IL-7-dependent and -independent lineages of IL-7R-dependent human T cells.

Infants with biallelic IL7R loss-of-function variants have severe combined immune deficiency (SCID) characterized by the absence of autologous T lymphocytes, but normal counts of circulating B and NK cells (T-B+NK+ SCID). We report 6 adults (aged 22 to 59 years) from 4 kindreds and 3 ancestries (Colombian, Israeli Arab, Japanese) carrying homozygous IL7 loss-of-function variants resulting in combined immunodeficiency (CID). Deep immunophenotyping revealed relatively normal counts and/or proportions of myeloid, B, NK, and innate lymphoid cells. By contrast, the patients had profound T cell lymphopenia, with low proportions of innate-like adaptive mucosal-associated invariant T and invariant NK T cells. They also had low blood counts of T cell receptor (TCR) excision circles, recent thymic emigrant T cells and naive CD4+ T cells, and low overall TCR repertoire diversity, collectively indicating impaired thymic output. The proportions of effector memory CD4+ and CD8+ T cells were high, indicating IL-7-independent homeostatic T cell proliferation in the periphery. Intriguingly, the proportions of other T cell subsets, including TCRγδ+ T cells and some TCRαß+ T cell subsets (including Th1, Tfh, and Treg) were little affected. Peripheral CD4+ T cells displayed poor proliferation, but normal cytokine production upon stimulation with mitogens in vitro. Thus, inherited IL-7 deficiency impairs T cell development less severely and in a more subset-specific manner than IL-7R deficiency. These findings suggest that another IL-7R-binding cytokine, possibly thymic stromal lymphopoietin, governs an IL-7-independent pathway of human T cell development.

Trajectory of Cognitive Decline Before and After Stroke in 14 Population Cohorts.

Importance: Poststroke cognitive impairment is common, but the cognitive trajectory following a first stroke, relative to prestroke cognitive function, remains unclear. Objective: To map the trajectory of cognitive function before any stroke and after stroke in global cognition and in 4 cognitive domains, as well as to compare the cognitive trajectory prestroke in stroke survivors with the trajectory of individuals without incident stroke over follow-up. Design, Setting, and Participants: The study used harmonized and pooled data from 14 population-based cohort studies included in the Cohort Studies of Memory in an International Consortium collaboration. These studies were conducted from 1993 to 2019 across 11 countries among community-dwelling older adults without a history of stroke or dementia. For this study, linear mixed-effects models were used to estimate trajectories of cognitive function poststroke relative to a stroke-free cognitive trajectory. The full model adjusted for demographic and vascular risk factors. Data were analyzed from July 2022 to March 2024. Exposure: Incident stroke. Main outcomes and measures: The primary outcome was global cognition, defined as the standardized average of 4 cognitive domains (language, memory, processing speed, and executive function). Cognitive domain scores were formed by selecting the most commonly administered test within each domain and standardizing the scores. Results: The study included 20 860 participants (12 261 [58.8%] female) with a mean (SD) age of 72.9 (8.0) years and follow-up of 7.51 (4.2) years. Incident stroke was associated with a substantial acute decline in global cognition (-0.25 SD; 95% CI, -0.33 to -0.17 SD), the Mini-Mental State Examination, and all cognitive domains (ranging from -0.17 SD to -0.22 SD), as well as accelerated decline in global cognition (-0.038 SD per year; 95% CI, -0.057 to -0.019 SD per year) and all domains except memory (ranging from -0.020 to -0.055 SD per year), relative to a stroke-free cognitive trajectory. There was no significant difference in prestroke slope in stroke survivors compared with the rate of decline in individuals without stroke in all cognitive measures. The mean rate of decline without a previous stroke was -0.049 SD per year (95% CI, -0.051 to -0.047 SD) in global cognition. Conclusions and relevance: In this cohort study using pooled data from 14 cohorts, incident stroke was associated with acute and accelerated long-term cognitive decline in older stroke survivors.

Health care providers' perceptions of burnout and moral distress during the COVID-19 pandemic: A qualitative study from Saskatchewan, Canada.

OBJECTIVES: This study sought to describe feelings and perceptions of burnout and moral distress experienced by health care providers in the Canadian province of Saskatchewan during the COVID-19 pandemic. METHODS: This study was part of a larger mixed methods project, and we here report on the qualitative results relating to burnout and moral distress experienced by medical doctors, registered nurses and respiratory therapists. We used an exploratory, qualitative descriptive design involving one-one-one interviews with 24 health care providers. Interview data were analysed using a reflexive thematic analysis approach. RESULTS: We identified three overarching themes each for health care provider burnout and moral distress. Interviews revealed that providers experienced burnout through (i) increased expectations and (ii) unfavourable work environments, which led most of them to recognise (iii) a need to step back. Regarding moral distress, key themes were: (i) a sense of compromised care, (ii) feelings of bumping heads with authorities and patient families, and (iii) seeing patients make difficult decisions. CONCLUSION: Our study found that medical doctors, registered nurses and respiratory therapists working during the COVID-19 pandemic experienced and continue to experience significant burnout and moral distress. This was often driven by both institution- and system-level factors. There is a need for sustained investment to build and support a motivated health care workforce to prepare for future pandemics and health emergencies.

Cajal's contributions to vestibular research.

The Spanish neurohistologist Santiago Ramón y Cajal (1852-1934) is widely regarded as the father of modern Neuroscience. In addition to identifying the individuality of cells in the nervous system (the neuron theory) or the direction followed by nerve impulses (the principle of dynamic polarization), he described numerous details regarding the organization of the different structures of the nervous system. This task was compiled in his magnum opus, "Textura del Sistema Nervioso del Hombre y los Vertebrados," first published in Spanish between 1899 and 1904, and later revised and updated in French as "Histologie du système nerveux de l'homme et des vertébrés" between 1909 and 1911 for wider distribution among the international scientific community. Some of Cajal's findings are fundamental to our understanding of the anatomy and histology of the vestibular system. He depicted the nerve endings in the sensory epithelia, the structure of the vestibular nerve and Scarpa ganglion, afferent vestibular fibers, vestibular nuclei, lateral vestibulospinal tract, vestibulocerebellar connections, and the fine structure of the cerebellum. However, most of these pioneering descriptions were published years earlier in Spanish journals with limited circulation. Our study aimed to gather Cajal's findings on the vestibular system and identify his original publications. After this endeavor, we claim a place for Cajal among the founders of anatomy and histology of the vestibular system.

Microalgae and phytase dietary supplementation improved growth and gut microbiota in juvenile European seabass (Dicentrarchus labrax).

Fishmeal and fish oil have been the main sources of protein and fatty acid for aquaculture fish. However, their increasing price and low sustainability have led the aquafeed industry to seek sustainable alternative feedstuffs to meet the nutritional requirements of fish and improve their health and performance. Plant proteins have been successfully used to replace fishery derivatives in aquafeeds, but the presence of anti-nutritional substances is a potential drawback of this approach. Thus, it has been reported that phytate breakdown can be caused by feed supplementation with exogenous phytase. The inclusion of microalgae has been proposed to improve gut functionality in fish fed diets with a high vegetable protein content. The aim of this study was to evaluate the effect on the growth and gut microbiota of European seabass (Dicentrarchus labrax) juveniles of a diet containing a blend of microalgae (Arthrospira platensis and Nannochloropsis gaditana) and different concentrations of phytase. An 83-day feeding trial was conducted, comprising four experimental diets with 2.5% microalgae and 500, 1,000, 2,000, or 10,000 phytase units (FTU)/kg feed and a microalgae- and phytase-free control diet. At the end of the trial, a significantly increased body weight was observed in fish fed the diet with the highest phytase concentration (10,000 FTU/kg) versus controls, although the gut bacterial composition did not differ from controls in alpha or beta diversity with either majority (Weighted UniFrac) or minority bacterial strains (Unweighted UniFrac). In comparison to the control group, the groups fed diets with 1,000 or 2,000 FTU/kg diets had a lower alpha diversity (Shannon's diversity index), while those fed diets with 500 FTU/kg or 1,000 FTU/kg showed distinct clusters in beta diversity (involving minority ASVs). According to these findings, the diet containing the 2.5% microalgae blend with 10,000 FTU/kg may be useful to increase the aquafeed quality and sustain the growth performance of juvenile European seabass.

Six and twelve-month respiratory outcomes in a cohort of severe and critical COVID-19 survivors: A prospective monocentric study in Latin America.

Introduction: Severe COVID-19 can result in long-term sequelae known as "chronic COVID," characterized by a wide range of persistent physical and mental symptoms. Chest imaging and pulmonary function test alterations have been observed in recovered patients. Most studies focus on up to a 3-month follow-up after symptom onset or hospital discharge, with few reports on long-term follow-up and limited evidence regarding disease progression in Latin America. Methods: This study aims to describe the clinical characteristics and changes in pulmonary function, imaging, and quality of life in severe and critical COVID-19 patients requiring ICU admission in a high-complexity hospital in Latin America. A prospective cohort of survivors underwent clinical, radiological, pulmonary function, and quality of life assessments 6 and 12 months post-discharge. Results: One hundred twelve patients were included, all of whom attended the 6-month follow-up, and 99 returned for the 12-month follow-up. Most subjects had no previous respiratory symptoms or significant medical history. At the end of the follow-up period, 74% of the patients showed interstitial infiltrates in chest tomography and a higher frequency of fibroatelectatic tracts and parenchymal bands. Pulmonary function tests returned to normal ranges, except for carbon monoxide diffusion, but no altered scores were reported in the questionnaires. Conclusion: Despite residual radiological findings, most parameters studied in severe and critical COVID-19 survivors improved over the 12-month follow-up period. Regardless of the imaging abnormalities, the improvement in variables such as symptomatic relief and normal pulmonary function suggests that these alterations are transient. Carbon monoxide diffusion did not normalize by the end of the follow-up, which is consistent with the abnormalities reported in multiple studies, indicating a potential disease-related pattern.

How does increasing temperature affect the toxicity of bisphenol A on Cryptomonas ovata and its consumer Daphnia magna?

The global rise in plastic production has led to significant plastic deposition in aquatic ecosystems, releasing chemical compounds as plastics degrade. Among these, bisphenol A (BPA) is a major global concern due to its endocrine-disrupting effects and widespread presence in aquatic environments. Furthermore, the toxicity of BPA on aquatic organisms can be modulated by global change stressors such as temperature, which plays an essential role in the metabolism of organisms, including the degradation and accumulation of toxic compounds. In this study, we aimed to understand how temperature can modulate the toxic effect of BPA on a phytoplankton species (Cryptomonas ovata) and how this effect can be transferred to its herbivorous consumer (Daphnia magna). To do this, we first determined the sensitivity of C. ovata over a BPA gradient (0-10 mg L-1). Subsequently, we experimentally determined how the increase in temperature (+5ºC) could modify the toxic effect of BPA on the physiology, metabolism and growth of the phytoplankton. Finally, we investigated how this effect transferred to the growth rate of D. magna through food. Our results show a negative effect of BPA on C. ovata from 5 mg BPA L-1, affecting its photosynthetic yield of photosystem II, net primary production, respiration, and growth. This effect was accelerated when the temperature was higher. Additionally, the growth rate of D. magna also decreased when fed on C. ovata grown in the presence of BPA and high temperature. Our results indicate that high temperature can accelerate the toxic effects of BPA on organisms located at the base of the food web and this effect could be transferred to higher levels through food.

Smoking, all-cause, and cause-specific mortality in individuals with diabetes in Mexico: an analysis of the Mexico city prospective study.

OBJECTIVE: Evidence from low- and middle-income countries regarding the effect of smoking in people with diabetes is lacking. Here, we report the association of smoking with mortality in a large cohort of Mexican adults with diabetes. METHODS: Participants with diabetes mellitus (self-reported diagnosis, use of antidiabetic medications or HbA1c ≥ 6.5%) aged 35-74 years when recruited into the Mexico City Prospective Study were included. Cox regression confounder-adjusted mortality rate ratios (RRs) associated with baseline smoking status were estimated. RESULTS: Among 15,975 women and 8225 men aged 35-74 years with diabetes but no other comorbidities at recruitment, 2498 (16%) women and 2875 (35%) men reported former smoking and 2753 (17%) women, and 3796 (46%) men reported current smoking. During a median of 17 years of follow-up there were 5087 deaths at ages 35-74 years. Compared with never smoking, all-cause mortality RR was 1.08 (95%CI 1.01-1.17) for former smoking, 1.11 (95%CI 1.03-1.20) for current smoking, 1.09 (95%CI 0.99-1.20) for non-daily smoking, 1.06 (95%CI 0.96-1.16) for smoking < 10 cigarettes/day (median during follow-up 4 cigarettes/day), and 1.28 (95% CI 1.14-1.43) for smoking ≥ 10 cigarettes/day (median during follow-up 15 cigarettes/day). Mortality risk among daily smokers was greatest for COPD, lung cancer, cardiovascular diseases, and acute diabetic complications. CONCLUSION: In this cohort of Mexican adults with diabetes, low-intensity daily smoking was associated with increased mortality, despite observing smoking patterns which are different from other populations, and over 5% of total deaths were associated with smoking.

Deformation dynamics of nanopores upon water imbibition.

Capillarity-driven transport in nanoporous solids is widespread in nature and crucial for modern liquid-infused engineering materials. During imbibition, curved menisci driven by high negative Laplace pressures exert an enormous contractile load on the porous matrix. Due to the challenge of simultaneously monitoring imbibition and deformation with high spatial resolution, the resulting coupling of solid elasticity to liquid capillarity has remained largely unexplored. Here, we study water imbibition in mesoporous silica using optical imaging, gravimetry, and high-resolution dilatometry. In contrast to an expected Laplace pressure-induced contraction, we find a square-root-of-time expansion and an additional abrupt length increase when the menisci reach the top surface. The final expansion is absent when we stop the imbibition front inside the porous medium in a dynamic imbibition-evaporation equilibrium, as is typical for transpiration-driven hydraulic transport in plants, especially in trees. These peculiar deformation behaviors are validated by single-nanopore molecular dynamics simulations and described by a continuum model that highlights the importance of expansive surface stresses at the pore walls (Bangham effect) and the buildup or release of contractile Laplace pressures as menisci collectively advance, arrest, or disappear. Our model suggests that these observations apply to any imbibition process in nanopores, regardless of the liquid/solid combination, and that the Laplace contribution upon imbibition is precisely half that of vapor sorption, due to the linear pressure drop associated with viscous flow. Thus, simple deformation measurements can be used to quantify surface stresses and Laplace pressures or transport in a wide variety of natural and artificial porous media.

Story of an infection: Viral dynamics and host responses in the Caenorhabditis elegans-Orsay virus pathosystem.

Orsay virus (OrV) is the only known natural virus affecting Caenorhabditis elegans, with minimal impact on the animal's fitness due to its robust innate immune response. This study aimed to understand the interactions between C. elegans and OrV by tracking the infection's progression during larval development. Four distinct stages of infection were identified on the basis of viral load, with a peak in capsid-encoding RNA2 coinciding with the first signs of viral egression. Transcriptomic analysis revealed temporal changes in gene expression and functions induced by the infection. A specific set of up-regulated genes remained active throughout the infection, and genes correlated and anticorrelated with virus accumulation were identified. Responses to OrV mirrored reactions to other biotic stressors, distinguishing between virus-specific responses and broader immune responses. Moreover, mutants of early response genes and defense-related processes showed altered viral load progression, uncovering additional players in the antiviral defense response.

Independent and joint associations of cardiometabolic multimorbidity and depression on cognitive function: findings from multi-regional cohorts and generalisation from community to clinic.

Background: Cardiometabolic multimorbidity (CMM) and depression are often co-occurring in older adults and associated with neurodegenerative outcomes. The present study aimed to estimate the independent and joint associations of CMM and depression on cognitive function in multi-regional cohorts, and to validate the generalizability of the findings in additional settings, including clinical. Methods: Data harmonization was performed across 14 longitudinal cohort studies within the Cohort Studies of Memory in an International Consortium (COSMIC) group, spanning North America, South America, Europe, Africa, Asia, and Australia. Three external validation studies with distinct settings were employed for generalization. Participants were eligible for inclusion if they had data for CMM and were free of dementia at baseline. Baseline CMM was defined as: 1) CMM 5, ≥2 among hypertension, hyperlipidemia, diabetes, stroke, and heart disease and 2) CMM 3 (aligned with previous studies), ≥2 among diabetes, stroke, and heart disease. Baseline depression was primarily characterized by binary classification of depressive symptom measurements, employing the Geriatric Depression Scale and the Center for Epidemiological Studies-Depression scale. Global cognition was standardized as z-scores through harmonizing multiple cognitive measures. Longitudinal cognition was calculated as changes in global cognitive z-scores. A pooled individual participant data (IPD) analysis was utilized to estimate the independent and joint associations of CMM and depression on cognitive outcomes in COSMIC studies, both cross-sectionally and longitudinally. Repeated analyses were performed in three external validation studies. Findings: Of the 32,931 older adults in the 14 COSMIC cohorts, we included 30,382 participants with complete data on baseline CMM, depression, and cognitive assessments for cross-sectional analyses. Among them, 22,599 who had at least 1 follow-up cognitive assessment were included in the longitudinal analyses. The three external studies for validation had 1964 participants from 3 multi-ethnic Asian older adult cohorts in different settings (community-based, memory clinic, and post-stroke study). In COSMIC studies, each of CMM and depression was independently associated with cross-sectional and longitudinal cognitive function, without significant interactions between them (Ps > 0.05). Participants with both CMM and depression had lower cross-sectional cognitive performance (e.g. ß = -0.207, 95% CI = (-0.255, -0.159) for CMM5 (+)/depression (+)) and a faster rate of cognitive decline (e.g. ß = -0.040, 95% CI = (-0.047, -0.034) for CMM5 (+)/depression (+)), compared with those without either condition. These associations remained consistent after additional adjustment for APOE genotype and were robust in two-step random-effects IPD analyses. The findings regarding the joint association of CMM and depression on cognitive function were reproduced in the three external validation studies. Interpretation: Our findings highlighted the importance of investigating age-related co-morbidities in a multi-dimensional perspective. Targeting both cardiometabolic and psychological conditions to prevent cognitive decline could enhance effectiveness. Funding: Natural Science Foundation of China and National Institute on Aging/National Institutes of Health.

L-Cysteine-Catalysed Hydration of Activated Alkynes.

Hydration reactions consist of the introduction of a molecule of water into a chemical compound and are particularly useful to transform alkynes into carbonyls, which are strategic intermediates in the synthesis of a plethora of compounds. Herein we demonstrate that L-cysteine can catalyse the hydration of activated alkynes in a very effective and fully regioselective manner to access important building blocks in synthetic chemistry such as ß-ketosulfones, amides and esters, in aqueous media. The mild reaction conditions facilitated the integration with enzyme catalysis to access chiral ß-hydroxy sulfones from the corresponding alkynes in a one-pot cascade process in good yields and excellent enantiomeric ratios. These findings pave the way towards establishing a general method for metal-free, cost-effective, and more sustainable alkyne hydration processes.

High-Resolution Cryo-EM Structure Determination of α-Synuclein - A Prototypical Amyloid Fibril.

The physiological role of α-synuclein (α-syn), an intrinsically disordered presynaptic neuronal protein, is believed to impact the release of neurotransmitters through interactions with the SNARE complex. However, under certain cellular conditions that are not well understood, α-syn will self-assemble into ß-sheet rich fibrils that accumulate and form insoluble neuronal inclusions. Studies of patient derived brain tissues have concluded that these inclusions are associated with Parkinson's disease, the second most common neurodegenerative disorder, and other synuclein related diseases called synucleinopathies. In addition, repetitions of and specific mutations to the SNCA gene, the gene that encodes α-syn, results in an increased disposition for synucleinopathies. The latest advances in cryo-EM structure determination and real-space helical reconstruction methods have resulted in over 60 in vitro structures of α-syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 Å. Here, we provide a protocol for α-syn protein expression, purification, and fibrilization. We detail how sample quality is assessed by negative stain transmission electron microscopy (NS-TEM) analysis and followed by sample vitrification using the Vitrobot Mark IV vitrification robot. We provide a detailed step by step protocol for high resolution cryo-EM structure determination of α-syn fibrils using RELION and a series of specialized helical reconstruction tools that can be run within RELION. Finally, we detail how ChimeraX, Coot, and Phenix are used to build and refine a molecular model into the high resolution cryo-EM map. This workflow resulted in a 2.04 Å structure of α-syn fibrils with excellent resolution of residues 36 to 97 and an additional island of density for residues 15 to 22 that had not been previously reported. This workflow should serve as a starting point for individuals new to the neurodegeneration and structural biology fields. Together, this procedure lays the foundation for advanced structural studies of α-synuclein and other amyloid fibrils. Key Features: In vitro fibril amplification method yielding twisting fibrils that span several micrometers in length and are suitable for cryo-EM structure determination. High-throughput cryo-EM data collection of neurodegenerative fibrils, such as alpha-synuclein.Use of RELION implementations of helical reconstruction algorithms to generate high-resolution 3D structures of a-synuclein fibrils.Brief demonstration of the use of ChimeraX, Coot, and Phenix for molecular model building and refinement.s. Graphical overview of α-synuclein fibrilization and cryo-EM structure determination: α-synuclein protein expression and purification is followed by a fibrilization protocol yielding twisting filaments that span several micrometers in length and are validated by negative stain transmission electron microscopy (NS-TEM). The sample is then vitrified, followed by cryo-EM data collection. Real-space helical reconstruction is performed in RELION to generate an electron potential map that is used for model building.

Nutrient competition predicts gut microbiome restructuring under drug perturbations.

Human gut commensal bacteria are routinely exposed to various stresses, including therapeutic drugs, and collateral effects are difficult to predict. To systematically interrogate community-level effects of drug perturbations, we screened stool-derived in vitro communities with 707 clinically relevant small molecules. Across ∼5,000 community-drug interaction conditions, compositional and metabolomic responses were predictably impacted by nutrient competition, with certain species exhibiting improved growth due to adverse impacts on competitors. Changes to community composition were generally reversed by reseeding with the original community, although occasionally species promotion was long-lasting, due to higher-order interactions, even when the competitor was reseeded. Despite strong selection pressures, emergence of resistance within communities was infrequent. Finally, while qualitative species responses to drug perturbations were conserved across community contexts, nutrient competition quantitatively affected their abundances, consistent with predictions of consumer-resource models. Our study reveals that quantitative understanding of the interaction landscape, particularly nutrient competition, can be used to anticipate and potentially mitigate side effects of drug treatment on the gut microbiota.

Chiral edge waves in a dance-based human topological insulator.

Topological insulators are insulators in the bulk but feature chiral energy propagation along the boundary. This property is topological in nature and therefore robust to disorder. Originally discovered in electronic materials, topologically protected boundary transport has since been observed in many other physical systems. Thus, it is natural to ask whether this phenomenon finds relevance in a broader context. We choreograph a dance in which a group of humans, arranged on a square grid, behave as a topological insulator. The dance features unidirectional flow of movement through dancers on the lattice edge. This effect persists when people are removed from the dance floor. Our work extends the applicability of wave physics to dance.

Update of the Spanish clinical practice guideline for urinary tract infection in infants and children. Summary of recommendations for diagnosis, treatment and follow-up.

The management of urinary tract infection (UTI) in infants and children has changed significantly over the past few decades based on scientific evidence that questioned the efficacy of strategies used to prevent kidney injury and subsequent progression to chronic kidney disease, which is very unlikely in most paediatric cases. However, there is still substantial heterogeneity in its management and uncertainty regarding the diagnosis, indication of imaging tests, treatment or follow-up in these patients. The Spanish clinical practice guideline has been updated through the review of the literature published since 2009 and a rigorous evaluation of current clinical practice aspects, taking into account the evidence on the benefits of each intervention in addition to its risks and drawbacks to attempt to provide more precise recommendations.

Near-Infrared Spectroscopic Determination of Pentacyclic Triterpenoid Concentrations in Additives for Animal Food.

The nutritional composition of food for animal production can be enhanced using olive tree and plant by-products due to their high content of bioactive compounds such as pentacyclic triterpenes. Here, we present a novel application of near-infrared spectroscopy (NIRS) for the prediction of the total or individual [maslinic acid (MA), oleanolic acid (OA), and uvaol (UO)] pentacyclic triterpene concentrations in a feed additive obtained from a plant mixture. The oxygen radical absorbance capacity of these types of samples demonstrated the existence of a high antioxidant capacity. The conventional determination methods of pentacyclic triterpene concentration are costly, labor-intensive, and not practical for analyzing several lines within a limited timeframe at the factory level. The optimal regression model developed in our work demonstrated high correlation values for the calibration and validation sets, along with a high residual prediction deviation value. We used 63 samples for the development of the model. The NIRS method can be applied directly to dried powder and makes extraction and high-performance liquid chromatography (HPLC) analysis unnecessary. Our results also demonstrate that NIRS can accurately quantify pentacyclic triterpenes even at low concentrations in food additives. It can be used at the factory level to directly determine the pentacyclic triterpene concentrations in the additive powder at the same time that the powder is produced.

Constant and fluctuating high temperatures interact with Saharan dust leading to contrasting effects on aquatic microbes over time.

Mediterranean lakes are facing heightened exposure to multiple stressors, such as intensified Saharan dust deposition, temperature increases and fluctuations linked to heatwaves. However, the combined impact of dust and water temperature on the microbial community in freshwater ecosystems remains underexplored. To assess the interactive effect of dust deposition and temperature on aquatic microbes (heterotrophic bacteria and phytoplankton), a combination of field mesocosm experiments covering a dust gradient (five levels, 0-320 mg L-1), and paired laboratory microcosms with increased temperature at two levels (constant and fluctuating high temperature) were conducted in a high mountain lake in the Spanish Sierra Nevada, at three points in time throughout the ice-free period. Heterotrophic bacterial production (HBP) increased with dust load regardless of the temperature regime. However, temperature regime affected the magnitude and nature of the interactive Dust×T effect on HBP. Specifically, constant and fluctuating high temperature showed opposing interactive effects in the short term that became additive over time. The relationships between HBP and predictor variables (soluble reactive phosphorus (SRP), excreted organic carbon (EOC), and heterotrophic bacterial abundance (HBA)), coupled with an evaluation of the mechanistic variable photosynthetic carbon use efficiency by bacteria (%CUEb), revealed that bacteria depended on primary production in nearly all treatments when dust was added. The %CUEb increased with dust load in the control temperature treatment, but it was highest at intermediate dust loads under both constant and fluctuating high temperatures. Overall, our results suggest that while dust addition alone strengthens algae-bacteria coupling, high temperatures lead to decoupling in the long term at intermediate dust loads, potentially impacting ecosystem function.

Purification and Cryo-Electron Microscopy Analysis of Bacterial Appendages.

A number of extracellular helical protein polymers are crucial for supporting bacterial motility. The bacterial flagellum is a polymeric appendage used to support cellular motility. Historically, structural studies of flagellar and other filaments were limited to those present as or locked into straightened states. Here, we present a robust workflow that produces biologically relevant high-resolution cryo-electron microscopy (cryo-EM) structures of bacterial flagellar filaments. We highlight how a simple purification method, centered around several centrifugation steps, exploits the process of filament ejection in Caulobacter crescentus and results in isolated filaments amenable to transmission electron microscopy (TEM) studies. The quality of the sample is validated by SDS-PAGE and negative stain TEM analysis before a sample is vitrified for cryogenic electron microscopy (cryo-EM) data collection. We provide a detailed protocol for reconstructing either straight or curved flagellar filaments by cryo-EM helical reconstruction methods, followed by an overview of model building and validation. In our hands, this workflow resulted in several flagellar structures below 3 Å resolution, with one data set reaching a global resolution of 2.1 Å. The application of this workflow supports structure-function studies to better understand the molecular interactions that regulate filament architecture in biologically relevant states. Future work will not only examine interactions that regulate bacterial flagellar and other filament organization but also provide a foundation for developing new helical biopolymers for biotech applications. Key features • Rapid high-quality purification of bacterial flagella via simple bacterial culturing, centrifugation, and resuspension methods. • High-throughput cryo-EM data collection of filamentous objects. • Use of cryoSPARC implementations of helical reconstruction algorithms to generate high-resolution 3D structures of bacterial flagella or other helical polymers.

NKG2C/KLRC2 tumor cell expression enhances immunotherapeutic efficacy against glioblastoma.

BACKGROUND: Activating and inhibitory receptors of natural killer (NK) cells such as NKp, NKG2, or CLEC are highly relevant to cold tumors including glioblastoma (GBM). Here, we aimed to characterize the expression of these receptors in GBM to gain insight into their potential role as modulators of the intratumoral microenvironment. METHODS: We performed a transcriptomic analysis of several NK receptors with a focus on the activating receptor encoded by KLRC2, NKG2C, among bulk and single-cell RNA sequencing GBM data sets. We also evaluated the effects of KLRC2-overexpressing GL261 cells in mice treated with or without programmed cell death protein-1 (PD-1) monoclonal antibody (mAb). Finally, we analyzed samples from two clinical trials evaluating PD-1 mAb effects in patients with GBM to determine the potential of NKG2C to serve as a biomarker of response. RESULTS: We observed significant expression of several inhibitory NK receptors on GBM-infiltrating NK and T cells, which contrasts with the strong expression of KLRC2 on tumor cells, mainly at the infiltrative margin. Neoplastic KLRC2 expression was associated with a reduction in the number of myeloid-derived suppressor cells and with a higher level of tumor-resident lymphocytes. A stronger antitumor activity after PD-1 mAb treatment was observed in NKG2Chigh-expressing tumors both in mouse models and patients with GBM whereas the expression of inhibitory NK receptors showed an inverse association. CONCLUSIONS: This study explored the role of neoplastic NKG2C/KLRC2 expression in shaping the immune profile of GBM and suggests that it is a predictive biomarker for positive responses to immune checkpoint inhibitor treatment in patients with GBM. Future studies could further validate this finding in prospective trials.