Taxonomic Impediment for Conservation: The Case of Bees in an Undersampled Tropical Mid-Elevation Site, San Martín, Peru.

In this first field survey of an entire bee fauna for any part of Peru, we report a total of 1796 bees belonging to 181 species or morphospecies in four families. The taxonomic impediment was pronounced with only 80 species of 181 that could be named. With such a high proportion of undetermined species, it is not possible to adequately compare pollinator communities across different studies, assess historical changes or analyze endemism patterns to document ecology, behavior and evolution of the species and genera. This information is required to provide a sound basis for policymakers to protect habitats for the conservation of native pollinators.

KLF17 is an important regulatory component of the transcriptomic response of Atlantic salmon macrophages to Piscirickettsia salmonis infection.

Piscirickettsia salmonis is the most important health problem facing Chilean Aquaculture. Previous reports suggest that P. salmonis can survive in salmonid macrophages by interfering with the host immune response. However, the relevant aspects of the molecular pathogenesis of P. salmonis have been poorly characterized. In this work, we evaluated the transcriptomic changes in macrophage-like cell line SHK-1 infected with P. salmonis at 24- and 48-hours post-infection (hpi) and generated network models of the macrophage response to the infection using co-expression analysis and regulatory transcription factor-target gene information. Transcriptomic analysis showed that 635 genes were differentially expressed after 24- and/or 48-hpi. The pattern of expression of these genes was analyzed by weighted co-expression network analysis (WGCNA), which classified genes into 4 modules of expression, comprising early responses to the bacterium. Induced genes included genes involved in metabolism and cell differentiation, intracellular transportation, and cytoskeleton reorganization, while repressed genes included genes involved in extracellular matrix organization and RNA metabolism. To understand how these expression changes are orchestrated and to pinpoint relevant transcription factors (TFs) controlling the response, we established a curated database of TF-target gene regulatory interactions in Salmo salar, SalSaDB. Using this resource, together with co-expression module data, we generated infection context-specific networks that were analyzed to determine highly connected TF nodes. We found that the most connected TF of the 24- and 48-hpi response networks is KLF17, an ortholog of the KLF4 TF involved in the polarization of macrophages to an M2-phenotype in mammals. Interestingly, while KLF17 is induced by P. salmonis infection, other TFs, such as NOTCH3 and NFATC1, whose orthologs in mammals are related to M1-like macrophages, are repressed. In sum, our results suggest the induction of early regulatory events associated with an M2-like phenotype of macrophages that drives effectors related to the lysosome, RNA metabolism, cytoskeleton organization, and extracellular matrix remodeling. Moreover, the M1-like response seems delayed in generating an effective response, suggesting a polarization towards M2-like macrophages that allows the survival of P. salmonis. This work also contributes to SalSaDB, a curated database of TF-target gene interactions that is freely available for the Atlantic salmon community.

Small RNA Differential Expression Analysis Reveals miRNAs Involved in Dormancy Progression in Sweet Cherry Floral Buds.

In sweet cherry (Prunus avium), as in other temperate woody perennials, bud dormancy allows for survival in adverse environmental conditions during winter. During this process, environmental signals such as short days and/or low temperatures trigger internal signals that enable buds to become tolerant to the cold. The process involves tracking chilling units up to chilling the requirement fulfillment to resume growth, a transition involving transcriptional regulation, metabolic signaling, and epigenetic-related regulatory events. Massive sequencing of small RNAs was performed to identify miRNAs involved in sweet cherry dormancy by comparing their expression in field (regular seasonal) and controlled non-stop (continuous) chilling conditions. miRNAs highlighted by sequencing were validated using specific stem-loop PCR quantification, confirming expression patterns for known miRNAs such as miR156e, miR166c, miR172d, miR391, miR482c, and miR535b, as well as for newly proposed miRNAs. In silico prediction of the target genes was used to construct miRNA/target gene nodes. In particular, the involvement of the sweet cherry version for the miR156/SQUAMOSA PROMOTER-BINDING-LIKE PROTEIN genes whose expression was opposite in the two conditions suggests their involvement on dormancy regulation in sweet cherry. miRNA levels indicate that the regulation of stress-related genes and hormone synthesis modulates the expression of calcium metabolism and cell development-associated genes. Understanding the regulatory networks involved in sweet cherry dormancy, particularly in the context of miRNA involvement, represents the first step in the development of new agricultural strategies that may help overcome the increasing challenges presented by global climate change.

Biological evaluation of a mechanical ventilator that operates by controlling an automated manual resuscitator. A descriptive study in swine.

The Covid-19 outbreak challenged health systems around the world to design and implement cost-effective devices produced locally to meet the increased demand of mechanical ventilators worldwide. This study evaluates the physiological responses of healthy swine maintained under volume- or pressure-controlled mechanical ventilation by a mechanical ventilator implemented to bring life-support by automating a resuscitation bag and closely controlling ventilatory parameters. Physiological parameters were monitored in eight sedated animals (t0) prior to inducing deep anaesthesia, and during the next six hours of mechanical ventilation (t1-7). Hemodynamic conditions were monitored periodically using a portable gas analyser machine (i.e. BEecf, carbonate, SaO2, lactate, pH, PaO2, PaCO2) and a capnometer (i.e. ETCO2). Electrocardiogram, echocardiography and lung ultrasonography were performed to detect in vivo alterations in these vital organs and pathological findings from necropsy were reported. The mechanical ventilator properly controlled physiological levels of blood biochemistry such as oxygenation parameters (PaO2, PaCO2, SaO2, ETCO2), acid-base equilibrium (pH, carbonate, BEecf), and perfusion of tissues (lactate levels). In addition, histopathological analysis showed no evidence of acute tissue damage in lung, heart, liver, kidney, or brain. All animals were able to breathe spontaneously after undergoing mechanical ventilation. These preclinical data, supports the biological safety of the medical device to move forward to further evaluation in clinical studies.

Metagenome-Assembled Genomes from Monte Cristo Cave (Diamantina, Brazil) Reveal Prokaryotic Lineages As Functional Models for Life on Mars.

Microbial communities have been explored in various terrestrial subsurface ecosystems, showing metabolic potentials that could generate noteworthy morphological and molecular biosignatures. Recent advancements in bioinformatic tools have allowed for descriptions of novel and yet-to-be cultivated microbial lineages in different ecosystems due to the genome reconstruction approach from metagenomic data. Using shotgun metagenomic data, we obtained metagenome-assembled genomes related to cultivated and yet-to-be cultivated prokaryotic lineages from a silica and iron-rich cave (Monte Cristo) in Minas Gerais State, Brazil. The Monte Cristo Cave has been shown to possess a high diversity of genes involved with different biogeochemical cycles, including reductive and oxidative pathways related to carbon, sulfur, nitrogen, and iron. Three genomes were selected for pangenomic analysis, assigned as Truepera sp., Ca. Methylomirabilis sp., and Ca. Koribacter sp. based on their lifestyles (radiation resistance, anaerobic methane oxidation, and potential iron oxidation). These bacteria exhibit genes involved with multiple DNA repair strategies, starvation, and stress response. Because these groups have few reference genomes deposited in databases, our study adds important genomic information about these lineages. The combination of techniques applied in this study allowed us to unveil the potential relationships between microbial genomes and their ecological processes with the cave mineralogy and highlight the lineages involved with anaerobic methane oxidation, iron oxidation, and radiation resistance as functional models for the search for extant life-forms outside our planet in silica- and iron-rich environments and potentially on Mars.

A comprehensive transcription factor and DNA-binding motif resource for the construction of gene regulatory networks in Botrytis cinerea and Trichoderma atroviride.

Botrytis cinerea and Trichoderma atroviride are two relevant fungi in agricultural systems. To gain insights into these organisms' transcriptional gene regulatory networks (GRNs), we generated a manually curated transcription factor (TF) dataset for each of them, followed by a GRN inference utilizing available sequence motifs describing DNA-binding specificity and global gene expression data. As a proof of concept of the usefulness of this resource to pinpoint key transcriptional regulators, we employed publicly available transcriptomics data and a newly generated dual RNA-seq dataset to build context-specific Botrytis and Trichoderma GRNs under two different biological paradigms: exposure to continuous light and Botrytis-Trichoderma confrontation assays. Network analysis of fungal responses to constant light revealed striking differences in the transcriptional landscape of both fungi. On the other hand, we found that the confrontation of both microorganisms elicited a distinct set of differentially expressed genes with changes in T. atroviride exceeding those in B. cinerea. Using our regulatory network data, we were able to determine, in both fungi, central TFs involved in this interaction response, including TFs controlling a large set of extracellular peptidases in the biocontrol agent T. atroviride. In summary, our work provides a comprehensive catalog of transcription factors and regulatory interactions for both organisms. This catalog can now serve as a basis for generating novel hypotheses on transcriptional regulatory circuits in different experimental contexts.

Multiplex TaqMan Real-Time PCR Assay for Sensitive Detection of Two Weevil Species (Coleoptera: Curculionidae).

Rapid and cost-efficient identification of Naupactus species is becoming a key process for the exportation of citrus fruit from Chile and other countries, considering the quarantine regulations for some species of the cosmopolitan genus Naupactus. This study deals with the development of a fast and sensitive detection protocol for Naupactus cervinus (Coleoptera: Curculionidae) (Boheman) and Naupactus xanthographus (Coleoptera: Curculionidae) (Germar) based on multiplex TaqMan Real-time polymerase chain reaction. Both N. cervinus and N. xanthographus primer and probe sets achieved species-specific detection in a linear range from 1 pg/µl to 1 × 10-6 pg/µl, allowing detection of as few as 160 copies of template DNA. Non-target amplifications were not detected and a panel composed of 480 test samples had 100% coincidence with the respective morphological identification.

Manifestaciones neurológicas asociadas a COVID-19 en el Hospital Edgardo Rebagliati Martins, Perú / Neurological manifestations associated with COVID-19 in Edgardo Rebagliati Martins Hospital, Peru

RESUMEN La enfermedad causada por el coronavirus 2 asociado al síndrome respiratorio agudo grave (SARS-CoV-2), se conoce formalmente como Coronavirus Disease 2019 (COVID-19). Su principal foco de infección es el sistema respiratorio, pero se han descrito también manifestaciones neurológicas. Objetivo: Describir las manifestaciones neurológicas en pacientes con COVID-19 en el hospital Edgardo Rebagliati Martins. Material y Métodos: Estudio observacional, descriptivo, transversal, empleando los registros médicos electrónicos de los pacientes hospitalizados con COVID -19 y dados de alta durante el periodo Abril-agosto 2020. Se clasificaron a los pacientes como portadores de manifestaciones neurológicas del sistema nervioso central y periférico. Resultados: De 1122 pacientes, 354 (31,5%) presentaron alguna manifestación neurológica, en tanto que 26,9% mostraron síntomas del sistema nervioso central (SNC) y 8,4% del sistema nervioso periféricos (SNP). El síntoma más frecuente del SNC fue cefalea (19,7%) y como patologías: encefalopatía (4,7%), el ataque cerebrovascular isquémico (2,7%), crisis epiléptica sintomática aguda (0,5%), hemorragia cerebral (0,4%), encefalitis (0,2%). Dentro de las manifestaciones del SNP predominaron mialgia (3,4%), anosmia (3,1%), disgeusia (2,9%) y, como patologías, rabdomiólisis (1,3%) y polirradiculoneuropatía aguda (0,3%). La tasa de letalidad alcanzó el 32,7% con un OR de 1,4. Conclusiones: Pacientes con infección por SARS-CoV-2 pueden presentar sintomatología neurológica como la descrita en el presente estudio, y sus manifestaciones deben ser consideradas en la atención oportuna e integral de los casos.

SUMMARY The disease caused by coronavirus 2 associated with severe acute respiratory syndrome (SARS-CoV-2) is formally known as Coronavirus Disease 2019 (COVID-19). Its main focus is the respiratory system, but neurological manifestations have also been described. Objective: To describe the neurological manifestations in patients with COVID-19 at the Edgardo Rebagliati Martins hospital. Material and Methods: An observational, descriptive, cross-sectional study, using the electronic medical records of COVID-19 hospitalized and discharged during the April-August 2020 period. The patients were classified as carrying neurological manifestations of the central and peripheral nervous system. Results: Of 1122 patients, 354 (31.5%) had some neurological manifestation while 26.9% showed symptoms of the central nervous system (CNS), and 8.4% of the peripheral nervous system (PNS). Among the CNS manifestations, the most frequent symptom was headache (19.7%), and as pathologies: encephalopathy (4.7%), ischemic stroke (2.7%), acute symptomatic seizure (0.5%), cerebral hemorrhage (0,4%), encephalitis (0.2%). From the PNS manifestations, myalgia (3.4%), anosmia (3.1%) and dysgeusia (2.9%) predominated and, as pathologies, rhabdomyolysis (1.3%) and acute polyradiculoneuropathy (0.3%). The fatality rate reached 32.7% with an OR of 1.4. Conclusions: Patients with SARS-CoV-2 infection can present neurological symptoms as those described in the present study, manifestations that should be considered in their timely and integrated care.

Diagnóstico y tratamiento de la etapa aguda del accidente cerebrovascular isquémico: Guía de práctica clínica del Seguro Social del Perú (EsSalud) / Diagnosis and therapy for the acute stage of ischemic cerebrovascular accident: A clinical practice guideline from Peruvian Social Security (EsSalud)

RESUMEN Objetivo: proveer recomendaciones clínicas basadas en evidencia para el diagnóstico y tratamiento de la etapa aguda del ataque cerebrovascular isquémico en EsSalud. Materiales y métodos: se conformó un grupo elaborador de la guía (GEG) que incluyó médicos especialistas y metodólogos. El GEG formuló 8 preguntas clínicas a ser respondidas por la presente GPC. Se realizó búsquedas sistemáticas de revisiones sistemáticas y -cuando fue considerado pertinente- estudios primarios en Medline y Cochrane Controlled Register of Trials durante el 2018. Se seleccionó la evidencia para responder cada una de las preguntas clínicas planteadas. La certeza de la evidencia fue evaluada usando la metodología Grading of Recommendations Assessment, Development, and Evaluation (GRADE). En reuniones de trabajo periódicas, el GEG usó la metodología GRADE para revisar la evidencia y formular las recomendaciones, los puntos de buenas prácticas clínicas y el flujograma de diagnóstico y tratamiento. Finalmente, la GPC fue aprobada con Resolución N° 128-IETSI-ESSALUD-2019. Resultados: la presente GPC abordó 8 preguntas clínicas, divididas en cuatro temas: tamizaje, diagnóstico, tratamiento, soporte y rehabilitación. En base a estas preguntas se formularon 28 recomendaciones (8 fuertes y 20 condicionales), 38 puntos de buena práctica clínica, 1 nota de implementación y 2 flujogramas. Conclusión: el presente artículo resume la metodología y las conclusiones basadas en evidencias de la GPC para el diagnóstico y tratamiento de la etapa aguda del ataque cerebrovascular isquémico en EsSalud.

ABSTRACT Objective: to provide evidence based clinical recommendations for the diagnosis and therapy of the acute stage of ischemic cerebrovascular accident in EsSalud. Materials and methods : a group for producing the guideline was conveyed, including specialized physicians and methodologists. The group prepared 8 clinical questions to be answered by the guideline. Systematic searches of previous reviews were performed, and - when it was deemed necessary - primary studies in Medline and the Cochrane Controlled Registry of Trials for 2018 were reviewed. Evidence was selected aiming to respond each one of the proposed clinical questions. Certainty of the evidence was determined using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) methodology. In periodic work sessions, the group used GRADE methodology for reviewing the evidence and generated recommendations, good clinical practice items, and designed flow charts for both diagnosis and therapy. Finally, the guide was approved with the decree N° 128-IETSI-ESSALUD-2019. Results : this clinical practice guideline focused in 8 clinical questions, which were divided into four topics: screening, diagnosis, therapy, support and rehabilitation. On the basis of these questions, 28 recommendations were formulated (8 strong and 20 conditional), 38 items for good clinical practice, 1 implementation note, and 2 flow charts. Conclusion : this paper summarizes both evidence based methodology and conclusions from a new clinical practice guide for diagnosis and therapy of the acute stage of a cerebrovascular accident in EsSalud.

Silencing of one copy of the translation initiation factor eIFiso4G in Japanese plum (Prunus salicina) impacts susceptibility to Plum pox virus (PPV) and small RNA production.

BACKGROUND: In plants, host factors encoded by susceptibility (S) genes are indispensable for viral infection. Resistance is achieved through the impairment or the absence of those susceptibility factors. Many S genes have been cloned from model and crop species and a majority of them are coding for members of the eukaryotic translation initiation complex, mainly eIF4E, eIF4G and their isoforms. The aim of this study was to investigate the role of those translation initiation factors in susceptibility of stone fruit species to sharka, a viral disease due to Plum pox virus (PPV). RESULTS: For this purpose, hairpin-inducing silencing constructs based on Prunus persica orthologs were used to generate Prunus salicina (Japanese plum) 4E and 4G silenced plants by Agrobacterium tumefaciens-mediated transformation and challenged with PPV. While down-regulated eIFiso4E transgenic Japanese plums were not regenerated in our conditions, eIFiso4G11-, but not the eIFiso4G10-, silenced plants displayed durable and stable resistance to PPV. We also investigated the alteration of the si- and mi-RNA profiles in transgenic and wild-type Japanese plums upon PPV infection and confirmed that the newly generated small interfering (si) RNAs, which are derived from the engineered inverted repeat construct, are the major contributor of resistance to sharka. CONCLUSIONS: Our results indicate that S gene function of the translation initiation complex isoform is conserved in Prunus species. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of the different isoforms of proteins involved in this complex to breed for resistance to sharka in fruit trees.

Permanencia del dispositivo intrauterino TCu 380A insertado después de algún procedmiento obstétrico / Stay of the TCU 380A IUD inserted after of the obstetric event

Resumen OBJETIVO: Determinar la permanencia del dispositivo intrauterino TCu 380A insertado después de algún procedimiento obstétrico efectuado a pacientes atendidas en el Hospital General Dr. Aurelio Valdivieso. MATERIAL Y MÉTODOS: Estudio transversal y descriptivo efectuado en pacientes que concluyeron el embarazo entre el 1 de octubre de 2016 y el 31 de marzo de 2017, en el Hospital General Dr. Aurelio Valdivieso y que aceptaron se les colocara el dispositivo intrauterino TCu 380A después de algún procedimiento obstétrico y antes de darlas de alta del hospital. Variables de estudio: cantidad de embarazos, terminación de estos, médico responsable de la colocación, técnica y permanencia. El análisis estadístico implementado fue el univariado. RESULTADOS: Se incluyeron 182 pacientes con media de edad de 23 años y límites de 14 y 43 años. En el seguimiento de la permanencia del dispositivo 42.3% (n = 77) se encontró in situ, 28.5% (n = 52) en mala posición, y 0.55% (n = 1) lo expulsó. CONCLUSIONES: La permanencia del dispositivo intrauterino parece tener una relación estrecha con la forma correcta de insertarlo y, desde luego, con la experiencia acumulada del médico. En las primeras inserciones el médico debe ser supervisado por otro de mayor experiencia o, simplemente, con la vigilancia de los médicos adscritos.

Abstract OBJECTIVE: To determine the permanence of the TCu 380A IUD inserted after the obstetric event in the Dr. Aurelio Valdivieso Hospital. MATERIAL AND METHODS: Cross-sectional and descriptive study carried out in patients who concluded their pregnancy, between October 1, 2016 and March 31, 2017, at the Dr. Aurelio Valdivieso General Hospital (Oaxaca, Mexico) and who accepted the placement of the TCu 380A intrauterine device during the postpartum period. and before discharge from the hospital. Study variables: number of pregnancies, termination of these, doctor responsible for the placement, technique and permanence. The statistical analysis implemented was univariate. RESULTS: The study was conducted in 182 patients with a mean age of 23 years, a minimum of 14 years and a maximum of 43 years. In the monitoring of the permanence of the IUD TCu 380A with 42.31% (77) was found in situ, 28.57% (52) in poor position, and 0.55% (1) was expelled. CONCLUSIONS: The permanence of the intrauterine device seems to have a close relationship with the correct way to insert it and, of course, with the accumulated experience of the doctor. In the first insertions the doctor must be supervised by another one of greater experience or, simply, with the surveillance of the assigned doctors.

A fast and efficient protocol for small RNA extraction in Japanese plum and other Prunus species

Background: Small ribonucleic acids represent an important repertoire of mobile molecules that exert key roles in several cell processes including antiviral defense. Small RNA based repertoire includes both small interfering RNA (siRNA) and microRNA (miRNA) molecules. In the Prunus genus, sharka disease, caused by the Plum pox virus (PPV), first occurred on European plum (Prunus domestica) and then spread over among all species in this genus and thus classified as quarantine pathogen. Next-generation sequencing (NGS) was used for the study of siRNA/miRNA molecules; however, NGS relies on adequate extraction protocols. Currently, knowledge of PPV-Prunus interactions in terms of siRNA populations and miRNA species is still scarce, and siRNA/miRNA extraction protocols are limited to species such as peach, almond, and sweet cherry. Results: We describe a reliable procedure for siRNA/miRNA purification from Prunus salicina trees, in which previously used protocols did not allow adequate purification. The procedure was based on a combination of commercially available RNA purification kits and specific steps that yielded high quality purifications. The resulting molecules were adequate for library construction and NGS, leading to the development of a pipeline for analysis of both siRNAs and miRNAs in the PPV­P. salicina interactions. Results showed that PPV infection led to altered siRNA profiles in Japanese plum as characterized by decreased 24-nt and increased 21- and 22-nt siRNAs. Infections showed miR164 and miR160 generation and increased miR166, miR171, miR168, miR319, miR157, and miR159. Conclusion: We propose this protocol as a reliable and reproducible small RNA isolation procedure for P. salicina and other Prunus species.

DNA methylation and small interference RNAs participate in the regulation of MADS-box genes involved in dormancy in sweet cherry (Prunus avium L.).

Epigenetic modifications can yield information about connections between genotype, phenotype variation and environmental conditions. Bud dormancy release in temperate perennial fruit trees depends on internal and environmental signals such as cold accumulation and photoperiod. Previous investigations have noted the participation of epigenetic mechanisms in the control of this physiological process. We examined whether epigenetic modifications were modulated in MADS-box genes, potential candidates for the regulation of bud dormancy and flowering in sweet cherry (Prunus avium L.). We identified and cloned two MADS-box genes homologous to the already-characterized dormancy regulators DORMANCY-ASSOCIATED MADS-box (DAM3 and DAM5) from Prunus persica (L.) Batsch. Bisulfite sequencing of the identified genes (PavMADS1 and PavMADS2), Methylated DNA Immunoprecipitation and small RNA deep sequencing were performed to analyze the presence of DNA methylations that could be guided by non-coding RNAs in the floral buds exposed to differential chilling hours. The results obtained reveal an increase in the level of DNA methylation and abundance of matching small interference RNAs (siRNAs) in the promoter of PavMADS1 when the chilling requirement is complete. For the first intron and 5' UTR of PavMADS1, de novo DNA methylation could be associated with the increase in the abundance of 24-nt siRNA matching the promoter area. Also, in the second large intron of PavMADS1, maintenance DNA methylation in all cytosine contexts is associated with the presence of homologous siRNAs in that zone. For PavMADS2, only maintenance methylation was present in the CG context, and no matching siRNAs were detected. Silencing of PavMADS1 and PavMADS2 coincided with an increase in Flowering Locus T expression during dormancy. In conclusion, DNA methylations and siRNAs appear to be involved in the silencing of PavMADS1 during cold accumulation and dormancy release in sweet cherry.

Candidate nematicidal proteins in a new Pseudomonas veronii isolate identified by its antagonistic properties against Xiphinema index.

The nematode Xiphinema index affects grape vines and transmits important viruses associated with fanleaf degeneration. Pseudomonas spp. are an extensive bacterial group in which important biodegradation and/or biocontrol properties can occur for several strains in the group. The aim of this study was to identify new Pseudomonas isolates with antagonist activity against X. index. Forty bacterial isolates were obtained from soil and root samples from Chilean vineyards. Thirteen new fluorescent pseudomonads were found and assessed for their antagonistic capability. The nematicide Pseudomonas protegens CHA0 was used as a control. Challenges of nematode individuals in King's B semi-solid agar Petri dishes facilitated the identification of the Pseudomonas veronii isolate R4, as determined by a 16S rRNA sequence comparison. This isolate was as effective as CHA0 as an antagonist of X. index, although it had a different lethality kinetic. Milk-induced R4 cultures exhibited protease and lipase activities in cell supernatants using both gelatin/tributyrin Petri dish assays and zymograms. Three proteins with these activities were isolated and subjected to mass spectrometry. Amino acid partial sequences enabled the identification of a 49-kDa protease similar to metalloprotease AprA and two lipases of 50 kDa and 69 kDa similar to LipA and ExoU, respectively. Electron microscopy analyses of challenged nematodes revealed degraded cuticle after R4 supernatant treatment. These results represent a new and unexplored property in this species associated with the presence of secretable lipases and protease, similar to characterized enzymes present in biocontrol pseudomonads.

Synthesis of an artificial Vitis vinifera miRNA 319e using overlapping long primers and its application for gene silencing.

The conserved mechanism of action of micro-RNAs (miRNAs) as regulators of gene expression has allowed the use of artificial miRNAs (amiRNAs) as a powerful tool for candidate gene evaluation in plants. Based on the use of a Vitis vinifera miRNA molecule (i.e., vvi-miR319e), the present work presents a new methodology for designing artificial miR319e precursors (pre-amiR319e). As a proof of concept, we silenced the green fluorescent protein (GFP) gene in transgenic Nicotiana benthamiana plants. This methodology includes a two-step PCR reaction in which overlapping long primers allow for the complete generation of pre-amiR319e-GFP molecules that are adequate for recombination into Gateway vectors with no further requirements. The seed region in amiRNA was directed against the 3'-end portion of the GFP gene. Three groups of transformed N. benthamiana plants were generated: GFP-, amiR319e-GFP-, and GFP plus miR319e-GFP-expressing vectors. A similar group of wild-type plants was included. Confocal microscopy evaluation of these groups revealed strong silencing of the GFP phenotype in the double GFP plus amiR319e-GFP group. The molecular characterization of silenced plants was achieved via modified 5'RACE of the GFP mRNA and revealed the occurrence of a partial, 3'-end GFP mRNA molecule that was generated in planta. In addition, large-scale small RNA sequencing confirmed the occurrence of the expected 21-nt miR319e-GFP species and other 22- and 24-nt species that exhibited sequence relationships with the expected amiRNA. These results highlight the possibility of using vvi-MIR319 as a template for the generation of single amiRNAs as a tool for gene silencing in plants.

Genetically engineered Thompson Seedless grapevine plants designed for fungal tolerance: selection and characterization of the best performing individuals in a field trial.

The fungi Botrytis cinerea and Erysiphe necator are responsible for gray mold and powdery mildew diseases, respectively, which are among the most devastating diseases of grapes. Two endochitinase (ech42 and ech33) genes and one N-acetyl-ß-D-hexosaminidase (nag70) gene from biocontrol agents related to Trichoderma spp. were used to develop a set of 103 genetically modified (GM) 'Thompson Seedless' lines (568 plants) that were established in open field in 2004 and evaluated for fungal tolerance starting in 2006. Statistical analyses were carried out considering transgene, explant origin, and plant response to both fungi in the field and in detached leaf assays. The results allowed for the selection of the 19 consistently most tolerant lines through two consecutive years (2007-2008 and 2008-2009 seasons). Plants from these lines were grafted onto the rootstock Harmony and established in the field in 2009 for further characterization. Transgene status was shown in most of these lines by Southern blot, real-time PCR, ELISA, and immunostrips; the most tolerant candidates expressed the ech42-nag70 double gene construct and the ech33 gene from a local Hypocrea virens isolate. B. cinerea growth assays in Petri dishes supplemented with berry juices extracted from the most tolerant individuals of the selected population was inhibited. These results demonstrate that improved fungal tolerance can be attributed to transgene expression and support the iterative molecular and physiological phenotyping in order to define selected individuals from a population of GM grapevines.

Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge.

Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.

Ictus recurrente isquémico según territorio vascular arterial, etiología y extensión tomográfica en tratados con estatinas dentro de los 6 meses de seguimiento en el HNERM - 2011 / Recurrent ischemic stroke by arterial vascular territory, etiology and tomographic extension in patients treated for 6 months follow-up in the HNERM-2011

INTRODUCCIÓN: El ictus es una de las principales causas de morbilidad y mortalidad en el mundo. Aproximadamente el 30 por ciento de ictus isquémicos son eventos recurrentes. Considerando a la recurrencia como: nuevo ictus después de 24 horas del primer ictus en un territorio vascular diferente o a > 21 días después del primer ictus en el mismo territorio, el cual no puede ser atribuido a edema, desviación de línea media, transformación hemorrágica, enfermedad intercurrente (infecciosa o metabólica), hipoxia o toxicidad de los medicamentos. Se requiere un empeoramiento súbito para considerarlo como un evento recurrente, y cuya progresión gradual del déficit agudo fuese excluido. Es importante, el estudio de estos eventos ya que son más propensos a ser incapacitante o fatales luego del primer ictus. OBJETIVOS: Identificar si existe alguna diferencia porcentual entre tipo de estatina empleada y la recurrencia de ictus isquémico dentro de los 6 meses de seguimiento en el HNERM-2011. Establecer los factores mayor frecuencia en la recurrencia del ictus. MATERIALES Y METODOS: Estudio descriptivo, observacional, prospectivo. Revisión de las historias clínicas de pacientes con ictus isquémico en >45 años hospitalizados en el Servicio de Neurología del HNERM. Los grupos muéstrales se conformaron en base al tipo de estatina recibida, donde el tipo y dosis administrada fue en función al criterio del médico tratante, pudiendo indicar atorvastatina a 10mg, 20mg, o simvastatina 20mg, 40mg o ninguna estatina, cuyo efecto a medir será la recurrencia del ictus (nuevo episodio de ictus) dentro de los 6 meses de seguimiento (la muestra se recolectó desde enero a diciembre del 2011). RESULTADOS: El 80 por ciento utilizó al menos una estatina. El 10.45 por ciento (37) de pacientes presentó recurrencia, siendo el porcentaje obtenido para cada grupo: 11.32 por ciento (6 de 53) sin estatina, 10.33 por ciento (19 de 184) simvastatina y 10.26 por ciento (12 de 117) con atorvastatina...

Negative plant-soil feedback predicts tree-species relative abundance in a tropical forest.

The accumulation of species-specific enemies around adults is hypothesized to maintain plant diversity by limiting the recruitment of conspecific seedlings relative to heterospecific seedlings. Although previous studies in forested ecosystems have documented patterns consistent with the process of negative feedback, these studies are unable to address which classes of enemies (for example, pathogens, invertebrates, mammals) exhibit species-specific effects strong enough to generate negative feedback, and whether negative feedback at the level of the individual tree is sufficient to influence community-wide forest composition. Here we use fully reciprocal shade-house and field experiments to test whether the performance of conspecific tree seedlings (relative to heterospecific seedlings) is reduced when grown in the presence of enemies associated with adult trees. Both experiments provide strong evidence for negative plant-soil feedback mediated by soil biota. In contrast, above-ground enemies (mammals, foliar herbivores and foliar pathogens) contributed little to negative feedback observed in the field. In both experiments, we found that tree species that showed stronger negative feedback were less common as adults in the forest community, indicating that susceptibility to soil biota may determine species relative abundance in these tropical forests. Finally, our simulation models confirm that the strength of local negative feedback that we measured is sufficient to produce the observed community-wide patterns in tree-species relative abundance. Our findings indicate that plant-soil feedback is an important mechanism that can maintain species diversity and explain patterns of tree-species relative abundance in tropical forests.

Violencia Intrafamiliar que afecta a Estudiantes de Educación Básica y Media en Bogotá / Intra-family violence affecting students attending basic- and middle-schools in Bogotá

Objetivo: Determinar el nivel de violencia intrafamiliar correspondiente a estudiantes de colegios de Ciudad Bolívar, Bogotá, Colombia. Material y métodos: La muestra está conformada por 3 226 alumnos de educación básica y media, de grados sexto a once, de colegios oficiales de la localidad Ciudad Bolívar de Bogotá. Los datos obtenidos a través de la aplicación de una encuesta, en la que se estableció su percepción de la violencia en sus hogares, fueron analizados estadísticamente utilizando SPSS 14.0. Resultados: un alto porcentaje de estudiantes tiene dificultades en los procesos de comunicación con sus padres y siente rechazo y falta de amor de algunos de los miembros de su hogar. Un 28,4 por ciento de los encuestados, 915 alumnos, son victimas de más de 20 de las situaciones de violencia y un 35 por ciento de los estudiantes encuestados tienen ambientes familiares caracterizados por altos niveles de violencia. El porcentaje de alumnos que afirman ser maltratados físicamente depende del grado escolar, presentándose los más altos porcentajes en grados 7 y 8. Conclusiones: La comunidad estudiantil de la localidad de Ciudad Bolívar, en Bogotá, Colombia está afectada por un alto número de situaciones de violencia intrafamiliar.

Objective: Determining levels of intra-family violence associated with students attending schools in Ciudad Bolivar, Bogotá, Colombia. Material and methods: The sample consisted of 3 226 students from the 6th to 11th grades attending state schools located in Ciudad Bolivar in Bogotá. The data was collected from a survey in which the students' perception of violence in their homes was determined; data was statistically analysed by using SPSS 14.0. Results: A high percentage of students had difficulty in communicating with their parents and felt rejected and a lack of love from some of the members in their homes. 28,4 percent of those surveyed (915 students) had been victims of more than 20 of the violent situations; 35 percent of the students in the sample had a family atmosphere characterised by high levels of violence. The percentage of students who stated that they had been physically mistreated depended on their education level; the highest percentages being presented in the 7th and 8th grades. Conclusions: The middle- and upper-school student community in Ciudad Bolivar in Bogotá, Colombia, is affected by many situations concerning intra-family violence.