Molecular Research on Stress Responses in Quercus spp.: From Classical Biochemistry to Systems Biology through Omics Analysis

The genus Quercus (oak), family Fagaceae, comprises around 500 species, being one of the most important and dominant woody angiosperms in the Northern Hemisphere. Nowadays, it is threatened by environmental cues, which are either of biotic or abiotic origin. This causes tree decline, dieback, and deforestation, which can worsen in a climate change scenario. In the 21st century, biotechnology should take a pivotal role in facing this problem and proposing sustainable management and conservation strategies for forests. As a non-domesticated, long-lived species, the only plausible approach for tree breeding is exploiting the natural diversity present in this species and the selection of elite, more resilient genotypes, based on molecular markers. In this direction, it is important to investigate the molecular mechanisms of the tolerance or resistance to stresses, and the identification of genes, gene products, and metabolites related to this phenotype. This research is being performed by using classical biochemistry or the most recent omics (genomics, epigenomics, transcriptomics, proteomics, and metabolomics) approaches, which should be integrated with other physiological and morphological techniques in the Systems Biology direction. This review is focused on the current state-of-the-art of such approaches for describing and integrating the latest knowledge on biotic and abiotic stress responses in Quercus spp., with special reference to Quercus ilex, the system on which the authors have been working for the last 15 years. While biotic stress factors mainly include fungi and insects such as Phytophthora cinnamomi, Cerambyx welensii, and Operophtera brumata, abiotic stress factors include salinity, drought, waterlogging, soil pollutants, cold, heat, carbon dioxide, ozone, and ultraviolet radiation. The review is structured following the Central Dogma of Molecular Biology and the omic cascade, from DNA (genomics, epigenomics, and DNA-based markers) to metabolites (metabolomics), through mRNA (transcriptomics) and proteins (proteomics). An integrated view of the different approaches, challenges, and future directions is critically discussed

Identification of Proteases and Protease Inhibitors in Seeds of the Recalcitrant Forest Tree Species Quercus ilex.

Proteases and protease inhibitors have been identified in the recalcitrant species Quercus ilex using in silico and wet methods, with focus on those present in seeds during germination. In silico analyses showed that the Q. ilex transcriptome database contained 2,240 and 97 transcripts annotated as proteases and protease inhibitors, respectively. They belonged to the different families according to MEROPS, being the serine and metallo ones the most represented. The data were compared with those previously reported for other Quercus species, including Q. suber, Q. lobata, and Q. robur. Changes in proteases and protease inhibitors alongside seed germination in cotyledon and embryo axis tissues were assessed using proteomics and in vitro and in gel activity assays. Shotgun (LC-MSMS) analysis of embryo axes and cotyledons in nonviable (NV), mature (T1) and germinated (T3) seeds allowed the identification of 177 proteases and 12 protease inhibitors, mostly represented by serine and metallo types. Total protease activity, as determined by in vitro assays using azocasein as substrate, was higher in cotyledons than in embryo axes. There were not differences in activity among cotyledon samples, while embryo axis peaked at germinated T4 stage. Gel assays revealed the presence of protease activities in at least 10 resolved bands, in the Mr range of 60-260 kDa, being some of them common to cotyledons and embryo axes in either nonviable, mature, and germinated seeds. Bands showing quantitative or qualitative changes upon germination were observed in embryo axes but not in cotyledons at Mr values of 60-140 kDa. Proteomics shotgun analysis of the 10 bands with protease activity supported the results obtained in the overall proteome analysis, with 227 proteases and 3 protease inhibitors identified mostly represented by the serine, cysteine, and metallo families. The combined use of shotgun proteomics and protease activity measurements allowed the identification of tissue-specific (e.g., cysteine protease inhibitors in embryo axes of mature acorns) and stage-specific proteins (e.g., those associated with mobilization of storage proteins accumulated in T3 stage). Those proteins showing differences between nonviable and viable seeds could be related to viability, and those variables between mature and germinated could be associated with the germination process. These differences are observed mostly in embryo axes but not in cotyledons. Among them, those implicated in mobilization of reserve proteins, such as the cathepsin H cysteine protease and Clp proteases, and also the large number of subunits of the CNS and 26S proteasome complex differentially identified in embryos of the several stages suggests that protein degradation via CNS/26S plays a major role early in germination. Conversely, aspartic proteases such as nepenthesins were exclusively identified in NV seeds, so their presence could be used as indicator of nonviability.This research was funded by the Spanish Ministry of Economy and Competitiveness in the framework of Projects BIO2015- 64737-R and PID2019-109038RB-I00.S

Classical Swine Fever Virus p7 Protein Interacts with Host Protein CAMLG and Regulates Calcium Permeability at the Endoplasmic Reticulum

We have previously shown that Classical Swine Fever Virus (CSFV) p7 is an essential nonstructural protein with a viroporin activity, a critical function in the progression of virus infection. We also identified p7 domains and amino acid residues critical for pore formation. Here, we describe how p7 specifically interacts with host protein CAMLG, an integral ER transmembrane protein involved in intracellular calcium release regulation and signal response generation. Detection of interaction as well as the identification of p7 areas mediating interaction with CAMLG was performed by yeast two-hybrid. p7-CAMLG interaction was further confirmed by confocal microscopy in eukaryotic cells, co-expressing both proteins. Mutant forms of p7 having substituted native residues identified as mediating interaction with CAMLG showed a decreased co-localization compared with the native forms of p7. Furthermore, it is shown that native p7, but not the mutated forms of p7 that fail to interact with CAMLG, efficiently mediates calcium permeability in the ER. Interestingly, viruses harboring some of those mutated forms of p7 have been previously shown to have a significantly decreased virulence in swine.ARS/USDA-University of Connecticut SCA# 58-1940-1-190 and ARS/USDA-University of the Basque Country NACA#8064-32000-056-18S

CITOTOXICIDAD EN CÉLULAS HELA DE EXTRACTOS DE TRES ESPECIES DE PLANTAS MEDICINALES DE HIDALGO, MÉXICO

Ethanolic extracts of three medicinal plants,Juniperus deppeana, Solanum rostratumand Bidens odorata, which are used in folk medicine in Hidalgo, Mexico, for thetreatment of wounds, ulcers, tumors andcancer, were tested in a HeLa cell line toevaluate their cytotoxic activity. The highestcytotoxicity was found in the extract of J.deppeana (IC50 = 4.63 μg/ml); hence, thisextract was separated via chromatographyon a silica gel plate, from which the mainfraction (Rf = 0.28) showed strong cytotoxic activity (IC50 = 0.79 μg/ml). Whereasthe extract of S. rostratum also exhibitedcytotoxicity (IC50 = 127.5 μg/ml), that ofB. odorata was inactive.Se evaluó la citotoxicidad en cultivos decélulas HeLa de los extractos etanólicosde tres especies de plantas, Juniperus deppeana, Solanum rostratum y Bidens odorata, que se utilizan tradicionalmente en dosregiones del estado de Hidalgo, México,para el tratamiento de heridas, úlceras, tumores y cáncer de matriz. La citotoxicidadmás elevada la presentó el extracto de J.deppeana (CI50 = 4.63 μg/ml), el cual fueseparado por cromatografía en placa de gelde sílice y la fracción principal (Rf = 0.28 )mostró actividad citotóxica (CI50 = 0.79 μg/ml). Aunque menor, el extracto de S. rostratum también presentó citotoxicidad (CI50= 127.5 μg/ml). B. odorata fue inactiva

Morfometría de la gónada masculina y espermatozoides de cobayos (Cavia porcellus) nativo y mejorado del sur de Ecuador

The aim of this research was to describe the morphometry of the testicles and sperm of guinea pigs (Cavia porcellus) of two genetic groups, native and improved, from southern Ecuador. Twenty-two native and 20 improved guinea pigs of the Peru line were used. The animals were reared in elevated cages and fed with forage and concentrate, until reaching the slaughter age of 97 to 112 days. After slaughtering, the testicles and epididymis were removed, and the epididymis was separated of the testis. Measurements of the testicles were done, and sperm was collected from the epididymis by the retrograde flow washing technique. Semen smears were prepared for sperm measurements. The data were processed by GLM analysis of variance and the LSMeans procedure was used to compare the means (SAS v. 9.0). The morphometric parameters of the testes were different (p<0.05) between subpopulations. Opposite testes from the same genetic group were similar, while the ipsilateral ones between subpopulations showed statistical differences (p<0.05). Testicular size was directly related to body weight, and the gonadosomatic index of native guinea pigs was higher than that of improved ones (p<0.05). Spermatozoa were different (p<0.05) between genetic groups in acrosome and head length and in head length / width ratio. In conclusion, this study showed morphological differences in the testes and sperm between the two genetic groups of guinea pigs.El objetivo de esta investigación fue describir la morfometría de los testículos y espermatozoides de cuyes (Cavia porcellus) de dos grupos genéticos, nativos y mejorados, del sur de Ecuador. Se utilizaron 22 cuyes nativos y 20 mejorados de la línea Perú, los cuales fueron criados en jaulas elevadas y alimentados con forraje y concentrado, hasta alcanzar la edad de sacrificio de 97 a 112 días. Luego de faenados, se retiraron los testículos y epidídimos, y mediante disección se separó el epidídimo del testículo para tomar sus dimensiones, y recolectar los espermatozoides del epidídimo por la técnica de lavado por flujo retrógrado. Se prepararon frotis de semen para las mediciones de espermatozoides. Los datos se procesaron por análisis de varianza GLM y se utilizó el procedimiento LSMeans para comparar las medias (SAS V.9.0). Los parámetros morfométricos de los testículos fueron diferentes (p<0.05) entre subpoblaciones; testículos opuestos del mismo grupo genético fueron similares, mientras que los ipsilaterales entre subpoblaciones mostraron diferencias estadísticas (p<0.05). El tamaño testicular tuvo relación directa con el peso corporal, y el índice gonadosomático de los cuyes nativos fue mayor que el de los mejorados (p<0.05). Los espermatozoides fueron diferentes (p<0.05) entre grupos genéticos en la longitud del acrosoma y cabeza y en la relación longitud/ancho de cabeza. En conclusión, se encontraron diferencias morfológicas en los testículos y en los espermatozoides entre los dos grupos de cuyes

Fodder, Nitrogen, and Energy Balances in Grasslands with Algarroba Trees (Prosopis juliflora (S.W.) DC.) under Dairy Cow Grazing

The purpose of this study was to evaluate the effect of algarroba (Prosopis juliflora (S.W.) DC.) on fodder, nitrogen and energy contents in Ecuadoran dairy farm grasslands. The study was made at ESPAM bovine facility, 15 meters above sea level, in Manabí, 00º49’23’’, south latitude, and 80º11’01” west longitude, with 962.4 mm of annual precipitation, between September 2011 and December 2014. The stocking rate was 1.09 LU/ha. The areas were populated with 1-4 algarroba trees/ha by 2011, and 8-35 trees/ha, in 2014. Fodder, nitrogen, and energy balances depended on the arborization degree. As a result, 52 t of DM were estimated in 2014, in comparison to the 21 t produced in 2011. Nitrogen was higher with increased arborization between 2011 (60.9 kg/ha), greater nutrient intake from external sources, and 2014 (39.3 kg/ha), with less use of supplements and mineral fertilizers, and greater N2 contribution by arborization. The energy values were higher in 2014, with an increase in algarroba population/ha. The rise in trees/ha in 2014 favored forage yields, with improved N2 and energy efficiency, which was linked to the benefits acquired by the grassland, the contribution of nitrogen to the ecosystem, and the reduction in feed and fertilizer consumption, which led to energy savings

The Adaptive Thermal Comfort Review from the 1920s, the Present, and the Future

The typical method for comfort analysis is the Predicted Mean Vote and Predicted Percentage Dissatisfied (PMV-PPD). However, they present limitations in accommodating the comfort of a disabled and elder group of people, which are the most vulnerable to climate change and energy poverty. The adaptive method can give flexibility and personalisation needed to overcome the problem due to the variability of the people's metabolism, historical and behavioural preferences. Investments to upgrade the indoor environmental quality and building design can then be effectively used and, for the first time, it will be possible to tailor the solutions for these particular groups of people. The adaptive approach uses Artificial Intelligence (AI), where it can introduce the imperfect learning process. Overcoming this, instead of going further for the Explainable AI, the PMV–PPD approach can be used for the learning validation and verification needed for the adaptive setting point and standards