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We aimed to infer ancestral area and historical colonization of Lobelia columnaris in the sky islands of Bioko and Cameroon through dated phylogeny using chloroplast genomes. Specifically, we aim to answer the following questions: (1) What are the phylogenetic relationships among Bioko Island and Cameroon populations? (2) Are the older populations found in the older sky islands? We assembled novel plastomes from 20 individuals of L. columnaris from 5 mountain systems. The plastome data were explored with phylogenetic analyses using Maximum Likelihood and Bayesian Inference. The populations of L. columnaris have a monophyletic origin, subdivided into three plastomes‐geographic clades. The plastid phylogenomic results and age of the sky islands indicate that L. columnaris colonized first along with the Cameroon Volcanic Line's young sky islands of Bioko. The crown group (1.54 Ma) split the population in Bioko and mainland Cameroon. It is possible that Bioko was the ancestral area and likely isolated during cold and dry conditions in forest refugia. Presumably, the colonization history occurred during the middle‐late Pleistocene from South Bioko's young sky island to North Bioko and the northern old sky islands in Cameroon. Furthermore, the central depression with lowland forest between North and South Bioko is a current geographic barrier that keeps separating the populations of Bioko from each other. Also, the shallow sea channel keeps isolated the populations of Bioko and the mainland populations. The Pleistocene climatic oscillations led to the divergence of the Cameroon and Bioko populations into three clades. L. columnaris colonized the older sky islands in mainland Cameroon after establishment in Bioko's younger sky islands. Contrary to expectations, the biogeography history was an inverse progression with respect to the age of the Afromontane sky islands., Lobelia columnaris colonized the sky island of West Africa during the Pleistocene. The colonization history of this giant lobelia was from the young sky islands through old sky islands in mainland Africa.

Leaf scald caused by Xanthomonas albilineans in sugarcane is one of the most important diseases around the world since it causes severe losses in both agricultural and industrial yields. In Cuba, resistance to this disease is considered a major selection criterion within the breeding program. The aim of this work was to detect sugarcane cultivars with stable reaction to X. albilineans by using both additive main effects and multiplicative interaction (AMMI) and linear general models. For this, 16 cultivars planted simultaneously in 2015, 2016, and 2017 in two locations: Jovellanos (Matanzas) and Florida (Camaguey) in Cuba were mechanically inoculated with a bacterial isolate previously characterized by sequencing its DNA fragment. Disease severity was recorded in plant cane and first ratoon, and results were consistent in both analyses and revealed that L55-5 and C323-68 were the most susceptible cultivars and C1051-73 was the most resistant; however, C1051-73, C275-80, C86-12, C88-382, C89-147, My5514, and Ty86-28 were the most stable across the years and localities evaluated. Results will allow adapting the methodology for the evaluation of the reaction to leaf scald of new sugarcane cultivars.

Numerous cells with very large and irregular nuclei (“monster” cells) have not hitherto been reported in desmoplastic melanoma (DM). Their prognostic significance in melanomas is a matter of debate, although some authors have associated them with more aggressive tumor behavior. We report a mixed DM on the scalp of an 88-year-old woman imitating an atypical fibroxanthoma. Tumor cells stained positive for SOX10, S100, and cyclin D1; BRAF mutation status was negative, and fluorescence in situ hybridization analysis showed copy number gains in 11q13 (cyclin D1) and 6p25 (RREB1), and loss in 6q23 (MYB). Cyclin D1 amplification is associated with poor prognosis in melanoma.

We detected the genome-wide pattern of DNA methylation and its association with gene expression in sexual and asexual progenies of mature Robinia pseudoacacia trees. DNA methylation plays an important role in plant reproduction and development. Although some studies on sexual reproduction have been carried out in model plants, little is known about the dynamic changes in DNA methylation and their effect on gene expression in sexual and asexual progeny of woody plants. Here, through whole-genome bisulfite sequencing, we revealed DNA methylation patterns in the sexual and asexual progenies of mature Robinia pseudoacacia to understand the regulation of gene expression by DNA methylation in juvenile seedlings. An average of 53% CG, 34% CHG and 5% CHH contexts was methylated in the leaves of mature and juvenile individuals. The CHH methylation level of asexually propagated seedlings was significantly lower than that of seed-derived seedlings and mature trees. The intergenic regions had the highest methylation level. Analysis of differentially methylated regions (DMRs) showed that most of them were hypermethylated and located in the gene upstream and introns. A total of 24, 108 and 162 differentially expressed genes containing DMRs were identified in root sprouts (RSs), root cuttings (RCs) and seed-derived seedlings (SSs), respectively, and a large proportion of them showed hypermethylation. In addition, DMRs were enriched within GO subcategories including catalytic activity, metabolic process and cellular process. The results reveal widespread DNA methylation changes between mature plants and their progenies through sexual/asexual reproduction, which provides novel insights into DNA methylation reprogramming and the regulation of gene expression in woody plants.

Classical swine fever is a highly contagious viral disease with a significant impact on food production worldwide. It currently represents one of the main limitations for the development of the pig industry in Cuba. PorvacÒ is a subunit marker vaccine that confers a very rapid onset of protection. Since there are different production systems in pig breeding, readjustments in the vaccination program are often required. This study compares the safety and efficacy in piglets of two vaccination schedules with PorvacÒ (0-2 weeks and 0-3 weeks), initiated at two or three weeks of age. Clinical monitoring was conducted, and a neutralization peroxidase-linked assay was used to measure the neutralization titers. All immunization regimens were safe and well-tolerated, without local or systemic adverse reactions in the vaccinated animals. Geometric mean neutralizing antibody titers higher than 1/1500 were detected in all groups during the six months of the trial. One month after the second immunization, piglets primed at two weeks of age, and boostered three weeks later, developed significantly higher neutralization titers (1/15644) compared to those vaccinated at a similar age but with a two-week interval between doses (1/5760). However, no significant differences in the titers were found three and six months after vaccination among the four regimens. In summary, all the variants studied are effective, but it is recommended to start vaccination at two weeks old, with the second dose at either two or three weeks later, depending on the production system and the purpose of the farm.

Rhipicephalus Boophilus microplus cattle tick is a scourge for livestock production. The infestations produced by this pathogen are incompletely contained by chemical treatments, with the associated environmental pollution risks. Vaccination against cattle ticks has emerged as a feasible and environmentally friendly strategy to control tick-borne diseases. In this setting, Gavac® vaccine has proven effective in decreasing cattle tick populations through antibody responses against the tick Bm86 antigen, as part of an Integrated Control Program. However, animal vaccination programs require easy and ready-to-use screening tests to follow up the immune response in vaccinated animals under field conditions. This study reports the evaluation HeberFast® Line Gavac, a lateral flow immunochromatographic system for the rapid detection of anti Bm86 antibodies in vaccinated cattle. The system was tested on 598 serum samples taken from immunized animals, arranged in three groups according to their anti-Bm86 antibody response in ELISA (209 high, 150 medium or 239 low and 100 samples from non-immunized animals. The HeberFast® Line Gavac system was assessed for sensitivity, specificity, and concordance against the ELISA reference test. Consistency was evaluated among production batches and inter-analyst reading-independent consistency at two moments: ten minutes after completing the test and after strip drying. The system showed high sensitivity (81.6%, 82.2%, and 81%), specificity (96.7, 94.6, and 93.3%), and agreement with the ELISA reference test (75%; 74%, and 71%) for high, medium and low anti-Bm86 sera, respectively. The effectiveness of the diagnosis was 87.6; 87.1; 85.9 for high, medium, and low antibody titers, respectively. Consistency among production batches and analysts was documented, and no significant differences between evaluation times were found. These results indicate that HeberFast® Line Gavac is a valuable tool for the serological surveillance of Gavac vaccinated cattle.

Bacteria from the rhizobia group have been previously identified as grass-endophytes in grassland with cropping history of legumes. In this paper we aimed to demonstrate that strains of the Rhizobium genus, isolated from the rhizosphere of a rice Cuban cultivar subjected to intensive monoculture management, behave as endophytes and promote its growth. Eleven bacteria previously isolated from the rhizosphere of rice plants, were screened for their plant growth promoting traits. We found that some of them were able to solubilize inorganic phosphate compounds, produce siderophores and biofilm, have exocellulase and protease activity and were able to inhibit the phytopathogen Pyricularia oryzae growth. The phylogenetic analysis using partial sequences of 16S rRNA gene showed that bacteria isolated from rice plant of cultivar INCA LP-5 belonged to Rhizobium, Agrobacterium and Pseudomonas genera. Two strains analyzed, Rhizobium sp. Rpr2 and Rdp16, increased the height and biomass of rice plants, 70 days post-inoculation in greenhouse conditions. According to the quantification of Rhizobium sp. strain Rpd16 in surface-disinfected tissues of root and sheath, together with the analysis of confocal microscopy images, we can conclude that the Rdp16 strain is a rice endophyte of the cultivar INCA LP-5. This investigation is the first evidence of endophytic rhizobia able to promote the rice growth of a Cuban cultivar subjected to intensive monoculture, without rotation with legume plants and without a previous history of rhizobia inoculation.

Chikungunya virus (CHIKV) nonstructural protein 1 (nsP1) harbors the methyltransferase (MTase) and guanylyltransferase (GTase) activities needed for viral RNA capping and represents a promising antiviral drug target. We compared the antiviral efficacies of nsP1 inhibitors belonging to the MADTP, CHVB, and FHNA series (69-fluoro- homoneplanocin A [FHNA], its 39-keto form, and 69-b-fluoro-homoaristeromycin). Cell-based phenotypic cross-resistance assays revealed that the CHVB and MADTP series had similar modes of action that differed from that of the FHNA series. In biochemical assays with purified Semliki Forest virus and CHIKV nsP1, CHVB compounds strongly inhibited MTase and GTase activities, while MADTP-372 had a moderate inhibitory effect. FHNA did not directly inhibit the enzymatic activity of CHIKV nsP1. The first-oftheir- kind molecular-docking studies with the cryo-electron microscopy (cryo-EM) structure of CHIKV nsP1, which is assembled into a dodecameric ring, revealed that the MADTP and CHVB series bind at the S-adenosylmethionine (SAM)-binding site in the capping domain, where they would function as competitive or noncompetitive inhibitors. The FHNA series was predicted to bind at the secondary binding pocket in the ring-aperture membrane-binding and oligomerization (RAMBO) domain, potentially interfering with the membrane binding and oligomerization of nsP1. Our cell-based and enzymatic assays, in combination with molecular docking and mapping of compound resistance mutations to the nsP1 structure, allowed us to group nsP1 inhibitors into functionally distinct classes. This study identified druggable pockets in the nsP1 dodecameric structure and provides a basis for the rational design, optimization, and combination of inhibitors of this unique and promising antiviral drug target., K.K. was supported by the Marie Sklodowska-Curie ETN European Training Network “ANTIVIRALS” (EU grant agreement 642434). M.G.G. was supported by funding from the Oncode Institute, the Netherlands. R.J. and J.R. were supported by the ATIP-Avenir program and the Bettencourt Shueller Foundation. M.-J.P.-P. acknowledges the financial support of AEI (grant PID2019-105117RR-C22).

8 páginas, 3 figuras, 1 tabla., The authors wish to acknowledge the collaboration of personnel from the Departamento de Sanidad Animal of the University of León and thank them for their help in the laboratory analyses, particularly to Eva Martín de Valmaseda. The authors were grateful to Falco Iberia S.L. Company for referring the falcon to the Pathological Diagnostic Service of the Faculty of Veterinary Sciences of León. Technical support provided by Marta Silva and Julio Benavides was also gratefully acknowledged. RV was supported by a predoctoral contract from the Spanish Ministry of Education. JE was the recipient of a postdoctoral contract Juan de la Cierva-Formación (FJC2019-042422-I) of the Spanish Ministry of Science and Innovation.

20 p.-6 fig.-3 tab., The regulators of complement activation (RCA) gene cluster in 1q31-1q32 includes most of the genes encoding complement regulatory proteins. Genetic variability in the RCA gene cluster frequently involve copy number variations (CNVs), a type of chromosome structural variation causing alterations in the number of copies of specific regions of DNA. CNVs in the RCA gene cluster often relate with gene rearrangements that result in the generation of novel genes, carrying internal duplications or deletions, and hybrid genes, resulting from the fusion or exchange of genetic material between two different genes. These gene rearrangements are strongly associated with a number of rare and common diseases characterized by complement dysregulation. Identification of CNVs in the RCA gene cluster is critical in the molecular diagnostic of these diseases. It can be done by bioinformatics analysis of DNA sequence data generated by massive parallel sequencing techniques (NGS, next generation sequencing) but often requires special techniques like multiplex ligation-dependent probe amplification (MLPA). This is because the currently used massive parallel DNA sequencing approaches do not easily identify all the structural variations in the RCA gene cluster. We will describe here how to use the MLPA assays and two computational tools to analyze NGS data, NextGENe and ONCOCNV, to detect CNVs and gene rearrangements in the RCA gene cluster., SRdeC is supported by the Spanish “Ministerio de Economía y Competitividad-FEDER” (SAF2015-66287R, PID2019-104912RB-I00, RTC-2016-4635-1 and the Autonomous Region of Madrid (S2017/BMD-3673). Secugen has received a soft loan from the Spanish “Ministerio de Economía y Competitividad” Retos Program RTC-2016-4635-1 cofinanced by FEDER funds.

18 p.-10 fig., Plant in vitro regeneration systems, like somatic embryogenesis, are essential in breeding; they permit to propagate elite genotypes, to produce doubled-haploids, and to convert gene editing or transformation events into plants. However, in many crop and forest species somatic embryogenesis is highly inefficient. We report a new strategy to improve in vitro embryogenesis using synthetic small molecule inhibitors of mammalian glycogen synthase kinase 3β (GSK-3β), never used in plants. These inhibitors increased in vitro embryo production in three different systems and species, microspore embryogenesis of Brassica napus and Hordeum vulgare, and somatic embryogenesis of Quercus suber. TDZD-8, representative compound of the molecules tested, inhibited GSK-3 activity in microspore cultures, and increased expression of embryogenesis- genes FUS3, LEC2 and AGL15. Plant GSK-3 kinase BIN2 is master regulator of brassinosteroid (BR) signalling. During microspore embryogenesis, BR biosynthesis and signalling genes CPD, GSK-3-BIN2, BES1 and BZR1 were upregulated and BAS1 catabolic gene was repressed, indicating activation of BR pathway. TDZD-8 increased expression of BR signalling elements, mimicking BR effects. The findings support that the small molecule inhibitors promoted somatic embryogenesis by activating the BR pathway, opening the way for new strategies using GSK-3β inhibitors that could be extended to other species., Work supported by the Spanish National Agency of Research (Agencia Estatal de Investigación, AEI) and European Regional Development Fund (ERDF/FEDER) [grants AGL2017-82447-R and PID2020-113018RB-I00 to PST; and SAF2016-76693-R and PID2019-105600RB-I00 to AM]

15 páginas., This study estimates the economic losses due to outbreaks of toxoplasma abortions in a dairy (1928 sheep) and a meat (700 sheep) flock in Spain raised under intensive and semi-extensive management conditions, respectively. In both flocks, sheep were divided into multiple groups to synchronise reproduction. The outbreaks resulted in abortion rates in individual lots of 12.6% (30/239) in the dairy flock and 33.3% (70/210) in the meat flock. Toxoplasma gondii was definitively diagnosed in most submitted cases and the only abortifacient pathogen identified despite extensive investigation. Upon completion of lambing and lactation, veterinarians and farmers completed a questionnaire to gather the data to determine the direct economic impact. The calculated total direct economic losses were €5154.5 (€171.8/abortion) in the dairy flock and €4456 (€63.6/abortion) in the meat flock. Results suggest that flock size, production system, abortion rate and control measures are the key factors influencing economic losses, which vary greatly between individual flocks., This work was supported by research projects from the “Junta de Castilla y León” (LE080U16) and Spanish Ministry of Sciences (AGL2016-75935-C2-1-R and AGL2016-75935-C2-2-R). Daniel Gutiérrez-Expósito is the recipient of a postdoctoral contract from the Ministry of Science and Innovation (grant no. FJCI-2017-32020). Mercedes Fernández was funded by a UCM-Santander/2017 pre-doctoral grant. Mark P. Dagleish was funded by the Scottish Government. We wish to acknowledge farmers of the flocks and especially the field veterinarian Luis Gómez for their help. We wish to acknowledge María Teresa Carro, María Murillas and Marta Silva for their excellent technical assistance.

Hybrid oncocytic/chromophobe tumor (HOCT) of the kidney arising from a precursor oncocytosis not associated with the Birt-Hogg-Dubé (BHD) syndrome is an unusual and highly interesting neoplasm. Immunohistochemical and molecular findings suggest that HOCT is an entity distinct from both oncocytoma and chromophobe carcinoma. Although uncertainty persists regarding the factors predisposing to the development of HOCT, experimental findings suggest that it may arise due to the effect of toxins or in association with chronic kidney failure. The potential role of prior renal lymphoma in the development of oncocytosis has not hitherto been examined. We present a morphological, immunohistochemical, and molecular analysis of an HOCT arising from renal oncocytosis in conjunction with CLL affecting the kidney. The findings suggest that this tumor belongs to a family of similar neoplasms including oncocytoma, the eosinophilic variant of chromophobe renal-cell carcinoma (CRCC), and low-grade oncocytic tumor, even though these neoplasms may arise from different precursor lesions. HOCT and oncocytosis revealed the same immunohistochemical profile consistent on positivity for epithelial membrane antigen (EMA), cytokeratin 7 (Ck7), E-cadherin, CAM 5.2 and negativity for Pax-8, vimentin, renal-cell carcinoma (RCC) antigen, CD117, racemase, progesterone receptor, and CD10. The Ki-67 proliferation index was

Rice biofertilization with Rhizobium increases the growth and yield of the crop. However, evidence for this has not been observed in Cuban rice cultivars. This research aimed to typify two Rhizobium isolates, considering the use of different carbon sources, their tolerance to stress conditions, and the ability to promote the growth and development of rice. Two Rhizobium sp. isolates (Rpr11 and 5P1) were used and their facility to grow on different carbon sources, pH, and salinity levels was determined. The effect of the inoculation of these bacteria on the growth and yield of rice was evaluated under controlled, greenhouse, and field conditions. Both isolates grew on mannitol, glycerol, maltose, and fructose at the highest concentrations of NaCl (1.0, 1.5 and 2.0%). The isolate 5P1 grew at all evaluated pH levels, especially at pH 5.0 and pH 8.0. The inoculation of both isolates increased the plant biomass and the potassium content. The plants inoculated with 5P1 had the highest contents of nitrogen, total chlorophyll, carbohydrates and proteins, and grain yield. This study is the first in Cuba that shows the beneficial effect of Rhizobium inoculation on the physiology and yield of rice.

Some plant cells are able to rebuild new organs after tissue damage or in response to definite stress treatments and/or exogenous hormone applications. Whole plants can develop through de novo organogenesis or somatic embryogenesis. Recent findings have enlarged our understanding of the molecular and cellular mechanisms required for tissue reprogramming during plant regeneration. Genetic analyses also suggest the key role of epigenetic regulation during de novo plant organogenesis. A deeper understanding of plant regeneration might help us to enhance tissue culture optimization, with multiple applications in plant micropropagation and green biotechnology. In this review, we will provide additional insights into the physiological and molecular framework of plant regeneration, including both direct and indirect de novo organ formation and somatic embryogenesis, and we will discuss the key role of intrinsic and extrinsic constraints for cell reprogramming during plant regeneration., This research was funded by the Ministerio de Economía, Industria y Competitividad (MINECO)of Spain (grant numbers BIO2015-64255-R and RTI2018-096505-B-I00 to JMP-P, and AGL2017-82447-R to PST),the Conselleria d’Educació, Cultura i Sport of the Generalitat Valenciana (grant numbers IDIFEDER 2018/016and PROMETEO/2019/117 to JMP-P), and the European Regional Development Fund (ERDF) of the EuropeanCommission. SI is a research fellow of the Generalitat Valenciana (grant number ACIF/2018/220).

11 páginas, 8 figuras, 2 tablas., Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and nonexpensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001). Under the conditions of this study, the use of centrifugation in density gradients allow for the simultaneous purification of PBMCs and neutrophils, with high purity of both populations, from the same sample of blood. The isolation of monocytes could be subsequently achieved through two different methods, i.e. based on immunomagnetic columns or adherence. The preference between both methods would depend on the necessities of the experiment, the initial sample with high purity of monocytes or a final population of MDMs required., We thank the staff of the Instituto de Ganadería de Montana ˜ (CSICULE) responsible for the handling of experimental animal as well as their help during the processing of the samples. We wish to acknowledge the staff of the laboratory of instrumental techniques of University of Leon. ´ Finally, we wish to acknowledge Carmen Agudín and María Teresa Carro for their excellent technical assistance., The Spanish Ministry of Science, Innovation and Universities (project AGL2015-66540-C2-1-R) financed this work. Noive Arteche Villasol has been financially supported by a predoctoral contract (BES-2016-076513) from the Spanish Ministry of Science, Innovation and Universities. Daniel Gutiérrez-Expósito is the recipient of a postdoctoral con- tract from the “Junta de Castilla y León” (LE080U16)

10 páginas, 2 tablas, 9 figuras., There is an unacknowledged clinical presentation of ovine toxoplasmosis characterized by early abortions and lesions of fetal leukoencephalomalacia. To investigate the pathogenesis of this condition, the extent and distribution of leukomalacia and the variations in the cell populations associated with it were characterized in 32 fetal brains from 2 previously published experimental studies of Toxoplasma gondii infection in pregnant sheep. Immunohistochemical labeling of βAPP allowed for the detection of leukomalacia in 100/110 (91%) studied samples. There was no clear influence of the challenge dose or the area of the brain (frontal lobe, corpus callosum, midbrain, and cerebellum). In tissues with leukomalacia, there was loss of oligodendrocytes and increased number of astrocytes and microglia both in the areas of necrosis but also in the surrounding area. These findings were similar to those described in ovine experimental models (inflammation syndrome and hypoxic models) of periventricular leukomalacia in humans. Thus, a fetal inflammatory syndrome may be involved in the pathogenesis of early abortion in ovine toxoplasmosis. However, further studies are needed to determine the pathogenesis of this clinical presentation because placental thrombosis and resulting hypoxia could also be responsible for the leukomalacia., This work was supported by research projects from the “Junta de Castilla y León” (LE080U16) and Spanish Ministry of Sciences (AGL2016-75935-C2- 1-R and AGL2016-75935-C2-2-R). Daniel Gutiérrez-Expósito is the recipient of a postdoctoral contract from the Ministry of Science and Innovation (Grant No. FJCI-2017-32020).

12 páginas, 5 figuras, 2 tablas., In order to gain further insight into the pathogenesis and transmission of ovine neosporosis, the serological response of 13 naturally infected pregnant sheep was monitored. All sheep were euthanized upon the detection of a sharp increase in the level of specific antibodies against N. caninum in order to study the maternal immune response after the recrudescence of a chronic infection. Ten sheep were euthanized between 84 and 118 days of gestation, whereas the three remaining and three control not infected, pregnant sheep were euthanized at 135 days of gestation after no sharp increase in antibodies was detected. Vertical transmission was confirmed in 11 sheep by detection of N. caninum-DNA in at least one fetus, confirming recrudescence. Not all of fetuses showed pathologic microscopic lesions, however, multifocal non-purulent encephalitis was the main finding. Furthermore, nine out of the 11 vertical transmission positive sheep had lesions in placentomes (mainly multifocal necrotic foci), and the parasite was detected in eight out of 11 placentas by PCR and/or immunohistochemestry. The placentomes from sheep that suffered recrudescence showed an increased number of T lymphocytes CD3+ (CD4/CD8 < 1) and macrophages (MHC-II+), assessed by immunohistochemestry, together with an upregulation of IFN-γ, IL-10, IL-4, TNFα, IL-2 and IL-18. IL-17 was only upregulated in the three infected sheep that did not have a sharp increase in antibody levels. In the sheep that showed fetal death at the time of euthanasia (n = 3) the placental microscopic lesions were more severe, the inflammatory infiltrate was higher, and the upregulation of cytokines was greater than in those sheep carrying viable fetuses. This study suggests that, similarly to bovine neosporosis, the time of gestation when recrudescence occurs determines the viability of the fetuses and, thus, seems to be related to the severity of lesions and immune response in the placenta. These results suggest that there might be a correlation, either as cause or as a consequence, between protection against vertical transmission of the parasite and a milder maternal serological response together with a high level of transcription of IL-17 in the placenta., The present study was financed by the Ministerio de Ciencia, Innovacion y Universidades of Spain (INIA project RTA2014-00013) and the European Regional Development Fund. Daniel Gutiérrez-Exposito ´ is the recipient of a postdoctoral contract from the Ministry of Science and Innovation (grant no. FJCI-2017-32020). We wish to acknowledge María del Carmen Carro, Xavier Cortizo, María Teresa Carro and Marta Silva for their excellent technical assistance. Finally, we wish to acknowledge Dr. Luis Miguel Ortega-Mora (SALUVET group) for kindly providing the in-house polyclonal antibody against N. caninum.

16 páginas, 5 figuras, 2 tablas., Paratuberculosis (PTB) is an enteric granulomatous disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) that mainly aects ruminants. Current vaccines have shown to be cost-eective control reagents, although they are restricted due to cross-interference with bovine tuberculosis (bTB). Therefore, novel vaccination strategies are needed and this study is focused on evaluating alternative vaccination routes and their eect on the local immune response. The MAP oral challenge rabbit model was used to evaluate and compare an experimental inactivated MAP vaccine through oral (VOR) and intradermal (VID) routes. The VID group presented the highest proportion of animals with no visible lesions and the lowest proportion of animals with MAP positive tissues. Immunohistochemistry analysis revealed that the VID group presented a dominantly M1 polarized response indicating an ability to control MAP infection. In general, all vaccinated groups showed lower calprotectin levels compared to the non-vaccinated challenged group suggesting less active granulomatous lesions. The VID group showed some degree of skin test reactivity, whereas the same vaccine through oral administration was completely negative. These data show that PTB vaccination has an eect on macrophage polarization and that the route influences infection outcome and can also have an impact on bTB diagnosis. Future evaluation of new immunological products against mycobacterial diseases should consider assaying dierent vaccination routes., The research was funded by the Department of Economy and Infrastructures (DEI) of the Basque Government and partially by grant RTA 2017-00089-00-00 of the National Institute for Agronomic Research (INIA). R.A. held a pre-doctoral fellowship (BFI-2012-237) from the Department of Education, Universities and Research of the Basque Government and IL is recipient of a pre-doctoral fellowship from the DEI of the Basque Government.

6 páginas, 4 figuras., The local expression of WC1 γδ T lymphocytes subset has been evaluated by immunohistochemical methods at the different types of lesions present in cows naturally infected with Mycobacterium avium subsp. paratuberculosis (Map) and in non-infected control animals. Infected cattle were either in the latent/subclinical (focal lesions) or clinical (diffuse paucibacillary and multibacillary forms) stage of paratuberculosis. To assess the cell distribution, a differential cell count was carried out at the lamina propria, gut-associated lymphoid tissue and submucosa. A significant increase in the number of WC1 γδ T cells was observed in all the infected animals, regardless of the type of lesion. Cows with focal lesions showed higher number of labeled cells than those with diffuse forms, where no differences were found between the two types. This increase in the number of positively immunolabelled lymphocytes in infected animals was seen in the lamina propria, with higher values in those with focal lesions. While in the lymphoid tissue no differences in the numbers were observed, in animals with focal lesions, WC1 γδ T cells tended to be located at the periphery of the granulomas. These findings suggest a proinflammatory action of WC1 γδ T lymphocytes in bovine paratuberculosis, which might play an important role in the containment of the Map-infection in the focal granulomas located in the lymphoid tissue, helping to prevent the progression toward diffuse forms responsible for the clinical signs., This study was funded by grant AGL2015-66540-C2-1-R from de Spanish Ministry of Economy and Competitiveness and LE259/P18 from “Junta de Castilla y León”. N. Arteche and R. Vallejo are supported by a predoctoral contract from the Spanish Ministry of Economy and Competitiveness, or Education, respectively. J. Espinosa and D. Gutiérrez are the recipients of a postdoctoral contract of “Junta de Castilla y León-Fondo Social Europeo” and “Juan de la Cierva” of the Spanish Ministry of Economy and Competitiveness, respectively. Technical and human support provided by Marta Silva is gratefully acknowledged.

6 páginas, 1 tabla, 3 figuras.-- Pesavento, P. A. et al., Ovine herpesvirus 2 (OvHV-2) is one of the gammaherpesviruses in the genus Macavirus that can cause malignant catarrhal fever (MCF) in ungulates. Sheep are the adapted host for OvHV-2 and it is generally assumed that infection is not associated with disease in this species. However, cases of "polyarteritis nodosa" or idiopathic systemic necrotizing vasculitis reported in sheep are similar to vascular lesions in clinically susceptible species with MCF. Using a recently developed in situ hybridization (ISH) method, we were able to identify OvHV-2 nucleic acids within lesions and correlate the viral distribution with systemic necrotizing vasculitis in 9 sheep, including both naturally and experimentally OvHV-2-infected animals. ISH, combined with polymerase chain reaction and histology, identify OvHV-2 as the likely agent responsible for sporadic, MCF-like vascular disease in sheep., The author(s) received the following financial support for the research, authorship, and/or publication of this article: Support funding to the Li laboratory for this project is from USDA-ARS CWU 2029-3200-037-00D. In the Pesavento laboratory, the study was supported by Boehringer Ingelheim Vetmedica and the Bernice Barbour Foundation for research in naturally occurring infectious disease.

Aquaculture production of turbot in Galicia (Spain) has grown significantly in the last 35 years. The evolution of the sector and the variations in its production technologies are the result of the...

El presente trabajo de investigación se centra en analizar la tendencia recesiva de la democracia en jóvenes mexicanos; se parte del supuesto que, cuando la actividad económica retrocede, se genera en la sociedad no solo una percepción de estancamiento económico sino democrático que se expresa en los niveles de abstinencia de las y los jóvenes a participar en la vida democrática de su país. El presente estudio busca determinar, mediante una muestra representativa de 400 jóvenes encuestados, correspondiente a 12 mil 884 electores de 18 a 23 años de la capital poblana, pertenecientes al distrito 12 electoral en México, si presentan una tendencia recesiva de su participación política. La relevancia de la investigación radica en que la Ciudad de Puebla, capital del Estado de Puebla ubicado en el centro de la República Mexicana, es una de las ciudades con mayor concentración de instituciones de educación superior -públicas y privadas- de México, para el año 2020, de acuerdo con la Secretaría de Educación Pública Federal, en Puebla operan 343 universidades, colocando a la entidad como la segunda a nivel nacional con más instituciones de este nivel educativo (SEP, 2020). Además de lo anterior, la entidad poblana recibe estudiantes de diferentes entidades federativas del país, de modo que es posible afirmar que los resultados que aquí se presentan, son representativos de las conductas observables en las y los jóvenes mexicanos frente la democracia como forma de vida.

Introduction Polycystic ovary syndrome (PCOS) is a complex metabolic disorder associated with ovulatory dysfunction, hyperandrogenism, obesity, and insulin resistance, that leads to subfertility. Sam68 is an RNA-binding protein with signaling functions that is ubiquitously expressed, including gonads. Sam68 is recruited to leptin signaling, mediating different leptin actions. Objective We aimed to investigate the role of Sam68 in leptin signaling, mediating the effect on aromatase expression in granulosa cells and the posible implication of Sam68 in the leptin resistance in PCOS. Materials and methods Granulosa cells were from healthy donors (n = 25) and women with PCOS (n = 25), within the age range of 20 to 40 years, from Valencian Infertility Institute (IVI), Seville, Spain. Sam68 expression was inhibited by siRNA method and overexpressed by expression vector. Expression level was analysed by qPCR and immunoblot. Statistical significance was assessed by ANOVA followed by different post-hoc tests. A P value of Results We have found that leptin stimulation increases phosphorylation and expression level of Sam68 and aromatase in granulosa cells from normal donors. Downregulation of Sam68 expression resulted in a lower activation of MAPK and PI3K pathways in response to leptin, whereas overexpression of Sam68 increased leptin stimulation of signaling, enhancing aromatase expression. Granulosa cells from women with PCOS presented lower expression of Sam68 and were resistant to the leptin effect on aromatase expression. Conclusions These results suggest the participation of Sam68 in leptin receptor signaling, mediating the leptin effect on aromatase expression in granulosa cells, and point to a new target in leptin resistance in PCOS.

Se presenta el estudio de la comunidad de líquenes epífitos de Juniperus flaccida, especie endémica de México. Se aplica la regla de Sturges para agrupar y ordenar los valores del diámetro altura pecho (DAP). Se comparan los grupos de DAP con la riqueza liquénica aplicando la prueba de Mann-Whitney U-test, los resultados no mostraron diferencias significativas. Se utilizó la composición de especies para clasificar a los grupos de DAP con el análisis de conglomerados de doble vía. Se determinó la diversidad alfa, beta y gamma. Se registran 39 especies de hongos liquenizados alrededor de J. flacciday 65 líquenes epífitos; de estos, 59 fueron nuevos registros asociados a J. flaccida. Se reportan cinco nuevos registros para Oaxaca (Caloplaca ferruginea, Dermatocarpon americanum, Lecanora albella, Lecanora helvay Ochrolechia mexicana) y tres nuevos registros para México (Diploschistes scruposus, Traponora variansy Chaenothecatrichialis).

In a continued effort to catalog the numerous phylogenetically detected and predicted species of Cora in the Americas, we focus here on the diversity of the genus in Mexico and the phylogenetic relationships of the taxa present in this area. Based on previous results and new collections, 12 taxa are recognized in Mexico, including eight new species and one new subspecies. The 12 taxa form 11 unrelated lineages within the genus, indicating multiple independent colonization from Central and South America. While the new subspecies is nested within a species known from the northern Andes in South America, the other species are all putative endemics for Mexico, resulting in endemism of 92% at species level and 100% at taxon level. Considering the rather narrow area of origin of the sequenced specimens in southeastern Mexico and the previously documented range of Cora including the northwestern part of the country, plus the underlying topography, we predict that the 12 species and subspecies now known represent only about 20% of the total richness of Cora in the country, and that many more endemic lineages are to be found in the western and northwestern parts (Sierra Madre Occidental). The new taxa from Mexico formally introduced in this study are Cora benitoana sp. nov., with a strongly projecting, cyphelloid hymenophore; C. buapana sp. nov., with elongate, finger-like and partly branched appendages on the lower medullary hyphae; C. dewisanti subsp. mexicana subsp. nov., with a marginally protruding hymenophore; C. guzmaniana sp. nov., with a partly setose lobe surface; C. ixtlanensis sp. nov., a phenotypically cryptic species similar but unrelated to C. terrestris; C. lawreyana sp. nov., with globose hyphal appendages; C. marusae sp. nov., a phenotypically cryptic species similar but unrelated to C. comaltepeca; C. totonacorum sp. nov., a phenotypically cryptic species similar but phylogenetically distant to C. davidia; and C. zapotecorum sp. nov., with a very thinly pilose lobe surface.

This study deals with the multiproduct valorization of the invasive macroalgae Sargassum muticum within a green biorefinery concept using microwave hydrothermal treatment. Temperatures of 160 and 180 ◦C for 0–60 min (severities 1.62–3.54) were evaluated, allowing a recovery of a liquid phase rich in fucoidan-derived compounds (up to 4.81 g/L), oligomers and phenolics with antioxidant capacity (up to 2.85 g TE/L by ABTS assay), and a high-enzymatically susceptible solid (glucan to glucose conversion 76–100% in 9 h) suitable for bioethanol production (20.5 g/L in 18 h, corresponding to 96% ethanol yield). Moreover, energy consumption of the pre-treatments’ temperature–time binomial was evaluated showing significant differences, demonstrating the ad-vantages of microwave as alternative heating pretreatment Financiado para publicación en acceso aberto: Universidade de Vigo/CISUG Agencia Estatal de Investigación | Ref. PID2019-110031RB-I00 Ministerio de Economía y Competitividad | Ref. CTM2015-68503-R Ministerio de Ciencia, Innovación y Universidades | Ref. RYC2018-026177-I Ministerio de Educación, Cultura y Deporte | Ref. FPU16/04077 Xunta de Galicia | Ref. ED431C 2017/62

When dealing with plant roots, a multiscale description of the functional root structure is needed. Since the beginning of 21st century, new devices such as laser confocal microscopes have been accessible for coarse root structure measurements, including three-dimensional (3D) reconstruction. Most researchers are familiar with using simple 2D geometry visualization that does not allow quantitative determination of key morphological features from an organ-like perspective. We provide here a detailed description of the quantitative methods available for 3D analysis of root features at single-cell resolution, including root asymmetry, lateral root analysis, cell size and nuclear organization, cell-cycle kinetics, and chromatin structure analysis. Quantitative maps of the root apical meristem (RAM) are shown for different species, including Arabidopsis thaliana (L.), Heynh, Nicotiana tabacum L., Medicago sativa L., and Setaria italica (L.) P. Beauv. The 3D analysis of the RAM in these species showed divergence in chromatin organization and cell volume distribution that might be used to study root zonation for each root tissue. Detailed protocols and possible pitfalls in the usage of the marker lines are discussed. Therefore, researchers who need to improve their quantitative root biology portfolio can use them as a reference.

Human endogenous retrovirus W family envelope proteins (pHERV-W ENV/syncytin-1) have been repeatedly associated with multiple sclerosis (MS). Here, we have focused on the study of pHERV-W ENV/syncytin-1 expression levels in MS patients (relapsing and progressive forms) and in healthy donors (HD) and on exploring their possible relationship with Epstein-Barr virus (EBV) and human herpesvirus-6A/B (HHV-6A/B). We included blood samples from 101 MS patients and 37 HD to analyze antiviral antibody titers by ELISA and pHERV-W ENV/syncytin-1 expression levels by flow cytometry as well as by qPCR. Patients with relapsing MS forms showed significantly higher pHERV-W ENV/syncytin-1 protein and gene expression levels than HD. Progressive MS patients also showed significantly higher protein and gene expression levels than both HD and relapsing MS patients. Regarding antiviral antibodies titers, anti-HHV-6A/B IgM levels were positively correlated with pHERV-W ENV/syncytin-1 protein expression levels in patients with relapsing MS, while in the progressive forms patients this correlation was found with anti-HHVA/B IgG levels. Therefore, pHERV-W ENV could be involved in MS pathogenesis, playing a role in relapsing and progressive forms. Besides, anti-HHV-6A/B antibodies positively correlated with pHERV-W ENV expression. Further studies are needed to better understand this possible relationship.

Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, particularly with respect to immune responses, we utilized the rhesus macaque model of SARS-CoV-2 infection. Two cohorts of eight older (16-23 years) and eight younger (3-5 years) rhesus macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at pre-defined timepoints in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication dynamics were limited between age cohorts. Transcriptional signatures of inflammation-associated genes in cells isolated from bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both younger and older animals. These findings suggested that age did not substantially skew major facets of acute disease in this model. However, age-specific divergence of immune responses emerged during the post-acute phase of infection (7-21 dpi). Older animals exhibited sustained local inflammatory innate responses while local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, in contrast to persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling in SARS-CoV-2-infected rhesus macaques suggests that age may delay or impair the induction of anti-viral cellular immune responses and delay efficient return to immune homeostasis following acute infection.

Background HD is a suitable model to monitor the whole neurodegeneration process. Huntington’s disease (HD) affects primarily the striatum, but loss of white matter (WM) integrity and iron homeostasis disruption have been also described. These anomalies together with metabolite profiles could unveil the pathophysiologic mechanisms involved. Aims To assess the temporal and spatial progression of neurodegeneration on both anterior thalamic radiations (ATR). Methods Thirty-one HD gene carriers and twenty-four controls underwent neuropsychological evaluation and were scanned at 3T-MRI unit. A multimodal study was conducted to measure relaxometry, diffusivity and spectroscopy as proxies of iron, WM microstructure and metabolite composition, respectively. Three statistical approaches (average, segmental, along-the-tract) were performed, with MANOVA and post-hoc Tukey test to evaluate differences among groups and Pearson test to assess correlations. Results ATR disintegration began in premanifest individuals and progressed in extent and severity in manifest patients. WM damage was more extensive in the right ATR that could translate a higher vulnerability and showed a spatial gradient from subcortical to deep WM in favour of the dying-back hypothesis. Iron was increased in the left ATR in premanifest individuals that might uncover a dysregulated myelination or an abnormal ferritin accumulation. NAA and creatine decreased exclusively in manifest patients suggesting neuronal loss and mitochondrial dysfunction. Furthermore, imaging parameters could be used as biomarkers given their links with clinical scores. Conclusions The complex neurodegeneration pattern of ATR in HD can help to understand the pathophysiological mechanisms underlying HD progression. The multimodal approach and along-the-tract analysis allow for a more comprehensive evaluation of neurodegeneration.

13 páginas, 3 tablas, 5 figuras., Macrophages have been confirmed to play a significant role in the behavior of human lymphomas, albeit no consistent data are so far available in canine lymphomas. The present study characterizes the macrophages present in cases of canine nodal lymphoma and their relationship with the histological grade and the immunophenotype. Samples from the lymph nodes of 25 dogs diagnosed with lymphoma were selected. Immunohistochemistry was used to determine the tumor immunophenotype (CD3 and CD20 antibodies) and macrophage characterization (Iba1, MAC387, CD204, CD163 and iNOS antibodies). Macrophage counting was performed in 10 randomly selected, high-power fields per sample. Generalized linear models with Poisson distribution were used for statistical analysis. A significantly greater number of macrophages (Iba1+) were detected in high-grade and B-cell lymphomas. The highest amount of both M1 (iNOS+) and M2 (CD204+ and CD163+) subtypes were observed in B-cell lymphomas. High-grade lymphomas showed a greater number of CD204+ and CD163+ cells and recently recruited MAC387+ macrophages. The latter were most abundant in T than in B-cell lymphomas. In conclusion, a significant population of macrophages is present in canine lymphomas, which constitute a heterogeneous population that shows variations in the amount and immunohistochemical profile according to the histological grade and immunophenotype., This work was awarded by the “D. Santos Ovejero del Agua” Foundation. The technical assistance of Marta Silva, Eva Martín de Valmaseda and María Murillas is also acknowledged.

Apicomplexans are a group of pathogenic protists that cause various diseases in humans and animals that cause economic losses worldwide. These unicellular eukaryotes are characterized by having a complex life cycle and the ability to evade the immune system of their host organism. Infections caused by some of these parasites affect millions of pregnant women worldwide, leading to various adverse maternal and fetal/placental effects. Unfortunately, the exact pathogenesis of congenital apicomplexan diseases is far from being understood, including the mechanisms of how they cross the placental barrier. In this review, we highlight important aspects of the diseases caused by species ofPlasmodium, Babesia, Toxoplasma, andNeospora, their infection during pregnancy, emphasizing the possible role played by the placenta in the host-pathogen interaction.

18 páginas, 6 figuras, 3 tablas., Toxoplasma gondii is an apicomplexan parasite that infects almost all warm-blooded animals. Little is known about how the parasite virulence in mice extrapolates to other relevant hosts. In the current study, in vitro phenotype and in vivo behavior in mice and sheep of a type II T. gondii isolate (TgShSp1) were compared with the reference type II T. gondii isolate (TgME49). The results of in vitro assays and the intraperitoneal inoculation of tachyzoites in mice indicated an enhanced virulence for the laboratory isolate, TgME49, compared to the recently obtained TgShSp1 isolate. TgShSp1 proliferated at a slower rate and had delayed lysis plaque formation compared to TgME49, but it formed more cyst-like structures in vitro. No mortality was observed in adult mice after infection with 1–105 tachyzoites intraperitoneally or with 25–2,000 oocysts orally of TgShSp1. In sheep orally challenged with oocysts, TgME49 infection resulted in sporadically higher rectal temperatures and higher parasite load in cotyledons from ewes that gave birth and brain tissues of the respective lambs, but no differences between these two isolates were found on fetal/lamb mortality or lesions and number of T. gondii-positive lambs. The congenital infection after challenge at mid-pregnancy with TgShSp1, measured as offspring mortality and vertical transmission, was different depending on the challenged host. In mice, mortality in 50% of the pups was observed when a dam was challenged with a high oocyst dose (500 TgShSp1 oocysts), whereas in sheep infected with the same dose of oocysts, mortality occurred in all fetuses. Likewise, mortality of 9 and 27% of the pups was observed in mice after infection with 100 and 25 TgShSp1 oocysts, respectively, while in sheep, infection with 50 and 10 TgShSp1 oocysts triggered mortality in 68 and 66% of the fetuses/lambs. Differences in vertical transmission in the surviving offspring were only found with the lower oocyst doses (100% after infection with 10 TgShSp1 oocysts in sheep and only 37% in mice after infection with 25 TgShSp1 oocysts). In conclusion, virulence in mice of T. gondii type II isolates may not be a good indicator to predict the outcome of infection in pregnant sheep., RS-S is supported by a fellowship from the Spanish Ministry of Education, Culture, and Sports (MECD), as a part of the Program of Training of University Teaching Staff (FPU, grant number FPU13/03438) and a mobility grant for predoctoral short stays in R+D centers (EST16/0719). DG-E is the recipient of a postdoctoral contract from the Junta de Castilla y León, partially funded by the European Social Fund (European Union). NA-V is the recipient of a predoctoral contract from the Ministerio de Economía, Industria y Competitividad (Ref. BES-2016-076513). This work was supported by the Ministry of Economy and Competitiveness (AGL2016-75935-C2-1-R and C2-2-R), the Community of Madrid, Spain (PLATESA, S2013/ABI2906), Junta de Castilla y León (LE080U16), and a grant of the Swiss National Science Foundation to AH (project No. 310030_165782).

When dealing with plant roots, a multi-scale description of the functional root structure is needed. Since the beginning of XXI century, new devices like laser confocal microscopes have been accessible for coarse root structure measurements, including 3D reconstruction. Most researchers are familiar with using simple 2D geometry visualization that does not allow quantitatively determination of key morphological features from an organ-like perspective. We provide here a detailed description of the quantitative methods available for three-dimensional (3D) analysis of root features at single cell resolution, including root asymmetry, lateral root analysis, xylem and phloem structure, cell cycle kinetics, and chromatin determination. Quantitative maps of the distal and proximal root meristems are shown for different species, including Arabidopsis thaliana, Nicotiana tabacum and Medicago sativa. A 3D analysis of the primary root tip showed divergence in chromatin organization and cell volume distribution between cell types and precisely mapped root zonation for each cell file. Detailed protocols are also provided. Possible pitfalls in the usage of the marker lines are discussed. Therefore, researchers who need to improve their quantitative root biology portfolio can use them as a reference.

Winemaking waste contain a high number of bioactive compounds with antimicrobial properties that can be exploited in agriculture. In the present study, hydrolysates from three wine grape (Vitis vinifera L.) marcs were characterized and their antifungal activities against phytopathogenic fungi (Fusarium oxysporum and Alternaria spp.) were evaluated. Wine grape marcs (red, pink and white wine) collected from Ensenada, Baja California, Mexico, were subjected to an acid hydrolysis treatment. Skin hydrolysates of pink and white marcs obtained high concentrations of reducing sugars (5.8 ± 0.1 and 5.5 ± 0.2 g L-1, respectively). Meanwhile, the highest concentration of total sugars was obtained for skin hydrolysates of white marc (9.7 ± 0.08 g L-1). The seed hydrolysates of white marc obtained high concentrations of phenolic compounds (0.52 ± 0.1 mg mL-1). In addition, the highest antioxidant activity was found for skin hydrolysates of red marc (96 ± 0.61%). Results of in vitro antifungal assays clearly indicated a marked inhibition of the mycelial growth and spore viability of F. oxysporum (100% inhibition using red and white hydrolysates) rather than Alternaria spp. (58% inhibition exposed to pink hydrolysates), due to high concentration of phenols. According to HPLC analysis, phenolic acids such as gallic acid, hydroxybenzoic acid, vanillic acid and p-coumaric acid were predominant in the hydrolysates. This study demonstrated that the grape marc hydrolysates exhibit a potential antifungal activity, and highlights that the hydrolysates can be exploited in agriculture as a safe alternative of antifungal agents.

Description, SARS-CoV-2 variants of concern B.1.1.7 and B.1.351 do not cause more severe disease in rhesus macaques., The emergence of several SARS-CoV-2 variants has caused global concerns about increased transmissibility, increased pathogenicity, and decreased efficacy of medical countermeasures. Animal models can be used to assess phenotypical changes in the absence of confounding factors. Here, we compared variants of concern (VOC) B.1.1.7 and B.1.351 to a recent B.1 SARS-CoV-2 isolate containing the D614G spike substitution in the rhesus macaque model. B.1.1.7 behaved similarly to D614G with respect to clinical disease and replication in the respiratory tract. Inoculation with B.1.351 resulted in lower clinical scores, lower lung virus titers, and less severe lung lesions. In bronchoalveolar lavages, cytokines and chemokines were up-regulated on day 4 in animals inoculated with D614G and B.1.1.7 but not with B.1.351. In nasal samples, cytokines and chemokines were up-regulated only in the B.1.1.7-inoculated animals. Together, our study suggests that circulation under diverse evolutionary pressures favors transmissibility and immune evasion rather than increased pathogenicity.

Surveillance and control activities for virus-transmitting mosquitoes have primarily focused on dwellings. There is little information about viral circulation in heavily trafficked places such as schools. We collected and analyzed data to assess the presence and prevalence of dengue, chikungunya, and Zika viruses in mosquitoes, and measured Aedes indices in schools in Medellín (Colombia) between 2016–2018. In 43.27% of 2632 visits we collected Aedes adults, creating 883 pools analyzed by RT-PCR. 14.27% of pools yielded positive for dengue or Zika (infection rates of 1.75–296.29 for Aedes aegypti). Ae. aegypti was more abundant and had a higher infection rate for all studied diseases. Aedes indices varied over time. There was no association between Aedes abundance and mosquito infection rates, but the latter did correlate with cases of arboviral disease and climate. Results suggest schools are important sources of arbovirus and health agencies should include these sites in surveillance programs, it is essential to know the source for arboviral diseases transmission and the identification of the most population groups exposed to these diseases to research and developing new strategies.

Rhizobia have been studied for the symbiosis that they establish with the roots of legumes. However, the colonization and promotion of growth in non-leguminous plants has also been demonstrated. The aim of this work was the biochemical and molecular identification of rhizosphere rhizobia present in the rhizosphere of two commercial maize cultivars. Cultivable isolates were obtained in yeast-mannitol-agar (YMA) medium from rhizospheric soil and the rhizoplane. The cultural (size, color, mucus, etc.), morphological, and staining (cell shape, response to staining and sporulation) characteristics were determined as well as isolate responses to eight biochemical tests (acid-base production, citrate, oxidase, catalase, H2S production, urease, gelatinase and the oxidative-fermentative assay) that are valuable for rhizobia identification. The genus was determined by 16S rDNA gene sequencing. We obtained 81 total isolates of which 30.86% showed the cultural, morphological and staining characteristics expected for rhizobia and only 20% of these corresponded to the genus Rhizobium. RESUMEN Los rizobios han sido estudiados por la simbiosis que establecen con las raíces de las leguminosas. Sin embargo, la colonización y promoción del crecimiento en plantas no leguminosas también ha sido demostrada. Este trabajo tuvo como objetivo la identificación bioquímica y molecular de rizobios rizosféricos presentes en la rizosfera de dos cultivares comerciales de maíz. Se obtuvieron aislamientos cultivables en medio Levadura-Manitol-Agar (YMA) a partir del suelo rizosférico y rizoplano de las plantas. Se determinaron las características culturales del cultivo (tamaño, color, mucosidad, etc.) y morfo-tintoriales (forma celular, respuesta a la tinción y esporulación), así como la respuesta de los aislados a ocho pruebas bioquímicas (producción ácido-base, citrato, oxidasa, catalasa, producción de H2S, ureasa, gelatinasa y ensayo oxidativo-fermentativo) con valor predictivo para la identificación de rizobios. Se determinó el género mediante la secuenciación del gen ADNr-16S. Se obtuvieron 81 aislados totales de los cuales el 30.86% mostró las características culturales, morfológicas y tintoriales esperadas para los rizobios y solamente el 20% de estos correspondió al género Rhizobium.

Although it is known that gestation could influence the clinical course of ovine toxoplasmosis, the precise effect of the term of gestation when sheep are infected are yet mostly unknown. The aim of this study was to evaluate the peripheral and placental immune responses developed in pregnant sheep after experimental infection with Toxoplasma gondii at different times of gestation. Thirty-six pregnant sheep were allocated in different groups, orally inoculated with sporulated oocysts of T. gondii at early, mid and late gestation and culled within 30 days post-infection. The peripheral humoral and cytokine responses were evaluated, as well as the transcription of cytokines at the placenta. Serological analysis revealed that, regardless the term of gestation when infected, specific IgG against T. gondii were detected from day 8 post-infection and there was an early peripheral release of IFN-γ at the first week post-infection followed by a short peak of IL10 and TNF-α at the second week post-infection. There were no significant differences in this response between infected groups. At the placenta, a similar increase in transcription of IFN-γ, and TNF-α was found at the three terms of gestation, while IL-4 increased mainly at the first and second terms and IL-10 transcription was higher at the last term. While these findings show that both Th1 and Th2 cytokines play a key role in the pathogenesis of ovine toxoplasmosis and that placental and peripheral immune responses do not closely correlate, there seems to be no clear modulation of these responses along the gestation., The authors wish also to thank to Dr Frank Katzer, from Moredun Research Institute for kindly providing the tachyzoites from M4 isolate, all the staff of the IGM (CSIC-ULE) responsible for handling the experimental animals, and the technical assistance of Mr. Panero, Mrs. Espiniella, Mrs. González and Mrs. Agudín. P. Castaño is founded by a predoctoral contract from the “Junta de Castilla y León”. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). This work was supported by Grants AGL2011-30205 and AGL2016-75935-C2-2-R and of the Spanish Ministry of Science and Innovation, and LE253U13, from the Junta de Castilla y León, Spain.

Introduction: The Zika virus has generated serious epidemics in the different countries where it has been reported and Colombia has not been the exception. Although in these epidemics Aedes aegypti traditionally has been the primary vector, other species could also be involved in the transmission. Methods: Mosquitoes were captured with entomological aspirators on a monthly ba sis between March and September of 2017, in four houses around each of the 250 entomological surveillance traps installed by the Secretaria de Sa lud de Medellin (Colombia). Additionally, 70 Educational Institutions and 30 Health Centers were visited each month. Results: 2 504 mosquitoes were captured and grouped into 1045 pools to be analyzed by RT-PCR for the detection of Zika virus. Twenty-six pools of Aedes aegypti, two pools of Ae. albopictus and one for Culex quinquefasciatus were positive for Zika virus. Conclusion: The presence of this virus in the three species and the abundance of these species in the different sectors of the city, suggests that the control strategies should be addressed to all species that could be potential vec tors until the vectorial role of Ae. albopictus and Culex quinquefasciatus is completely elucidated in the transmission of the Zika virus. Resumen Introducción: El virus Zika ha generado serias epidemias en los diferentes países en donde se ha reportado, Colombia no ha sido la excepción. Aunque en estas epidemias el vector presuntamente involucrado ha sido Aedes aegypti, otras especies también podrían estar implicadas en su transmisión. Meto dología: en el marco del Programa de vigilancia virológica en mosquitos de la Secretaría de Salud de Medellín (Colombia). Resultados: entre marzo y septiembre de 2017 se capturaron 2 504 mosquitos en predios visitados durante los levantamientos de índices entomológicos. El material entomo lógico fue agrupado en 1 045 pooles para ser analizados por RT-PCR para la detección de virus Zika. Resultaron positivos para virus Zika 26 pooles de Aedes aegypti, dos pooles de Ae. albopictus y uno para Culex quinquefasciatus. Conclusión: la presencia de este virus en las tres especies y su abundancia en los distintos sectores de la ciudad sugieren que las estrategias de control deban dirigirse a todos los potenciales vectores, hasta tanto no se dilucide comple tamente el papel vectorial de Ae. albopictus y Cx. quinquefasciatus en la transmisión del virus Zika.

Wetlands have been considered a feasible technology for wastewater treatment in the last decades; however, information on the kinetics and microbial structure of nitrogen cycle bacteria involved on the rhizosphere activity of natural wetlands polluted with chromium is still scarce. The goal was to evaluate the kinetic behavior of nitrification, denitrification, and ANAMMOX on rhizosphere sludge, with and without chromium, through batch cultures, as well as the microbial structure using Next Generation Sequencing (NGS). The microbial sludge was able to nitrify (3.8 ± 0.2 mg NO3--N/gVSS-h) and carry out the ANAMMOX (0.67 ± 0.05 mg NH4+-N/gVSS-h), however, denitrifying activity was not observed. Chromium inhibited the nitrifying process, and the IC50 obtained for the nitrifying activity was of 7.9 mg CrVI/L. ANAMMOX activity was stopped in the presence of chromium, even to the lowest chromium concentration tested. Recovery cultures showed that ANAMMOX bacteria suffered some damage by chromium presence since they required more than 5 days to recover the activity. Microbial results indicated that Xanthomonadaceae (17.17%), Ignavibacteriaceae (16.52%), Trueperaceae (10.66%) and Chitinophagaceae (10.06%) dominated in the microbial sludge, whereas Nitrosomonas and Planctomycetaceae were in lesser proportion. This research improves the understanding of bacteria behavior on natural wetlands polluted with metals.

Microspore embryogenesis is a model system of plant cell reprogramming, totipotency acquisition, stress response and embryogenesis initiation. This in vitro system constitutes an important biotechnological tool for haploid and doubled-haploid plant production, very useful for crop breeding. In this process, microspores (cells that produce pollen grains in planta) are reprogrammed toward embryogenesis by specific stress treatment, but many microspores die after the stress. The occurrence of cell death is a serious limiting problem that greatly reduces microspore embryogenesis yield. In animals, increasing evidence has revealed caspase proteolytic activities as essential executioners of programmed cell death (PCD) processes, however, less is known in plants. Although plant genomes do not contain caspase homologues, caspase-like proteolytic activities have been detected in many plant PCD processes. In the present study, we have analysed caspase 3-like activity and its involvement in stress-induced cell death during initial stages of microspore embryogenesis of Brassica napus. After stress treatment to induce embryogenesis, isolated microspore cultures showed high levels of cell death and caspase 3-like proteolytic activity was induced. Treatments with specific inhibitor of caspase 3-like activity reduced cell death and increased embryogenesis induction efficiency. Our findings indicate the involvement of proteases with caspase 3-like activity in the initiation and/or execution of cell death at early microspore embryogenesis in B. napus, giving new insights into the pathways of stress-induced cell death in plants and opening a new way to improve in vitro embryogenesis efficiency by using chemical modulators of cell death proteases.

Microtubule targeting agents represent a very active arena in the development of anticancer agents. In particular, compounds binding at the colchicine site in tubulin are being deeply studied, and the structural information recently available on this binding site allows structure-directed design of new ligands. Structural comparison of our recently reported high resolution X-Ray structure of the cyclohexanedione derivative TUB075 bound to tubulin and the tubulin-DAMA-colchicine complex has revealed a conformational change in the aT5 loop. By a grid-based computational analysis of the tubulinDAMA-colchicine binding site, we have identified a new favourable binding area in the colchicine-site that was unexplored by our lead TUB075. Thus, based on a structure-guided design, new cyclohexanedione derivatives have been synthesized and tested for tubulin binding and in cellular assays. As a result, we have identified diphenyl ether derivatives with IC50 values around 10e40 nM against three different tumor cell lines and affinity constants for tubulin similar to that of colchicine around 107 M 1 . As expected, they halted the cell cycle progression at G2/M phase at concentrations as low as 0.08 mM., This project has been supported by the Spanish Ministerio de Economía y Competitividad SAF2015-64629-C2-1-R (to EMP, MJPP. and MJC) and BFU2016-75319-R (AEI/FEDER, UE) (JFD.) Financial support from CSIC (Proyecto Intramural PIE-201668E079 to MJPP) is also acknowledged. The authors acknowledge networking contribution by COST action CM1407 “Challenging synthesis inspired by nature e from natural products chemistry to drug discovery” (to MJPP, SL, and JFD). JFD. is a member of the CIB Intramural Program ‘‘Molecular Machines for Better Life’’ (MACBET). We acknowledge Lizette Van Berckelaer, Eef Meyen and Sam Noppen for excellent technical assistance. We thank ganadería Fernando Díaz for calf brains for tubulin purification.

Background Adventitious root (AR) formation in excised plant parts is a bottleneck for survival of isolated plant fragments. AR formation plays an important ecological role and is a critical process in cuttings for the clonal propagation of horticultural and forestry crops. Therefore, understanding the regulation of excision-induced AR formation is essential for sustainable and efficient utilization of plant genetic resources. Scope Recent studies of plant transcriptomes, proteomes and metabolomes, and the use of mutants and transgenic lines have significantly expanded our knowledge concerning excision-induced AR formation. Here, we integrate new findings regarding AR formation in the cuttings of diverse plant species. These findings support a new system-oriented concept that the phytohormone-controlled reprogramming and differentiation of particular responsive cells in the cutting base interacts with a co-ordinated reallocation of plant resources within the whole cutting to initiate and drive excision-induced AR formation. Master control by auxin involves diverse transcription factors and mechanically sensitive microtubules, and is further linked to ethylene, jasmonates, cytokinins and strigolactones. Hormone functions seem to involve epigenetic factors and cross-talk with metabolic signals, reflecting the nutrient status of the cutting. By affecting distinct physiological units in the cutting, environmental factors such as light, nitrogen and iron modify the implementation of the genetically controlled root developmental programme. Conclusion Despite advanced research in the last decade, important questions remain open for future investigations on excision-induced AR formation. These concern the distinct roles and interactions of certain molecular, hormonal and metabolic factors, as well as the functional equilibrium of the whole cutting in a complex environment. Starting from model plants, cell type- and phase-specific monitoring of controlling processes and modification of gene expression are promising methodologies that, however, need to be integrated into a coherent model of the whole system, before research findings can be translated to other crops.

SUMMARYPlants have remarkable regenerative capacity, which allows them to survive tissue damaging after biotic and abiotic stress. Some of the key transcription factors and the hormone crosstalk involved in wound-induced organ regeneration have been extensively studied in the model plantArabidopsis thaliana. However, little is known about the role of metabolism in wound-induced organ regeneration.Here, we performed detailed transcriptome analysis and targeted metabolomics approach duringde novoorgan formation in tomato hypocotyl explants and found tissue-specific metabolic differences and divergent developmental pathways after wounding.Our results indicate that callus growth in the apical region of the hypocotyl depends on a specific metabolic switch involving the upregulation of the photorespiratory pathway and the differential regulation of photosynthesis-related genes and of the gluconeogenesis pathway.The endogenous pattern of ROS accumulation in the apical and basal region of the hypocotyl during the time-course were dynamically regulated, and contributed to tissue-specific wound-induced regeneration.Our findings provide a useful resource for further investigation on the molecular mechanisms involved in wound-induced organ formation in a crop species such as tomato.One-sentence SummaryMetabolic switch during wound-induced regeneration

Epstein–Barr virus (EBV), human herpesvirus 6A/B (HHV-6A/B) and multiple sclerosis (MS)-associated retrovirus (MSRV) have been described as possible MS triggers. We analysed antibody titres against EBV and HHV-6, and MSRV envelope (env) mRNA expression, in the serum of pregnant multiple sclerosis patients (P-MS) to study their possible link to the clinical activity of MS during pregnancy and postpartum and their possible role as relapse predictors. For that purpose, serum samples were collected from 71 pregnant women (50 pregnant MS and 21 pregnant healthy controls—P-HC) during pregnancy and postpartum. Relating to antibody titres, IgM antibody titres against HHV-6A/B were significantly higher in P-MS than in P-HC both in each pregnancy trimester and in the postpartum period. Moreover, IgM antibody titres against HHV-6A/B were higher in P-MS who suffered a relapse during the postpartum. Regarding MSRV env mRNA expression, the prevalence in the first trimester of pregnancy was significantly higher in P-MS who suffered relapses during pregnancy. Summing it up, high IgM antibody titres against HHV-6A/B and MSRV env mRNA expression during the first trimester of pregnancy could act as relapse predictors for the gestation/postpartum periods.