56 research outputs found
Wheat Germ Agglutinin Conjugated Fluorescent pH Sensors for Visualizing Proton Fluxes [preprint]
Small molecule fluorescent wheat germ agglutinin (WGA) conjugates are routinely used to demarcate mammalian plasma membranes because they bind to the cellâs glycocalyx. Here we describe the derivatization of WGA with a pH sensitive rhodamine fluorophore (pHRho: pKa = 7) to detect proton channel fluxes and extracellular proton accumulation and depletion from primary cells. We found that WGA-pHRho labeling was uniform, did not appreciably alter the voltage-gating of glycosylated ion channels, and the extracellular changes in pH directly correlated with proton channel activity. Using single plane illumination techniques, WGA-pHRho was used to detect spatiotemporal differences in proton accumulation and depletion over the extracellular surface of cardiomyocytes, astrocytes, and neurons. Because WGA can be derivatized with any small molecule fluorescent ion sensor, WGA conjugates should prove useful to visualize most electrogenic and non-electrogenic events on the extracellular side of the plasma membrane
Water and agriculture
This chapter shows the strong links between water, agriculture and the economy in Latin America and Caribbean (LAC). Both green and blue water are vital for LAC's economies and for its food security. Awareness of LAC's virtual water trade volumes and water footprints alone will not solve the local or global water problems. However, the awareness gained increases the likelihood that optimized water allocation decisions, which consider the hydrological and economical aspects of water resources, are made. âą Agriculture is a significant economic sector for many LAC countries with some being major world players in the agricultural commodities world markets, such is the case for Brazil and Argentina who contribute to 13% of the global green water export. At the micro level, agriculture still plays a significant role for the food security of the population. âą The consumptive water use of agricultural production was on average 1,057Gm 3 / yr for the period 1996â2005; of which, 95% corresponds to the green water footprint, whereas 5% refers to the blue component. This indicates that LAC relies heavily on green water for agricultural production, i.e. rain-fed agriculture. âą Maize is a fundamental crop in Argentina, Brazil, Chile, Mexico and Peru, representing 15% of the total agricultural blue and green water footprint (773,408hm 3 /yr) and contributing to 35% of the agricultural nitrogen pollution, estimated as grey water footprint, in Argentina, Brazil, Chile, Colombia, Mexico and Peru. Only in Mexico, maize contributes 60% of the agricultural grey water footprint. âą Grazing represents 24% of the total green water footprint of agriculture in these countries. The blue water consumption by the animal water supply is very significant in Argentina, Brazil, Chile, Mexico and Peru, which amounts to 13% (38,825hm 3 /yr) of the total consumption
Near-Membrane Dynamics and Capture of TRPM8 Channels within Transient Confinement Domains
The cold and menthol receptor, TRPM8, is a non-selective cation channel expressed in a subset of peripheral neurons that is responsible for neuronal detection of environmental cold stimuli. It was previously shown that members of the transient receptor potential (TRP) family of ion channels are translocated toward the plasma membrane (PM) in response to agonist stimulation. Because the spatial and temporal dynamics of cold receptor cell-surface residence may determine neuronal activity, we hypothesized that the movement of TRPM8 to and from the PM might be a regulated process. Single particle tracking (SPT) is a useful tool for probing the organization and dynamics of protein constituents in the plasma membrane.We used SPT to study the receptor dynamics and describe membrane/near-membrane behavior of particles containing TRPM8-EGFP in transfected HEK-293T and F-11 cells. Cells were imaged using total internal reflection fluorescence (TIRF) microscopy and the 2D and 3D trajectories of TRPM8 molecules were calculated by analyzing mean-square particle displacement against time. Four characteristic types of motion were observed: stationary mode, simple Brownian diffusion, directed motion, and confined diffusion. In the absence of cold or menthol to activate the channel, most TRPM8 particles move in network covering the PM, periodically lingering for 2â8 s in confined microdomains of about 800 nm radius. Removing cholesterol with methyl-beta-cyclodextrin (MÎČCD) stabilizes TRPM8 motion in the PM and is correlated with larger TRPM8 current amplitude that results from an increase in the number of available channels without a change in open probability.These results reveal a novel mechanism for regulating TRPM8 channel activity, and suggest that PM dynamics may play an important role in controlling electrical activity in cold-sensitive neurons
Physico-chemical, biological and geological study of an underwater volcano in a degassing stage: Island of El Hierro
El objetivo principal del proyecto âPhysico-chemical, biological and geological study of an underwater volcano in a degassing stage: Island of El Hierroâ, (VULCANO-II) es estudiar, desde un punto de vista totalmente interdisciplinar, la fase de desgasificaciĂłn activa del Ășnico volcĂĄn submarino monitoreado desde su nacimiento en aguas españolas. De esta forma, se pretende ademĂĄs, dar continuidad a los estudios multidisciplinares realizados sobre el volcĂĄn submarino de la isla de El Hierro en el contexto del proyecto del Plan Nacional VULCANO-I, (CTM2012-36317) y VULCANA (VulcanologĂa Canaria Submarina, IEO).
Para ello, se realizarĂĄ la monitorizaciĂłn de las propiedades fĂsico-quĂmicas, biolĂłgicas y geolĂłgicas del proceso eruptivo submarino de la isla de El Hierro y otros puntos sensibles, como el volcĂĄn de Enmedio entre Gran Canaria y Tenerif
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field
Recommended from our members
Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.
Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (nâ=â143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (nâ=â152), or no hydrocortisone (nâ=â108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (nâ=â137), shock-dependent (nâ=â146), and no (nâ=â101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707
Measurement of the Higgs boson production rate in association with top quarks in final states with electrons, muons, and hadronically decaying tau leptons at sâ=13TeV
The rate for Higgs (H) bosons production in association with either one (tH) or two (ttÂŻH) top quarks is measured in final states containing multiple electrons, muons, or tau leptons decaying to hadrons and a neutrino, using protonâproton collisions recorded at a center-of-mass energy of 13TeV by the CMS experiment. The analyzed data correspond to an integrated luminosity of 137fbâ1. The analysis is aimed at events that contain HâWW, HâÏÏ, or HâZZ decays and each of the top quark(s) decays either to lepton+jets or all-jet channels. Sensitivity to signal is maximized by including ten signatures in the analysis, depending on the lepton multiplicity. The separation among tH, ttÂŻH, and the backgrounds is enhanced through machine-learning techniques and matrix-element methods. The measured production rates for the ttÂŻH and tH signals correspond to 0.92±0.19(stat)+0.17â0.13(syst) and 5.7±2.7(stat)±3.0(syst) of their respective standard model (SM) expectations. The corresponding observed (expected) significance amounts to 4.7 (5.2) standard deviations for ttÂŻH, and to 1.4 (0.3) for tH production. Assuming that the Higgs boson coupling to the tau lepton is equal in strength to its expectation in the SM, the coupling yt of the Higgs boson to the top quark divided by its SM expectation, Îșt=yt/ySMt, is constrained to be within â0.9<Îșt<â0.7 or 0.7<Îșt<1.1, at 95% confidence level. This result is the most sensitive measurement of the ttÂŻH production rate to date.SCOAP
The Tiny Drosophila Melanogaster for the Biggest Answers in Huntingtonâs Disease
The average life expectancy for humans has increased over the last years. However, the quality of the later stages of life is low and is considered a public health issue of global importance. Late adulthood and the transition into the later stage of life occasionally leads to neurodegenerative diseases that selectively affect different types of neurons and brain regions, producing motor dysfunctions, cognitive impairment, and psychiatric disorders that are progressive, irreversible, without remission periods, and incurable. Huntingtonâs disease (HD) is a common neurodegenerative disorder. In the 25 years since the mutation of the huntingtin (HTT) gene was identified as the molecule responsible for this neural disorder, a variety of animal models, including the fruit fly, have been used to study the disease. Here, we review recent research that used Drosophila as an experimental tool for improving knowledge about the molecular and cellular mechanisms underpinning HD
- âŠ