Chemogenetic activation of VGLUT3-expressing neurons decreases movement.

Vesicular glutamate transporters (VGLUTs) are responsible for the storage of glutamate into secretory vesicles. The VGLUT3 isoform is mainly expressed in neurons that secrete other classical neurotransmitters, including the cholinergic interneurons in the striatum, and VGLUT3-expressing neurons often secrete two distinct neurotransmitters. VGLUT3 is discretely distributed throughout the brain and is found in subpopulations of spinal cord interneurons, in subset of neurons in the dorsal root ganglion, and in Merkel cells. Mice with a global loss of VGLUT3 are hyperactive and the modulation of specific VGLUT3-expressing circuits can lead to changes in movement. In this study, we tested the hypothesis that increased activity of VGLUT3-expressing neurons is associated with decreased movement. Using a mouse line expressing excitatory designer receptor exclusively activated by designer drugs (hM3Dq-DREADD) on VGLUT3-expressing neurons, we showed that activation of hM3Dq signalling acutely decreased locomotor activity. This decreased locomotion was likely not due to circuit changes mediated by glutamate nor acetylcholine released from VGLUT3-expressing neurons, as activation of hM3Dq signalling in mice that do not release glutamate or acetylcholine from VGLUT3-expressing neurons also decreased locomotor activity. This suggests that other neurotransmitters are likely driving this hypoactive phenotype. We used these mouse lines to compare the effects of DREADD agonists in vivo. We observed that clozapine-N-oxide (CNO), clozapine, compound 21 and perlapine show small differences in the speed at which they prompt behavioural responses but the four of them are selective DREADD ligands.

Common surgical training program: standardization of learning quality. / Programa de formación quirúrgica común: uniformidad en la calidad del aprendizaje.

INTRODUCTION: The various surgical specialties in our center have used the simulation and experimental surgery resources available for their training tasks in minimally invasive surgery (MIS) in an individualized manner. With this learning model, a great dispersion of effort and expense was observed, so it was decided to create a unified program based on the following: shared learning, synergy among specialties, moderation of the economic cost, and rational use of the facilities. OBJECTIVE: To describe and assess our consensually designed training program in order to consolidate a shared learning strategy that will enable our residents to acquire and perfect surgical skills in MIS. MATERIALS AND METHODS: The program consists of various increasingly complex phases implemented on a continuous basis throughout the period of specialized training in the virtual laboratory and experimental operating room. The assessment methods were based on quantifiable criteria: percentage of efficiency and completion time of the "McGill Inanimate System for Training and Evaluation of Laparoscopic Skills" (MISTELS) exercises at the beginning and end of the program. An economic study was also conducted. RESULTS: 20 residents have completed the program. Mean times show a significant reduction in each of the exercises. The efficiency percentages at the end of the program were higher than at the beginning (p < 0.001). The cost of the program represented a saving of 67.89%. CONCLUSION: The new MIS training program improved the quality of learning in a safe environment, establishing common criteria among the different specialties and an improved use of resources.

INTRODUCCION: Las diferentes especialidades quirúrgicas de nuestro centro han usado los recursos de simulación y cirugía experimental para sus tareas de formación en cirugía mínimamente invasiva (CMI) de manera individualizada. Con este modelo de aprendizaje se detectó una gran dispersión de esfuerzos y gasto, por lo que se decidió crear un programa unificado basado en: aprendizaje compartido, sinergia entre especialidades, moderación del coste económico y uso racional de las instalaciones. OBJETIVO: Describir y evaluar nuestro programa de entrenamiento diseñado por consenso de cara a la consolidación de una estrategia de aprendizaje compartido que permita a nuestros residentes adquirir y perfeccionar habilidades quirúrgicas en CMI. MATERIAL Y METODOS: El programa consta de diferentes fases con complejidad creciente desarrolladas durante todo el periodo de formación especializada de forma continuada en laboratorio virtual y quirófano experimental. Los criterios de evaluación se basaron en criterios cuantificables: porcentaje de eficiencia y tiempo de realización de los ejercicios de McGill Inanimate System for Training and Evaluation of Laparoscopic Skills (MISTELS) al inicio y final del programa. Se realizó también el estudio económico. RESULTADOS: Han completado el programa 20 residentes. Los tiempos medios demuestran una reducción significativa en cada uno de los ejercicios. Los porcentajes de eficiencia al final fueron mayores que al inicio del programa (p < 0,001). El coste del programa supuso un ahorro del 67,89%. CONCLUSION: El nuevo programa de entrenamiento en CMI mejoró la calidad de aprendizaje en un entorno seguro, estableciendo criterios comunes entre las diferentes especialidades y un mayor aprovechamiento de los recursos.

Programa de formación quirúrgica común: uniformidad en la calidad del aprendizaje / Common surgical training program: standardization of learning quality

Introducción: Las diferentes especialidades quirúrgicas de nuestrocentro han usado los recursos de simulación y cirugía experimentalpara sus tareas de formación en cirugía mínimamente invasiva (CMI)de manera individualizada. Con este modelo de aprendizaje se detectóuna gran dispersión de esfuerzos y gasto, por lo que se decidió crear unprograma unificado basado en: aprendizaje compartido, sinergia entreespecialidades, moderación del coste económico y uso racional de lasinstalaciones. Objetivo: Describir y evaluar nuestro programa de entrenamientodiseñado por consenso de cara a la consolidación de una estrategia deaprendizaje compartido que permita a nuestros residentes adquirir yperfeccionar habilidades quirúrgicas en CMI. Material y métodos: El programa consta de diferentes fases concomplejidad creciente desarrolladas durante todo el periodo de forma-ción especializada de forma continuada en laboratorio virtual y quiró-fano experimental. Los criterios de evaluación se basaron en criterioscuantificables: porcentaje de eficiencia y tiempo de realización de losejercicios de McGill Inanimate System for Training and Evaluation ofLaparoscopic Skills (MISTELS) al inicio y final del programa. Se realizótambién el estudio económico. Resultados: Han completado el programa 20 residentes. Los tiem-pos medios demuestran una reducción significativa en cada uno de losejercicios. Los porcentajes de eficiencia al final fueron mayores queal inicio del programa (p < 0,001). El coste del programa supuso unahorro del 67,89%. Conclusión: El nuevo programa de entrenamiento en CMI mejoróla calidad de aprendizaje en un entorno seguro, estableciendo criterioscomunes entre las diferentes especialidades y un mayor aprovechamientode los recursos.(AU)

Introduction: The various surgical specialties in our center haveused the simulation and experimental surgery resources availablefor their training tasks in minimally invasive surgery (MIS) in anindividualized manner. With this learning model, a great dispersionof effort and expense was observed, so it was decided to create aunified program based on the following: shared learning, synergyamong specialties, moderation of the economic cost, and rationaluse of the facilities. Objective: To describe and assess our consensually designed train-ing program in order to consolidate a shared learning strategy that willenable our residents to acquire and perfect surgical skills in MIS. Materials and methods: The program consists of various increas-ingly complex phases implemented on a continuous basis throughout theperiod of specialized training in the virtual laboratory and experimentaloperating room. The assessment methods were based on quantifiablecriteria: percentage of efficiency and completion time of the “McGillInanimate System for Training and Evaluation of Laparoscopic Skills”(MISTELS) exercises at the beginning and end of the program. Aneconomic study was also conducted. Results: 20 residents have completed the program. Mean timesshow a significant reduction in each of the exercises. The efficiencypercentages at the end of the program were higher than at the begin-ning (p < 0.001). The cost of the program represented a saving of67.89%.(AU)

Filifactor alocis: Recent Insights and Advances.

Filifactor alocis, a fastidious Gram-positive obligate anaerobic bacterium, is a newly appreciated member of the periodontal community that is now proposed to be a diagnostic indicator of periodontal disease. Its pathogenic characteristics are highlighted by its ability to survive in the oxidative stress-rich environment of the periodontal pocket and to significantly alter the microbial community dynamics by forming biofilms and interacting with several oral bacteria. Here, we describe the current understanding of F. alocis virulence attributes, such as its comparative resistance to oxidative stress, production of unique proteases and collagenases that can cause structural damage to host cells, and dysregulation of the immune system, which enable this bacterium to colonize, survive, and outcompete other traditional pathogens in the inflammatory environment of the periodontal pocket. Furthermore, we explore the recent advancements and future directions for F. alocis research, including the potential mechanisms for oxidative stress resistance and our evolving understanding of the interactions and mechanisms of bacterial survival inside neutrophils. We also discuss the current genetic tools and challenges involved in manipulating the F. alocis genome for the functional characterization of the putative virulence genes. Collectively, this information will expedite F. alocis research and should lead to the identification of prime targets for the development of novel therapeutics to aid in the control and prevention of periodontal disease.

Chronic hM3Dq signaling in microglia ameliorates neuroinflammation in male mice.

Microglia express muscarinic G protein-coupled receptors (GPCRs) that sense cholinergic activity and are activated by acetylcholine to potentially regulate microglial functions. Knowledge about how distinct types of muscarinic GPCR signaling regulate microglia function in vivo is still poor, partly due to the fact that some of these receptors are also present in astrocytes and neurons. We generated mice expressing the hM3Dq Designer Receptor Exclusively Activated by Designer Drugs (DREADD) selectively in microglia to investigate the role of muscarinic M3Gq-linked signaling. We show that activation of hM3Dq using clozapine N-oxide (CNO) elevated intracellular calcium levels and increased phagocytosis of FluoSpheres by microglia in vitro. Interestingly, whereas acute treatment with CNO increased synthesis of cytokine mRNA, chronic treatment attenuated LPS-induced cytokine mRNA changes in the brain. No effect of CNO on cytokine expression was observed in DREADD-negative mice. Interestingly, CNO activation of M3Dq in microglia was able to attenuate LPS-mediated decrease in social interactions. These results suggest that chronic activation of M3 muscarinic receptors (the hM3Dq progenitor) in microglia, and potentially other Gq-coupled GPCRs, can trigger an inflammatory-like response that preconditions microglia to decrease their response to further immunological challenges. Our results indicate that hM3Dq can be a useful tool to modulate neuroinflammation and study microglial immunological memory in vivo, which may be applicable for manipulations of neuroinflammation in neurodegenerative and psychiatric diseases.

The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1-S2 linkage.

The voltage-gated potassium channel Kv1.5 belongs to the Shaker superfamily. Kv1.5 is composed of four subunits, each comprising 613 amino acids, which make up the N terminus, six transmembrane segments (S1-S6), and the C terminus. We recently demonstrated that, in HEK cells, extracellularly applied proteinase K (PK) cleaves Kv1.5 channels at a single site in the S1-S2 linker. This cleavage separates Kv1.5 into an N-fragment (N terminus to S1) and a C-fragment (S2 to C terminus). Interestingly, the cleavage does not impair channel function. Here, we investigated the role of the N terminus and S1 in Kv1.5 expression and function by creating plasmids encoding various fragments, including those that mimic PK-cleaved products. Our results disclosed that although expression of the pore-containing fragment (Frag(304-613)) alone could not produce current, coexpression with Frag(1-303) generated a functional channel. Immunofluorescence and biotinylation analyses uncovered that Frag(1-303) was required for Frag(304-613) to traffic to the plasma membrane. Biochemical analysis revealed that the two fragments interacted throughout channel trafficking and maturation. In Frag(1-303)+(304-613)-coassembled channels, which lack a covalent linkage between S1 and S2, amino acid residues 1-209 were important for association with Frag(304-613), and residues 210-303 were necessary for mediating trafficking of coassembled channels to the plasma membrane. We conclude that the N terminus and S1 of Kv1.5 can attract and coassemble with the rest of the channel (i.e. Frag(304-613)) to form a functional channel independently of the S1-S2 linkage.

Size-Dependent Consequences of Exogenous Cortisol Manipulation on Overwinter Survival and Condition of Largemouth Bass.

Little is known about the size-dependent consequences of stressors on wild animals, which is particularly relevant during winter where size-specific trends in survival are common. Here, exogenous cortisol manipulation was used to investigate the effect of a physiological challenge on overwinter mortality and spring condition of largemouth bass (Micropterus salmoides) across a range of body sizes. Fish were wild-caught in the fall, assigned into either control or cortisol manipulated treatments, and held in replicated experimental ponds. For bass that survived the winter, length, mass, and health metrics (e.g., gonadosomatic index [GSI], hepatosomatic index [HSI], and water content) were determined in the spring. Winter survival was marginally lower for cortisol treated bass; however, there was no influence of initial length, mass, or condition on overwinter survival. When bass were grouped by size, survival was significantly higher for bass 300-350 mm in length compared to those <200 mm. The treatment did not strongly influence spring health metrics, suggesting that largemouth bass that survived the winter were able to recover from the effects of the cortisol elevation. Initial size and sex were linked to some spring health metrics, with large females having the highest GSI and HSI scores. Overall, results from this study do not support the notion that there are size-dependent responses to cortisol manipulation in a teleost fish. Rather, this type of physiological challenge may modulate the natural rates of winter mortality that are primarily driven by starvation and predation, independent of body size, in subadult and adult largemouth bass.

Viral shedding and immune responses to respiratory syncytial virus infection in older adults.

BACKGROUND: Comprehensive analyses of host, viral, and immune factors associated with severe respiratory syncytial virus (RSV) infection in adults have not been performed. METHODS: Adults with RSV infection identified in both outpatient and inpatient settings were evaluated. Upper and lower respiratory tract virus load, duration of virus shedding, select mucosal chemokine and cytokine levels, humoral and mucosal immunoglobulin responses, and systemic T-cell responses were measured. RESULTS: A total of 111 RSV-infected adults (61 outpatients and 50 hospitalized patients) were evaluated. Hospitalized subjects shed virus in nasal secretions at higher titers and for longer durations than less ill outpatients, had greater mucosal interleukin 6 (IL-6) levels throughout infection, and had higher macrophage inflammatory protein 1α (MIP-1α) levels early in infection. Persons >64 years old and those with more severe disease had a higher frequency of activated T cells in the blood than younger, less ill subjects at infection. Multivariate analysis found that the presence of underlying medical conditions, female sex, increased mucosal IL-6 level, and longer duration of virus shedding were associated with severe disease. Older age and increased nasal MIP-1α levels were of borderline statistical significance. CONCLUSIONS: Multiple factors, but not older age, are independently associated with severe RSV infection in adults. The presence of underlying medical conditions had the greatest influence on disease severity.

Características genéticas, poblacionales y fenotípicas de pacientes con enfermedad de Hirschsprung / Genetic, population and phenotypic characterístics of patients with hirschsprung disease

La enfermedad de Hirschsprung (HSCR) está causada por la ausencia de células ganglionares en el intestino, debido a defectos en la migración de las células del sistema nervioso entérico durante el desarrollo embrionario. La incidencia es aproximadamente de uno en cada 5000 nacimientos, más frecuente en hombres que en mujeres. Hay dos fenotipos según la longitud del segmento aganglionar: corto (S-HSCR, 80% de los enfermos) y largo (L-HSCR, el 20%). Se han detectado variaciones en la secuencia codificante del proto-oncogén rEt en enfermos con HCSR, lo que sugiere predisposición genética a padecer la enfermedad. Nuestro trabajo ha consistido en encontrar y analizar polimorfismos (SNPs) asociados a la enfermedad, así como su distribución poblacional (sexo y tipo de segmento). En el estudio genético se han analizado dos polimorfismos presentes en el promotor del gen, así como un polimorfismo en el exón 13 fuertemente asociado con la enfermedad. Como poblaciones en este estudio se establecieron una de enfermos con HSCR esporádico y un grupo de individuos sanos.Los resultados obtenidos corroboran que la enfermedad es más frecuente en hombres que en mujeres. El genotipado de rEt indica que los alelos A y G del promotor (c.-200A>G y c.-196C>A) y G del exón 13 (c.2307T>G) están asociados a la población enferma. Los datos apuntan a que no existe relación entre el fenotipo de la enfermedad y la distribución de los polimorfismos analizados. Concluimos que la presencia de ciertos polimorfismos en la secuencia de rEt indica predisposición genética (combinada con otros factores genéticos o ambientales) a padecer la enfermedad (AU)

Hirschsprung disease (HSCR) is caused by the absence of ganglion cells in the intestine due to defects in the migration of enteric nervous system cells during embryologic development. The incidence is one in every 5000 births, more common in men than women. There are two main phenotypes according to the aganglionic segment length: Short (S-HSCR, (80% of patients) and Long (L-HSCR, 20%). Variations have been detected in the coding sequence of the rEt proto-oncogene in patients with HSCR, suggesting a genetic predisposition to the disease. Our aim is to find and analyze polymorphisms (SNPs) associated with the disease. We are interested also in stablish an association between sex and type of aganglionic segment. We analyzed the rEt promoter as well a polymorphism in exon 13 strongly associated to the disease. The populations for the study were a group of 56 patients with sporadic HSCR and 178 healthy controls.The results obtained show that the disease is more common in men than in women (3:1). The rEt genotype shows that alleles A and G of the promoter (c.-200A> G and c.-196C> A) and G of exon 13 (c.2307T> G) are associated with the affected population. Our data suggest neither association between the disease phenotype and the distribution of the polymorphisms analyzed nor with the sex of the patients. The presence of certain polymorphisms in the rEt sequence indicates a genetic predisposition (combined with other genetic or environmental factors) to the disease (AU)

[Genetic, population and phenotypic characteristics of patients with Hirschsprung disease]. / Características genéticas, poblacionales y fenotípicas de pacientes con enfermedad de Hirschsprung.

Hirschsprung disease (HSCR) is caused by the absence of ganglion cells in the intestine due to defects in the migration of enteric nervous system cells during embryologic development. The incidence is one in every 5000 births, more common in men than women. There are two main phenotypes according to the aganglionic segment length: Short (S-HSCR, (80% of patients) and Long (L-HSCR, 20%). Variations have been detected in the coding sequence of the RET proto-oncogene in patients with HSCR, suggesting a genetic predisposition to the disease. Our aim is to find and analyze polymorphisms (SNPs) associated with the disease. We are interested also in stablish an association between sex and type of aganglionic segment. We analyzed the RET promoter as well a polymorphism in exon 13 strongly associated to the disease. The populations for the study were a group of 56 patients with sporadic HSCR and 178 healthy controls. The results obtained show that the disease is more common in men than in women (3:1). The RET genotype shows that alleles A and G of the promoter (c.-200A > G and c.-196C > A) and G of exon 13 (c.2307T > G) are associated with the affected population. Our data suggest neither association between the disease phenotype and the distribution of the polymorphisms analyzed nor with the sex of the patients. The presence of certain polymorphisms in the RET sequence indicates a genetic predisposition (combined with other genetic or environmental factors) to the disease.

Estudio de polimorfi smos asociadosa la enfermedad de Hirschsprung / Study of polymorphisms associated to hirschsprung’s disease

La enfermedad de Hirschsprung (HSCR) es un desorden congénito caracterizado por la ausencia de células ganglionares a lo largo del tracto gastrointestinal. Está causada por defectos en la migración de las células del sistema nervioso entérico durante el desarrollo embrionario. La mejora de tratamientos quirúrgicos ha disminuido la mortalidad de los pacientes, lo que facilita el estudio genético de enfermos y sus familiares. Aunque se desconoce la causa genética de la enfermedad, se sospecha que el oncogén RET es el principal involucrado. Se han encontrado alteraciones en este gen en enfermos con HSCR, por lo que algunos autores sugieren que ciertos polimorfismos (SNPs) en el gen podrían causar cierta predisposición genética a padecer esta enfermedad. Nuestro trabajo ha consistido en el análisis del gen RET en pacientes con diagnóstico de HSCR mediante secuenciación directa y genotipado con sondas Taqman®.Los resultados obtenidos indican que ciertos alelos de los polimorfismos p.Leu769Leu (c.2307T>G, Exón 13) p.Gly691Ser (c.2071G>A, Exón 11) y p.Ser904Ser (c.2712C>G, Exón 15) están asociados a losenfermos HSCR, ya que existen diferencias significativas respecto ala población sana (AU)

Hirschsprung’s disease (HSCR) is a developmental disorder characterized by the absence of the enteric ganglia along the intestine. Is regarded as the consequence of the premature arrest of the migration of neural crest cells in the hindgut during the embryonic development to form the enteric nervous system (ENS). Is considered, therefore, a neurocristopathy. The development of surgical approaches has importantly decreased mortality and morbility of Hirschsprung’s patients, which has allowed the emergence of genetic studies of patients and their families. Although the genetic cause of the disease is still unknown, the RET oncogene is the main involved. Alterations in this gene have been found in HSCR patients, so many authors suggest that certain polymorphisms(SNPs) in this gene could be responsible of genetic predisposition to have the disease. Our work has consisted in the genetic analysis of the RET gene in HSCR patients using direct sequencing and genotyping with Taq-Man® probes. Our results show that some alleles of the polymorphismsp.Leu769Leu (c.2307T>G, Exon 13) p.Gly691Ser (c.2071G>A, Exon11) and p.Ser904Ser (c.2712C>G, Exon 15) are associated to the disease since there are significant differences from the healthy population (AU)

[Study of polymorphisms associated to Hirschsprung's disease]. / Estudio de polimorfismos asociados a la enfermedad de Hirschsprung.

Hirschsprung's disease (HSCR) is a developmental disorder characterised by the absence of the enteric ganglia along the intestine. Is regarded as the consequence of the premature arrest of the migration of neural crest cells in the hindgut during the embryonic development to form the enteric nervous system (ENS). Is considered, therefore, a neurocristopathy. The development of surgical approaches has importantly decreased mortality and morbility of Hirschsprung's patients, which has allowed the emergence of genetic studies of patients and their families. Although the genetic cause of the disease is still unknown, the RET oncogene is the main involved. Alterations in this gene have been found in HSCR patients, so many authors suggest that certain polymorphisms (SNPs) in this gene could be responsible of genetic predisposition to have the disease. Our work has consisted in the genetic analysis of the RET gene in HSCR patients using direct sequencing and genotyping with TaqMan probes. Our results show that some alleles of the polymorphisms p.Leu769Leu (c.2307T>G, Exon 13) p.Gly691Ser (c.2071G>A, Exon 11) and p.Ser904Ser (c.2712C>G, Exon 15) are associated to the disease since there are significant differences from the healthy population.

The inability of vaccinia virus A33R protein to form intermolecular disulfide-bonded homodimers does not affect the production of infectious extracellular virus.

The orthopoxvirus protein A33 forms a disulfide-bonded high molecular weight species that could be either a homodimer or a heteromultimer. The protein is a major target for neutralizing antibodies and the majority of antibodies raised against A33 only recognize the disulfide-bonded form. Here, we report that A33 is present as a disulfide-bonded homodimer during infection. Additionally, we examined the function of intermolecular disulfide bonding in A33 homodimerization during infection. We show that the cysteine at amino acid 62 is required for intermolecular disulfide bonding, but not dimerization as this mutant was still able to form homodimers. To investigate the role of disulfide-bonded homodimers during viral morphogenesis, recombinant viruses that express an A33R with cysteine 62 mutated to serine were generated. The recombinant viruses had growth characteristics similar to their parental viruses, indicating that intermolecular disulfide-bonded homodimerization of A33 is not required for its function.