Analysis of genetic diversity of mezcalero agaves from central México

"En el Altiplano Potosino, un ecosistema árido y semiárido, Agave salmiana Otto Salm Dick ssp. crassispina (Trel Gentry) es una especie utilizada para la recolección de insectos comestibles y para producción de mezcal, bebida alcohólica tradicional con denominación de origen. El aprovechamiento para producción de mezcal de las poblaciones silvestres de Agave salmiana ssp. crassispina carece de un sistema de explotación basado en el conocimiento biológico de la especie. Esto tiene efectos demográficos, y a su vez podría causar el deterioro de sus recursos fitogenéticos y colocar a la especie en situación de riesgo. Este trabajo analizó diversidad genética y estructura poblacional de tres poblaciones silvestres de Agave salmiana ssp. crassispina en San Luis Potosí, México, mediante polimorfismos de longitud de los fragmentos amplificados, AFLP. La evidencia mostró un alto nivel de diversidad genética dentro de las poblaciones analizadas y un bajo nivel de diferenciación entre ellas, probablemente debido a una fragmentación del hábitat producida por actividades antropogénicas. Postulamos que la diversidad genética en las poblaciones analizadas, a pesar de la constante explotación, se origina por el sinergismo entre la polinización cruzada y la actividad de elementos genéticos transponibles. El alto grado de diversidad genética encontrado en las poblaciones silvestres analizadas en San Luis Potosí demuestra la importancia agroecológica de Agave salmiana ssp. crassispina en el Altiplano Mexicano, una región geográfica que abarca casi la mitad del territorio mexicano. Sin embargo, uso descontrolado y manejo inapropiado de las magueyeras silvestres podría arriesgar los recursos fitogenéticos de Agave salmiana ssp. crassispina.""The Potosino Upland Plateau is an arid and semiarid ecosystem. Agave salmiana Otto Salm Dick ssp. crassispina (Trel Gentry), which grows in this environment, is a species used for gathering of edible insects and for production of mezcal. Mezcal is a Mexican alcoholic beverage with denomination of origin. Production of mezcal with wild populations of Agave salmiana ssp. crassispina lacks an exploitation system based on biological knowledge of the species. The lack of organized use has demographic effects, which leads to degradation of genetic resources and risks to this species. This study analyzed genetic diversity and population structure of three wild populations of Agave salmiana ssp. crassispina from San Luis Potosí, Mexico by AFLP's (amplified fragment length polymorphism). The evidence showed a high level of genetic diversity within populations and low level of genetic differentiation among populations, probably as a result of habitat fragmentation produced by anthropogenic activities. We postulate that the genetic diversity in the wild populations analyzed, despite their constant exploitation, is originated by the synergism between cross-pollination and the activity of transposable genetic elements. The high level of genetic diversity found in the wild populations in San Luis Potosí shows agro-ecological relevance of Agave salmiana ssp. crassispina in the Mexican upland plateau. However, uncontrolled use and inappropriate management of wild populations could risk the genetic resources of Agave salmiana ssp. crassispina.

Sodium N-methyldithiocarbamate impact on soil bacterial diversity in greenhouse tomato (Solanum lycopersicum L.) crop

The constant use of sodium N-methyldithiocarbamate (metam sodium: MS) in protected agriculture in México has attracted the attention of researchers and producers on their effects on the environment. The objective of this study was to evaluate the impact of MS on the bacterial community structure in an agricultural soil with tomato crop (Solanum lycopersicum L.) considering the different phenological stages of the crop. The experiment was carried out in a greenhouse, with a completely randomized block design with two treatments: 1) without MS and 2) with application of 400 L·ha-1 of MS. For the determination of the bacterial structure, the biodiversity indexes of richness (S), diversity (H') and equity (J'), identification of operational taxonomic units (OTU) were used through the T-RFLP technique. Application of MS in soil showed no significant effect on bacterial richness. However, the application of MS does alter the structure of the bacterial community (H' and J') in each of the tomato phenological stages. Finally, future studies which include the evaluation of the effects of MS on the physiology of intensive crops and functions in the different soil types are need.The constant use of sodium N-methyldithiocarbamate (metam sodium: MS) in protected agriculture in México has attracted the attention of researchers and producers on their effects on the environment. The objective of this study was to evaluate the impact of MS on the bacterial community structure in an agricultural soil with tomato crop (Solanum lycopersicum L.) considering the different phenological stages of the crop. The experiment was carried out in a greenhouse, with a completely randomized block design with two treatments: 1) without MS and 2) with application of 400 L·ha-1 of MS. For the determination of the bacterial structure, the biodiversity indexes of richness (S), diversity (H') and equity (J'), identification of operational taxonomic units (OTU) were used through the T-RFLP technique. Application of MS in soil showed no significant effect on bacterial richness. However, the application of MS does alter the structure of the bacterial community (H' and J') in each of the tomato phenological stages. Finally, future studies which include the evaluation of the effects of MS on the physiology of intensive crops and functions in the different soil types are need

Evaluation of a SUMO E2 conjugating enzyme involved in resistance to Clavibacter michiganensis subsp. michiganensis in Solanum peruvianum, through a tomato mottle virus VIGS assay

"Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker of tomato. Currently, no Solanum lycopersicum resistant varieties are commercially available, but some degree of Cmm resistance has been identified in Solanum peruvianurn. Previous research showed up regulation of a SUMO E2 conjugating enzyme (SCEI) transcript in S. peruvianum compared to S. lycopersicum following infection with Cmm. In order to test the role of SCEI in resistance to Cmm, a fragment of SCEI from S. peruvianum was cloned into a novel virus induced gene silencing (VIGS) vector based on the geminivirus, Tomato Mottle Virus (ToMoV). Using biolistic inoculation, the ToMoV-based VIGS vector was shown to be effective in S. peruvianum by silencing the magnesium chelatase gene, resulting in leaf bleaching. VIGS with the ToMoV_SCEI construct resulted in 61% silencing of SCEI in leaves of S. peruvianum as determined by quantitative RT-PCR. The SCEI -silenced plants showed unilateral wilting (15 dpi) and subsequent death (20 dpi) of the entire plant after Cmm inoculation, whereas the empty vector-treated plants only showed wilting in the Cmm-inoculated leaf. The SCEI-silenced plants showed higher Cmm colonization and an average of 4.5 times more damaged tissue compared to the empty vector control plants. SCEI appears to play an important role in the innate immunity of S. peruvianum against Cmm, perhaps through the regulation of transcription factors, leading to expression of proteins involved in salicylic acid dependent defense responses.

Neuroprotective effects of insulin-like growth factor II in a mouse model of Parkinson's disease

Progressive degeneration of the nigrostriatal dopaminergic pathway is a core, currently irreversible pathological hallmark of Parkinson’s disease (PD) that leads to a variety of motor and non-motor symptoms. Here, we aimed to study the potential neuroprotective effects of insulin-like growth factor II (IGF-II) in a PD mouse model based on the chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTP/p), which induces loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNc). Male C57BL6/J mice (N=36) received a 5-week treatment with MPTP/p (or vehicle) and were co-treated with chronic IGF-II (or saline) from either the beginning of the procedure (plus an additional week, days 1-44) or once the MPTP/p insult was already triggered (days 21-44). Baseline and post-treatment measurements for motor performance in the Rotarod and self-grooming in an Open Field were taken. Likewise, dopaminergic (TH, DAT) and neuroinflammatory-related (GFAP) markers in the SNc and the dorsal striatum were studied by immunohistochemistry. Our results revealed that both early and delayed IGF-II co-treatment were successful in preventing motor and behavioral impairment in the MPTP/p model. Moreover, chronic IGF-II protected against MPTP/p-induced loss of dopaminergic neurons in the SNc and promoted a significant recovery of dopaminergic activity in the terminals located in the dorsal striatum, further reducing reactive astrocytosis in these brain regions. Thus, we demonstrated the neuroprotective role of IGF-II in a mouse model of PD, highlighting its potential as a promising therapeutical target for treating this disease. Funding: UMA18-FEDERJA-004, PID2020-113806RB-I00. Universidad de Málaga, Campus de Excelencia Internacional Andalucía Tech.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Effect of viral storm in patients admitted to intensive care units with severe COVID-19 in Spain: a multicentre, prospective, cohort study

Background: The contribution of the virus to the pathogenesis of severe COVID-19 is still unclear. We aimed to evaluate associations between viral RNA load in plasma and host response, complications, and deaths in critically ill patients with COVID-19. Methods: We did a prospective cohort study across 23 hospitals in Spain. We included patients aged 18 years or older with laboratory-confirmed SARS-CoV-2 infection who were admitted to an intensive care unit between March 16, 2020, and Feb 27, 2021. RNA of the SARS-CoV-2 nucleocapsid region 1 (N1) was quantified in plasma samples collected from patients in the first 48 h following admission, using digital PCR. Patients were grouped on the basis of N1 quantity: VIR-N1-Zero ([removed]2747 N1 copies per mL). The primary outcome was all-cause death within 90 days after admission. We evaluated odds ratios (ORs) for the primary outcome between groups using a logistic regression analysis. Findings: 1068 patients met the inclusion criteria, of whom 117 had insufficient plasma samples and 115 had key information missing. 836 patients were included in the analysis, of whom 403 (48%) were in the VIR-N1-Low group, 283 (34%) were in the VIR-N1-Storm group, and 150 (18%) were in the VIR-N1-Zero group. Overall, patients in the VIR-N1-Storm group had the most severe disease: 266 (94%) of 283 patients received invasive mechanical ventilation (IMV), 116 (41%) developed acute kidney injury, 180 (65%) had secondary infections, and 148 (52%) died within 90 days. Patients in the VIR-N1-Zero group had the least severe disease: 81 (54%) of 150 received IMV, 34 (23%) developed acute kidney injury, 47 (32%) had secondary infections, and 26 (17%) died within 90 days (OR for death 0·30, 95% CI 0·16–0·55; p<0·0001, compared with the VIR-N1-Storm group). 106 (26%) of 403 patients in the VIR-N1-Low group died within 90 days (OR for death 0·39, 95% CI 0·26–0·57; p[removed]11 página

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Background A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. Methods This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. Findings Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0–75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4–97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8–80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3–4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. Interpretation ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials

Análisis de la expresión génica durante la interacción de jitomate y especies silvestres con Clavibacter michiganensis subsp. michiganensis

Tesis (Doctorado en Ciencias en Biología Molecular)"Clavibacter michiganensis subsp. michiganensis (Cmm) es un fitopatógeno Gram-positivo causante de la enfermedad conocida como cáncer bacteriano del jitomate (Solanum Iycopersicum), una enfermedad para la cual no existe tratamientos curativos o preventivos, lo que ha provocado pérdidas económicas importantes en México y en el mundo entero. En este trabajo, se analizó la expresión génica de varias especies silvestres resistente relacionadas al jitomate como S. peruvianum LA2157, S. peruvianum LA2172, y S. habrochaites LA2128, y una especie susceptible, con el fin de identificar genes involucrados en las repuestas de defensa y/o susceptibilidad. Mediante el estudio del transcriptoma con cDNA-AFLP (cDNA-Amplified Fragment Length Polymorphism), se logró la identificación de 403 transcritos diferencialmente expresados. Entre ellos, algunos genes mostraron un patrón de expresión opuesto entre las especies resistentes y la susceptible, dentro de los que se incluyen genes involucrados en la ruta de degradación de proteínas mediada por ubiquitina, y una peroxidasa secretoria. Estos genes mostraron una inducción en las especies resistentes, y fueron reprimidos en la especie susceptible, lo que sugiere una posible participación en las etapas tempranas de las respuestas de defensa posteriores a la infección por Cmm. Los genes identificados en las especies silvestres son nuevos candidatos para análisis funcionales posteriores para determinar su contribución en las respuestas de defensa contra Cmm, y determinar las bases moleculares de incompatibilidad entre las especies silvestres resistentes y Cmm. Esto contribuirá en el desarrollo de mejores métodos de control del cáncer bacteriano del jitomate,""Clavibacter michiganensis subsp. michiganensis (Cmm) is a Gram-positive bacterial pathogen causing bacterial wilt and canker of tomato (Solanum lycopersicum), an economically important disease producing economic losses worldwide. In this study, gene expression analysis was conducted using several resistant tomato-related wild species, including S. peruvianum LA2157, S. peruvianum LA2172, and S. habrochaites LA2128, and a susceptible tomato species, to identify genes involved in the disease response. Using cDNAamplified fragment length polymorphism (AFLP), 403 differentially expressed transcripts were identified. Among those, several genes showed contrasting expression patterns among resistant and susceptible species, including genes involved in the ubiquitin-mediated protein degradation pathway and secretory peroxidase. These genes were up-regulated in resistant species, but downregulated in susceptible species, suggesting their likely involvement in early plant defense responses following Cmm infection. These identified genes would serve as new candidate bacterial wilt disease resistance genes and should be subjected to further functional analyses to determine the molecular basis of incompatibility between wild species of tomato and Cmm. This would then contribute to the development of more effective and sustainable Cmm-control methods.

Obtención de la huella genética de agaves mezcladores de San Luis Potosi por RAPD y AFLP.

Tesis (Maestría en Ciencias en Biología Molecular)"En el Altiplano Potosino, la especie vegetal utilizada para la producción de mezcal es Agave salmiana. La industria mezcalera, que carece de sistemas de explotación basados en el conocimiento biológico de la especie que utiliza como materia prima, puede causar estragos tanto demográficos como en la diversidad genética de A. salmiana. Los marcadores moleculares RAPD y AFLP basados en PCR, son ampliamente utilizados para el análisis de diversidad genética ya que su principal ventaja es que no requieren un conocimiento previo de la especie bajo estudio. En el presente trabajo, los resultados del análisis de la diversidad genética de tres poblaciones de A. salmiana (Ipiña, Aquiles Serdán y El Cuervo) utilizando extractos de DNA total de hoja y del meristemo central de los especímenes colectados, mostraron que la diversidad genética dentro de cada población es mayor que la variabilidad interpoblacional. La diversidad interpoblacional se puede observar en los dendrogramas, donde existe una tendencia a la formación de grupos de acuerdo al origen geografico. El método de AFLP mostró ser más robusto que el de RAPD al aplicarse a A. salmiana, en base a los valores de “bootstrap” de los dendrogramas generados; en tanto que la prueba de Mantel mostró que al menos en dos poblaciones, El Cuervo e Ipiña; AFLP y RAPD no son métodos correlacionados. Se sugiere que el origen de la variabilidad genética de las poblaciones analizadas se debe a que las plantas madre de los diferentes hijuelos colectados son producto de polinizaciones cruzadas que tuvieron lugar anteriormente en esas localidades. Sin embargo, en A. fourcroydes se ha reportado variabilidad genética asexual al comparar los patrones de bandeo de AFLP de una planta madre con el de sus hijuelos, y se ha sugerido que la planta progenitora acumula mutaciones somáticas que se heredan a los descendientes. En A. tequilana se han encontrado secuencias terminales de retrotransposones Ty-copia, que podrían utilizarse para inferir relaciones filogenéticos con otras especies de Agave. Con estos antecedentes podemos sugerir que parte de la variabilidad genética encontrada en A, salmiana puede deberse a la actividad de elementos de transposición, por lo que es necesario realizar estudios pareados hoja-meristemo (in vitro), con el fin de determianr si existen diferencias en el patrón de bandeo de un tejido a otro en el mismo individuo, y demostrar la presencia de estos elementos. Este ers el primer trabajo de marcadores moleculares en A. salmiana de San Luis Potosí y la diversidad genética encontrada es importante desde el punto de vista ecológico, sin embargo el uso irracional de las magueyeras pudiera afectar esta diversidad en un fututro cercano si no se implementan programas de conservación de germoplasma in situ e in vitro.""In the “Altiplano Potosino”, the plant species used for “mezcal” production (a Mexican spirit) is Agave salmiana. The industry of mezcal production lacks a sustainable exploitation system that is based on the biological knowledge of the species used as a carbohydrates source and this may reduce dramatically the population and genetic diversity of A. salmiana. The molecular markers based on polymerase chain reaction (PCR), such as RADP and AFLP, are widely used for genetic diversity assays; applying these markers has the great advantage that no additional genetic or molecular information (e.g. specific DNA sequences) of A. salmiana is necessary. In this work, the analyses of genetic diversity of three populations of A. salmiana (located at Ipiña, Aquiles Serdán and El Cuervo in San Luis Potosí), using total DNA extracts from leaves and plants propagated from central meristematic tissues, showed that the genetic diversity within populations is higher than that among populations, however a population cluster group trend was observed in the dendrograms produced. The AFLP method was more robust than the RAPD method based on the bootstrap values obtained from the dendrograms; on the other hand, the Mantel test showed that at least in two populations (El Cuervo and Ipiña) AFLP and RAPD were not correlated. We propose that the source of genetic variability of the populations analyzed, originates from cross pollination of the progenitor plants of the samples collected. However, in previous studies, it has been reported that A. fourcroydes showed genetic diversity in plants that were asexually produced when AFLP fingerprints of mother plants and daughter ramets (hijuelos) were compared. It has been suggested that the mother plant accumulates somatic mutations which can be inherited to the daughter ramets when a rhizome is produced. In A. tequilana, terminal sequences of retrotransposons (Ty-copia) have been found and these elements can be used to track phylogenetic relations with other Agave species. Based on this information, we suggest that part of the genetic variability found in A. salmiana is due to the transposition elements, however it is necessary to perform additional experiments with paired analysis [plants propagated from central meristem (in vitro) – leaf] to determine if differences in AFLP fingerprints are present in mother plants and daughter ramets of the same plant and also if retrotransposon sequences are present.