Construction of bicistronic lentiviral vectors for tracking the expression of CDNF in transduced cells.

CDNF is a recently described evolutionary conserved neurotrophic factor reported to be of relevance for the treatment of Parkinson's disease. Treatment with recombinant CDNF showed neurorestorative and neuroprotective effects on dopaminergic neurons in Parkinsonian animal models. Similar results are obtained using adeno-associated viral (AAV) vectors for CDNF expression in these animal models; however, the extent of the transduced brain tissue is difficult to assess due to the lack of reporter genes in the vectors used. Here, we describe two bicistronic lentiviral plasmids based on the Δ1D/2A and IRES elements for the expression of EGFP and rat CDNF, in order to track the transduced cells expressing CDNF with EGFP fluorescence. Transfected heterologous cells or transduced neurons with these vectors are easily identified by EGFP fluorescence and CDNF expression results in its recruitment to the endoplasmic reticulum (ER) by both bicistronic vectors. CDNF immunostaining is also observed in the Golgi apparatus when expressed in heterologous cells or hippocampal neuronal cultures; however, colocalization with a dense core secretory vesicle marker was scarce. Additionally, we showed that the expression of CDNF inhibited dendrite formation in hypothalamic neurons, suggesting that CDNF expressed by these bicistronic lentiviral vectors is functional and could have a role in neuronal morphology. The bicistronic lentiviral plasmids developed here could be of use to study the effect of rat CDNF at the cellular level or to better delineate the perikarya of neurons transduced with lentiviral vectors in animal models of Parkinson's disease.

Serine-Arginine Protein Kinase SRPK2 Modulates the Assembly of the Active Zone Scaffolding Protein CAST1/ERC2.

Neurons release neurotransmitters at a specialized region of the presynaptic membrane, the active zone (AZ), where a complex meshwork of proteins organizes the release apparatus. The formation of this proteinaceous cytomatrix at the AZ (CAZ) depends on precise homo- and hetero-oligomerizations of distinct CAZ proteins. The CAZ protein CAST1/ERC2 contains four coiled-coil (CC) domains that interact with other CAZ proteins, but also promote self-assembly, which is an essential step for its integration during AZ formation. The self-assembly and synaptic recruitment of the Drosophila protein Bruchpilot (BRP), a partial homolog of CAST1/ERC2, is modulated by the serine-arginine protein kinase (SRPK79D). Here, we demonstrate that overexpression of the vertebrate SRPK2 regulates the self-assembly of CAST1/ERC2 in HEK293T, SH-SY5Y and HT-22 cells and the CC1 and CC4 domains are involved in this process. Moreover, the isoform SRPK2 forms a complex with CAST1/ERC2 when co-expressed in HEK293T and SH-SY5Y cells. More importantly, SRPK2 is present in brain synaptic fractions and synapses, suggesting that this protein kinase might control the level of self-aggregation of CAST1/ERC2 in synapses, and thereby modulate presynaptic assembly.

The Exocyst Component Exo70 Modulates Dendrite Arbor Formation, Synapse Density, and Spine Maturation in Primary Hippocampal Neurons.

Neurons are highly polarized cells displaying an elaborate architectural morphology. The design of their dendritic arborization and the distribution of their synapses contribute importantly to information processing in the brain. The growth and complexity of dendritic arbors are driven by the formation of synapses along their lengths. Synaptogenesis is augmented by the secretion of factors, like BDNF, Reelin, BMPs, or Wnts. Exo70 is a component of the exocyst complex, a protein complex that guides membrane addition and polarized exocytosis. While it has been linked to cytokinesis and the establishment of cell polarity, its role in synaptogenesis is poorly understood. In this report, we show that Exo70 plays a role in the arborization of dendrites and the development of synaptic connections between cultured hippocampal neurons. Specifically, while the overexpression of Exo70 increases dendritic arborization, synapse number, and spine density, the inhibition of Exo70 expression reduces secondary and tertiary dendrite formation and lowers synapse density. Moreover, increasing Exo70 expression augmented synaptic vesicle recycling as evaluated by FM4-64 dye uptake and the inverse was observed with downregulation of endogenous Exo70. Monitoring the formation of dendritic spines by super-resolution microscopy, we also observed that mRFP-Exo70 accumulates at the tip of EGFP-ß-actin-positive filopodia. Together, these results suggest that Exo70 is essentially involved in the formation of synapses and neuronal dendritic morphology.

Patrones de susceptibilidad antimicrobiana "in vitro" de bacterias Gram negatives asilades de infección de vías urinarias en pacientes ambulatorios de una clínica del sur de la Ciudad de México / Patterns of in vitro antimicrobial susceptibility of Gram-negative bacteria isolated from urinary tract infections in outpatients of a clinic in southern Ciudad de México

OBJETIVO: Determinar los patrones de susceptibilidad antimicrobiana de bacterias Gram negativas aisladas de cultivos de orina de pacientes ambulatorios, y asociarlos con variables como edad, sexo, infección urinaria previa y presencia de diabetes mellitus tipo 2. MATERIAL Y MÉTODOS: Estudio descriptivo observacional y trasversal, con cepas aisladas de 278 pacientes con infección urinaria baja, que acudieron a consulta externa a la CMF Dr. Ignacio Chávez del Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado, del sur de la Ciudad de México, entre los meses de marzo de 2018 a febrero de 2019. Se utilizó el sistema Phoenix 100 de Becton Dickinson, tanto para identificación de cepas, como para determinación de susceptibilidad antimicrobiana. Se probaron 16 antimicrobianos. Se utilizó estadística descriptiva para determinar proporción de resistencias. Programa estadístico SPSS versión 22. RESULTADOS: Se incluyeron 278 cepas: 231 Escherichia coli, 24 Klebsiella spp; 8 Enterobacter spp, 7 Proteus spp, 7 Citrobacter spp, 1 Serratia spp. La mayor resistencia fue para: ampicilina con 74,1 %, y la mayor sensibilidad para amikacina con 100 %. Del total de cepas, 140 (50,4 %) fueron Multi-Drogo-Resistentes, no hubo cepas Pan-Drogo-Resistentes. Al asociar las variables de estudio con la resistencia a cada antimicrobiano, sólo se obtuvo significancia estadística entre las cefalosporinas cefaxolina y cefoxitin, y el imipenem con el sexo de los pacientes, con mayor porcentaje en los hombres. CONCLUSIONES: Se obtuvo una alta resistencia en prácticamente todos los grupos de antimicrobianos, lo que hace necesario estar al tanto de los patrones de susceptibilidad en cada zona o país

OBJECTIVES: To determine patterns of antimicrobial susceptibility of Gram-negative bacteria isolated from urine cultures of outpatients, and to associate them with variables such as age, sex, previous urinary infection, and presence of type-2 diabetes mellitus. MATERIAL AND METHODS: Descriptive, observational, cross-sectional study, with strains isolated from 278 patients with low urinary infection, who had outpatient consultation in CMF Dr. Ignacio Chávez of the Instituto de Seguridad y Servicios Sociales de los Trabajadoresdel Estado, in southern Ciudad de México, between March 2018 and February 2019. The Becton-Dickinson Phoenix 100 system was used both for identifying strains, and for determining antimicrobial susceptibility. 16 antimicrobial agents were tested. Descriptive statistics was used for determining resistance proportion. Statistical programme SPSS version 22. RESULTS: 278 strains were included: 231 Escherichia coli, 24 Klebsiella spp., 8 Enterobacter spp., 7 Proteus spp., 7 Citrobacter spp., 1 Serratia spp. The greatest resistance was for: ampicillin with 74.1% and the greatest sensitivity was for amikacin with 100%. Of all strains, 140 (50.4%) were multidrug resistant, and none was pandrug resistant. When the study variables were associated with resistance to each antimicrobial, there was statistical significance only between cephalosporins cefazolin and cefoxitin, as well as imipenem with patient sex, with higher percentage in men. CONCLUSIONS: High resistance was obtained in virtually all the groups of antimicrobials. This makes it necessary to be aware of susceptibility patterns in each area or country