Real-time monitoring of PtaHMGB activity in poplar transactivation assays

Precise control of gene expression is essential to synchronize plant development with the environment. In perennial plants, transcriptional regulation remains poorly understood, mainly due to the long time required to perform functional studies. Transcriptional reporters based on luciferase have been useful to study circadian and diurnal regulation of gene expression, both by transcription factors and chromatin remodelers. The high mobility group proteins are considered transcriptional chaperones that also modify the chromatin architecture. They have been found in several species, presenting in some cases a circadian expression of their mRNA or protein. Results: Transactivation experiments have been shown as a powerful and fast method to obtain information about the potential role of transcription factors upon a certain reporter. We designed and validated a luciferase transcriptional reporter using the 5? sequence upstream ATG of Populus tremula × alba LHY2 gene. We showed the robustness of this reporter line under long day and continuous light conditions. Moreover, we confirmed that pPtaLHY2::LUC activity reproduces the accumulation of PtaLHY2 mRNA. We performed transactivation studies by transient expression, using the reporter line as a genetic background, unraveling a new function of a high mobility group protein in poplar, which can activate the PtaLHY2 promoter in a gate-dependent manner. We also showed PtaHMGB2/3 needs darkness to produce that activation and exhibits an active degradation after dawn, mediated by the 26S proteasome. Conclusions: We generated a stable luciferase reporter poplar line based on the circadian clock gene PtaLHY2, which can be used to investigate transcriptional regulation and signal transduction pathway. Using this reporter line as a genetic background, we established a methodology to rapidly assess potential regulators of diurnal and circadian rhythms. This tool allowed us to demonstrate that PtaHMGB2/3 promotes the transcriptional activation of our reporter in a gate-dependent manner. Moreover, we added new information about the PtaHMGB2/3 protein regulation along the day. This methodology can be easily adapted to other transcription factors and reporters

Effects of dietary chromium-yeast level on growth performance, blood metabolites, meat traits and muscle fatty acids profile, and microminerals content in liver and bone of lambs

To assess the effect of dietary supplement levels of chromium-yeast (Cr-yeast) on growth performance, blood glucose and triglycerides, fatty acid (FA) profile in intramuscular fat, carcase and meat traits, iron, copper, chromium and zinc concentrations in liver and bone, 24 Rambouillet male lambs (29.2 ± 0.17 kg body weight) were randomly assigned to four diets with 0, 0.2, 0.4 and 0.6mg Cr/kg DM. The growth performance trial lasted 49 d. Supplemental Cryeast did not affect growth performance and carcase characteristics (p>.05), but reduced (p<.05) perirenal and intramuscular fat, as well as 3 h post-feeding blood glucose and triglycerides concentration. In liver, Fe and Cu concentration decreased (p<.05), while Cr concentrations in liver increased with increasing Cr-yeast dietary levels. In bone, Fe decreased (p<.05) as Cryeast dietary levels increasing, and Cr-yeast supplementation increased Cr concentrations (p<.05). As Cr-yeast dietary level increased, palmitic (C16:0) and stearic (C18:0) SFA decreased linearly (p<.05), while palmitoleic (C16:1n-7), vaccenic (C18:1n-7), linoleic (C18:2n-6) and arachidic (C20:4) unsaturated fatty acids (UFA) increased linearly (p<.01). In conclusion, Cr-yeast did not affect growth performance and carcase quality, but decreased the perirenal and intramuscular fat, blood glucose and triglyceride content, and Fe and Cu concentrations in liver as increased Cr-yeast levels in the diet. Because supplemental Cr-yeast improved index of atherogenicity and unsaturated to saturated FA ratio in muscle of lambs, it could be of human nutritional interest

Library of Seleno-Compounds as Novel Agents against Leishmania Species

The in vitro leishmanicidal activities of a series of 48 recently synthesized selenium derivatives against Leishmania infantum and Leishmania braziliensis parasites were tested using promastigotes and intracellular amastigote forms. The cytotoxicity of the tested compounds for J774.2 macrophage cells was also measured in order to establish their selectivity. Six of the tested compounds (compounds 8, 10, 11, 15, 45, and 48) showed selectivity indexes higher than those of the reference drug, meglumine antimonate (Glucantime), for both Leishmania species; in the case of L. braziliensis, compound 20 was also remarkably selective. Moreover, data on infection rates and amastigote numbers per macrophage showed that compounds 8, 10, 11, 15, 45, and 48 were the most active against both Leishmania species studied. The observed changes in the excretion product profile of parasites treated with these six compounds were also consistent with substantial cytoplasmic alterations. On the other hand, the most active compounds were potent inhibitors of Fe superoxide dismutase (Fe-SOD) in the two parasite species considered, whereas their impact on human CuZn-SOD was low. The high activity, low toxicity, stability, low cost of the starting materials, and straightforward synthesis make these compounds appropriate molecules for the development of affordable antileishmanicidal agents

ISGylation controls exosome secretion by promoting lysosomal degradation of MVB proteins

Exosomes are vesicles secreted to the extracellular environment through fusion with the plasma membrane of specific endosomes called multivesicular bodies (MVB) and mediate cell-to-cell communication in many biological processes. Posttranslational modifications are involved in the sorting of specific proteins into exosomes. Here we identify ISGylation as a ubiquitin-like modification that controls exosome release. ISGylation induction decreases MVB numbers and impairs exosome secretion. Using ISG15-knockout mice and mice expressing the enzymatically inactive form of the de-ISGylase USP18, we demonstrate in vitro and in vivo that ISG15 conjugation regulates exosome secretion. ISG15 conjugation triggers MVB co-localization with lysosomes and promotes the aggregation and degradation of MVB proteins. Accordingly, inhibition of lysosomal function or autophagy restores exosome secretion. Specifically, ISGylation of the MVB protein TSG101 induces its aggregation and degradation, being sufficient to impair exosome secretion. These results identify ISGylation as a novel ubiquitin-like modifier in the control of exosome production.We thank Dr K. Knobeloch, Dr A. Garcia-Sastre and Dr M.A. Alonso for providing reagents, and Dr S. Bartlett for assistance with English editing. C.E. is thankful to electron microscopy facility (campus Casanova), CCiT-University of Barcelona. This study was supported by grants SAF2014-55579-R from the Spanish Ministry of Economy and Competitiveness, INDISNET-S2011/BMD-2332 from the Comunidad de Madrid, Cardiovascular Network RD12-0042-0056 and PIE13/00041 from Instituto de Salud Carlos III (Fondo de Investigacion Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional FEDER), ERC-2011-AdG 294340-GENTRIS and COST-Action BM1202 to F.S.-M.; grant SAF2014-54623-R, FIS grant PI11/00127 (Fondo de Investigacion Sanitaria del Instituto de Salud Carlos III and Ministry of Health of Spain, State secretary of R+D and FEDER/FSE) and Bayer Group Grants4Grants (ID 2013-08-0982) to S.G.; and grant BFU2015-66785-P from the Spanish Ministry of Economy and Competitiveness to C.E.; Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). C.V.-B. was supported by FPU programme (Spanish Ministry of Education). M. M. is supported by MS14/00219 from Instituto de Salud Carlos III.S

Atlantic circulation change still uncertain

Deep oceanic overturning circulation in the Atlantic (Atlantic Meridional Overturning Circulation, AMOC) is projected to decrease in the future in response to anthropogenic warming. Caesar et al. 1 argue that an AMOC slowdown started in the 19 th century and intensified during the mid-20th century. Although the argument and selected evidence proposed have some merits, we find that their conclusions might be different if a more complete array of data available in the North Atlantic region had been considered. We argue that the strength of AMOC over recent centuries is still poorly constrained and the expected slowdown may not have started yet.K.H.K. acknowledges funding from NOAA grant NA20OAR4310481. D.E.A. and B.L.O.-B. acknowledge support from the National Center for Atmospheric Research, which is a major facility sponsored by the National Science Foundation under cooperative agreement no. 1852977. N.M.W. acknowledges support from a NOAA Climate and Global Change Postdoctoral Fellowship. M.F.J. acknowledges support from NSF award OCE-1846821 and C.M.L. acknowledges support from NSF award OCE-1805029. This is UMCES contribution 6062.Peer ReviewedArticle signat per 17 autors/es: University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons, MD, USA: K. Halimeda Kilbourne / Department of Geological and Atmospheric Sciences, Iowa State University, Ames, IA, USA: Alan D. Wanamaker / Geography Department, Durham University, Durham, UK: Paola Moffa-Sanchez / Centre for Geography and Environmental Sciences, University of Exeter, Penryn, UK: David J. Reynolds, Paul G. Butler & James Scourse / Climate and Global Dynamics Laboratory, National Center for Atmospheric Research, Boulder, CO, USA: Daniel E. Amrhein & Bette L. Otto-Bliesner / Woods Hole Oceanographic Institution, Falmouth, MA, USA: Geoffrey Gebbie & Nina M. Whitney / Cooperative Institute for Marine and Atmospheric Studies, University of Miami, Miami, FL, USA: Marlos Goes / Atlantic Oceanographic and Meteorological Laboratory, National Oceanic and Atmospheric Administration, Miami, FL, USA: Marlos Goes / Department of the Geophysical Sciences, The University of Chicago, Chicago, IL, USA: Malte F. Jansen / Oceanography Department, Atmospheric and Environmental Research, Inc., Texas, TX, USA: Christopher M. Little / US Geological Survey, St Petersburg Coastal and Marine Science Center, St Petersburg, FL, USA: Madelyn Mette / Barcelona Supercomputing Center, Barcelona, Spain: Eduardo Moreno-Chamarro & Pablo Ortega / Graduate School of Oceanography, University of Rhode Island, Kingston, RI, USA: Thomas Rossby / University Corporation of Atmospheric Research, Boulder, CO, USA: Nina M. WhitneyPostprint (author's final draft)Matters Arising published on 17 February 2022. The Original Article was published on 25 February 2021

Laparoscopic choledochal cyst resection with simplified common bile duct reconstruction in an adult population: A case series

Introducción: Los quistes de colédoco (CC) son una patología congénita poco frecuente en la población adulta. Desde 1995, el manejo laparoscópico ha sido descrito para esta entidad. Sin embargo, su manejo se considera un tema controvertido debido al aumento del riesgo de colangiocarcinoma. Materiales y métodos: se realizó un estudio retrospectivo, observacional y descriptivo considerando pacientes diagnosticados de CC que fueron operados en un centro de referencia de cirugía hepatobiliar desde enero de 2013 hasta junio de 2018. Los pacientes fueron llevados a laparoscopia simplificada hepaticoyeyunostomía con reconstrucción en Y de Roux. Se presenta un análisis retrospectivo de los datos obtenidos. Resultados: Diez pacientes adultos con CC fueron sometidos a reconstrucción biliar quirúrgica a una edad media de 34,5 años; 75% tenía CC Todani tipo I y 25% Todani tipo IV-B CC. Aproximadamente el 50% de los pacientes fueron diagnosticados mediante colangiopancreatografía retrógrada endoscópica (CPRE) y el 50% de ellos mediante colangiopancreatografía por resonancia magnética. Ninguno requirió reintervención, no se informó mortalidad; y la estancia media en el hospital fue 5 días, ningún paciente tuvo fuga biliar posoperatoria, ninguno se convirtió a cirugía abierta y todos los pacientes tuvieron una tolerancia adecuada a la alimentación oral 2 días postoperatorios. El seguimiento a largo plazo no mostró incidencia de colangiocarcinoma después de un seguimiento de 2 años. Conclusión: Los quistes de colédoco en adultos son una patología poco frecuente que tiene una alta probabilidad de desarrollar malignidad cuando no se realiza de forma adecuada quirúrgicamente manejado y debido al reflujo biliar secundario. Estos factores hacen que el manejo quirúrgico sea una decisión crítica. El abordaje laparoscópico simplificado presentado en este trabajo parece ser una alternativa eficaz y segura a la cirugía reconstructiva de la vía biliar.Introduction: Choledochal cysts (CC) are rare congenital pathology in adult population. Since 1995, laparoscopic management has been described for this entity. Nevertheless, its management is considered to be a controversial matter due to the augmented risk of associated cholangiocarcinoma. Materials and methods: A retrospective, observational, and descriptive study was conducted considering patients diagnosed with CC who were operated at a hepatobiliary surgery referral center from January 2013 to June 2018. Patients were taken to simplified laparoscopic hepaticojejunostomy with a Roux-en-Y reconstruction. A retrospective analysis of the data obtained is presented. Results: Ten adult patients with CC underwent surgical biliary reconstruction at a mean age of 34.5 years; 75% had Todani type I CC and 25% Todani type IV-B CC. About 50% of the patients were diagnosed via endoscopic retrograde holangiopancreatography (ERCP) and 50% of them via magnetic resonance cholangiopancreatography. None required re-intervention, no mortality was reported; and the mean hospital stay was 5 days, no patient had postoperative biliary leakage, none was converted to open surgery, and all patients had adequate oral feeding tolerance 2 days postoperative. Long-term follow-up showed no incidence of cholangiocarcinoma after 2-year follow-up. Conclusion: Choledochal cysts in adults is a rare pathology that has a high probability of developing malignancy when not adequately surgically managed and because of secondary bile reflux. These factors make surgical management a critical decision. The simplified laparoscopic approach presented in this paper seems to be an effective and safe alternative to biliary duct reconstructive surgery

Atopic Dermatitis and Erythrodermia Associated With Systemic Lupus Erythematosus: A Case Report

La eritrodermia es una dermatosis inflamatoria que involucra capas parciales o totales de la piel. La mortalidad es del 16%. Los síntomas sistémicos incluyen fiebre, taquicardia, hinchazón de las extremidades, adenomegalia y hepatomegalia. Las causas infecciosas son las más frecuentes, estando presentes hasta en un 40%, seguidas de ictiosis y dermatitis en el 15% de los casos

A palaeoecological approach to understanding the past and present of Sierra Nevada, a Southwestern European biodiversity hotspot

Mediterranean mountainous environments are biodiversity hotspots and priority areas in conservation agendas. Although they are fragile and threatened by forecasted global change scenarios, their sensitivity to long-term environmental variability is still understudied. The Sierra Nevada range, located in southern Spain on the north-western European flanks of the Mediterranean basin, is a biodiversity hotspot. Consequently, Sierra Nevada provides an excellent model system to apply a palaeoecological approach to detect vegetation changes, explore the drivers triggering those changes, and how vegetation changes link to the present landscape in such a paradigmatic mountain system. A multi-proxy strategy (magnetic susceptibility, grain size, loss-on-ignition, macroremains, charcoal and palynological analyses) is applied to an 8400-year long lacustrine environmental archive from the Laguna de la Mosca (2889 masl). The long-term ecological data show how the Early Holocene pine forests transitioned towards mixed Pinus-Quercus submediterranean forests as a response to a decrease in seasonality at ~7.3 cal. kyr BP. The mixed Pinus-Quercus submediterranean forests collapsed drastically giving way to open evergreen Quercus formations at ~4.2 cal. kyr BP after a well-known aridity crisis. Under the forecasted northward expansion of the Mediterranean area due to global change-related aridity increase, mountain forests inhabiting territories adjacent to the Mediterranean Region could experience analogous responses to those detected in the Sierra Nevada forests to the Mid to Late Holocene aridification, moving from temperate to submediterranean and then Mediterranean formations

The Interplay of the Tree and StandLevel Processes Mediate DroughtInduced Forest Dieback: Evidence from Complementary Remote Sensing and Tree-Ring Approaches

Moreno-Fernández, D., Camarero, J.J., García, M. et al. The Interplay of the Tree and Stand-Level Processes Mediate Drought-Induced Forest Dieback: Evidence from Complementary Remote Sensing and Tree-Ring Approaches. Ecosystems 25, 1738-1753 (2022).Drought-induced forest dieback can lead to a tipping point in community dominance, but the coupled response at the tree and stand-level response has not been properly addressed. New spatially and temporally integrated monitoring approaches that target different biological organization levels are needed. Here, we compared the temporal responses of dendrochronological and spectral indices from 1984 to 2020 at both tree and stand levels, respectively, of a drought-prone Mediterranean Pinus pinea forest currently suffering strong dieback. We test the influence of climate on temporal patterns of tree radial growth, greenness and wetness spectral indices; and we address the influence of major drought episodes on resilience metrics. Tree-ring data and spectral indices followed different spatio-temporal patterns over the study period (1984?2020). Combined information from tree growth and spectral trajectories suggests that a reduction in tree density during the mid-1990s could have promoted tree growth and reduced dieback risk. Additionally, over the last decade, extreme and recurrent droughts have resulted in crown defoliation greater than 40% in most plots since 2019. We found that tree growth and the greenness spectral index were positively related to annual precipitation, while the wetness index was positively related to mean annual temperature. The response to drought, however, was stronger for tree growth than for spectral indices. Our study demonstrates the value of long-term retrospective multiscale analyses including tree and stand-level scales to disentangle mechanisms triggering and driving forest dieback.Ministerio de Ciencia, Innovacion y UniversidadesUniversidad de AlcaláMinisterio de Ciencia e InnovaciónComunidad de MadridUK Research and Innovatio

Novel winter-associated regulators of the circadian clock in poplar

Background Winter dormancy is an adaptive mechanism that allows trees from temperate and cold regions to survive the harsh conditions of this season. Critical steps of this process are strongly influenced by environmental cues, mainly daylength and temperature. The mechanism that integrates these signals is the circadian clock. Despite the importance of the correct functioning of the clock for the healthy state of the plant [1], low temperatures cause the disruption of the circadian clock in trees, which consists in a transcriptional activation followed by an arrhythmic expression [2-5]. In this work we uncover winter-associated regulators of the circadian clock in poplar. Methods Firstly, we made a transcriptional fusion with the promoter of LHY2, a circadian clock gene, and the luciferase gene. This construct was used to generate transgenic poplars (717-1B4, INRA clone). With these events we characterized the expression of this promoter under different conditions of photoperiod and temperature. To this aim we have set up a circadian luminiscence assay registering luciferase activity from leaf discs with a luminometer. Then we carried out a Yeast One Hybrid (Y1H) screening with a library enriched in winter-associated factors and using this promoter as bait. Candidate regulators are tested in vivo using Golden Braid technology [6] and transient assays in poplar, by which we overexpressed and silenced the candidate genes. Results and Conclusions Here we present the characterization of the Populus tremula x alba LHY2 promoter under three different photoperiod conditions. Our results indicate the selected promoter region contains the circadian elements as well as the luciferase activity shows the expected expression under both long and short days. In the Y1H screening, we found several candidates that are classified either as transcription factors or chromatin remodelers. We will discuss the possible role of these proteins as regulators of the poplar circadian clock