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Recent evidences highlight the importance of mitochondria-nucleus communication for the clinical phenotype of oxidative phosphorylation (OXPHOS) diseases. However, the participation of small non-coding RNAs (sncRNAs) in this communication has been poorly explored. We asked whether OXPHOS dysfunction alters the production of a new class of sncRNAs, mitochondria] tRNA fragments (mt tRFs), and, if so, whether mt tRFs play a physiological role and their accumulation is controlled by the action of mt tRNA modification enzymes. To address these questions, we used a cybrid model of MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), an OXPHOS disease mostly caused by mutation m.3243A > G in the mitochondrial tRNA(Leu(UUR)) gene. High-throughput analysis of small-RNA-Seq data indicated that m.3243A > G significantly changed the expression pattern of mt tRFs. A functional analysis of potential mt tRFs targets (performed under the assumption that these tRFs act as miRNAs) indicated an association with processes that involve the most common affected tissues in MELAS. We present evidences that mt tRFs may be biologically relevant, as one of them (mt i-tRF GluUUC), likely produced by the action of the nuclease Dicer and whose levels are Ago2 dependent, down-regulates the expression of mitochondria] pyruvate carrier 1 (MPC1), promoting the build-up of extracellular lactate. Therefore, our study underpins the idea that retrograde signaling from mitochondria is also mediated by mt tRFs. Finally, we show that accumulation of mt i-tRF GluUUC depends on the modification status of mt tRNAs, which is regulated by the action of stress-responsive miRNAs on mt tRNA modification enzymes.

The pathomechanisms underlying oxidative phosphorylation (OXPHOS) diseases are not well-understood, but they involve maladaptive changes in mitochondria-nucleus communication. Many studies on the mitochondria nucleus cross-talk triggered by mitochondrial dysfunction have focused on the role played by regulatory proteins, while the participation of miRNAs remains poorly explored. MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) is mostly caused by mutation m.3243A > G in mitochondrial tRNA(Leu(UUR))) gene. Adverse cardiac and neurological events are the commonest causes of early death in m.3243A > G patients. Notably, the incidence of major clinical features associated with this mutation has been correlated to the level of m.3243A > G mutant mitochondrial DNA (heteroplasmy) in skeletal muscle. In this work, we used a transmitochondrial cybrid model of MELAS (100% m.3243A > G mutant mitochondrial DNA) to investigate the participation of miRNAs in the mitochondria-nucleus cross-talk associated with OXPHOS dysfunction. High-throughput analysis of small-RNA-Seq data indicated that expression of 246 miRNAs was significantly altered in MELAS cybrids. Validation of selected miRNAs, including miR-4775 and miR-218-5p, in patient muscle samples revealed miRNAs whose expression declined with high levels of mutant heteroplasmy. We show that miR-218-5p and miR-4775 are direct regulators of fetal cardiac genes such as NODAL, RHOA, ISL1 and RXRB, which are up-regulated in MELAS cybrids and in patient muscle samples with heteroplasmy above 60%. Our data clearly indicate that TGF-beta superfamily signaling and an epithelial-mesenchymal transition-like program are activated in MELAS cybrids, and suggest that down-regulation of miRNAs regulating fetal cardiac genes is a risk marker of heart failure in patients with OXPHOS diseases.

Study Objective: To compare operative and postoperative results of ovarian cortex retrieval by conventional laparoscopy (1cm umbilical site and 3 accessory 5-mm-reusable working ports) (HASS) versus single site laparoscopy (SSL). Design: Prospective cohort study. Setting: Fertility Preservation Programme at La Fe University Hospital-University of Valencia, Valencia, Spain, 2011 to 2012. Fertility Preservation Programme at La Fe University Hospital of Valencia, Valencia, Spain. Patients: Twenty-one patients with cancer (breast cancer: n = 17; Hodgkin's lymphoma: n = 3; and non-Hodgkin's lymphoma: n = 1). Intervention: Ovarian cortex retrieval either by conventional laparoscopy using an umbilical Hasson port and 3 accessory ports (HASS group: n = 11) or by SSL (SSL group: n = 10). Measurements and Main Results: Operative length, blood loss, postoperative pain (visual analog scale for pain at 6, 24, and 48 hours), need of additional analgesia, quality of life (European Quality of Life-5 Dimensions), cosmesis of the scar, and patient's self-perception were assessed at 24 and 48 hours and 3 months after surgery. Baseline characteristics were similar between groups. Estimated blood loss, operative length, and postoperative pain did not differ between groups. The start of chemotherapy was not delayed in either group, and cosmesis and image self-perception were also similar. Conclusion: The SSL approach can be considered a safe option compared with the classic multisite approach. (C) 2015 AAGL. All rights reserved.

MnmE is an evolutionarily conserved, three domain GTPase involved in tRNA modification. In contrast to Ras proteins, MnmE exhibits a high intrinsic GTPase activity and requires GTP hydrolysis to be functionally active. Its G domain conserves the GTPase activity of the full protein, and thus, it should contain the catalytic residues responsible for this activity. In this work, mutational analysis of all conserved arginine residues of the MnmE G-domain indicates that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis. In addition, we show that residues in the G2 motif (249GTTRD253), which resides in the switch I region, are not important for GTP binding but play some role in stabilizing the transition state, specially Gly249 and Thr251. On the other hand, G2 mutations leading to a minor loss of the GTPase activity result in a non-functional MnmE protein. This indicates that GTP hydrolysis is a required but non-sufficient condition so that MnmE can mediate modification of tRNA. The conformational change of the switch I region associated with GTP hydrolysis seems to be crucial for the function of MnmE, and the invariant threonine (Thr251) of the G2 motif would be essential for such a change, because it cannot be substituted by serine. MnmE defects result in impaired growth, a condition that is exacerbated when defects in other genes involved in the decoding process are simultaneously present. This behavior is reminiscent to that found in yeast and stresses the importance of tRNA modification for gene expression.

The Escherichia coli MnmE protein is a three-domain protein that exhibits a very high intrinsic GTPase activity and low affinity for GTP and GDP. The middle GTPase domain, when isolated, conserves the high intrinsic GTPase activity of the entire protein, and the C-terminal domain contains the only cysteine residue present in the molecule. MnmE is an evolutionarily conserved protein that, in E. coli, has been shown to control the modification of the uridine at the wobble position of certain tRNAs. Here we examine the biochemical and functional consequences of altering amino acid residues within conserved motifs of the GTPase and C-terminal domains of MnmE. Our results indicate that both domains are essential for the MnmE tRNA modifying function, which requires effective hydrolysis of GTP. Thus, it is shown for the first time that a confirmed defect in the GTP hydrolase activity of MnmE results in the lack of its tRNA modifying function. Moreover, the mutational analysis of the GTPase domain indicates that MnmE is closer to classical GTPases than to GTP-specific metabolic enzymes. Therefore, we propose that MnmE uses a conformational change associated with GTP hydrolysis to promote the tRNA modification reaction, in which the C-terminal Cys may function as a catalytic residue. We demonstrate that point mutations abolishing the tRNA modifying function of MnmE confer synthetic lethality, which stresses the importance of this function in the mRNA decoding process.

The first phases of the development of pines could be affected by weed competition. The use of herbicides is fundamental for weed control but herbicide treatment must be safe for pines. This work study the effect of two herbicides, hexazinone and simazine, over three pine species Pinus halepensis, P. nigra and P. pinaster by means of measures of the fast phase of the chlorophyll fluorescence induction kinetics. The study is conducted in early caulinars leaves of pine treated for 48 hours with doses of 2 and 4 ppm of each herbicide. Herbicide treatment and recovery of plants was done under hydroponics conditions in climatic chamber. P. nigra was more tolerant than P. pinaster and P. halepensis to hexazinone. There were no differences between species in simazine response. The three pine species show better recovery of photosynthetic activity in hexazinone treatments than in simazine treatments, indicating better tolerance of tree to hexazinone than to simazine., Las primeras fases del desarrollo de pinos pueden verse afectadas por la competencia de las malas hierbas, siendo la utilización de herbicidas fundamental para su control, aunque este tratamiento no debe afectar al pino. En este trabajo, se estudia el efecto de dos herbicidas simazina y hexazinona en tres especies de pino, Pinus halepensis, P. nigra y P. pinaster, mediante medidas de la fase rápida de la cinética de inducción de fluorescencia clorofílica. El estudio se lleva a cabo sobre las primeras hojas caulinares de pino, sometidos a tratamiento con dosis de 2 y 4 ppm de cada uno de los herbicidas durante 48 horas. El tratamiento y la posterior recuperación, se lleva a cabo en cultivo hidropónico en cámara climática. En los tratamientos con hexazinona, P. nigra ha resultado más tolerante que P. pinaster y P. halepensis. No se han encontrado diferencias entre especies en la respuesta a simazina. En las tres especies de pino se produce una mayor recuperación de la actividad fotosintética, lo que indica una mayor tolerancia del árbol, a hexazinona que a simazina.

The first phases of the development of pines could be affected by weed competition. The use of herbicides is fundamental for weed control but herbicide treatment must be safe for pines. This work study the effect of two herbicides, hexazinone and simazine, over three pine species Pinus halepensis, P. nigra and P. pinaster by means of measures of the fast phase of the chlorophyll fluorescence induction kinetics. The study is conducted in early caulinars leaves of pine treated for 48 hours with doses of 2 and 4 ppm of each herbicide. Herbicide treatment and recovery of plants was done under hydroponics conditions in climatic chamber. P. nigra was more tolerant than P. pinaster and P. halepensis to hexazinone. There were no differences between species in simazine response. The three pine species show better recovery of photosynthetic activity in hexazinone treatments than in simazine treatments, indicating better tolerance of tree to hexazinone than to simazine. Las primeras fases del desarrollo de pinos pueden verse afectadas por la competencia de las malas hierbas, siendo la utilización de herbicidas fundamental para su control, aunque este tratamiento no debe afectar al pino. En este trabajo, se estudia el efecto de dos herbicidas simazina y hexazinona en tres especies de pino, Pinus halepensis, P. nigra y P. pinaster, mediante medidas de la fase rápida de la cinética de inducción de fluorescencia clorofílica. El estudio se lleva a cabo sobre las primeras hojas caulinares de pino, sometidos a tratamiento con dosis de 2 y 4 ppm de cada uno de los herbicidas durante 48 horas. El tratamiento y la posterior recuperación, se lleva a cabo en cultivo hidropónico en cámara climática. En los tratamientos con hexazinona, P. nigra ha resultado más tolerante que P. pinaster y P. halepensis. No se han encontrado diferencias entre especies en la respuesta a simazina. En las tres especies de pino se produce una mayor recuperación de la actividad fotosintética, lo que indica una mayor tolerancia del árbol, a hexazinona que a simazina.

The use of herbicides in croplands forestation is needed in order to control weed competition for water, light and nutrients with tree seedlings. As selectivity of our forest species to herbicides and mode of aplication are scarcely known, we have studied the effect of three herbicides, hexazinone, pendimethalin and simazine on emergency and growth of Pinus halepensis and P. pinaster sowed in Forest-Pot during November and February inside a non-climatized greenhouse. Treatments were made in pine preemergency, one week after each sowing. Mean time emergency of two species of pine was minor in February than November sowing, however, thermic integrals (sumatory of temperatures > 5 °C since sowing time until mean time emergency) are not differents. Herbicide treatments did not influence emergency of pines, but later some of then caused mortality in seedlings. Hexazinone at doses of 1.5 kg a.i./ha caused mortalities of 84 % and 71 % in P. halepensis seedlings belonging to November and February sowings, respectively, and in P. pinaster, 93 % in seedlings of both sowings. Hexazinone at doses of 0.75 kg a.i/ha caused mortalities of 31 % and 18 % in P. halepensis seedlings belonging to November and February sowings, respectively, and 49 % and 61 %, in P. pinaster, respectively. Pendimethalin at 2 kg m.a./ha did not cause mortality in pine seedlings and simazine at 3 kg m.a./ha was tolerated for two pine species in February sowings, but in November sowings caused mortalities of 29 % in P. halepensis and 32 % in P. pinaster., El empleo de herbicidas en la forestación de tierras de cultivo se hace necesario para controlar la invasión de malas hierbas que compiten por agua, luz y nutrientes con los árboles que se intentan implantar. Como la selectividad de nuestras especies arbóreas a los herbicidas y su modo de aplicación son poco conocidos, se ha estudiado el efecto de tres herbicidas, hexazinona, pendimetalina y simazina sobre la emergencia y crecimiento de Pinus halepensis y P. pinaster que fueron sembrados en noviembre y en febrero en bandejas Forest-Pot. Los tratamientos se hicieron en preemergencia de los pinos, una semana después de cada siembra. El tiempo medio de emergencia de las dos especies fue menor en la siembra de febrero que en la de noviembre; sin embargo, no hubo diferencias en las integrales térmicas necesarias para alcanzar la emergencia. Los tratamientos herbicidas no afectaron a la germinación de los pinos, pero algunos causaron mortalidad cuando se alcanzó el estado de plántula. Dosis de 1,5 kg m.a./ha de hexazinona provocaron mortalidades del 84 % y 71 % en P. halepensis procedentes de las siembras de noviembre y febrero, respectivamente, y en P. pinaster del 93 % en ambas siembras. Este mismo herbicida a dosis de 0,75 kg m.a/ha determinó mortalidades del 31 % y 18 % en P. halepensis en las siembras de noviembre y febrero, respectivamente, y del 49 % y 61 %, en P. pinaster, respectivamente. Pendimetalina a 2 kg m.a./ha no causó mortalidad y simazina a 3 kg m.a./ha fue tolerada por las dos especies en las siembras de febrero, pero en las de noviembre provocó mortalidades del 29 % en P. halepensis y del 32 % en P. pinaster.

The use of herbicides in croplands forestation is needed in order to control weed competition for water, light and nutrients with tree seedlings. As selectivity of our forest species to herbicides and mode of aplication are scarcely known, we have studied the effect of three herbicides, hexazinone, pendimethalin and simazine on emergency and growth of Pinus halepensis and P. pinaster sowed in Forest-Pot during November and February inside a non-climatized greenhouse. Treatments were made in pine preemergency, one week after each sowing. Mean time emergency of two species of pine was minor in February than November sowing, however, thermic integrals (sumatory of temperatures > 5 °C since sowing time until mean time emergency) are not differents. Herbicide treatments did not influence emergency of pines, but later some of then caused mortality in seedlings. Hexazinone at doses of 1.5 kg a.i./ha caused mortalities of 84 % and 71 % in P. halepensis seedlings belonging to November and February sowings, respectively, and in P. pinaster, 93 % in seedlings of both sowings. Hexazinone at doses of 0.75 kg a.i/ha caused mortalities of 31 % and 18 % in P. halepensis seedlings belonging to November and February sowings, respectively, and 49 % and 61 %, in P. pinaster, respectively. Pendimethalin at 2 kg m.a./ha did not cause mortality in pine seedlings and simazine at 3 kg m.a./ha was tolerated for two pine species in February sowings, but in November sowings caused mortalities of 29 % in P. halepensis and 32 % in P. pinaster. El empleo de herbicidas en la forestación de tierras de cultivo se hace necesario para controlar la invasión de malas hierbas que compiten por agua, luz y nutrientes con los árboles que se intentan implantar. Como la selectividad de nuestras especies arbóreas a los herbicidas y su modo de aplicación son poco conocidos, se ha estudiado el efecto de tres herbicidas, hexazinona, pendimetalina y simazina sobre la emergencia y crecimiento de Pinus halepensis y P. pinaster que fueron sembrados en noviembre y en febrero en bandejas Forest-Pot. Los tratamientos se hicieron en preemergencia de los pinos, una semana después de cada siembra. El tiempo medio de emergencia de las dos especies fue menor en la siembra de febrero que en la de noviembre; sin embargo, no hubo diferencias en las integrales térmicas necesarias para alcanzar la emergencia. Los tratamientos herbicidas no afectaron a la germinación de los pinos, pero algunos causaron mortalidad cuando se alcanzó el estado de plántula. Dosis de 1,5 kg m.a./ha de hexazinona provocaron mortalidades del 84 % y 71 % en P. halepensis procedentes de las siembras de noviembre y febrero, respectivamente, y en P. pinaster del 93 % en ambas siembras. Este mismo herbicida a dosis de 0,75 kg m.a/ha determinó mortalidades del 31 % y 18 % en P. halepensis en las siembras de noviembre y febrero, respectivamente, y del 49 % y 61 %, en P. pinaster, respectivamente. Pendimetalina a 2 kg m.a./ha no causó mortalidad y simazina a 3 kg m.a./ha fue tolerada por las dos especies en las siembras de febrero, pero en las de noviembre provocó mortalidades del 29 % en P. halepensis y del 32 % en P. pinaster.