasitic wasp.

We report basic biochemical characteristics of a parasitism-specific protein (PSP) expressed in hosts (Trichopluisa ni) of a parasitic wasp (Chelonus near curvimaculatus). Size exclusion HPLC and SDS-PAGE analysis of cross-linked products indicated the native conformation of the protein is as a monomeric polypeptide with an estimated M(r) of 185,000. Resolution by non-denaturing PAGE revealed two major PSP bands with different charges, PSP-1 and PSP-2, each of which corresponded to several isoforms on native IEF with a pl range of 4.57-5.45. Sequences for the N-terminus region and internal peptides after fragmentation of purified preparations of PSP-1 and PSP-2 did not resemble the sequence of any reported protein. Immunological characterization of PSP using antibodies generated against virus proteins from C. near curvimaculatus female wasps revealed that PSP shares a common epitope(s) with some structural components of the wasp polydnavirus.

Expression of a parasitism-specific protein in lepidopteran hosts of Chelonus sp.

The hemolymph of each noctuid species successfully parasitized by Chelonus near curvimaculatus possessed a parasitism-specific protein (PSP) previously identified in host T. ni (Insect Biochem. 19:445;21:845). Expression of PSP occurred in a stage-specific manner in the stadium during which the host undergoes precocious metamorphosis. The appearance of the protein was not due to nutritional stress associated with parasitism of hosts, since starved nonparasitized larvae did not produce the protein, or to low juvenile hormone titers occurring in precociously metamorphosing hosts, but rather was dependent on the presence of the endoparasite larva. Results of an in vivo incorporation experiments with [35S]-methionine showed that synthesis and subsequent appearance of the protein in the hemolymph of parasitized hosts was abrogated by prior surgical removal of endoparasite. Immunoprecipitation analysis of proteins from C. near curvimaculatus larvae cultured in vitro using antibodies specific to PSP indicated that the source of the protein was the endoparasite. Synthesis of PSP by the endoparasitic larvae with its subsequent secretion into the hemocoel of hosts was specific to the advanced stages of parasite development prior to its regression from the host.