Mitochondria and Endoplasmic Reticulum Stress in Retinal Organoids from Patients with Vision Loss.

Activating transcription factor 6 (ATF6), a key regulator of the unfolded protein response, plays a key role in endoplasmic reticulum function and protein homeostasis. Variants of ATF6 that abrogate transcriptional activity cause morphologic and molecular defects in cones, clinically manifesting as the human vision loss disease achromatopsia (ACHM). ATF6 is expressed in all retinal cells. However, the effect of disease-associated ATF6 variants on other retinal cell types remains unclear. Herein, this was investigated by analyzing bulk RNA-sequencing transcriptomes from retinal organoids generated from patients with ACHM, carrying homozygous loss-of-function ATF6 variants. Marked dysregulation in mitochondrial respiratory complex gene expression and disrupted mitochondrial morphology in ACHM retinal organoids were identified. This indicated that loss of ATF6 leads to previously unappreciated mitochondrial defects in the retina. Next, gene expression from control and ACHM retinal organoids were compared with transcriptome profiles of seven major retinal cell types generated from recent single-cell transcriptomic maps of nondiseased human retina. This indicated pronounced down-regulation of cone genes and up-regulation in Müller glia genes, with no significant effects on other retinal cells. Overall, the current analysis of ACHM patient retinal organoids identified new cellular and molecular phenotypes in addition to cone dysfunction: activation of Müller cells, increased endoplasmic reticulum stress, disrupted mitochondrial structure, and elevated respiratory chain activity gene expression.

Establishment of the Daucus carota SMC-1 Cell Suspension Line for Poliovirus Vaccine Development.

The development of virus-free, oral vaccines against poliovirus capable of inducing mucosal protective immunity is needed to safely combat this pathogen. In the present study, a carrot cell line expressing the poliovirus VP2 antigen was established at the level of callus and cell suspensions, exploring the effects of culture media (MS and B5), supplementation with urea, phytoregulators (2,4-D : KIN), and light conditions (continuous light, photoperiod, and total darkness). The best callus growth was obtained on B5 medium supplemented with 2 mg/L of 2,4-D + 2 mg/L kinetin and 0.0136 g/L of urea and in continuous light conditions. Suspension cultures of the SMC-1 line in 250 mL Erlenmeyer flasks had a maximum growth of 16.07 ± 0.03 g/L DW on day 12 with a growth rate of µ=0.3/d and a doubling time of 2.3 days. In a 2 L airlift bioreactor, the biomass yield achieved was 25.6 ± 0.05 g/L DW at day 10 with a growth rate of µ= 0.58/d and doubling time of 1.38 d. Cell growth was 1.5 times higher in bioreactors than in shake flasks, highlighting that both systems resulted in the accumulation of VP2 throughout the time in culture. The maximum VP2 yield in flasks was 387.8 µg/g DW at day 21, while in the reactor it was 550.2 µg/g DW at day 18. In conclusion, bioreactor-based production of the VP2 protein by the SMC-1 suspension cell line offers a higher productivity when compared to flask cultures, offering a key perspective to produce low-cost vaccines against poliomyelitis.

Statistical Experimental Designs for cLTB-Syn Vaccine Production Using Daucus carota Cell Suspension Cultures.

The carrot-made LTB-Syn antigen (cLTB-Syn) is a vaccine candidate against synucleinopathies based on carrot cells expressing the target antigen LTB and syn epitopes. Therefore, the development of an efficient production process is required with media culture optimization to increase the production yields as the main goal. In this study, the effect of two nitrogen sources (urea and glutamate) on callus cultures producing cLTB-Syn was studied, observing that the addition of 17 mM urea to MS medium favored the biomass yield. To optimize the MS media composition, the influence of seven medium components on biomass and cLTB-Syn production was first evaluated by a Plackett-Burman design (PBD). Then, three factors were further analyzed using a central composite design (CCD) and response surface methodology (RSM). The results showed a 1.2-fold improvement in biomass, and a 4.5-fold improvement in cLTB-Syn production was achieved at the shake-flask scale. At the bioreactor scale, there was a 1.5-fold increase in biomass and a 2.8-fold increase in cLTB-Syn yield compared with the standard MS medium. Moreover, the cLTB-Syn vaccine induced humoral responses in BALB/c mice subjected to either oral or subcutaneous immunization. Therefore, cLTB-Syn is a promising vaccine candidate that will aid in developing immunotherapeutic strategies to combat PD and other neurodegenerative diseases without the need for cold storage, making it a financially viable option for massive immunization.

Use of renin-angiotensin-aldosterone system inhibitors and risk of COVID-19 requiring admission to hospital: a case-population study.

BACKGROUND: Concerns have been raised about the possibility that inhibitors of the renin-angiotensin-aldosterone system (RAAS) could predispose individuals to severe COVID-19; however, epidemiological evidence is lacking. We report the results of a case-population study done in Madrid, Spain, since the outbreak of COVID-19. METHODS: In this case-population study, we consecutively selected patients aged 18 years or older with a PCR-confirmed diagnosis of COVID-19 requiring admission to hospital from seven hospitals in Madrid, who had been admitted between March 1 and March 24, 2020. As a reference group, we randomly sampled ten patients per case, individually matched for age, sex, region (ie, Madrid), and date of admission to hospital (month and day; index date), from Base de datos para la Investigación Farmacoepidemiológica en Atención Primaria (BIFAP), a Spanish primary health-care database, in its last available year (2018). We extracted information on comorbidities and prescriptions up to the month before index date (ie, current use) from electronic clinical records of both cases and controls. The outcome of interest was admission to hospital of patients with COVID-19. To minimise confounding by indication, the main analysis focused on assessing the association between COVID-19 requiring admission to hospital and use of RAAS inhibitors compared with use of other antihypertensive drugs. We calculated odds ratios (ORs) and 95% CIs, adjusted for age, sex, and cardiovascular comorbidities and risk factors, using conditional logistic regression. The protocol of the study was registered in the EU electronic Register of Post-Authorisation Studies, EUPAS34437. FINDINGS: We collected data for 1139 cases and 11 390 population controls. Among cases, 444 (39·0%) were female and the mean age was 69·1 years (SD 15·4), and despite being matched on sex and age, a significantly higher proportion of cases had pre-existing cardiovascular disease (OR 1·98, 95% CI 1·62-2·41) and risk factors (1·46, 1·23-1·73) than did controls. Compared with users of other antihypertensive drugs, users of RAAS inhibitors had an adjusted OR for COVID-19 requiring admission to hospital of 0·94 (95% CI 0·77-1·15). No increased risk was observed with either angiotensin-converting enzyme inhibitors (adjusted OR 0·80, 0·64-1·00) or angiotensin-receptor blockers (1·10, 0·88-1·37). Sex, age, and background cardiovascular risk did not modify the adjusted OR between use of RAAS inhibitors and COVID-19 requiring admission to hospital, whereas a decreased risk of COVID-19 requiring admission to hospital was found among patients with diabetes who were users of RAAS inhibitors (adjusted OR 0·53, 95% CI 0·34-0·80). The adjusted ORs were similar across severity degrees of COVID-19. INTERPRETATION: RAAS inhibitors do not increase the risk of COVID-19 requiring admission to hospital, including fatal cases and those admitted to intensive care units, and should not be discontinued to prevent a severe case of COVID-19. FUNDING: Instituto de Salud Carlos III.

Determination of histone epigenetic marks in Arabidopsis and tomato genes in the early response to Botrytis cinerea.

KEY MESSAGE: Determination of histone epigenetic marks in Arabidopsis and tomato genes in the early response to Botrytis cinerea may contribute to find biomarkers of the early detection of this devastating pathogen. Recent studies have linked epigenetic modifications with plant responses to biotic stresses. Information about specific histone marks upon necrotrophic pathogens is scarce. Here we wondered whether the altered responsiveness of specific genes in plants infected with Botrytis cinerea was associated with changes in chromatin structure. We performed a chromatin immunoprecipitation analysis that obtained differential epigenetic signature of activating marks H3K4me3, H3K9ac, and the repressor one H3K27me3 on both the promoter and the body of the highly induced PR1 in Arabidopsis plants infected with B. cinerea at 24 and 33 h after inoculation. We also determined the histone marks' profile in two differentially expressed genes in response to B. cinerea, as well as to oxidative stress, given its relevance in this infection. These are both the induced CYP71A13, which encodes a cytochrome P450 involved in camalexin synthesis, and is essential against this necrotroph and the repressed EXL7 (Exordium-like 1). We also adapted our protocol in tomato plants infected with B. cinerea. At 24 hpi, H3K4me3 level increased on the promoter and at different locations of the body of the genes induced upon B. cinerea, including DES (divinyl ethyl synthase), LoxD (lipoxygenase D), DOX1 (α-dioxygenase 1), PR2 (pathogenesis-related protein2), WRKY53 and WRKY33. The histone modifications determined herein will allow future studies on epigenetic marks and their transgenerational inheritance in plants infected with B. cinerea. In addition, the analyzed genes are potential biomarkers of B. cinerea infection that could contribute to its early detection in tomato and related crops.

Raltegravir pharmacokinetics in HIV/HCV-coinfected patients with advanced liver cirrhosis (Child-Pugh C).

OBJECTIVES: To describe raltegravir pharmacokinetics at steady-state in HIV/hepatitis C virus (HCV)-coinfected patients under antiretroviral (ARV) treatment with (n = 5) and without (n = 5) advanced liver cirrhosis (Child-Pugh C). METHODS: This was a non-randomized, Phase I, parallel-assignment, open-label pharmacokinetic study in HIV/HCV-coinfected patients with Child-Pugh grade C hepatic cirrhosis. We recruited clinically stable HIV/HCV-coinfected adult patients with controlled HIV viraemia (<50 copies/mL) for at least 6 months. Raltegravir (400 mg twice daily) was added under fasting conditions for 5 days to the successful ritonavir-boosted protease inhibitor-based ARV regimen. The trial was registered in the ClinicalTrials.gov database (NCT01289951) (LIVERAL). RESULTS: Raltegravir AUC0-12 and C12 were increased 1.72-fold (90% CI, 1.02 to 2.92) and 6.58-fold (90% CI, 2.92 to14.85), respectively, in patients with advanced liver cirrhosis. No safety issues were identified and raltegravir was well tolerated by all patients. CONCLUSIONS: Raltegravir plasma levels are increased in HIV/HCV-coinfected patients with advanced liver cirrhosis (Child-Pugh C). Despite the higher exposure, raltegravir was safe and well tolerated.

The Genetic Variant TNFA (rs361525) Is Associated with Increased Susceptibility to Developing Dengue Symptoms.

Dengue virus (DENV) is the causal agent of dengue fever. The symptoms and signs of dengue vary from febrile illness to hemorrhagic syndrome. IFITM3 and TNFA are genes of the innate immune system. Variants IFITM3 (rs12252 T>C) and TNFA (rs1800629 G > A and rs361525 G>A) might alter gene expression and change the course of the disease. Our first objective was to determine whether these variants were associated with the susceptibility and severity of dengue. The second was to assess the association of these variants with each symptom. We studied 272 cases with suspected dengue infection, of which 102 were confirmed dengue cases (DENV+) and 170 were dengue-like cases without DENV infection (DENV-). Samples of 201 individuals from the general population of Mexico were included as a reference. Genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism technique. Odds ratios and confidence intervals were calculated using Pearson's chi-square test and later adjusted for age and sex with a binary logistic regression model. Haldane correction is applied when necessary. We found a significantly higher frequency of the A allele of TNFA rs361525 in both the DENV+ and DENV- groups compared with the general population. Focusing on DENV+ and DENV-, the frequency of the A allele of TNFA rs361525 was higher in the DENV+ group. A broad spectrum of symptoms was related to the A allele of both TNFA variants. We conclude that TNFA rs361525 increases the susceptibility to symptomatic dengue but can also be associated with susceptibility to other dengue-like symptoms from unknown causes.

Implementing a Comprehensive Preceptor Development Program Through Professional Governance.

Precepting is a demanding but important role for successful onboarding in healthcare institutions. However, preceptors face many challenges, including inconsistent formal training, selection, and evaluation. One health system took a unique approach to this challenge by developing a transition to practice preceptor program through the institution's professional governance culture. Program components include clearly outlined selection criteria, formal initial preparation, competency verification, resources, and recognition for preceptors.

Influence of vintage, geographic location and cultivar on the structure of microbial communities associated with the grapevine rhizosphere in vineyards of San Juan Province, Argentina.

Soil microbiomes, as a primary reservoir for plant colonizing fungi and bacteria, play a major role in determining plant productivity and preventing invasion by pathogenic microorganisms. The use of 16S rRNA and ITS high-throughput amplicon sequencing for analysis of complex microbial communities have increased dramatically in recent years, establishing links between wine specificity and, environmental and viticultural factors, which are framed into the elusive terroir concept. Given the diverse and complex role these factors play on microbial soil structuring of agricultural crops, the main aim of this study is to evaluate how external factors, such as vintage, vineyard location, cultivar and soil characteristics, may affect the diversity of the microbial communities present. Additionally, we aim to compare the influence these factors have on the structuring of bacterial and fungal populations associated with Malbec grapevine rhizosphere with that of the more widespread Cabernet Sauvignon grapevine cultivar. Samples were taken from Malbec and Cabernet Sauvignon cultivars from two different vineyards in the San Juan Province of Argentina. Total DNA extracts from the rhizosphere soil samples were sequenced using Illumina's Miseq technology, targeting the V3-V4 hypervariable 16S rRNA region in prokaryotes and the ITS1 region in yeasts. The major bacterial taxa identified were Proteobacteria, Bacteroidetes and Firmicutes, while the major fungal taxa were Ascomycetes, Basidiomycetes, Mortierellomycetes and a low percentage of Glomeromycetes. Significant differences in microbial community composition were found between vintages and vineyard locations, whose soils showed variances in pH, organic matter, and content of carbon, nitrogen, and absorbable phosphorus.

Effect of long-acting aripiprazole monohydrate on inpatient encounters: A retrospective mirror image study.

INTRODUCTION: Antipsychotics improve symptoms associated with schizophrenia and bipolar disorder. To improve medication adherence and decrease the need for hospitalization, many antipsychotics have been developed into long-acting injectable (LAI) formulations. Though mirror-image studies have demonstrated significantly decreased hospitalization rates with LAI use, there is limited data when suboptimal use parameters are present. METHODS: A retrospective chart review was conducted on patients who were administered aripiprazole monohydrate long-acting injectable (AM-LAI) in an adult mental health unit. Demographics and AM-LAI use parameters were analyzed descriptively. Endpoints compared the days between encounters pre and post AM-LAI administration and number of inpatient encounters between the 180 days pre and post AM-LAI administration. Effects of AM-LAI on inpatient encounters were analyzed using a Wilcoxon signed rank test with an alpha set to <0.05 for significance. RESULTS: Fifty-eight patients met inclusion criteria. Mean (± SD) age was 39.4 (11.4) years with 55.2% of the sample male. Most patients were diagnosed with schizophrenia or unspecified psychotic disorder and admitted involuntarily. The mean number of days from last admission to the date of initial AM-LAI administration was 109.3 (75.2), compared with 131.3 (69.8) days to next encounter (P = .044) post AM-LAI. Total inpatient encounters were also reduced (P = .004), although no differences in encounters for psychiatric reasons were detected. DISCUSSION: Use of AM-LAI was associated with a prolonged time to next inpatient encounter and reduced total inpatient encounters, however its use failed to demonstrate reductions in psychiatric encounters.

Effect of Polymorphisms in CYP2C9 and CYP2C19 on the Disposition, Safety and Metabolism of Progesterone Administrated Orally or Vaginally.

INTRODUCTION: Exogenous progesterone is prescribed for a variety of conditions with endogenous progesterone deficiency, e.g. menstrual alterations, primary or secondary infertility or premenopause. To the best of our knowledge, no pharmacogenetic studies have been published in relation to exogenous progesterone pharmacokinetic safety or progesterone metabolites so far. METHODS: Candidate-gene study where we evaluated whether five single-nucleotide polymorphisms (CYP2C9*2, *3, CYP2C19*2, *3 and *17) were related to the pharmacokinetics, safety and metabolism of progesterone in 24 healthy volunteers who received a 200-mg progesterone formulation either orally or vaginally. RESULTS: The vaginal formulation had an average AUCt value approximately 18 times greater than the oral formulation. CYP2C19 intermediate metabolizers (IM) consistently showed higher adjusted AUCt and adjusted Cmax than extensive metabolizers (EM) (P < 0.05); CYP2C9 EM incongruently exhibited higher adjusted Cmax and longer half-life than IM (p < 0.05). CONCLUSION: This is the first study that reports variability in progesterone disposition according to the CYP2C19 and CYP2C9 phenotype. We suggest that CYP2C19 may condition progesterone disposition and that it may be more relevant than CYP2C9. This study lays the foundations for further in-depth research to evaluate the pharmacogenetics of progesterone. TRIAL REGISTRATION: EudraCT numbers are 2012-005105-43 and 2012-005011-10.

Quantification of event-related desynchronization/synchronization at low frequencies in a semantic memory task.

Although several techniques have been developed for the visualization of EEG event-related desynchronization/synchronization (ERD/ERS) in both time and frequency domains, none of the quantification methods takes advantage of the time and frequency resolution at the same time. Existing techniques for the quantification of the ERD/ERS changes compute the average EEG power increase/decrease relative to certain reference value, over fixed time intervals and/or frequency bands (either fixed or individualized). Inaccuracy in the computation of these frequency bands (where the process is actually measured) in combination with the averaging process over time may lead to errors in the computation of any ERD/ERS quantification parameter. In this paper, we present a novel method for the automatic, individual and exact quantification of the most significant ERD/ERS region within a given window of the time-frequency domain. The method is exemplified by quantifying the ERS at low frequencies of 10 subjects performing a semantic memory task, and compared with existing techniques.

A data-driven process for the development of an eyes-closed EEG normative database.

A normative database constitutes a representative sample of a neurologically and clinically healthy population. The practical utility of a normative EEG database is to evaluate the clinical status of a subject whose EEG patterns statistically diverge from average population patterns. These normative data are daily used in clinical practice and in the evaluation of therapeutical interventions. The main obstacle of all normative databases developed to date is inter-individual variability. Such difficulty has been addressed by stratifying the population by age and then using regression in the EEG groups to bound variability, which is always an approximation. This paper describes the first data-driven EEG normative database that explicitly deals with EEG variability by stratifying the population based on their EEG patterns. The database has been constructed for 84 subjects in eyes-closed condition and has been validated by cross validation, leading to a global specificity of 100%.

EEG-based upper alpha neurofeedback training improves working memory performance.

Neurofeedback (NF) training has revealed its therapeutical effects to treat a variety of neurological and psychological disorders, and has demonstrated its feasibility to improve certain cognitive aptitudes in healthy users. Although promising results of NF training exist in recent literature, the reliability of its effects remains questioned due to a lack of deep studies examining its impact on the human electrophysiology. This paper presents a NF training aimed at improving working memory performance in healthy users by the enhancement of upper alpha band. A user-specific training was used (upper alpha was determined for each user using the individual alpha frequency) to reduce the unspecific factors of training the entire classical alpha band as traditional NF usually does. EEG assessments in active and passive open-eyes state were conducted pre/post the NF training. The EEG analyses reveal the UA enhancement during the active tasks which is independent of other frequency bands. UA was also enhanced in the passive state but independence could not be obtained in lower alpha band. Finally, significant improvement in working memory was obtained with regard to a control group.

Funciones cognoscitivas en la orientación sexual / Cognitive functions in sexual orientation

En México existe poca evidencia respecto a cognición y orientación sexual, por lo tanto, el objetivo de esta investigación fue describir y comparar el desempeño cognoscitivo de atención, memoria y Funciones Ejecutivas (FE) de personas con diversas Orientaciones Sexuales. Participaron en total setenta y tres personas divididas (edad= 20.7+/-2.4)en cinco grupos: Hombres heterosexuales (H), Mujeres heterosexuales (M), Hombres Homosexuales (HH), Mujeres Homosexuales (MH) y Mujeres Bisexuales (MB). Evaluados individualmente con la prueba NEUROPSI Atención y Memoria. Los resultados (p<.05) mostraron diferencias en las subpruebas: funciones motoras en el cambio de la mano derecha, lectura congruente de Stroop, memoria en la etapa de codificación en las subpruebas de: memoria verbal en resencia, figura de Rey-Osterreith; asimismo en memoria lógica. Estos hallazgos indican que la evocación de información episódica de eventos o sucesos sin un vínculo autobiográfico y la codificación de información visoespacial tienen una influencia del sexo y la orientación sexual en esta muestra. Finalmente,los procesos de atención, memoria y FE en personas con diversa orientación sexual son normales.

In México there are few sexual orientation evidences related to cognition, therefore the aim of the study was to describe and compare the cognitive performance of attention, memory and executive functions in different sexual orientations. Total sample were seventy-three people divided (age=20.7+/-2.4) in five groups: heterosexual Men (M), heterosexual Women (W), Homosexual Men (HM), Homosexual Women (HW) and Bisexual Women (BW). Participants were individually assessed with the NEUROPSI Attention and Memory. The results (p<.05) showed differences in subtests: motor functions in the change of the right hand, congruent reading Stroop, encoding memory subtests: verbal memory resence, figure of Rey-Osterreith; retrieval logical Memory. These findings indicate that the retrieval of episodic event information (without an autobiographical link) and visuospatial coding information have an influence of sex and sexual orientation in this sample. Finally, attentional processes, memory and executive functions in people with different sexual orientation are normal.

Índices de calidad de agua del río Cucabaj ubicado en el municipio de Santa Cruz del Guiché, Guiché y la influencia en los costos de tratamientos de potabilización / Water quality index of cucabaj river located in the municipality of Santa Cruz del Quiché, Quiché and influence in treatment costs purification

El río Cucabaj es una de las fuentes principales de agua que abastece junto a los ríos Tabil y Aguacate a 1,352 hogares del área urbana del municipio de Santa Cruz del Quiché. Este rio recorre parte de los municipios de Santa Cruz del Quiché y Chiché. Es tributario del río Durazno y pertenece a esta microcuenca. En el periodo de estudio el caudal osciló entre 0.009-0.1004 m3 /seg. Gran proporción de este caudal se capta y recorre alrededor de 9 km. hacia la planta de tratamiento para su potabilización ubicada en la salida a San Pedro Jocopilas. La presente investigación tiene como objetivo mostrar la variabilidad temporal del recurso hídrico, determinando el índice de calidad del agua e índice de contaminación del río Cucabaj, así como los costos de desinfección utilizando hipoclorito de calcio. El monitoreo del rio fue realizado por un periodo de diez meses, de abril 2012 a enero 2013, según el índice de calidad de agua que muestra en una escala de 0-100 puntos, que suman los resultados de nueve parámetros y clasifica el agua del río Cucabaj en la categoría de media a buena (contaminada a levemente contaminada para uso humano, por lo que antes de su consumo debe de aplicarse tratamientos de potabilización). El mes con mayor caudal fue septiembre con 0.1004 m3/seg, particularidad que influyó considerablemente en presentar el valor más bajo en calidad y uno de los valores más altos en contaminación por materia orgánica. Esta característica se asocia principalmente a la presencia de coliformes totales. En cuanto a los costos de desinfección por hipoclorito de calcio, no se establecieron diferencias significativas en función de la cantidad de caudal.

Cucabaj River is a major source of water supplies along the Aguacate and Tabil Rivers to 1,352 households in the urban area of Santa Cruz del Quiché. This river runs through the municipality of Santa Cruz del Quiché and Chiche communities. It is a tributary of Durazno River and belongs to this watershed. In the study period the rate ranged from 0.009 - 0.1004 m3/s. A large proportion of this flow is captured and about 9 km transported to the treatment plant for purification in located in San Pedro Jocopilas. This investigation aims to show the temporal variability of water resources, determining the rates of water quality and pollution index Cucabaj River, as well as the cost of disinfection by means of calcium hypochlorite. The measurements were performed for a period of ten months, from April 2012 to January 2013. According to the water quality index which is a 100-point scale resulting of a total of nine different measurements, it was determined that the water quality of Cucabaj river is classified as moderate to good (which means that is slightly polluted to polluted for human use, so should receive purification treatment before consumption). The month with highest flow was September with rate 0.1004 m3/s, a feature that greatly influenced present the lowest value in quality and one of the highest values in organic pollution. This feature is mainly associated with the presence of total coliforms. As for costs calcium hypochlorite disinfection, no significant differences in terms of the amount of flow over other months were established.

Análise molecular da expressão do fenótipo multi-droga resistente (MDR) em enterobactérias isoladas de amostras clínicas após exposição in vitro ao Imipenem / Molecular analysis of multi-drug resistant phenotype expression (MDR) in enterobacteria isolated from clinical specimens after exposure in vitro to imipenem

Após o surgimento e disseminação das ß-lactamases de amplo espectro em membros da família Enterobacteriaceae, os antibióticos carbapenêmicos (imipenem, meropenemeertapenem) têm sido considerados a terapia de escolha devido à estabilidade apresentada contra estas enzimas. A desvantagem destes antibióticos é a sua capacidade de induzir resistência aos ß-lactâmicos e a outros antibióticos quimicamente não relacionados. O imipenem tem favorecido a indução de cefalosporinases cromossômicas (AmpC) e também tem sido relacionado, in vivo, com a seleção de mecanismos intrínsecos de resistência, contribuindo com o perfil multi -droga resistente (MDR). Esse perfil é freqüentemente associado à diminuição da permeabilidade por alteração na síntese de porinas em conjunto com um aumento da atividade de bombas de efluxo, as quais não permitem o estabelecimento de uma concentração ativa do antibiótico no interior da célula bacteriana. O presente trabalho teve como objetivo avaliar o estabelecimento do perfil MDR em enterobactérias provenientes de isolados clínicos em função da exposição a diferentes concentrações de imipenemin vitro. A seleção do grupo das amostras estudadas foi feito por meio da determinação do perfil de sensibilidade dos isolados, tipagem molecular e ensaio de hidrólise de Imipenem. Nos isolados selecionados para a indução foi realizada numa etapa inicial (etapa basal) a análise de porinas de membrana externa por SDS-PAGE e o estudo de genes codificadores de ß-lactamases pela técnica de PCR. O estudo do estabelecimento do perfil MDR foi feito por meio de passagens sucessivas das amostras em meio contendo concentrações sub-inibitórias de imipenem seguido de análise fenotípica (CIM e acúmulo do antibiótico intracelular e SDS-PAGE), e a análise da expressão gênica de genes associados a permeabilidade de membrana (ompC, ompF eAcrA) e genes reguladores(marA e ompR). Após a indução com o imipenem, 77% dos isolados induzidos aumentaram a CIM para os carbapenêmicos, mudando assim o perfil de resistência observado na etapa basal Também foi afetado o perfil de resistência para outros antibióticos não relacionados a ß-lactámicos, porém numa percentagem menor. Com relação à alteração da permeabilidade, a perda de porina foi observada apenas para um isolado, no entanto a diminuição na expressão gênica de Omp36 foi significativa desde o começo da indução. A expressão da bomba de efluxoAcrAB foi afetada pela indução com imipenem, aumentando significativamente a expressão de AcrA, enquanto os reguladores estudados, MarA e OmpR tiveram a sua expressão induzida pelo imipenem. Foi possível observar também associação do nível de expressão gênica do regulador MarA com a expressão de AcrA,porém não foi possível observar uma associação estatisticamente significativa deste regulador com o perfil de expressão de OMPs. A indução de OmpR foi associado com um aumento da expressão de RNAm de Omp35, já para Omp36 foi possível observar apenas uma tendência na repressão deste gene. O estudo da resposta destes genes reguladores e determinantes de resistência, em resposta à exposição ao com o imipenem in vitro, permitiu reportar o comportamento molecular da bactéria numa resposta adaptativa no estagio inicial do estabelecimento do fenótipo MDR. A utilização de isolados clínicos com diversos determinantes de resistência permitiu observar a variabilidade nas respostas adaptativas das enterobacterias, o que é fundamental para a compreensão dos mecanismos de adaptação da bactéria e sua contribuição na falha terapêutica

After emergence and broad dissemination of extended spectrum ß-lactamases into the Enterobacteriaceae family, the carbapenemic antibiotics (imipenem, meropenem and ertapenem) have been considered the chosen therapy in the treatment of nosocomial infections by the stability that these antibiotics show to these enzymes. The disadvantage of carbapenems is theirs capacity to induce resistance against ß-lactamics and to other chemically unrelated antibiotics. The imipenem has been shown to induce chromosomal cephalosporinases (AmpC) and it was also related, in vivo, with the selection of intrinsic mechanism leading to multi-drug resistance profile (MDR). This profile is usually associated with membrane impermeability due to reduced outer membrane porin synthesis with an incremented activity of efflux pumps, which results in a reduced concentration of antibiotics inside the bacteria. This study aimed to evaluate the establishment of the MDR profile in Enterobacteriaceae from clinical isolates by exposure to different concentrations of imipenem in vitro. The selection of the study group was performed by determination of antibiotic susceptibility profile,molecular typing and hydrolysis assay of imipenem. In the selected isolates submitted to induction, in an initial step (baseline), was performed the outer membrane porin analysis by SDS-PAGE and the gene-specific amplification of B-lactamase enzymes by PCR. The study of the establishment of MDR was performed by progressive passages with subclinical concentrations of imipenem, followed each one by the evaluation of phenotypic profile (MIC, accumulation antibiotic in celland SDS-PAGE) and gene expression analysisof genes related to membrane permeability (ompC, ompF and acrA) and regulatory genes(MarA and ompR). After induction with imipenem, 77 % of the isolates increased the MIC for the carbapenems, changing the resistance profile at the baseline. In a lesser percentage, the resistance profile to other ß-lactams-unrelated antibiotics was also affected. Loss of porin was observed only for an isolated, however a significantly decreased Omp36 mRNA expression was observed from the start of induction. The expression of the efflux pump AcrAB ,was also affected by the imipenem induction, significantly increasing the AcrA gene expression, whereas the studied regulatory genes,MarA and OmpR,were induced by the imipenem. It was also possible to observe an association between the expression of the regulator MarA and the expression of AcrA, nevertheless no association was observed between this regulator and OMPs . OmpR induction was associated with an increased Omp35mRNA expression, however only a trend for the repression of Omp36was observed. The study of the response of these regulatory genes and genetic determinants of resistance, in response to the imipenem exposure in vitro, allowed to report the molecular behavior of the bacteria in an adaptive response in the initial stage of the establishment of a MDR phenotype. The use of clinical isolates with diverse resistance determinants allowed observing the variability in adaptive responses in enterobacteria, which is important to understand the adaptive mechanisms of bacteria to this antibiotic, the involvement in the emergence of the MDR profile and its contribution to the treatment failure

Cultivo de blastocitos con medios secuenciales / Human blastocysts culture with sequential media

Son bien conocidas las ventajas de cultivar preembriones humanos hasta etapa de blastocisto. Esto no es nuevo, lo que es reciente es el empleo de medios de cultivo secuenciales sin el apoyo de células somáticas. El objetivo fue cultivar preembriones humanos hasta etapa de blastocisto para determinar las tasas de recuperación, tasa de implantación y tasa de embarazo en pacientes sometidas a FIVT/TE o ICSI. Una vez obtenidos los óvulos de las pacientes, fueron inseminados/inyectados para posteriormente ser cultivados por 72 horas. usando medio P1 al 10 por ciento de SSS bajo aceite mineral para posteriormente ser transferidos a un medio complejo para cultivo de blastocistos por 48 horas. y finalmente ser transferidos. Se incluyeron 13 pacientes (9 de FIV y 4 de ICSI) obteniendo 205 óvulos, fertilizaron 143 y 131 tuvieron división celular. Se cultivaron 121 preembriones a etapa de blastocisto de los cuales 53 alcanzaron esa etapa (43.8 por ciento), transfiriendo 28 y congelando 25. En promedio se transfirieron 2.1 blastocistos por paciente. Se presentaron cuatro embarazos y un recién nacido vivo a término para una tasa de implantación de 14.2 por ciento y una tasa de embarazo de 30 por ciento. El estudio muestra nuestra experiencia inicial, la cual demostró una aceptable tasa de recuperación de blastocistos y de embarazo.