Abolishing Retro-Transduction of Producer Cells in Lentiviral Vector Manufacturing.

Transduction of producer cells during lentiviral vector (LVV) production causes the loss of 70-90% of viable particles. This process is called retro-transduction and it is a consequence of the interaction between the LVV envelope protein, VSV-G, and the LDL receptor located on the producer cell membrane, allowing lentiviral vector transduction. Avoiding retro-transduction in LVV manufacturing is crucial to improve net production and, therefore, the efficiency of the production process. Here, we describe a method for quantifying the transduction of producer cells and three different strategies that, focused on the interaction between VSV-G and the LDLR, aim to reduce retro-transduction.

Architecture of the ESCPE-1 membrane coat.

Recycling of membrane proteins enables the reuse of receptors, ion channels and transporters. A key component of the recycling machinery is the endosomal sorting complex for promoting exit 1 (ESCPE-1), which rescues transmembrane proteins from the endolysosomal pathway for transport to the trans-Golgi network and the plasma membrane. This rescue entails the formation of recycling tubules through ESCPE-1 recruitment, cargo capture, coat assembly and membrane sculpting by mechanisms that remain largely unknown. Herein, we show that ESCPE-1 has a single-layer coat organization and suggest how synergistic interactions between ESCPE-1 protomers, phosphoinositides and cargo molecules result in a global arrangement of amphipathic helices to drive tubule formation. Our results thus define a key process of tubule-based endosomal sorting.

Supplementation of culture medium with quercetin improves mouse blastocyst quality and increases the expression of HIF-1α protein.

Regarding the low number of embryos that reach the blastocyst stage when cultured in vitro, this study aimed to evaluate the effects of quercetin on pre-implantation mouse (Mus musculus) embryos obtained using in vitro fertilization, especially during the passage from morula to blastocyst. Furthermore, we studied whether quercetin also affected the expression of hypoxia-inducible factor 1α (HIF-1α). The culture medium for the embryos was supplemented with quercetin, for long or short periods of time, and then the development potential, total cell number, apoptosis rates and expression of HIF-1α were studied to determine the effect of quercetin. Embryos failed to develop when cultured for long periods of time with quercetin, implying the possible toxic effects of this, alternatively antioxidant, compound. However, a short culture from morula to blastocyst significantly improved the development potential of in vitro produced embryos, increasing the final total cell number and reducing the apoptosis rate, observing similar results to those embryos cultured in low-oxygen concentrations or developed in utero. Furthermore, in embryos treated with quercetin for 2 or 4 h we found an increase in HIF-1α compared with untreated embryos. This work could imply a way to use quercetin in fertility clinics to improve the production of healthy blastocysts and, consequently, increase the success rates in assisted reproduction techniques.

Effect of the addition of 4OHE2 and quercetin in culture media on ROS levels and gene expression in mouse blastocysts.

AIMS: The main objective of this work is to elucidate whether Quercetin (Qc) and 4-Hidroxistradiol (4OHE2 ) decrease the level of reactive oxygen species (ROS) in in vitro obtained embryos and to analyse which genes are activated under the treatments that could explain this improvement. METHODS: Oxidative stress was induced during embryo culture by H2 O2 treatment and ROS production was measured and compared with embryos treated with Qc or 4OHE2 . Gene expression was analysed by Q-PCR in control embryos obtained in utero (IU) or by IVF and compared with the levels found in embryos cultured with Qc or 4OHE2 to determine the effect of these compounds. KEY RESULTS: Qc strongly reduces ROS levels in embryos after a treatment of 4h. On the contrary, 4OHE2 had no effect in reducing ROS levels in embryos. The addition of these molecules to the culture media upregulate several hypoxia-related genes when Qc is added to the culture media, and implantation-related genes when 4OHE2 is used. CONCLUSIONS: Qc is a very strong antioxidant molecule that when used for short periods of time during culture can reduce ROS levels and improve embryo quality by activating antioxidant enzymes. 4OHE2 supplementation, despite having no effects in reducing ROS levels, acts directly in the molecular signalling implicated in the implantation process and could be also considered as a supplement for embryo culture during IVF. IMPLICATIONS: Proper supplementation of the culture media could greatly improve the quality of embryos cultured in vitro , resulting in better results in IVF clinics.

4-Hydroxyestradiol improves mouse embryo quality, epidermal growth factor-binding capability in vitro and implantation rates.

Embryo implantation in the uterus is a critical step to achieve success following ART. Despite favorable uterine conditions, a great number of good quality embryos fail to implant, often for reasons that are unknown. Hence, improving the implantation potential of embryos is a subject of great interest. 4-Hydroxyestradiol (4-OH-E2), a metabolic product of estradiol produced by endometrial cells, plays a key role in endometrial-embryonic interactions that are necessary for implantation. Nonetheless, the effects of 4-OH-E2 on embryos obtained in vitro have not been yet described. This study was designed to determine whether culture media enriched in 4-OH-E2 could improve the quality and implantation rate of embryos obtained in vitro, using both in vitro and in vivo models. We also analyzed its effects on the epidermal growth factor (EGF)-binding capability of the embryos. Our results showed that the presence of 4-OH-E2 in the culture media of embryos during the morula to blastocyst transition increases embryo quality and attachment to endometrial cells in vitro. 4-OH-E2 can also improve viable pregnancy rates of mouse embryos produced in vitro, reaching success rates that are similar to those from embryos obtained directly from the uterus. 4-OH-E2 improved the embryos' ability to bind EGF, which could be responsible for the increased embryo implantation potential observed. Therefore, our results strongly suggest that 4-OH-E2 is a strong candidate molecule to supplement human IVF culture media in order to improve embryo implantation. However, further research is required before these findings can be translated with efficacy and safety to fertility clinics.

Histological cut of a paraffin-embedded blastocyst: Optimized protocol for murine blastocysts.

Paraffin-embedded tissues have been used for research and therapeutic applications for decades, as they represent a valuable tool in histology and for molecular analysis, as well as being a way to preserve tissue samples for long periods at a low cost. For tissues such as the liver, lungs, kidney, heart or brain, there are many protocols available, already optimized. The purpose of this work is to optimize and simplify the protocols already available to take a single blastocyst from a mouse, fix it and embed it into a paraffin block without using gelatin, to then perform histological cuts using a microtome, with no need of sophisticated equipment or trained personnel. •The protocol presented here preserves well the morphology of the blastocyst.•Paraffin-embedded sections of the sample can be used for studies such as in situ hybridization, immunohistochemistry, enzyme histochemistry, DNA, RNA or protein extractions, analysis of biomarkers, characterization of surface markers of stem cells integrated into the embryo, to prepare histological material for educational purposes, etc.•Some of these studies could represent a valuable source of new information for the field of reproductive biology.

VPS29, a tweak tool of endosomal recycling.

The endolysosomal system is a highly dynamic network of membranes for degradation and recycling. During endosomal maturation, cargo molecules destined for lysosomal degradation are progressively concentrated through continuous rounds of fusion and fission reactions concomitant with inbound and outbound membrane fluxes. Of the cargo molecules delivered to endosomes, about two-thirds are rescued from degradation and recycled for reuse. This balance between degradation and recycling is essential to preserve the proteostatic plasticity of the cell under variable physiological demands. Cargo retrieval from endosomes involves several sorting complexes with stable core compositions that associate with multidomain regulatory proteins, consequently displaying complex interaction networks. The vacuolar protein sorting 29 (VPS29) has emerged as a central scaffold that coordinates the physical assembly of retrieval complexes with regulatory components in what appears to be an elegant solution for regulating distinct retrieval stations. This review summarizes the VPS29-binding partners and its integration into retrieval complexes for endosomal sorting and trafficking.

High-fidelity DNA replication in Mycobacterium tuberculosis relies on a trinuclear zinc center.

High-fidelity DNA replication depends on a proofreading 3'-5' exonuclease that is associated with the replicative DNA polymerase. The replicative DNA polymerase DnaE1 from the major pathogen Mycobacterium tuberculosis (Mtb) uses its intrinsic PHP-exonuclease that is distinct from the canonical DEDD exonucleases found in the Escherichia coli and eukaryotic replisomes. The mechanism of the PHP-exonuclease is not known. Here, we present the crystal structure of the Mtb DnaE1 polymerase. The PHP-exonuclease has a trinuclear zinc center, coordinated by nine conserved residues. Cryo-EM analysis reveals the entry path of the primer strand in the PHP-exonuclease active site. Furthermore, the PHP-exonuclease shows a striking similarity to E. coli endonuclease IV, which provides clues regarding the mechanism of action. Altogether, this work provides important insights into the PHP-exonuclease and reveals unique properties that make it an attractive target for novel anti-mycobacterial drugs.The polymerase and histidinol phosphatase (PHP) domain in the DNA polymerase DnaE1 is essential for mycobacterial high-fidelity DNA replication. Here, the authors determine the DnaE1 crystal structure, which reveals the PHP-exonuclease mechanism that can be exploited for antibiotic development.

[The 1, 2, 3 of laboratory animal experimentation]. / El 1, 2, 3 de la experimentación con animales de laboratorio.

The slow scientific development in Latin America in recent decades has delayed the incorporation of laboratory animal experimentation; however, this situation has started to change. Today, extraordinary scientific progress is evident, which has promoted the introduction and increased use of laboratory animals as an important tool for the advancement of biomedical sciences. In the aftermath of this boom, the need to provide the scientific community with training and guidance in all aspects related to animal experimentation has arisen. It is the responsibility of each country to regulate this practice, for both bioethical and legal reasons, to ensure consideration of the animals' rights and welfare. The following manuscript is the result of papers presented at the International Workshop on Laboratory Animal Testing held at the Technical University of Ambato, Ecuador; it contains information regarding the current state of affairs in laboratory animal testing and emphasizes critical aspects such as main species used, ethical and legal principles, and experimental and alternative designs for animal use. These works aim to ensure good practices that should define scientific work. This document will be relevant to both researchers who aim to newly incorporate animal testing into their research and those who seek to update their knowledge.

El 1, 2, 3 de la experimentación con animales de laboratorio / The 1, 2, 3 of laboratory animal experimentation

RESUMEN El lento desarrollo científico experimentado en América Latina en las últimas décadas ha retrasado la incorporación de la experimentación con animales de laboratorio, sin embargo, esta realidad ha comenzado a cambiar. En la actualidad, se evidencia un extraordinario progreso científico que ha promovido la introducción e incremento del uso de animales de laboratorio como una importante herramienta para el avance de las ciencias biomédicas. A raíz de este auge, surge la necesidad de proporcionar a la comunidad científica la formación y directrices en todos los aspectos relacionados con la experimentación animal. Es responsabilidad de cada país legislar esta práctica para que no solo por razones bioéticas, sino también legales, se considere el derecho de los animales y con ello su bienestar. El siguiente manuscrito es el resultado de las comunicaciones presentadas en el Taller Internacional en Ciencias de Animales de Laboratorio celebrado en la Universidad Técnica de Ambato, Ecuador; contiene la actualidad en la ciencia de animales de laboratorio, haciendo énfasis en aspectos fundamentales, tales como: principales especies utilizadas y su biología, principios éticos y legales, diseño experimental y alternativas al uso de animales, todas ellas encaminadas a garantizar las buenas prácticas que deben caracterizar el quehacer científico. Sin duda, este documento será relevante tanto para aquellos investigadores que pretenden incorporar la experimentación animal a sus investigaciones, como para aquellos que, aun teniendo experiencia, pretendan actualizar sus conocimientos.

ABSTRACT The slow scientific development in Latin America in recent decades has delayed the incorporation of laboratory animal experimentation; however, this situation has started to change. Today, extraordinary scientific progress is evident, which has promoted the introduction and increased use of laboratory animals as an important tool for the advancement of biomedical sciences. In the aftermath of this boom, the need to provide the scientific community with training and guidance in all aspects related to animal experimentation has arisen. It is the responsibility of each country to regulate this practice, for both bioethical and legal reasons, to ensure consideration of the animals' rights and welfare. The following manuscript is the result of papers presented at the International Workshop on Laboratory Animal Testing held at the Technical University of Ambato, Ecuador; it contains information regarding the current state of affairs in laboratory animal testing and emphasizes critical aspects such as main species used, ethical and legal principles, and experimental and alternative designs for animal use. These works aim to ensure good practices that should define scientific work. This document will be relevant to both researchers who aim to newly incorporate animal testing into their research and those who seek to update their knowledge.

The guanine-exchange factor Ric8a binds to the Ca²âº sensor NCS-1 to regulate synapse number and neurotransmitter release.

The conserved Ca(2+)-binding protein Frequenin (homolog of the mammalian NCS-1, neural calcium sensor) is involved in pathologies that result from abnormal synapse number and probability of neurotransmitter release per synapse. Both synaptic features are likely to be co-regulated but the intervening mechanisms remain poorly understood. We show here that Drosophila Ric8a (a homolog of mammalian synembryn, which is also known as Ric8a), a receptor-independent activator of G protein complexes, binds to Frq2 but not to the virtually identical homolog Frq1. Based on crystallographic data on Frq2 and site-directed mutagenesis on Frq1, the differential amino acids R94 and T138 account for this specificity. Human NCS-1 and Ric8a reproduce the binding and maintain the structural requirements at these key positions. Drosophila Ric8a and Gαs regulate synapse number and neurotransmitter release, and both are functionally linked to Frq2. Frq2 negatively regulates Ric8a to control synapse number. However, the regulation of neurotransmitter release by Ric8a is independent of Frq2 binding. Thus, the antagonistic regulation of these two synaptic properties shares a common pathway, Frq2-Ric8a-Gαs, which diverges downstream. These mechanisms expose the Frq2-Ric8a interacting surface as a potential pharmacological target for NCS-1-related diseases and provide key data towards the corresponding drug design.

Frq2 from Drosophila melanogaster: cloning, expression, purification, crystallization and preliminary X-ray analysis.

Drosophila melanogaster contains two calcium-binding proteins, Frq1 and Frq2, in the nervous system that control the number of synapses and the probability of release. To understand the differential function of the two proteins, whose sequence is only 5% dissimilar, the crystal structures of Frq1 and Frq2 are needed. Here, the cloning, expression, purification, crystallization and preliminary crystallographic analysis of Frq2 are presented. The full-length protein was purified using a two-step chromatographic procedure. Two different diffracting crystal forms were obtained using a progressive streak-seeding method and detergents.

[Rational use of antibiotics in the Department of Internal Medicine from a university hospital: results of a pilot experience]. / Uso racional de antimicrobianos en el departamento de medicina interna de un hospital universitario: resultados de una experiencia piloto.

INTRODUCTION: The programs of rational use of antibiotics are designed to optimize antimicrobial therapy and minimize the emergence of bacterial resistance. In order to optimize the use of antibiotics we implemented an educational program based on the application of a checklist criteria for the rational use of these drugs. METHOD: We performed a cohort study unpaired in the Department of Internal Medicine, during three months. We compared a prospective cohort (A) which used a checklist, with a retrospective cohort (B) in wich prescription was based on usual clinical practice. RESULTS: We included 227 prescriptions of antibiotics. In cohort A compared to B, there was a higher proportion of switch to oral antibiotics agents and adjustment of the antimicrobial therapy to the susceptibility in the antibiogram and reduced use of associated antibiotics. Total antibiotic consumption was 117.7 DDD/100 bed-days (Defined Daily Doses). Consumption in cohorts A and B was 46.1 DDD/100 bed-days and 71.6 DDD/100 bed-days (reduction, 35.6%). There was also a reduction in consumption of ceftriaxone, ceftazidime, quinolones, vancomycin and carbapenems. Costs were reduced by 55%. There was no difference in the average hospital stay. CONCLUSIONS: The implementation of an educational strategy based on a checklist allowed the optimum use of antibiotics.

Uso racional de antimicrobianos en el departamento de medicina interna de un hospital universitario: resultados de una experiencia piloto / Rational use of antibiotics in the department of internal medicine from a university hospital: results of a pilot experience

Introduction: The programs of rational use of antibiotics are designed to optimize antimicrobial therapy and minimize the emergence of bacterial resistance. In order to optimize the use of antibiotics we implemented an educational program based on the application of a checklist criteria for the rational use of these drugs. Method: We performed a cohort study unpaired in the Department of Internal Medicine, during three months. We compared a prospective cohort (A) which used a checklist, with a retrospective cohort (B) in wich prescription was based on usual clinical practice. Results: We included 227 prescriptions of antibiotics. In cohort A compared to B, there was a higher proportion of switch to oral antibiotics agents and adjustment of the antimicrobial therapy to the susceptibility in the antibiogram and reduced use of associated antibiotics. Total antibiotic consumption was 117.7 DDD/100 bed-days (Defined Daily Doses). Consumption in cohorts A and B was 46.1 DDD/100 bed-days and 71.6 DDD/100 bed-days (reduction, 35.6%). There was also a reduction in consumption of ceftriaxone, ceftazidime, quinolones, vancomycin and carbapenems. Costs were reduced by 55%. There was no difference in the average hospital stay. Conclusions: The implementation of an educational strategy based on a checklist allowed the optimum use of antibiotics.

Introducción: Los programas de uso racional de antimicrobianos tienen la finalidad de optimizar la terapia antimicrobiana y minimizar la aparición de resistencia bacteriana. Con el objetivo de optimizar el uso de antimicrobianos se implementó un programa educativo basado en la aplicación de una lista de verificación (check list) conteniendo criterios establecidos de uso racional de estos fármacos. Método: Se realizó un estudio de cohortes no pareadas en el Departamento de Medicina Interna, durante tres meses. Se comparó una cohorte prospectiva (A) en que se aplicó la lista de verificación, con una cohorte retrospectiva (B) con prescripción de acuerdo a la práctica clínica habitual. Resultados: Se incluyeron 227 prescripciones de antimicrobianos. En la cohorte A, hubo mayor proporción de paso a vía oral y adecuación del antimicrobiano al antibiograma y menor uso de asociación de antimicrobianos, con respecto a la cohorte B. El consumo total de antimicrobianos fue de 117,7 DDD/100 días-cama (Dosis Diaria Definida). El consumo en las cohortes A y B fue de 46,1 DDD/100 días-cama y 71,6 DDD/100 días-cama respectivamente (reducción de un 35,6%). También hubo una reducción en el consumo de ceftriaxona, ceftazidima, quinolonas, vancomicina y carbapenem. Los costos se redujeron en 55%. No hubo diferencias en la estadía media hospitalaria. Conclusiones: La aplicación de una estrategia educativa basada en una lista de verificación permitió optimizar el uso de antimicrobianos.

Circadian levels of serotonin in plasma and brain after oral administration of tryptophan in rats.

Serotonin, one of the most important neurotransmitters in the central nervous system, is synthesized by the amino acid, tryptophan. Given that this essential amino acid is consumed in the diet, the aim of this study was to evaluate the effect of orally administered L-tryptophan (125 mg/kg) on circadian variations in the levels of serotonin in brain and plasma. We used male Wistar rats of 14 +/- 2 weeks of age (n = 240), maintained under conditions of a 12-hr light:dark cycle, and food and water ad libitum. Tryptophan administration was by gavage in a daily single dose at 7 p.m. for 7 days. The serotonin levels were measured by ELISA every hour at night (8 p.m. to 8 a.m.) and every 4 hr during daytime (8 a.m. to 8 p.m.). The results show that in both the tryptophan-treated and untreated groups the highest values appeared during the beginning of the darkness with a peak at 9, 10 and 11 p.m. in controls, and at 9 p.m. in the tryptophan-treated group. After tryptophan administration, the levels of serotonin were significantly higher in the plasma and all the brain regions analysed than in the control group. This increase of serotonin levels was greater in the pineal gland than in other brain regions, and the least in plasma. In conclusion, oral administration of tryptophan during 7 days enhances serotonin levels over a 24-hr period, and produces an advance in the peak of serotonin in both plasma and different brain regions.

Surveillance of antibiotic resistance evolution and detection of class 1 and 2 integrons in human isolates of multi-resistant Salmonella Typhimurium obtained in Uruguay between 1976 and 2000.

OBJECTIVES: To study the evolution of antibiotic resistance in isolates of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) obtained in Uruguay between the years 1976 and 2000, and to determine the incidence of class 1 and 2 integrons in the multi-resistant isolates. METHODS: We studied 258 strains of Salmonella Typhimurium from various sources, isolated between 1976 and 2000. We determined the evolution of antibiotic resistance and the distribution of class 1 and 2 integrons in all isolates by means of disk diffusion assays and PCR. RESULTS: During the period 1989-2000 resistance to streptomycin was 56.8%, tetracycline 13.6%, sulfonamides 11.2%, and ampicillin 7.2%. Resistance to gentamicin, kanamycin, chloramphenicol, and nalidixic acid were lower than 5%; no resistance was detected to fluoroquinolones, oxyiminocephalosporins, and amikacin. These results show a dramatic decrease with respect to values found in the period 1976-1988. In this period, resistance to streptomycin was 63.2%, tetracycline 36.8%, sulfonamides 32.3%, and ampicillin 27.8%. Throughout the two periods, 29 multi-resistant Salmonella Typhimurium strains were isolated harboring some class of integron: 15 strains had only intI2, 11 strains presented both intI1 and intI2, and three isolates only intI1. CONCLUSIONS: Our results show a marked decrease in resistance throughout these years, along with a correlation between resistance to different antibiotics and the presence of integrons.

[A strategy for the management of hospitalized children with acute lower respiratory infections]. / Estrategia de atención de niños hospitalizados por infecciones respiratorias agudas bajas.

OBJECTIVES: To improve the quality of care provided to hospitalized children having acute lower respiratory infections (ALRI), to increase the knowledge on this health condition, and to broaden the utilization of health care resources through a program called "Winter Plan". METHODS: The program comprised the use of guidelines for diagnosis and treatment, disease-oriented hospitalizations to provide an increased level of care, management of health care resources and implementation of computerized medical records. Systematic investigation of viral etiology was performed in order to rationalize the use of medications and reduce nosocomial infections. RESULTS: During program implementation (19/V-19/IX/99), 3,317 children were admitted; 1,347 (40.61%) had ALRI, of which 1,096 (81%) were included in the study. Of them, 71% aged less than 1 year. Most ALRI were viral (68%). Admission criteria were: oxygen saturation <95%, tachypnea, retractions or pleural effusion (92.4% of the children). The demand magnitude prevented compliance with isolation guidelines in all cases. Treatment guidelines were followed in a high percentage of cases: 73% of children having bronchiolitis and 72% of those with viral pneumonia received no antibiotics and 96% of children with bacterial pneumonia were put on antibiotics as recommended; use of bronchodilators and corticosteroids was reduced. Medication costs were reduced especially in the corticosteroid group, which meant a greater impact on hospitalization costs. CONCLUSIONS: To decrease ALRI morbidity and mortality there is a need to continue improving the quality of health care during hospitalization and to reinforce health promotion actions and preventive programs at the primary level.