Autophagy is activated and involved in cell death with participation of cathepsins during stress-induced microspore embryogenesis in barley.

Microspores are reprogrammed towards embryogenesis by stress. Many microspores die after this stress, limiting the efficiency of microspore embryogenesis. Autophagy is a degradation pathway that plays critical roles in stress response and cell death. In animals, cathepsins have an integral role in autophagy by degrading autophagic material; less is known in plants. Plant cathepsins are papain-like C1A cysteine proteases involved in many physiological processes, including programmed cell death. We have analysed the involvement of autophagy in cell death, in relation to cathepsin activation, during stress-induced microspore embryogenesis in Hordeum vulgare. After stress, reactive oxygen species (ROS) and cell death increased and autophagy was activated, including HvATG5 and HvATG6 up-regulation and increase of ATG5, ATG8, and autophagosomes. Concomitantly, cathepsin L/F-, B-, and H-like activities were induced, cathepsin-like genes HvPap-1 and HvPap-6 were up-regulated, and HvPap-1, HvPap-6, and HvPap-19 proteins increased and localized in the cytoplasm, resembling autophagy structures. Inhibitors of autophagy and cysteine proteases reduced cell death and promoted embryogenesis. The findings reveal a role for autophagy in stress-induced cell death during microspore embryogenesis, and the participation of cathepsins. Similar patterns of activation, expression, and localization suggest a possible connection between cathepsins and autophagy. The results open up new possibilities to enhance microspore embryogenesis efficiency with autophagy and/or cysteine protease modulators.

Academic outcomes and cognitive performance in problematic Internet users. / Rendimiento académico y cognitivo en el uso problemático de Internet.

Only few studies have examined the relationship between problematic Internet use (PIU) and cognitive and academic performance in adolescents. The aim of this study was to analyze the differences in academic and cognitive performance (perception, attention, memory, verbal fluency and abstract reasoning) between adolescents with and without PIU. A total of 575 students from different high schools of the region of Alicante participated. Students were divided into two groups: adolescents with and without PIU (PIU and NPIU, respectively). Several questionnaires were administered to assess problematic Internet use, as well as students' academic performance. Substance use (alcohol / cannabis) was also assessed as exclusion criteria. A battery of neuropsychological tests was used to assess cognitive abilities. On the one hand, PIU users group obtained poorer academic results than NPIU, in terms of lower marks and more failed subjects. On the other hand, PIU group had a better hit ratio in the perception test than NPIU group. However, PIU adolescents got higher error rates for the abstract reasoning test. This greater number of errors, plus a similar number of hits compared to the NPIU group, could indicated a higher response rate for the PIU group, which may might be associated with greater impulsivity. As occurs in other addictive and non-substance-related problems studies, these results could mean difficulties in impulse control and regulation of response inhibition circuits in PIU users group. Future research is needed to analyze in depth the results presented in this paper.

Son escasos los estudios que hayan analizado la relación entre el uso problemático de Internet (UPI) y el rendimiento cognitivo y académico en adolescentes. El objetivo de este estudio fue analizar las diferencias en rendimiento académico y cognitivo (percepción, atención, memoria, fluidez verbal y razonamiento abstracto) en una muestra de estudiantes de Secundaria con y sin UPI. Participaron un total de 575 estudiantes de diferentes institutos de la provincia de Alicante, que fueron divididos en dos grupos: adolescentes con y sin uso problemático de Internet (UPI y NUPI, respectivamente). Se administraron varios cuestionarios para evaluar el uso problemático de Internet de los sujetos, su rendimiento académico, su consumo de sustancias (alcohol/cannabis) como criterios de exclusión, así como una batería de pruebas neuropsicológicas para evaluar sus habilidades cognitivas. Por un lado, los adolescentes con UPI mostraron un peor rendimiento académico que los estudiantes del grupo NUPI, presentando una nota media más baja y un mayor número de asignaturas suspendidas. Por otro lado, el grupo UPI obtuvo una mayor tasa de aciertos en el test de percepción que el grupo NUPI. Sin embargo, los adolescentes con UPI obtuvieron una mayor tasa de errores para el test de razonamiento abstracto. Este mayor número de errores, sumado a un número similar de aciertos que NUPI, indicaría una mayor tasa de respuesta total para el grupo UPI, que podría estar asociada a mayor impulsividad. Concretamente, tal y como se ha observado en otros problemas adictivos con y sin sustancia, estos resultados podrían indicar en los sujetos del grupo UPI dificultades en el control de impulsos y en la regulación de los circuitos de inhibición de respuesta. Resultan necesarios, no obstante, futuros estudios que profundicen en las conclusiones presentadas en este trabajo.

Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control.

Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria.

Evidence for a role of VIPP1 in the structural organization of the photosynthetic apparatus in Chlamydomonas.

The vesicle-inducing protein in plastids (VIPP1) was suggested to play a role in thylakoid membrane formation via membrane vesicles. As this functional assignment is under debate, we investigated the function of VIPP1 in Chlamydomonas reinhardtii. Using immunofluorescence, we localized VIPP1 to distinct spots within the chloroplast. In VIPP1-RNA interference/artificial microRNA cells, we consistently observed aberrant, prolamellar body-like structures at the origin of multiple thylakoid membrane layers, which appear to coincide with the immunofluorescent VIPP1 spots and suggest a defect in thylakoid membrane biogenesis. Accordingly, using quantitative shotgun proteomics, we found that unstressed vipp1 mutant cells accumulate 14 to 20% less photosystems, cytochrome b(6)f complex, and ATP synthase but 30% more light-harvesting complex II than control cells, while complex assembly, thylakoid membrane ultrastructure, and bulk lipid composition appeared unaltered. Photosystems in vipp1 mutants are sensitive to high light, which coincides with a lowered midpoint potential of the Q(A)/Q(A)(-) redox couple and increased thermosensitivity of photosystem II (PSII), suggesting structural defects in PSII. Moreover, swollen thylakoids, despite reduced membrane energization, in vipp1 mutants grown on ammonium suggest defects in the supermolecular organization of thylakoid membrane complexes. Overall, our data suggest a role of VIPP1 in the biogenesis/assembly of thylakoid membrane core complexes, most likely by supplying structural lipids.

Novel adenovirus detected in captive bottlenose dolphins (Tursiops truncatus) suffering from self-limiting gastroenteritis.

BACKGROUND: Adenoviruses are common pathogens in vertebrates, including humans. In marine mammals, adenovirus has been associated with fatal hepatitis in sea lions. However, only in rare cases have adenoviruses been detected in cetaceans, where no clear correlation was found between presence of the virus and disease status. CASE PRESENTATION: A novel adenovirus was identified in four captive bottlenose dolphins with self-limiting gastroenteritis. Viral detection and identification were achieved by: PCR-amplification from fecal samples; sequencing of partial adenovirus polymerase (pol) and hexon genes; producing the virus in HeLa cells, with PCR and immunofluorescence detection, and with sequencing of the amplified pol and hexon gene fragments. A causative role of this adenovirus for gastroenteritis was suggested by: 1) we failed to identify other potential etiological agents; 2) the exclusive detection of this novel adenovirus and of seropositivity for canine adenoviruses 1 and 2 in the four sick dolphins, but not in 10 healthy individuals of the same captive population; and 3) the virus disappeared from feces after clinical signs receded. The partial sequences of the amplified fragments of the pol and hexon genes were closest to those of adenoviruses identified in sea lions with fatal adenoviral hepatitis, and to a Genbank-deposited sequence obtained from a harbour porpoise. CONCLUSION: These data suggest that adenovirus can cause self-limiting gastroenteritis in dolphins. This adenoviral infection can be detected by serology and by PCR detection in fecal material. Lack of signs of hepatitis in sick dolphins may reflect restricted tissue tropism or virulence of this adenovirus compared to those of the adenovirus identified in sea lions. Gene sequence-based phylogenetic analysis supports a common origin of adenoviruses that affect sea mammals. Our findings suggest the need for vigilance against adenoviruses in captive and wild dolphin populations.

First molecular detection and characterization of herpesvirus and poxvirus in a Pacific walrus (Odobenus rosmarus divergens).

BACKGROUND: Herpesvirus and poxvirus can infect a wide range of species: herpesvirus genetic material has been detected and amplified in five species of the superfamily Pinnipedia; poxvirus genetic material, in eight species of Pinnipedia. To date, however, genetic material of these viruses has not been detected in walrus (Odobenus rosmarus), another marine mammal of the Pinnipedia clade, even though anti-herpesvirus antibodies have been detected in these animals. CASE PRESENTATION: In February 2013, a 9-year-old healthy captive female Pacific walrus died unexpectedly at L'Oceanografic (Valencia, Spain). Herpesvirus was detected in pharyngeal tonsil tissue by PCR. Phylogenetic analysis revealed that the virus belongs to the subfamily Gammaherpesvirinae. Poxvirus was also detected by PCR in skin, pre-scapular and tracheobronchial lymph nodes and tonsils. Gross lesions were not detected in any tissue, but histopathological analyses of pharyngeal tonsils and lymph nodes revealed remarkable lymphoid depletion and lymphocytolysis. Similar histopathological lesions have been previously described in bovine calves infected with an alphaherpesvirus, and in northern elephant seals infected with a gammaherpesvirus that is closely related to the herpesvirus found in this case. Intracytoplasmic eosinophilic inclusion bodies, consistent with poxviral infection, were also observed in the epithelium of the tonsilar mucosa. CONCLUSION: To our knowledge, this is the first molecular identification of herpesvirus and poxvirus in a walrus. Neither virus was likely to have contributed directly to the death of our animal.

Unusual striped dolphin mass mortality episode related to cetacean morbillivirus in the Spanish Mediterranean sea.

BACKGROUND: In the last 20 years, Cetacean Morbillivirus (CeMV) has been responsible for many die-offs in marine mammals worldwide, as clearly exemplified by the two dolphin morbillivirus (DMV) epizootics of 1990-1992 and 2006-2008, which affected Mediterranean striped dolphins (Stenella coeruleoalba). Between March and April 2011, the number of strandings on the Valencian Community coast (E Spain) increased. CASE PRESENTATION: Necropsy and sample collection were performed in all stranded animals, with good state of conservation. Subsequently, histopathology, immunohistochemistry, conventional reverse transcription polymerase chain reaction (RT-PCR) and Universal Probe Library (UPL) RT-PCR assays were performed to identify Morbillivirus. Gross and microscopic findings compatible with CeMV were found in the majority of analyzed animals. Immunopositivity in the brain and UPL RT-PCR positivity in seven of the nine analyzed animals in at least two tissues confirmed CeMV systemic infection. Phylogenetic analysis, based on sequencing part of the phosphoprotein gene, showed that this isolate is a closely related dolphin morbillivirus (DMV) to that responsible for the 2006-2008 epizootics. CONCLUSION: The combination of gross and histopathologic findings compatible with DMV with immunopositivity and molecular detection of DMV suggests that this DMV strain could cause this die-off event.

Two cases of pseudohermaphroditism in loggerhead sea turtles Caretta caretta.

Two juvenile (curved carapace lengths: 28 and 30 cm) loggerhead sea turtles Caretta caretta with precocious male external characteristics were admitted to the ARCA del Mar rescue area at the Oceanogràfic Aquarium in Valencia, Spain, in 2009 and 2010. Routine internal laparoscopic examination and subsequent histopathology confirmed the presence of apparently healthy internal female gonads in both animals. Extensive tissue biopsy and hormone induction assays were consistent with female sex. To the best of our knowledge, this is the first report of pseudohermaphroditism in loggerhead sea turtles based on sexual external characteristics and internal laparoscopic examination. Our findings suggest that the practice of using external phenotypical characteristics as the basis for gender identification in sea turtles should be reevaluated. Future research should focus on detecting more animals with sexual defects and their possible effects on the sea turtle population.

First case of erysipelas in a free-ranging bottlenose dolphin (Tursiops truncatus) stranded in the Mediterranean Sea.

An adult female bottlenose dolphin (Tursiops truncatus) stranded alive and subsequently died several minutes later on the Mediterranean coast of Spain on 14 July 2010. Clinical examination revealed foam through the blowhole and rales upon lung auscultation. On venipuncture, the blood was abnormally dense and dark. Hematological and biochemical abnormalities included dehydration, leukocytosis (48 600 leukocytes microl(-1)) characterized by neutrophilia (48 200 neutrophils microl(-1)), and elevated bilirubin (4.38 mg dl(-1)), alanine aminotransferase (382.3 U l(-1)), aspartate aminotransferase (1449.3 U l(-1)), lactate dehydrogenase (1631.3 U l(-1)), and creatine kinase (404.7 U l(-1)). The most relevant findings of the gross examination were rhomboid-shaped skin lesions, stable froth in the trachea, pulmonary congestion, abnormally thick and rough pleura with adhesions, edematous and congestive superficial cervical and tracheobronchial lymph nodes, red-tinged urine, and severe brain congestion. Histopathology of the kidney, lung, skin, and brain revealed multisystemic intravascular bacterial emboli. Samples of skin, brain, and lung were cultured on Columbia blood agar under both aerobic and anaerobic conditions, and pure and heavy bacterial cultures were obtained from skin and brain samples. The microorganism isolated was Gram-positive, catalase-negative, facultatively anaerobic, and rod-shaped. The isolates were identified as Erysipelothrix rhusiopathiae by the API Coryne biochemical system. Based on the gross and microscopic findings, a diagnosis of acute E. rhusiopathiae septicemia was made. To the best of our knowledge, this is the first report of E. rhusiopathiae septicemia in a free-ranging bottlenose dolphin.

Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii.

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.

Family predictors of parent participation in an adolescent drug abuse prevention program.

Low participation rates constitute a serious problem faced by family drug abuse prevention programs. In this study we analyse the factors related to participation in a Life Skills Training program implemented in three schools in Spain. Participants in the study were 485 pupils aged 12 - 14 years and their respective parents. The variables that predicted participation in the program were: number of children and educational level of parents, children's drug use, family conflict, parental rearing style, relationships between parents and children and family communication. The results from Spain are similar to those found in international studies, and indicate that the families most at risk of drug use are those least likely to participate in prevention programs. There is a need for strategies to increase participation in prevention programs of the families most at risk.

Simultaneous diagnosis of Cetacean morbillivirus infection in dolphins stranded in the Spanish Mediterranean sea in 2011 using a novel Universal Probe Library (UPL) RT-PCR assay.

A highly sensitive and specific real-time (rt) RT-PCR assay has been developed for rapid, simultaneous detection of three strains of cetacean morbillivirus (CeMV). In this assay, two PCR primers and a hydrolysis probe from a commercially available Universal Probe Library (UPL) are used to amplify a highly conserved region within the fusion protein gene. RT-PCR is carried out on the same sample using two primer sets in parallel: one set detects the more virulent strains, dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV), and the other set detects the least virulent and least common strain, pilot whale morbillivirus (PWMV). Sensitivity analysis using dilute samples containing purified DMV, PMV and PWMV showed that viral RNA detection limits in this UPL RT-PCR assay were lower than in a conventional RT-PCR assay. Our method gave no amplification signal with field samples positive for viruses related and unrelated to CeMV, such as phocine distemper virus (PDV). The reliability and robustness of the UPL RT-PCR assay were verified using tissue samples previously analyzed by conventional methods, as well as a panel of clinical samples suspected of containing CeMV. Using the UPL RT-PCR assay, we were able to associate DMV with a mass stranding of striped dolphins in the Spanish Mediterranean in 2011 with greater reliability than was possible with a conventional RT-PCR method. These results suggest that this UPL RT-PCR method is more sensitive and specific than the conventional approach, and that it may be an affordable and rapid test for routine diagnosis of three CeMV strains.

Review of 793 facial fractures treated from 2001 to 2008 in a coruña university hospital: types and etiology.

The purpose of this article is to analyze the incidence, demographic distribution, type, and etiology of all facial fractures treated by the Department of Oral and Maxillofacial Surgery in A Coruña University Hospital (Spain) from 2001 to 2008. A descriptive and analytic retrospective study evaluated 643 patients treated for facial fracture (excluding nasal and dento-alveolar) by the Department of Oral and Maxillofacial Surgery in A Coruña University Hospital from January 2001 to December 2008. Five parameters were studied: year of the injury, gender, age, fracture type, and etiology. Six hundred and forty-three patients with 793 fractures were included. Of these, 83.2% were males and 16.8% were females. The patients' age ranged between 18 months and 89 years, with a mean of 37.6 and a median of 33. The major cause of injury was traffic accidents (27%), followed by assaults (20.5%), accidental traumas (20.1%), sports (11%), syncopes (7.8%), rural accidents (6.1%), industrial accidents (5.1%), and suicide attempts (0.3%). In 1.1% of the patients, it was impossible to verify the etiology. The etiology of facial fractures varies from one country to another, depending on the cultural, environmental, and socioeconomic factors. In our study, the most common cause was traffic accidents, closely followed by assaults. The number of fractures due to traffic accidents has decreased in the last 3 years. Rural accidents accounted for a significantly higher percentage of fractures than that observed in other series. The number of fractures receiving a surgical treatment from 2005 to 2008 has progressively decreased.

Recruitment characteristics influencing parental participation in family-based drug-abuse prevention programs: the Spoth and Redmond model in Spain.

Low rates of participation constitute a serious problem for family-based drug-use prevention programs. This study analyzes the characteristics of calls for parents' participation in such programs through manipulation of the variables indicated by the Spoth and Redmond (1995) model as involved in the process (severity of the drug problem, susceptibility of the family's own children and existence of barriers to participation). Participants were 485 school pupils ages 12 to 14 years and their respective parents. Results show that the attempt to manipulate such variables did not have behavioral consequences in relation to participation in the family-based prevention program. There is a need for strategies that increase attendance of high-risk families in this type of intervention. The study's limitations are noted.