An internal ribosome entry site element directs the synthesis of the 80 kDa isoforms of protein 4.1R

<p>Abstract</p> <p>Background</p> <p>In red blood cells, protein 4.1 (4.1R) is an 80 kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane through its FERM domain. While the expression pattern of 4.1R in mature red cells is relatively simple, a rather complex array of 4.1R protein isoforms varying in N-terminal extensions, internal sequences and subcellular locations has been identified in nucleated cells. Among these, 135 kDa and 80 kDa isoforms have different N-terminal extensions and are expressed either from AUG1- or AUG2-containing mRNAs, respectively. These two types of mRNAs, varying solely by presence/absence of 17 nucleotides (nt) which contain the AUG1 codon, are produced by alternative splicing of the 4.1R pre-mRNA. It is unknown whether the 699 nt region comprised between AUG1 and AUG2, kept as a 5' untranslated region in AUG2-containing mRNAs, plays a role on 4.1R mRNA translation.</p> <p>Results</p> <p>By analyzing the <it>in vitro </it>expression of a panel of naturally occurring 4.1R cDNAs, we observed that all AUG1/AUG2-containing cDNAs gave rise to both long, 135 kDa, and short, 80 kDa, 4.1R isoforms. More importantly, similar results were also observed in cells transfected with this set of 4.1R cDNAs. Mutational studies indicated that the short isoforms were not proteolytic products of the long isoforms but products synthesized from AUG2. The presence of a cryptic promoter in the 4.1R cDNA sequence was also discounted. When a 583 nt sequence comprised between AUG1 and AUG2 was introduced into bicistronic vectors it directed protein expression from the second cistron. This was also the case when ribosome scanning was abolished by introduction of a stable hairpin at the 5' region of the first cistron. Deletion analysis of the 583 nt sequence indicated that nucleotides 170 to 368 are essential for expression of the second cistron. The polypyrimidine tract-binding protein bound to the 583 nt active sequence but not to an inactive 3'-fragment of 149 nucleotides.</p> <p>Conclusion</p> <p>Our study is the first demonstration of an internal ribosome entry site as a mechanism ensuring the production of 80 kDa isoforms of protein 4.1R. This mechanism might also account for the generation of 60 kDa isoforms of 4.1R from a downstream AUG3. Our results reveal an additional level of control to 4.1R gene expression pathways and will contribute to the understanding of the biology of proteins 4.1R and their homologues, comprising an ample family of proteins involved in cytoskeletal organization.</p

Reprogramación Metabólica en Cáncer: Caracterización de un modelo celular knock out para genoma mitocondrial como fenotipo metabólico extremo

Durante la agresiva fase proliferativa en tumores primarios, las células tumorales deben adaptarse y evolucionar frente a unas condiciones extremas, en un nicho celular hipóxico y con una disponibilidad mínima de nutrientes (1). Incluso en estas condiciones, las células son capaces de evolucionar y sobrevivir gracias a un proceso extremadamente eficiente de reprogramación metabólica (2), manteniendo una elevada tasa anabólica-proliferativa (3). Para comprobar el nivel de eficiencia de la reprogramación metabólica, generamos una línea celular de osteosarcoma knockout para el genoma mitocondrial (ρ0), como modelo de fenotipo metabólico extremo al carecer de fosforilación oxidativa mitocondrial. En primer lugar, se comprobó que la línea ρ0 mantiene la viabilidad celular y una capacidad proliferativa similar a la línea parental 143B. Para caracterizar la reprogramación metabólica que permite a las células ρ0 mantener la homeostasis y capacidad proliferativa, se realizó un estudio metabolómico comparativo con la línea parental 143B. Mediante análisis isotopomérico de flujo metabólico con 13C-glucosa y utilizando cultivos selectivos para glucosa, glutamina y piruvato, descubrimos que  las células ρ0 no presentan un ciclo de ácidos tricarboxílicos (TCA) activo. Sin embargo, este déficit en TCA se compensa mediante una fuerte dependencia metabólica de piruvato hacia la vía de transaminación, lo cual les permite obtener aminoácidos no esenciales, y compensar los niveles de NAD/NADH+ que no pueden obtener por la ausencia de metabolismo oxidativo mitocondrial. Del mismo modo, se observó que la glutamina puede jugar un papel importante en la supervivencia de ρ0, mediante su reprogramación hacia síntesis de nucleótidos, aminoácidos y lípidos. Por el contrario, la línea 143B presentó un metabolismo autosuficiente mediante la ruta glucolítica y el aporte de intermediarios del TCA, que le permite adaptarse a medios sin glutamina o piruvato. En conclusión, la línea celular ρ0 representa un fenotipo metabólico extremo. Mediante una eficiente reprogramación metabólica consiguen adaptarse y superar la presión selectiva que supone la ausencia de genoma mitocondrial, y por tanto de metabolismo oxidativo mitocondrial. El fenotipo ρ0 es por tanto un modelo experimental con gran potencial para el estudio de la reprogramación metabólica tumoral.   

Overexpression of the X-Linked Inhibitor of Apoptosis Protein (XIAP) in Neurons Improves Cell Survival and the Functional Outcome after Traumatic Spinal Cord Injury

Mechanical trauma to the spinal cord causes extensive neuronal death, contributing to the loss of sensory-motor and autonomic functions below the injury location. Apoptosis affects neurons after spinal cord injury (SCI) and is associated with increased caspase activity. Cleavage of X-linked inhibitor of apoptosis protein (XIAP) after SCI may contribute to this rise in caspase activity. Accordingly, we have shown that the elevation of XIAP resulted in increased neuronal survival after SCI and improved functional recovery. Therefore, we hypothesise that neuronal overexpression of XIAP can be neuroprotective after SCI with improved functional recovery. In line with this, studies of a transgenic mice with overexpression of XIAP in neurons revealed that higher levels of XIAP after spinal cord trauma favours neuronal survival, tissue preservation, and motor recovery after the spinal cord trauma. Using human SH-SY5Y cells overexpressing XIAP, we further showed that XIAP reduced caspase activity and apoptotic cell death after pro-apoptotic stimuli. In conclusion, this study shows that the levels of XIAP expression are an important factor for the outcome of spinal cord trauma and identifies XIAP as an important therapeutic target for alleviating the deleterious effects of SCI

Abundancia de depredadores en setos de frutales en el Parc Agrari del Baix Llobregat

Se ha estudiado la abundancia de la entomofauna auxiliar asociada a los setos de un arboretum de frutales ecológicos perteneciente al "Parc Agrari del Baix Llobregat", cuya gestión técnica está realizada por la "ADV de Fruita del Baix Llobregat. Las especies vegetales estudiadas fueron lentisco (Pistacia Lentiscus L.), genista (Genista sp.), durillo (Viburnum tinus L.), adelfa (Nerium oleander L.), madroño (Arbutus unedo L.), salvia (Salvia sp.), romero (Rosmarinus oficinalis L.), así como tres tipos de macizos de aromáticas con salvia, romero, espliego (Lavandula sp.) o tomillo (Thymus oficinalis L.) como principales especies. El muestreo se ha efectuado con aspiración (soplador ECHO PB 46-LN,+ kit aspirador). El análisis de las aspiraciones muestran como resultado un total de 67.717 artrópodos, de los cuales 14.236 pueden considerarse depredadores generalistas (un 21% del total). Arácnidos y hormigas son con mucho los más habituales, con una población identificada de 6.934 y 4.999 individuos, respectivamente. Del resto de depredadores, los más numerosos fueron los coccinélidos con 618 individuos, seguidos de heterópteros con 557 (nábidos, míridos y antocóridos, por este orden), dípteros con 290 (de los cuáles 283 eran cecidómidos) y neurópteros con 224 (fundamentalmente crisópidos, con 199). Existen variaciones notables de la abundancia entre las especies de setos estudiadas. Así, contando el conjunto de depredadores, salvia, madroño y aromáticas destacan del resto, mientras que baladre y genista son las de menor cantidad de depredadores. Si dejamos aparte hormigas, serían salvia, aromáticas y durillo los mejores. En neurópteros destacan lentisco y madroño, mientras que en coccinélidos, dípteros, heterópteros y arácnidos depredadores salvia y aromáticas

Efecto de cubiertas vegetales permanentes en la fertilidad del cultivo de cítricos ecológicos

Se han estudiado diferentes cubiertas vegetales permanentes en mandarinos ecológicos y convencionales de Alzira, en suelo arenoso, para comprobar su comportamiento fertilizante y su crecimiento. En plantación joven, con aspersión, se sembró alfalfa (Medicago sativa), sola y junto a ray-grass inglés (Lolium perenne), trévoles (Trifolium subterraneum+T. repens) y mielgas (Medicago rugosa+M. truncatula+M. polymorpha). En Clemenules adultos a goteo se estudiaron las silvestres en la conducción ecológica, y el no laboreo con herbicidas en la convencional. La evolución muestra como trévoles y mielgas degeraron muy deprisa, dando paso a silvestres (grama -Cynodon dactylon- en verano, y Bromus spp. y otras en invierno), descartándolos como coberturas en estas condiciones. La alfalfa es la que mejor ha resistido la competencia de las hierbas en condiciones de insolación alta y aspersión. En biomasa y cobertura no se han encontrado diferencias entre alfalfa y grama, mientras que las demás eran menores, sobre todo en los adultos, por su sombreado

Enalapril reduces proliferation and hyaluronic acid release in orbital fibroblasts

BACKGROUND: Orbital fibroblast proliferation and hyaluronic acid (HA) release are responsible for some of the clinical features of Graves' ophthalmopathy (GO). Thus, inhibition of these processes may be a possible therapeutic approach to this syndrome. Enalapril, a widely used antihypertensive drug, was found to have some inhibitory actions on fibroblast proliferation in cheloid scars in vivo, based on which we investigated its effects in primary cultures of orbital fibroblasts from GO patients and control subjects. METHODS: Primary cultures of GO and control fibroblasts were treated with enalapril or with a control compound (lisinopril). Cell proliferation assays, lactate dehydrogenase release assays (as a measure of cell necrosis), apoptosis assays, and measurement of HA in the cell media were performed. RESULTS: Enalapril significantly reduced cell proliferation in both GO and control fibroblasts. Because enalapril did not affect cell necrosis and apoptosis, we concluded that its effects on proliferation reflected an inhibition of cell growth and/or a delay in cell cycle. Enalapril significantly reduced HA concentrations in the media from both GO and control fibroblasts. CONCLUSIONS: Enalapril has antiproliferative and HA suppressing actions in both GO and control fibroblasts. Clinical studies are needed to investigate whether enalapril has any effects in vivo in patients with GO

Wide Range Applications of Spirulina: From Earth to Space Missions

Spirulina is the most studied cyanobacterium species for both pharmacological applications and the food industry. The aim of the present review is to summarize the potential benefits of the use of Spirulina for improving healthcare both in space and on Earth. Regarding the first field of application, Spirulina could represent a new technology for the sustainment of long-duration manned missions to planets beyond the Lower Earth Orbit (e.g., Mars); furthermore, it could help astronauts stay healthy while exposed to a variety of stress factors that can have negative consequences even after years. As far as the second field of application, Spirulina could have an active role in various aspects of medicine, such as metabolism, oncology, ophthalmology, central and peripheral nervous systems, and nephrology. The recent findings of the capacity of Spirulina to improve stem cells mobility and to increase immune response have opened new intriguing scenarios in oncological and infectious diseases, respectively

Reversal of mitochondrial malate dehydrogenase 2 enables anaplerosis via redox rescue in respiration-deficient cells

Inhibition of the electron transport chain (ETC) prevents the regeneration of mitochondrial NAD+, resulting in cessation of the oxidative tricarboxylic acid (TCA) cycle and a consequent dependence upon reductive carboxylation for aspartate synthesis. NAD+ regeneration alone in the cytosol can rescue the viability of ETC-deficient cells. Yet, how this occurs and whether transfer of oxidative equivalents to the mitochondrion is required remain unknown. Here, we show that inhibition of the ETC drives reversal of the mitochondrial aspartate transaminase (GOT2) as well as malate and succinate dehydrogenases (MDH2 and SDH) to transfer oxidative NAD+ equivalents into the mitochondrion. This supports the NAD+-dependent activity of the mitochondrial glutamate dehydrogenase (GDH) and thereby enables anaplerosis—the entry of glutamine-derived carbon into the TCA cycle and connected biosynthetic pathways. Thus, under impaired ETC function, the cytosolic redox state is communicated into the mitochondrion and acts as a rheostat to support GDH activity and cell viability.P.A.-M was supported by a Marie Skłodowska-Curie Actions individual fellowship and the Beug Foundation. A.V. was supported by Fonds Wetenschappelijk Onderzoek (FWO Vlaanderen). J.E.-H. was supported by an MRC studentship. J.C.A was supported by a Cancer Research UK Career Development Fellowship (C47559/A16243). S.-M.F. acknowledges funding from the European Research Council under the ERC Consolidator grant agreement no. 771486–MetaRegulation, FWO Projects, Fonds Baillet Latour, KU Leuven-FTBO/Internal Funding, Stichting Tegen Kanker and the King Baudouin Foundation. Work in the A.J.F. group was supported by a Wellcome Trust-ISSF grant, funding from Barts Charity (MGU0404), and by a Cancer Research UK Centre Grant to Barts Cancer Institute (C355/A25137). The illustrations in the graphical abstract and Figure 5F were created using BioRender.com

Evaluation of Poly(N-Ethyl Pyrrolidine Methacrylamide) (EPA) and Derivatives as Polymeric Vehicles for miRNA Delivery to Neural Cells

MicroRNAs (miRNAs) are endogenous, short RNA oligonucleotides that regulate the expression of hundreds of proteins to control cells' function in physiological and pathological conditions. miRNA therapeutics are highly specific, reducing the toxicity associated with off-target effects, and require low doses to achieve therapeutic effects. Despite their potential, applying miRNA-based therapies is limited by difficulties in delivery due to their poor stability, fast clearance, poor efficiency, and off-target effects. To overcome these challenges, polymeric vehicles have attracted a lot of attention due to their ease of production with low costs, large payload, safety profiles, and minimal induction of the immune response. Poly(N-ethyl pyrrolidine methacrylamide) (EPA) copolymers have shown optimal DNA transfection efficiencies in fibroblasts. The present study aims to evaluate the potential of EPA polymers as miRNA carriers for neural cell lines and primary neuron cultures when they are copolymerized with different compounds. To achieve this aim, we synthesized and characterized different copolymers and evaluated their miRNA condensation ability, size, charge, cytotoxicity, cell binding and internalization ability, and endosomal escape capacity. Finally, we evaluated their miRNA transfection capability and efficacy in Neuro-2a cells and rat primary hippocampal neurons. The results indicate that EPA and its copolymers, incorporating β-cyclodextrins with or without polyethylene glycol acrylate derivatives, can be promising vehicles for miRNA administration to neural cells when all experiments on Neuro-2a cells and primary hippocampal neurons are considered together.This research was supported by the Council of Education, Culture and Sports of the Regional Government of Castilla La Mancha (Spain) and Co-financed by the European Union (FEDER) “A way to make Europe” (project references SBPLY/17/000376 and SBPLY/21/180501/000097) and by the Ministerio de Ciencia, Innovación y Universidades (RTI2018-096328-B-I00). Altea Soto was funded by the Council of Education, Culture and Sports of the Regional Government of Castilla La Mancha (Spain) M. Asunción Barreda-Manso is funded by the Council of Health of the Regional Government of Castilla La Mancha (Spain), through: “Convocatoria de Ayudas Regionales a la Investigación en Biomedicina y Ciencias de la Salud” (II-2020_05). Irene Novillo Algaba is funded by the Next Generation Funds of the European Union through the “Programa Investigo”.Peer reviewe