Unsaturation in the air spaces of leaves and its implications.

Modern plant physiological theory stipulates that the resistance to water movement from plants to the atmosphere is overwhelmingly dominated by stomata. This conception necessitates a corollary assumption-that the air spaces in leaves must be nearly saturated with water vapour; that is, with a relative humidity that does not decline materially below unity. As this idea became progressively engrained in scientific discourse and textbooks over the last century, observations inconsistent with this corollary assumption were occasionally reported. Yet, evidence of unsaturation gained little traction, with acceptance of the prevailing framework motivated by three considerations: (1) leaf water potentials measured by either thermocouple psychrometry or the Scholander pressure chamber are largely consistent with the framework; (2) being able to assume near saturation of intercellular air spaces was transformational to leaf gas exchange analysis; and (3) there has been no obvious mechanism to explain a variable, liquid-phase resistance in the leaf mesophyll. Here, we review the evidence that refutes the assumption of universal, near saturation of air spaces in leaves. Refining the prevailing paradigm with respect to this assumption provides opportunities for identifying and developing mechanisms for increased plant productivity in the face of increasing evaporative demand imposed by global climate change.

The interplay of short-term mesophyll and stomatal conductance responses under variable environmental conditions.

Understanding the short-term responses of mesophyll conductance (gm ) and stomatal conductance (gsc ) to environmental changes remains a challenging yet central aspect of plant physiology. This review synthesises our current knowledge of these short-term responses, which underpin CO2 diffusion within leaves. Recent methodological advances in measuring gm using online isotopic discrimination and chlorophyll fluorescence have improved our confidence in detecting short-term gm responses, but results need to be carefully evaluated. Environmental factors like vapour pressure deficit and CO2 concentration indirectly impact gm through gsc changes, highlighting some of the complex interactions between the two parameters. Evidence suggests that short-term responses of gm are not, or at least not fully, mechanistically linked to changes in gsc , cautioning against using gsc as a reliable proxy for gm . The overarching challenge lies in unravelling the mechanistic basis of short-term gm responses, which will contribute to the development of accurate models bridging laboratory insights with broader ecological implications. Addressing these gaps in understanding is crucial for refining predictions of gm behaviour under changing environmental conditions.

Evidence for an RNAi-independent role of Arabidopsis DICER-LIKE2 in growth inhibition and basal antiviral resistance.

Flowering plant genomes encode four or five DICER-LIKE (DCL) enzymes that produce small interfering RNAs (siRNAs) and microRNAs, which function in RNA interference (RNAi). Different RNAi pathways in plants effect transposon silencing, antiviral defense, and endogenous gene regulation. DCL2 acts genetically redundantly with DCL4 to confer basal antiviral defense. However, DCL2 may also counteract DCL4 since knockout of DCL4 causes growth defects that are suppressed by DCL2 inactivation. Current models maintain that RNAi via DCL2-dependent siRNAs is the biochemical basis of both effects. Here, we report that DCL2-mediated antiviral resistance and growth defects cannot be explained by the silencing effects of DCL2-dependent siRNAs. Both functions are defective in genetic backgrounds that maintain high levels of DCL2-dependent siRNAs, either with specific point mutations in DCL2 or with reduced DCL2 dosage because of heterozygosity for dcl2 knockout alleles. Intriguingly, all DCL2 functions require its catalytic activity, and the penetrance of DCL2-dependent growth phenotypes in dcl4 mutants correlates with DCL2 protein levels but not with levels of major DCL2-dependent siRNAs. We discuss this requirement and correlation with catalytic activity but not with resulting siRNAs, in light of other findings that reveal a DCL2 function in innate immunity activation triggered by cytoplasmic double-stranded RNA.

The interaction of the ribotoxin α-sarcin with complex model lipid vesicles.

Fungal ribotoxins are extracellular RNases that inactivate ribosomes by cleaving a single phosphodiester bond at the universally conserved sarcin-ricin loop of the large rRNA. However, to reach the ribosomes, they need to cross the plasma membrane. It is there where these toxins show their cellular specificity, being especially active against tumoral or virus-infected cells. Previous studies have shown that fungal ribotoxins interact with negatively charged membranes, typically containing phosphatidylserine or phosphatidylglycerol. This ability is rooted on their long, non-structured, positively charged loops, and its N-terminal ß-hairpin. However, its effect on complex lipid mixtures, including sphingophospholipids or cholesterol, remains poorly studied. Here, wild-type α-sarcin was used to evaluate its interaction with a variety of membranes not assayed before, which resemble much more closely mammalian cell membranes. The results confirm that α-sarcin is particularly sensitive to charge density on the vesicle surface. Its ability to induce vesicle aggregation is strongly influenced by both the lipid headgroup and the degree of saturation of the fatty acid chains. Acyl chain length is indeed particularly important for lipid mixing. Finally, cholesterol plays an important role in diluting the concentration of available negative charges and modulates the ability of α-sarcin to cross the membrane.

Pembrolizumab as first-line treatment for advanced NSCLC in older adults: A phase II clinical trial evaluating geriatric and quality-of-life outcomes.

OBJECTIVES: Since specific data on immunotherapy in older adults with advanced non-small cell lung cancer (aNSCLC) are scarce, we designed this study to determine the overall survival (OS) at one year of first-line pembrolizumab in patients older than 70 years with aNSCLC expressing PD-L1. Secondary objectives included progression-free survival, disease-specific survival, response rate, tolerability, quality of life (QoL) changes, and geriatric assessments. MATERIALS AND METHODS: A single-arm, open-label, phase II clinical trial was carried out by the Spanish Lung Cancer Group between February 2018 and November 2019 at ten active sites in Spain. We included patients 70 years old and older with histological or cytological documented stage IIIB or IV aNSCLC and PD-L1 expression ≥ 1%. Each subject received 200 mg of intravenous pembrolizumab every three weeks for a maximum of two years. RESULTS: 83 patients were recruited for the study and 74 were finally analysed. Most were male (N = 64, 86.5%) and former smokers (N = 51, 68.9%). 24 patients (32.4%) completed at least one year of treatment, 62 (83.7%) discontinued treatment, and 30 (40.5%) experienced disease progression. The median follow-up of our cohort was 18.0 months [range: 0.1-47.7] and 46 patients (62.2%) died during the period of study. The estimated OS at one year was 61.7% (95% CI: 49.6-71.8%) and the median OS of our cohort was 19.2 months (95% CI: 11.3-25.5). QoL tended to improve throughout the study, although the differences were not statistically significant. The main geriatric scores remained stable, except for a worsening in nutritional status (P = 0.004) and an improvement in frailty (P = 0.028). CONCLUSION: Our results support treating older adults with aNSCLC expressing PD-L1 with pembrolizumab in monotherapy. The stability of most geriatric scores and the positive trend on the patients' QoL should be highlighted, although our results did not reach statistical significance.

Regulation of plant immunity via small RNA-mediated control of NLR expression.

Plants use different receptors to detect potential pathogens: membrane-anchored pattern recognition receptors (PRRs) activated upon perception of pathogen-associated molecular patterns (PAMPs) that elicit pattern-triggered immunity (PTI); and intracellular nucleotide-binding leucine-rich repeat proteins (NLRs) activated by detection of pathogen-derived effectors, activating effector-triggered immunity (ETI). The interconnections between PTI and ETI responses have been increasingly reported. Elevated NLR levels may cause autoimmunity, with symptoms ranging from fitness cost to developmental arrest, sometimes combined with run-away cell death, making accurate control of NLR dosage key for plant survival. Small RNA-mediated gene regulation has emerged as a major mechanism of control of NLR dosage. Twenty-two nucleotide miRNAs with the unique ability to trigger secondary siRNA production from target transcripts are particularly prevalent in NLR regulation. They enhance repression of the primary NLR target, but also bring about repression of NLRs only complementary to secondary siRNAs. We summarize current knowledge on miRNAs and siRNAs in the regulation of NLR expression with an emphasis on 22 nt miRNAs and propose that miRNA and siRNA regulation of NLR levels provides additional links between PTI and NLR defense pathways to increase plant responsiveness against a broad spectrum of pathogens and control an efficient deployment of defenses.

Post intensive care syndrome in survivors of COVID-19 who required mechanical ventilation during the third wave of the pandemic: A prospective study.

BACKGROUND: Post intensive care syndrome is defined as the presence of any impairment affecting the physical, psychiatric, or cognitive domains as a result of critical illnesses. OBJECTIVES: To explore functional, cognitive and psychological outcomes at 30 days post hospital discharge among survivors of COVID-19-associated acute respiratory distress syndrome, who required mechanical ventilation. METHODS: Prospective cohort study. We included adult patients with COVID-19-associated acute respiratory distress syndrome, invasively ventilated in two ICUs in Buenos Aires. We measured functional, cognitive and psychological impairments with Barthel index, Montreal Cognitive Assessment test, Patient Health Questionnaire-9 and General Anxiety Disorder-7. Primary outcome was post-intensive care syndrome. Secondary outcome was mortality at 60 days. RESULTS: We admitted 40 patients, median age was 69 (60-75) and mostly male (75%). Mortality at 60 days was 37%. Cox regression analysis identified diabetes and Apache II as independent predictors of mortality. Out of 22 patients studied, 14 (64%) developed PICS after discharge. With a physical, cognitive and psychological impairment in 64%, 41% and 32% of patients, respectively. Obesity, days of mechanical ventilation, Apache II, vasopressors use, delirium duration and cumulative midazolam dose were associated with functional dependence. CONCLUSIONS: We identified a high prevalence of functional, cognitive and mental impairment at 30 days after hospital discharge in COVID-19-associated acute respiratory distress syndrome survivors, invasively ventilated. The physical domain was the most frequently affected. These findings suggest the need for long-term follow-up of this population.

Sticholysin recognition of ceramide-phosphoethanolamine.

Actinoporins are pore-forming toxins produced by sea anemones. They exert their activity by binding to the membranes of target cells. There, they oligomerize, forming cation-selective pores, and inducing cell death by osmotic shock. In the early days of the field, it was shown that accessible sphingomyelin (SM) in the bilayer is required for the activity of actinoporins. While these toxins can also act on membranes composed solely of phosphatidylcholine (PC) with a high amount of cholesterol (Chol), consensus is that SM acts as a lipid receptor for actinoporins. It has been shown that the 2NH and 3OH moieties of SM are essential for actinoporin recognition. Hence, we wondered if ceramide-phosphoethanolamine (CPE) could also be recognized. Like SM, CPE has the 2NH and 3OH groups, and a positively charged headgroup. While actinoporins have been observed to affect membranes containing CPE, Chol was always also present, with the recognition of CPE remaining unclear. To test this possibility, we used sticholysins, produced by the Caribbean Sea anemone Stichodactyla helianthus. Our results show that sticholysins can induce calcein release on vesicles composed only of PC and CPE, in absence of Chol, in a way that is comparable to that induced on PC:SM membranes.

Assessing the CO2 concentration at the surface of photosynthetic mesophyll cells.

We present a robust estimation of the CO2 concentration at the surface of photosynthetic mesophyll cells (cw ), applicable under reasonable assumptions of assimilation distribution within the leaf. We used Capsicum annuum, Helianthus annuus and Gossypium hirsutumas model plants for our experiments. We introduce calculations to estimate cw using independent adaxial and abaxial gas exchange measurements, and accounting for the mesophyll airspace resistances. The cw was lower than adaxial and abaxial estimated intercellular CO2 concentrations (ci ). Differences between cw and the ci of each surface were usually larger than 10 µmol mol-1 . Differences between adaxial and abaxial ci ranged from a few µmol mol-1 to almost 50 µmol mol-1 , where the largest differences were found at high air saturation deficits (ASD). Differences between adaxial and abaxial ci and the ci estimated by mixing both fluxes ranged from -30 to +20 µmol mol-1 , where the largest differences were found under high ASD or high ambient CO2 concentrations. Accounting for cw improves the information that can be extracted from gas exchange experiments, allowing a more detailed description of the CO2 and water vapor gradients within the leaf.

Position statement on use of pharmacological combinations in a single pill for treatment of hypertension by Argentine Federation of Cardiology (FAC) and Argentine Society of Hypertension (SAHA).

The present document provides scientific evidence reviewed and analysed by a group of specialist clinicians in hypertension that aims to give an insight into a pharmacological strategy to improve blood pressure control. Evidence shows that most hypertensive patients will need at least two drugs to achieve blood pressure goals. There is ample evidence showing that treatment adherence is inversely related to the number of drugs taken. Observational studies show that use of drug combinations to initiate treatment reduces the time to reach the treatment goal and reduces CVD, especially with single pill combinations (SPCs). This work, based on recommendations of the Argentine Federation of Cardiology and Argentine Society of Hypertension as a reference, aims to review the more recent evidence on SPC, and to serve as guidelines for health professionals in their clinical practice and to the wider use of SPCs for the treatment of hypertension. Evidence from clinical trials on the effectiveness and adverse effects of using SPCs are provided. An analysis is also made of the main contributions of SPCs in special populations, e.g., elderly and diabetic patients, and its use in high risk and resistant hypertension. The effects of SPCs on hypertensive-mediated organ damage is also examined. Finally, we provide some aspects to consider when choosing treatments in the economic context of Latin-America for promoting the most efficient use of resources in a scarce environment and to provide quality information to decision makers to formulate safe, cost-effective, and patient-centered health policies. Finally, future perspectives and limitations in clinical practice are also discussed.

Brachiocephalic Vein and Superior Vena Cava Reconstruction with a Superficial Femoral Vein Graft

Abstract Superior vena cava syndrome (SVCS) is an entity that has become more frequent due to the increasing use of indwelling central venous catheters. Surgical management is considered in patients with extensive venous thrombosis and when endovascular therapy is not feasible. The use of superficial femoral vein is an excellent technique for reconstruction of the brachiocephalic vein and superior vena cava (SVC) in cases with benign and malignant etiologies. We describe two cases of SVCS that were managed surgically at our institution with replacement of the SVC and brachiocephalic veins with a superficial femoral vein graft technique.

Determination of the boundary lipids of sticholysins using tryptophan quenching.

Sticholysins are α-pore-forming toxins produced by the sea-anemone Stichodactyla helianthus. These toxins exert their activity by forming pores on sphingomyelin-containing membranes. Recognition of sphingomyelin by sticholysins is required to start the process of pore formation. Sphingomyelin recognition is coupled with membrane binding and followed by membrane penetration and oligomerization. Many features of these processes are known. However, the extent of contact with each of the different kinds of lipids present in the membrane has received little attention. To delve into this question, we have used a phosphatidylcholine analogue labeled at one of its acyl chains with a doxyl moiety, a known quencher of tryptophan emission. Here we present evidence for the contact of sticholysins with phosphatidylcholine lipids in the sticholysin oligomer, and for how each sticholysin isotoxin is affected differently by the inclusion of cholesterol in the membrane. Furthermore, using phosphatidylcholine analogs that were labeled at different positions of their structure (acyl chains and headgroup) in combination with a variety of sticholysin mutants, we also investigated the depth of the tryptophan residues of sticholysins in the bilayer. Our results indicate that the position of the tryptophan residues relative to the membrane normal is deeper when cholesterol is absent from the membrane.

Hypertension mediated kidney and cardiovascular damage and risk stratification: Redefining concepts / Daño renal y cardiovascular mediado por hipertensión y estratificación del riesgo: redefinición de conceptos

Hypertension mediated organ damage (HMOD) refers to structural or functional changes in arteries or target organs that can be present in long-standing hypertension, but it can be also found in naïve never treated patients. Traditionally, cardiovascular risk is stratified with charts or calculators that tend to underestimate the real cardiovascular risk. The diagnosis of HMOD automatically reclassifies patients to the highest level of cardiovascular risk. Subclinical HMOD can be present already at the diagnosis of hypertension and more than 25% of hypertensives are misclassified with the routine tests recommended by hypertension guidelines. Whether HMOD regression improves cardiovascular outcomes has never been investigated in randomized clinical trials and remains controversial. However, different drugs have been probed with promising results in high cardiovascular risk patients, such as the new antidiabetic or the novel non-steroid mineralocorticoid antagonists. Accordingly, trials have shown that lowering blood pressure reduces cardiovascular events. In this narrative review, we will discuss the role of HMOD in cardiovascular risk stratification, the different types of organ damage, and the evidence available to define whether HMOD can be used as a therapeutic target. (AU)

El daño orgánico mediado por hipertensión (HMOD) se refiere a cambios estructurales o funcionales de larga duración en las arterias u órganos diana de la hipertensión, pero también se puede encontrar en pacientes que nunca han recibido tratamiento antihipertensivo previo. Tradicionalmente, el riesgo cardiovascular se ha estratificado utilizando tablas, calculadoras o algoritmos que tienden a subestimar el riesgo cardiovascular real. El diagnóstico del HMOD reclasifica automáticamente a los pacientes al nivel más alto de riesgo cardiovascular. El HMOD subclínico ya puede estar presente en el momento del diagnóstico de hipertensión y más del 25% de los hipertensos están mal clasificados con las pruebas de rutina recomendadas por las guías de hipertensión. Sin embargo, si la regresión del HMOD mejora los resultados cardiovasculares no suele ser un objeto de investigación en ensayos clínicos aleatorizados y sigue siendo un aspecto controvertido. A pesar de ello, se han probado diferentes fármacos con resultados prometedores en pacientes de alto riesgo cardiovascular, como los nuevos antidiabéticos o los nuevos antagonistas de mineralocorticoides no esteroides. De hecho, diferentes estudios han demostrado que bajar la presión arterial reduce los eventos cardiovasculares. En esta revisión narrativa, se discutirá el papel del HMOD en la estratificación del riesgo cardiovascular, los diferentes tipos de daño orgánico y la evidencia disponible para definir si HMOD puede usarse como un objetivo terapéutico. (AU)

Humidity gradients in the air spaces of leaves.

Stomata are orifices that connect the drier atmosphere with the interconnected network of more humid air spaces that surround the cells within a leaf. Accurate values of the humidities inside the substomatal cavity, wi, and in the air, wa, are needed to estimate stomatal conductance and the CO2 concentration in the internal air spaces of leaves. Both are vital factors in the understanding of plant physiology and climate, ecological and crop systems. However, there is no easy way to measure wi directly. Out of necessity, wi has been taken as the saturation water vapour concentration at leaf temperature, wsat, and applied to the whole leaf intercellular air spaces. We explored the occurrence of unsaturation by examining gas exchange of leaves exposed to various magnitudes of wsat - wa, or Δw, using a double-sided, clamp-on chamber, and estimated degrees of unsaturation from the gradient of CO2 across the leaf that was required to sustain the rate of CO2 assimilation through the upper surface. The relative humidity in the substomatal cavities dropped to about 97% under mild Δw and as dry as around 80% when Δw was large. Measurements of the diffusion of noble gases across the leaf indicated that there were still regions of near 100% humidity distal from the stomatal pores. We suggest that as Δw increases, the saturation edge retreats into the intercellular air spaces, accompanied by the progressive closure of mesophyll aquaporins to maintain the cytosolic water potential.

Sea Anemones, Actinoporins, and Cholesterol.

Spanish or Spanish-speaking scientists represent a remarkably populated group within the scientific community studying pore-forming proteins. Some of these scientists, ourselves included, focus on the study of actinoporins, a fascinating group of metamorphic pore-forming proteins produced within the venom of several sea anemones. These toxic proteins can spontaneously transit from a water-soluble fold to an integral membrane ensemble because they specifically recognize sphingomyelin in the membrane. Once they bind to the bilayer, they subsequently oligomerize into a pore that triggers cell-death by osmotic shock. In addition to sphingomyelin, some actinoporins are especially sensible to some other membrane components such as cholesterol. Our group from Universidad Complutense of Madrid has focused greatly on the role played by sterols in this water-membrane transition, a question which still remains only partially solved and constitutes the main core of the article below.

PAMP-triggered genetic reprogramming involves widespread alternative transcription initiation and an immediate transcription factor wave.

Immune responses triggered by pathogen-associated molecular patterns (PAMPs) are key to pathogen defense, but drivers and stabilizers of the growth-to-defense genetic reprogramming remain incompletely understood in plants. Here, we report a time-course study of the establishment of PAMP-triggered immunity (PTI) using cap analysis of gene expression. We show that around 15% of all transcription start sites (TSSs) rapidly induced during PTI define alternative transcription initiation events. From these, we identify clear examples of regulatory TSS change via alternative inclusion of target peptides or domains in encoded proteins, or of upstream open reading frames in mRNA leader sequences. We also find that 60% of PAMP response genes respond earlier than previously thought. In particular, a cluster of rapidly and transiently PAMP-induced genes is enriched in transcription factors (TFs) whose functions, previously associated with biological processes as diverse as abiotic stress adaptation and stem cell activity, appear to converge on growth restriction. Furthermore, examples of known potentiators of PTI, in one case under direct mitogen-activated protein kinase control, support the notion that the rapidly induced TFs could constitute direct links to PTI signaling pathways and drive gene expression changes underlying establishment of the immune state.

Sticholysin I-II oligomerization in the absence of membranes.

Sticholysins are pore-forming toxins produced by the sea anemone Stichodactyla helianthus. When they encounter a sphingomyelin-containing membrane, these proteins bind to it and oligomerize, a process that ends in pore formation. Mounting evidence indicates that StnII can favour the activity of StnI. Previous results have shown that these two isotoxins can oligomerize together. Furthermore, StnII appeared to potentiate the activity of StnI through the membrane-binding step of the process. Hence, isotoxin interaction should occur prior to membrane encounter. Here, we have used analytical ultracentrifugation to investigate the oligomerization of Stns in solution, both separately and together. Our results indicate that while StnI seems to be more prone to oligomerize in water solution than StnII, a small percentage of StnII in StnI-StnII mixtures promotes oligomerization.

Cuticular conductance of adaxial and abaxial leaf surfaces and its relation to minimum leaf surface conductance.

Cuticular conductance to water (gcw ) is difficult to quantify for stomatous surfaces due to the complexity of separating cuticular and stomatal transpiration, and additional complications arise for determining adaxial and abaxial gcw . This has led to the neglect of gcw as a separate parameter in most common gas exchange measurements. Here, we describe a simple technique to simultaneously estimate adaxial and abaxial values of gcw , tested in two amphistomatous plant species. What we term the 'Red-Light method' is used to estimate gcw from gas exchange measurements and a known CO2 concentration inside the leaf during photosynthetic induction under red light. We provide an easy-to-use web application to assist with the calculation of gcw . While adaxial and abaxial gcw varies significantly between leaves of the same species we found that the ratio of adaxial/abaxial gcw (γn ) is stable within a plant species. This has implications for use of generic values of gcw when analysing gas exchange data. The Red-Light method can be used to estimate total cuticular conductance (gcw-T ) accurately with the most common setup of gas exchange instruments, i.e. a chamber mixing the adaxial and abaxial gases, allowing for a wide application of this technique.