Direct conversion of human pluripotent stem cells into cranial motor neurons using a piggyBac vector

Human pluripotent stem cells (PSCs) are widely used for in vitro disease modeling. One of the challenges in the field is represented by the ability of converting human PSCs into specific disease-relevant cell types. The nervous system is composed of a wide variety of neuronal types with selective vulnerability in neurodegenerative diseases. This is particularly relevant for motor neuron diseases, in which different motor neurons populations show a different susceptibility to degeneration. Here we developed a fast and efficient method to convert human induced Pluripotent Stem Cells into cranial motor neurons of the branchiomotor and visceral motor subtype. These populations represent the motor neuron subgroup that is primarily affected by a severe form of amyotrophic lateral sclerosis with bulbar onset and worst prognosis. This goal was achieved by stable integration of an inducible vector, based on the piggyBac transposon, allowing controlled activation of Ngn2, Isl1 and Phox2a (NIP). The NIP module effectively produced electrophysiologically active cranial motor neurons. Our method can be easily extended to PSCs carrying disease-associated mutations, thus providing a useful tool to shed light on the cellular and molecular bases of selective motor neuron vulnerability in pathological conditions

FUS mutant human motoneurons display altered transcriptome and microRNA pathways with implications for ALS pathogenesis

The FUS gene has been linked to amyotrophic lateral sclerosis (ALS). FUS is a ubiquitous RNA-binding protein, and the mechanisms leading to selective motoneuron loss downstream of ALS-linked mutations are largely unknown. We report the transcriptome analysis of human purified motoneurons, obtained from FUS wild-type or mutant isogenic induced pluripotent stem cells (iPSCs). Gene ontology analysis of differentially expressed genes identified significant enrichment of pathways previously associated to sporadic ALS and other neurological diseases. Several microRNAs (miRNAs) were also deregulated in FUS mutant motoneurons, including miR-375, involved in motoneuron survival. We report that relevant targets of miR-375, including the neural RNA-binding protein ELAVL4 and apoptotic factors, are aberrantly increased in FUS mutant motoneurons. Characterization of transcriptome changes in the cell type primarily affected by the disease contributes to the definition of the pathogenic mechanisms of FUS-linked ALS

Time trend prevalence of artificial nutrition counselling in a university hospital.

Abstract Objectives The negative effects of malnutrition on the prognosis of hospitalized patients are well documented; however, less known is the awareness and knowledge of health care professionals about this complication. The aim of this study was to evaluate the trend of the requests for nutritional consultation in years and the prescription of artificial nutrition (AN), for adult patients at a university hospital in southern Italy in the years 2004, 2008, 2012, and 2016 to assess the progress of medical teams concerning awareness of hospital malnutrition. Methods This was a retrospective study that evaluated the time trend of nutritional consultation requests and related prescription of AN, for adult patients at a university hospital in southern Italy in the years 2004, 2008, 2012, and 2016. Of 112 233 inpatients, 2505 received a nutritional consultation with the prescription of AN. Results The number of patients on AN increased from 507 of 33 240 (1.52%) in 2004 to 730 of 29 195 (2.5%) in 2008 (P The request for AN was quite equally distributed between surgical (51.5%) and medical wards (48.5%), with a prevalence among patients with oncologic diseases (806 patients [65.6%]). As for nononcologic diseases, 20.4% involved the gastrointestinal tract and 6.3% the nervous system. Throughout the 12 y of observation, parenteral nutrition was the main prescribed support (59.8%) followed by oral nutritional supplements (26.1%) and enteral nutrition (9.3%). Mean nutritional intervention duration was 11 d (±10.8 d). Conclusions The request of AN for hospitalized patients increased over time, probably owing to improved medical consciousness of the potential risks for malnutrition and the availability of a specialized clinical nutrition team

Ultrasound assessment of diaphragmatic dysfunction and its improvement with levosimendan in patients with chronic obstructive pulmonary disease

Diaphragmatic Dysfunction (DD) is a clinical condition in which the diaphragm becomes weak or paralyzed, because of muscle strength reduction. It can be due to muscular issues or loss of proper innervation, but, also, to pulmonary hyperinflation or air trapping, such as in Chronic Obstructive Pulmonary Disease (COPD). DD impacts on COPD induced dyspnea, determining its progressive worsening, but levosimendan, an inodilator better known as Ca2+ sensitizer, may limit this phenomenon and diaphragmatic ultrasound assessment can be useful in monitoring its effect. Here, we show the case of a 77-year-old woman admitted to the Emergency department for acute exacerbation of chronic dyspnea in COPD, related to right ventricular failure and DD, which did not respond to medical therapy and non-invasive mechanical ventilation but did experience a favorable outcome after intravenous administration of levosimendan

Do acesso à justiça pleno do brasileiro perante a corte interamericana de Direitos Humanos

Using bibliographic research methods, this study will demonstrate how indivuals, especially Brazilians, do not have full access to justice in Interamerican Court of Human Rights because they are not legitimize to address a formal petition direct to the Court, which constitute as an obstacle to the protection an effectiveness of Human Rights, and can generate discontent and injustice feeling. This article proposes, by the end, an evolution to the American Regional System of Human Rights Protection, able to provide this lack of legitimation, inspired with the European System.Através da metodologia de pesquisa bibliográfica e documental, com método indutivo, o presente artigo verificará a impossibilidade dos indivíduos, principalmente dos brasileiros, de ter o pleno acesso à justiça perante o Sistema Regional Americano de Proteção aos Direitos Humanos ante o impedimento de peticionamento direto e representação própria perante a Corte Interamericana de Direitos Humanos, o que constitui um obstáculo na proteção e efetivação dos próprios Direitos Humanos per se à eles consagrados, capaz de gerar o sentimento de insatisfação e injustiça, propondo-se, ao final, a evolução do Sistema, inspirado no Europeu como modelo, capaz de suprir essa falta de legitimação.Através da metodologia de pesquisa bibliográfica e documental, com método indutivo, o presente artigo verificará a impossibilidade dos indivíduos, principalmente dos brasileiros, de ter o pleno acesso à justiça perante o Sistema Regional Americano de Proteção aos Direitos Humanos ante o impedimento de peticionamento direto e representação própria perante a Corte Interamericana de Direitos Humanos, o que constitui um obstáculo na proteção e efetivação dos próprios Direitos Humanos per se à eles consagrados, capaz de gerar o sentimento de insatisfação e injustiça, propondo-se, ao final, a evolução do Sistema, inspirado no Europeu como modelo, capaz de suprir essa falta de legitimação

An innovative protocol to select the best growth phase for astaxanthin biosynthesis in H. pluvialis.

H. pluvialis is a green unicellular microalgae and it is the first producer of natural astaxanthin in the world if subjected to stress conditions such as high light, high salinity and nutrient starvation. Astaxanthin is a powerful antioxidant used in many fields, such as aquaculture, pharmaceutical, food supplements and cosmetic. To obtain a large amount of astaxanthin, researcher focused on the optimisation of H. pluvialis growth. H. pluvialis has four different size growth stage (macrozooids, microzooids, palmelloid and “red non-motile astaxanthin accumulated encysted”), and astaxanthin production occur in the last phase. Recent studies shown that non-motile cells can produce more astaxanthin than motile cells if subjected to light stress. For these reasons, the aim of this study is to find a new and innovative methodology to select and recovery H. pluvialis in his last growth phase thanks to an electrophoretic run, and optimize, in this way, astaxanthin production

FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons

The RNA-binding protein FUS participates in several RNA biosynthetic processes and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Here we report that FUS controls back-splicing reactions leading to circular RNA (circRNA) production. We identified circRNAs expressed in in vitro -derived mouse motor neurons (MNs) and determined that the production of a considerable number of these circRNAs is regulated by FUS. Using RNAi and overexpression of wild-type and ALS-asso- ciated FUS mutants, we directly correlate the modulation of circRNA biogenesis with alteration of FUS nuclear levels and with putative toxic gain of function activities. We also demonstrate that FUS regulates circRNA biogenesis by binding the introns flanking the back-splicing junctions and that this control can be reproduced with artificial constructs. Most circRNAs are conserved in humans and specific ones are deregulated in human-induced pluripotent stem cell-derived MNs carrying the FUS P525L mutation associated with AL

ATM inhibition blocks glucose metabolism and amplifies the sensitivity of resistant lung cancer cell lines to oncogene driver inhibitors

Background: ATM is a multifunctional serine/threonine kinase that in addition to its well-established role in DNA repair mechanisms is involved in a number of signaling pathways including regulation of oxidative stress response and metabolic diversion of glucose through the pentose phosphate pathway. Oncogene-driven tumorigenesis often implies the metabolic switch from oxidative phosphorylation to glycolysis which provides metabolic intermediates to sustain cell proliferation. The aim of our study is to elucidate the role of ATM in the regulation of glucose metabolism in oncogene-driven cancer cells and to test whether ATM may be a suitable target for anticancer therapy. Methods: Two oncogene-driven NSCLC cell lines, namely H1975 and H1993 cells, were treated with ATM inhibitor, KU55933, alone or in combination with oncogene driver inhibitors, WZ4002 or crizotinib. Key glycolytic enzymes, mitochondrial complex subunits (OXPHOS), cyclin D1, and apoptotic markers were analyzed by Western blotting. Drug-induced toxicity was assessed by MTS assay using stand-alone or combined treatment with KU55933 and driver inhibitors. Glucose consumption, pyruvate, citrate, and succinate levels were also analyzed in response to KU55933 treatment. Both cell lines were transfected with ATM-targeted siRNA or non-targeting siRNA and then exposed to treatment with driver inhibitors. Results: ATM inhibition deregulates and inhibits glucose metabolism by reducing HKII, p-PKM2Tyr105, p-PKM2Ser37, E1α subunit of pyruvate dehydrogenase complex, and all subunits of mitochondrial complexes except ATP synthase. Accordingly, glucose uptake and pyruvate concentrations were reduced in response to ATM inhibition, whereas citrate and succinate levels were increased in both cell lines indicating the supply of alternative metabolic substrates. Silencing of ATM resulted in similar changes in glycolytic cascade and OXPHOS levels. Furthermore, the driver inhibitors amplified the effects of ATM downregulation on glucose metabolism, and the combined treatment with ATM inhibitors enhanced the cytotoxic effect of driver inhibitors alone by increasing the apoptotic response. Conclusions: Inhibition of ATM reduced both glycolytic enzymes and OXPHOS levels in oncogene-driven cancer cells and enhanced apoptosis induced by driver inhibitors thus highlighting the possibility to use ATM and the driver inhibitors in combined regimens of anticancer therapy in vivo