Very Rare Complementation between Mitochondria Carrying Different Mitochondrial DNA Mutations Points to Intrinsic Genetic Autonomy of the Organelles in Cultured Human Cells

In the present work, a large scale investigation was done regarding the capacity of cultured human cell lines (carrying in homoplasmic form either the mitochondrial tRNALys A8344G mutation associated with the myoclonic epilepsy and ragged red fiber (MERRF) encephalomyopathy or a frameshift mutation, isolated in vitro, in the gene for the ND4 subunit of NADH dehydrogenase) to undergo transcomplementation of their recessive mitochondrial DNA (mtDNA) mutations after cell fusion. The presence of appropriate nuclear drug resistance markers in the two cell lines allowed measurements of the frequency of cell fusion in glucose-containing medium, non-selective for respiratory capacity, whereas the frequency of transcomplementation of the two mtDNA mutations was determined by growing the same cell fusion mixture in galactose-containing medium, selective for respiratory competence. Transcomplementation of the two mutations was revealed by the re-establishment of normal mitochondrial protein synthesis and respiratory activity and by the relative rates synthesis of two isoforms of the ND3 subunit of NADH dehydrogenase. The results of several experiments showed a cell fusion frequency between 1.4 and 3.4% and an absolute transcomplementation frequency that varied between 1.2 × 10^-5 and 5.5 × 10^-4. Thus, only 0.3-1.6% of the fusion products exhibited transcomplementation of the two mutations. These rare transcomplementing clones were very sluggish in developing, grew very slowly thereafter, and showed a substantial rate of cell death (22-28%). The present results strongly support the conclusion that the capacity of mitochondria to fuse and mix their contents is not a general intrinsic property of these organelles in mammalian cells, although it may become activated in some developmental or physiological situations

Spatial variability in floodplain sedimentation: the use of generalized linear mixed-effects models

14 Pag., 4 Tabl., 5 Fig. © Author(s) 2010. This work is distributed under the Creative Commons Attribution 3.0 License.Sediment, Total Organic Carbon (TOC) and total nitrogen (TN) accumulation during one overbank flood (1.15 y return interval) were examined at one reach of the Middle Ebro River (NE Spain) for elucidating spatial patterns. To achieve this goal, four areas with different geomorphological features and located within the study reach were examined by using artificial grass mats. Within each area, 1 m2 study plots consisting of three pseudo-replicates were placed in a semi-regular grid oriented perpendicular to the main channel. TOC, TN and Particle-Size composition of deposited sediments were examined and accumulation rates estimated. Generalized linear mixed-effects models were used to analyze sedimentation patterns in order to handle clustered sampling units, specific-site effects and spatial self-correlation between observations. Our results confirm the importance of channel-floodplain morphology and site micro-topography in explaining sediment, TOC and TN deposition patterns, although the importance of other factors as vegetation pattern should be included in further studies to explain small-scale variability. Generalized linear mixed-effect models provide a good framework to deal with the high spatial heterogeneity of this phenomenon at different spatial scales, and should be further investigated in order to explore its validity when examining the importance of factors such as flood magnitude or suspended sediment concentration.Field works were funded by the Department of Environmental Science, Technology and University – Government of Aragon (Research group E-61 on Ecological Restoration)– and MEC (CGL2005-07059). The Spanish Research Council (CSIC) granted Álvaro Cabezas through the I3P program(I3P-EPD2003-2), which was financed by European Social Funds (UE). Research of M. Angulo-Martínez is supported by a JAE-Predoc Research Grant from the Spanish National Research Council (Consejo Superior de Investigaciones Científicas – CSIC).Peer reviewe

Endemic and epidemic Acinetobacter baumannii clones: a twelve-year study in a tertiary care hospital

BACKGROUND: Nosocomial outbreaks of multidrug-resistant Acinetobacter baumannii are of worldwide concern. Using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and multiple locus variable number tandem repeat sequence (VNTR) analysis (MLVA), the present work examines the genetic diversity of the endemic and epidemic A. baumannii clones isolated in a single hospital over a twelve-year period. RESULTS: PFGE analysis of 405 A. baumannii-calcoaceticus complex isolates detected 15 A. baumannii endemic/epidemic PFGE types (EE1 to EE15) that grouped into five clusters: EE1-EE8, EE9, EE10, EE11 and EE12-EE15. The MLST sequence type (ST) distributions were: international clone II (ST-2) 60%, international clone III (ST-3) 26.7%, ST-15 6.7%, and ST-80 6.7%. MLVA-8Orsay returned 17 allelic profiles. The large (L) VNTR marker profiles were fully concordant with the detected STs, and concordant with 14 up to 15 PFGE types. Imipenem resistance was detected in five PFGE types; the prevalence of the bla OXA-58-like and bla OXA-40-like genes was 60% and 40% respectively. CONCLUSIONS: PFGE proved to be a vital tool for analysis of the temporal and spatial distribution of the clones. MLST and the VNTR L-markers grouped the isolates into clonal clusters. The wide diversity of MLVA small (S)-markers, however, did not permit clustering. The present results demonstrate the persistence of several endemic PFGE types in the hospital, the involvement of some of them in outbreaks, and the inter hospital transmission of extensively drug-resistant ST-15 and ST-80.S

Inverse problems in high pressure processes and food engineering

Depto. de Análisis Matemático y Matemática AplicadaInstituto de Matemática Interdisciplinar (IMI)Fac. de Ciencias MatemáticasTRUEpu

Plataformas emergentes para monitorear la ocurrencia y amenazas de aves marinas críticamente en peligro: El albatros de Galápagos en Chile y en el Pacífico Sudeste

Long-lived seabird species such as albatrosses and petrels, which are widely distributed at sea a nd have small populations are threatened globally by bycatch in fisheries. Among these, the waved albatross Phoebastria irrorata is scarcely detected in the Exclusive Economic Zones (EEZs) of countries beyond its traditional distribution throughout the Southeast Pacific. In this article, in situ distributional records on the waved albatross obtained from non-systematic (pelagic birdwatching) and systematic (fishery monitoring) records were compiled. Occurrence of sightings was carried out in relation to recording traits as presence/absence of breeding period and El Niño proxies such as the Southern Oscillation Index (SOI), among others. A total of 13 sightings of waved albatrosses was described which 76.9% of sightings were related to non-breeding period (December to April). Records indicated waved albatrosses reached 4,391 km south of the Galapagos Islands (38°S) and 920 km west of the South American coast (79°W). The waved albatross was associated with industrial pelagic longline and demersal trawl fisheries, and although there were no records of bycatch, the observation of individuals feeding on discards and offal suggests a potential risk of mortality for this species. 53.8% of sightings were during positive readings of the Southern Oscillation Index (SOI), representing colder conditions than neutral El Niño Southern Oscillation (ENSO).Las especies de aves marinas longevas, tales como albatros y petreles ampliamente distribuidos en el mar y con poblaciones reducidas, tienen a la captura incidental en pesquerías como una de las principales amenazas para su conservación. Entre estas, el albatros de Galápagos Phoebastria irrorata es escasamente detectado en las Zonas Económicas Exclusivas (ZEE) de países más allá de su distribución tradicional a lo largo del Pacífico Sudeste. En este artículo se recopilan registros distribucionales del albatros de Galápagos in situ y obtenidos desde observaciones no sistemáticas en el mar, tales como actividades de turismo (i.e., observación pelágica de aves) desde la década de 1980s en adelante. A su vez, se incorporan registros desde monitoreos sistemáticos en pesquerías de palangre y arrastre, operando entre el norte y centro-sur de Chile (Pacífico Sudeste). La ocurrencia de avistamientos fue analizada según características ambientales relacionadas a estos, como presencia/ausencia de periodo reproductivo e indicadores de eventos El Niño, tal como valores negativos en el Índice de Oscilación Sur (IOS), entre otros. Se describió un total de 13 avistamientos del albatros de Galápagos y con 76,9% de estos fuera de su periodo reproductivo (diciembre a abril). Los registros evidencian una distribución meridional del albatros de Galápagos hasta el centro-sur de Chile (38°S), alcanzando 4.391 km al sur de Islas Galápagos, así como hasta 920 km al oeste (79°O) desde la costa Sudamericana. El albatros de Galápagos fue registrado en asociación con pesquerías industriales de palangre pelágico y arrastre demersal. No hubo registros de captura incidental, aunque la observación de individuos asociados a barcos de estas pesquerías para alimentarse de descartes y eviscerados sugiere un potencial riesgo de mortalidad para esta especie. El 53,8% de los avistamientos fueron durante lecturas positivas del Índice de Oscilación Sur (IOS), representando condiciones más frías que las neutrales de El Niño Oscilación Sur (ENOS)

Patrones de abundancia de la macrofauna asociada a macroalgas marinas a largo de la Península Ibérica

macroalgae were studied on a spatial scale along the Iberian Peninsula. Nineteen stations and four dominant algae were selected (intertidal zone: Corallina elongata and Asparagopsis armata; subtidal zone: Stypocaulon scoparium and Cladostephus spongiosus). Five environmental factors were also considered (seawater temperature, conductivity, dissolved oxygen, turbidity and pH). The Atlantic coast was characterized by lower temperature and conductivity as well as higher values of oxygen and turbidity than the Mediterranean coast. A total of 106274 macrofaunal specimens were sorted and examined (68% arthropods, 27% molluscs, 4% annelids and 1% echinoderms). Crustaceans were the dominant group in all the macroalgae (ca. 80% in C. elongata and A. armata, ca. 50% in S. scoparium and C. spongiosus) followed by molluscs, which were more abundant in the subtidal algae (ca. 40%) than in intertidal ones (ca.10%). Abundance patterns of macrofauna along the Iberian Peninsula were similar in the four studied algae. Most of crustaceans belonged to the order Amphipoda, which showed high densities (>1000 ind/1000 ml algae) along the whole Peninsula; isopods showed the highest abundances in the Atlantic, while tanaids, cumaceans and decapods were more abundant in the Mediterranean. Among molluscs, gasteropods showed highest abundances along the Atlantic coasts, whereas bivalves showed higher densities along the MediterraneanSe llevó a cabo un estudio espacial de los patrones de abundancia y distribución de la macrofauna asociada a macroalgas a lo largo de la Península Ibérica. Se seleccionaron 19 estaciones y 4 algas dominantes (zona intermareal: Corallina elongata y Asparagopsis armata; zona submareal: Stypocaulon scoparium y Cladostephus spongiosus). Se consideraron también cinco variables ambientales (temperatura del agua, conductividad, oxígeno disuelto, turbidez y pH). La costa atlántica se caracterizó por valores más bajos de temperatura y conductividad, y más altos de oxígeno y turbidez. Se examinaron 106274 individuos de la macrofauna (68% artrópodos, 27% moluscos, 4% anélidos y 1% equinodermos). Los crustáceos fueron dominantes en todas las macroalgas (alrededor del 80% en C. elongata y A. armata, y en torno al 50% en S. scoparium y C. spongiosus), seguidos por los moluscos, que fueron más abundantes en el submareal (40%) que en el intermareal (10%). Los patrones de abundancia de la macrofauna a lo largo de la Península Ibérica fueron similares en las cuatro algas estudiadas. La mayoría de los crustáceos pertenecieron al orden Amphipoda, que mostró densidades muy altas (>1000 ind/1000 ml alga) en toda la Península; los isópodos mostraron las mayores densidades en el Atlántico, mientras que los tanaidáceos, cumáceos y decápodos fueron más abundantes en el Mediterráneo. Entre los moluscos, los gasterópodos mostraron abundancias mayores en el Atlántico, mientras que los bivalvos dominaron en el Mediterráneo. Teniendo en cuenta que todas las estaciones seleccionadas no tenían influencia antrópica importante, los patrones de abundancia obtenidos podrían explicarse en base a diferencias naturales en la temperatura del agua, oxígeno, conductividad y turbidez, existiendo un gradiente transicional entre taxones de aguas más cálidas (del norte de Africa y del Mediterráneo) y taxones de aguas más frías (del Mar del Norte y el Ártico)

Proteins and pathways in atrial fibrillation and atrial cardiomyopathy underlying cryptogenic stroke

Atrial fibrillation (AF) is one of the most prevalent causes of cryptogenic stroke. Also, apart from AF itself, structural and remodelling changes in the atria might be an underlying cause of cryptogenic stroke. We aimed to discover circulating proteins and reveal pathways altered in AF and atrial cardiomyopathy, measured by left atrial volume index (LAVI) and peak atrial longitudinal strain (PALS), in patients with cryptogenic stroke. An aptamer array (including 1310 proteins) was measured in the blood of 20 cryptogenic stroke patients monitored during 28 days with a Holter device as a case-control study of the Crypto-AF cohort. Protein levels were compared between patients with (n = 10) and without AF (n = 10) after stroke, and the best candidates were tested in 111 patients from the same cohort (44 patients with AF and 67 without AF). In addition, in the first 20 patients, proteins were explored according to PALS and LAVI values. Forty-six proteins were differentially expressed in AF cases. Of those, four proteins were tested in a larger sample size. Only DPP7, presenting lower levels in AF patients, was further validated. Fifty-seven proteins correlated with LAVI, and 270 correlated with PALS. NT-proBNP was common in all the discovery analyses performed. Interestingly, many proteins and pathways were altered in patients with low PALS. Multiple proteins and pathways related to AF and atrial cardiomyopathy have been revealed. The role of DPP7 as a biomarker for stroke aetiology should be further explored. Moreover, the present study may be considered hypothesis-generatin

Improving zebrafish embryo xenotransplantation conditions by increasing incubation temperature and establishing a proliferation index with ZFtool

Background Zebrafish (Danio rerio) is a model organism that has emerged as a tool for cancer research, cancer being the second most common cause of death after cardiovascular disease for humans in the developed world. Zebrafish is a useful model for xenotransplantation of human cancer cells and toxicity studies of different chemotherapeutic compounds in vivo. Compared to the murine model, the zebrafish model is faster, can be screened using high-throughput methods and has a lower maintenance cost, making it possible and affordable to create personalized therapies. While several methods for cell proliferation determination based on image acquisition and quantification have been developed, some drawbacks still remain. In the xenotransplantation technique, quantification of cellular proliferation in vivo is critical to standardize the process for future preclinical applications of the model. Methods This study improved the conditions of the xenotransplantation technique – quantification of cellular proliferation in vivo was performed through image processing with our ZFtool software and optimization of temperature in order to standardize the process for a future preclinical applications. ZFtool was developed to establish a base threshold that eliminates embryo auto-fluorescence and measures the area of marked cells (GFP) and the intensity of those cells to define a ‘proliferation index’. Results The analysis of tumor cell proliferation at different temperatures (34 °C and 36 °C) in comparison to in vitro cell proliferation provides of a better proliferation rate, achieved as expected at 36°, a maintenance temperature not demonstrated up to now. The mortality of the embryos remained between 5% and 15%. 5- Fluorouracil was tested for 2 days, dissolved in the incubation medium, in order to quantify the reduction of the tumor mass injected. In almost all of the embryos incubated at 36 °C and incubated with 5-Fluorouracil, there was a significant tumor cell reduction compared with the control group. This was not the case at 34 °C. Conclusions Our results demonstrate that the proliferation of the injected cells is better at 36 °C and that this temperature is the most suitable for testing chemotherapeutic drugs like the 5-FluorouracilThis research was funded by the Fondo de Investigación Sanitaria (Instituto Carlos III) - FIS project (PI13/01388). The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing of this manuscriptS

Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells

© The Author(s) 2020Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7–9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.We acknowledge and thank Dr. Nuria Malats and Jaime Villarreal from the Spanish National Cancer Research Center (CNIO) for RNA sequencing and analysis, funded by Fondo de Investigaciones Sanitarias (FIS) grant PI18/01347. We thank Patricia Sánchez-Tomero and Marina Ochando-Garmendia for technical assistance and support and Dr. Raúl Sánchez Lanzas for assistance with autophagy experiments. We want to particularly acknowledge the patients and the BioBank Hospital Ramón y Cajal-IRYCIS (PT13/0010/0002) integrated in the Spanish National Biobanks Network for its collaboration and, in particular, Adrián Povo Retana for macrophage isolation. We would also like to thank the Transmission Electron Microscopy Unit Laboratory, part of the UAM Interdepartmental Investigation Service (SIdI); Coral Pedrero for exceptional help with in vivo experiments; and the laboratories of Dr. Amparo Cano and Dr. José González Castaño for reagents and helpful discussions. S.V. was a recipient of an Ayuda de Movilidad del Personal Investigador del IRYCIS, a mobility grant from the Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain, and a pre-doctoral fellowship from the Comunidad de Madrid, Ayudas Para La Contratación De Investigadores Predoctorales Y Posdoctorales (PEJD-2017-PRE/BMD-5062), Madrid, Spain. This study was supported by a Rámon y Cajal Merit Award (RYC-2012-12104) from the Ministerio de Economía y Competitividad, Spain (to B.S.); funding from la Beca Carmen Delgado/Miguel Pérez-Mateo from AESPANC-ACANPAN Spain (to B.S.); a Conquer Cancer Now Grant from the Concern Foundation (Los Angeles, CA, USA) (to B.S.); a Coordinated grant (GC16173694BARB) from the Fundación Asociación Española Contra el Cáncer (AECC) (to B.S.); FIS grants PI18/00757 (to B.S.), PI16/00789 (to M.A.F.-M.), PI18/00267 (to L.G.-B.; co-financed through Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa”); a Miguel Servet award (CP16/00121) (to P.S.); a Max Eder Fellowship of the German Cancer Aid (111746) (to P.C.H.); and the German Research Foundation (DFG, CRC 1279 “Exploiting the human peptidome for Novel Antimicrobial and Anticancer Agents”; to P.C.H.)

Paclitaxel Plus Cetuximab as Induction Chemotherapy for Patients With Locoregionally Advanced Head and Neck Squamous Cell Carcinoma Unfit for Cisplatin-Based Chemotherapy

ObjectivesInduction chemotherapy (ICT) followed by definitive treatment is an accepted non-surgical approach for locoregionally advanced head and neck squamous cell carcinoma (LA-HNSCC). However, ICT remains a challenge for cisplatin-unfit patients. We evaluated paclitaxel and cetuximab (P-C) as ICT in a cohort of LA-HNSCC patients unfit for cisplatin. Materials and MethodsThis is a retrospective analysis of patients with newly diagnosed LA-HNSCC considered unfit for cisplatin-based chemotherapy (age >70 and/or ECOG >= 2 and/or comorbidities) treated with weekly P-C followed by definitive radiotherapy and cetuximab (RT-C) between 2010 and 2017. Toxicity and objective response rate (ORR) to ICT and RT-C were collected. Median overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method. Cox regression analysis was performed to determine baseline predictors of OS and PFS. ResultsA total of 57 patients were included. Grade 3-4 toxicity rate to ICT was 54.4%, and there was a death deemed treatment-related (G5). P-C achieved an ORR of 66.7%, including 12.3% of complete responses (CR). After P-C, 45 patients (78.9%) continued with concomitant RT-C. Twenty-six patients (45.6%) achieved a CR after definitive treatment. With a median follow-up of 21.7 months (range 1.2-94.6), median OS and PFS were 22.9 months and 10.7 months, respectively. The estimated 2-year OS and PFS rates were 48.9% and 33.7%, respectively. Disease stage had a negative impact on OS (stage IVb vs. III-IVa: HR = 2.55 [1.08-6.04], p = 0.03), with a trend towards worse PFS (HR = 1.92 [0.91-4.05], p = 0.09). Primary tumor in the larynx was associated with improved PFS but not OS (HR = 0.45 [0.22-0.92], p = 0.03, and HR = 0.69 [0.32-1.54], p = 0.37, respectively). ConclusionP-C was a well-tolerated and active ICT regimen in this cohort of LA-HNSCC patients unfit for cisplatin-based chemotherapy. P-C might represent a valid ICT option for unfit patients and may aid patient selection for definitive treatment