Use of human MAR elements to improve retroviral vector production.

Retroviral vectors have many favorable properties for gene therapies, but their use remains limited by safety concerns and/or by relatively lower titers for some of the safer self-inactivating (SIN) derivatives. In this study, we evaluated whether increased production of SIN retroviral vectors can be achieved from the use of matrix attachment region (MAR) epigenetic regulators. Two MAR elements of human origin were found to increase and to stabilize the expression of the green fluorescent protein transgene in stably transfected HEK-293 packaging cells. Introduction of one of these MAR elements in retroviral vector-producing plasmids yielded higher expression of the viral vector RNA. Consistently, viral titers obtained from transient transfection of MAR-containing plasmids were increased up to sixfold as compared with the parental construct, when evaluated in different packaging cell systems and transfection conditions. Thus, use of MAR elements opens new perspectives for the efficient generation of gene therapy vectors.

The use of ECMO in ICU. Recommendations of the Spanish Society of Critical Care Medicine and Coronary Units. / Empleo de ECMO en UCI. Recomendaciones de la Sociedad Española de Medicina Intensiva Crítica y Unidades Coronarias.

The use of extracorporeal membrane oxygenation systems has increased significantly in recent years; given this reality, the Spanish Society of Critical Intensive Care Medicine and Coronary Units (SEMICYUC) has decided to draw up a series of recommendations that serve as a framework for the use of this technique in intensive care units. The three most frequent areas of extracorporeal membrane oxygenation systems use in our setting are: as a cardiocirculatory support, as a respiratory support and for the maintenance of the abdominal organs in donors. The SEMICYUC appointed a series of experts belonging to the three working groups involved (Cardiological Intensive Care and CPR, Acute Respiratory Failure and Transplant work group) that, after reviewing the existing literature until March 2018, developed a series of recommendations. These recommendations were posted on the SEMICYUC website to receive suggestions from the intensivists and finally approved by the Scientific Committee of the Society. The recommendations, based on current knowledge, are about which patients may be candidates for the technique, when to start it and the necessary infrastructure conditions of the hospital centers or, the conditions for transfer to centers with experience. Although from a physiopathological point of view, there are clear arguments for the use of extracorporeal membrane oxygenation systems, the current scientific evidence is weak, so studies are needed that define more precisely which patients benefit most from the technique and when they should start.

Development of a child with Joubert syndrome.

The article describes the development of a child with Joubert Syndrome who, since the age of 16 months, has received personalized stimulation therapy at home and in the Early Intervention Unit (EIU) of the Faculty, in each of the five areas considered by the Portage Guide to Early Education: socialization, language, self-help, cognition, and motoricity. Repeated evaluations during the treatment (up to age 40 months) showed show progress in all developmental areas, as well as in general attitude to and capacity for learning. During treatment, greatest progress was made in the areas of cognition and communication.

Prothymosin alpha, a mammalian c-myc-regulated acidic nuclear protein, provokes the decondensation of human chromosomes in vitro.

Prothymosin (ProT alpha) is an acidic nuclear protein, widely distributed in mammalian cells, whose expression is regulated by c-myc and linked to cell proliferation. ProT alpha interacts with histone H1 via its acidic domain, and its overexpression provokes the unfolding of chromatin fibers. Here we show that incubation of human native metaphase chromosomes with ProT alpha induces their extensive unravelling suggesting a function of this protein in chromosome decondensation.

Kidney ATPase response in seawater-transferred rainbow trout (Oncorhynchus mykiss). Effect of salinity and fish size.

Two sizes of domesticated rainbow trout (Oncorhynchus mykiss, 40 +/- 0.67 and 180 +/- 3.9 g) were directly transferred to brackish water (9 ppt) and seawater (28 ppt). Kidney Na(+)-K(+)-ATPase and Mg(2+)-ATPase activities were measured in fresh water, and after long-term seawater adaptation (up to 21 days). Renal Na(+)-K(+)-ATPase activity increased after saltwater loading in small trout, while large trout displayed an unmodified ATPase activity. The smallest trout showed a low but progressive increase in renal Mg(2+)-ATPase activity after the transfer to both salinities. However, ATPase activity remained unchanged or significantly decreased in large trout after the transfer to seawater or brackish water, respectively.

A c-myc antisense oligonucleotide inhibits human retinal pigment epithelial cell proliferation.

The purpose of this work was to investigate if MYC-dependent intracellular mitogenic pathway is active in cultures of human retinal pigment epithelial (hRPE) cells and whether myc antisense phosphorotioate oligonucleotides (c-myc-AS-ODN) are useful tools for inhibiting the proliferation of hRPE cells. Cultures of hRPE cells were established from adult human corneal donors. These cells were positively stained for cytokeratins and vimentin. Myc mRNA expression was determined by Northern blot analysis and it was determined by means of immunofluorescence if MYC was expressed. C-myc-AS-ODN effect on cell proliferation was estimated by evaluating the incorporation of 5-bromo-2'-deoxy-uridine into cellular DNA. Cell number was estimated by using a tetrazolium bromide based colorimetric method. Human RPE cells in culture expressed MYC and myc mRNA as well as prothymosin alpha mRNA--a gene whose transcription is under MYC control--indicating that MYC-dependent intracellular mitogenic pathway is active in these cells. In accordance with this, we found that blocking the expression of myc by the addition of c-myc-AS-ODN to the culture medium inhibited hRPE cell proliferation. The effect of the c-myc-AS-ODN was found to be sequence specific (the use of a control oligonucleotide with the same sequence but in an opposite direction had no effect) and dose-dependent (4 microM was the lowest effective dose tested). By using RT-PCR we found that the c-myc-AS-ODN inhibition of cell proliferation was related to a diminution in c-myc mRNA expression, and by immunofluorescence we detected a diminution in c-MYC protein staining in RPE cells after 48 hr of treatment with c-myc-AS-ODN. Furthermore, growth inhibition remained for at least 5 days after addition of a single dose of the c-myc-AS-ODN to the culture. We conclude that hRPE cell proliferation is under MYC control. Blocking the expression of myc by c-myc-AS-ODN inhibited hRPE cell proliferation. These findings establish a rationale for investigating the potential use of a c-myc-AS-ODN as a novel therapeutical tool in the treatment of Proliferative Vitreoretinopathy.

Overexpression of prothymosin alpha accelerates proliferation and retards differentiation in HL-60 cells.

Prothymosin alpha (ProTalpha) is an acidic nuclear protein the expression of which is related to the proliferation and differentiation processes in mammalian cells. In the present study we have stably transfected HL-60 cells, a biological system that allows the study of both proliferation and differentiation, with recombinant vectors encoding sense and antisense ProTalpha mRNA. In the HL-60 cell clones overexpressing ProTalpha we observed an acceleration in the growth rate, whereas expression of the antisense orientation showed the opposite effect. Moreover, cell-cycle analysis demonstrated that the G1-phase was shortened in the cells expressing the sense construct. Before studying how ProTalpha affects differentiation, we showed that the down-regulation of ProTalpha gene during differentiation occurs in all mammalian cell lines (HL-60, K562, U937, MEL C88, N2A and PC12) analysed. The biological effect evoked by the induction of the ProTalpha sense vector was the retardation of cell differentiation, although expression of the antisense construct showed no effect on differentiation. In conclusion, our findings provide evidence that ProTalpha is directly implicated in cellular proliferation and that the maintenance of high levels of ProTalpha inside HL-60 cells is incompatible with their ability to differentiate.