Neonatal kaempferol exposure attenuates impact of cerebral palsy model on neuromotor development, cell proliferation, microglia activation, and antioxidant enzyme expression in the hippocampus of rats.

OBJECTIVES: This study aims to assess the effect of neonatal treatment with kaempferol on neuromotor development, proliferation of neural precursor cells, the microglia profile, and antioxidant enzyme gene expression in the hippocampus. METHODS: A rat model of cerebral palsy was established using perinatal anoxia and sensorimotor restriction of hindlimbs during infancy. Kaempferol (1 mg/ kg) was intraperitoneally administered during the neonatal period. RESULTS: Neonatal treatment with kaempferol reduces the impact of the cerebral palsy model on reflex ontogeny and on the maturation of physical features. Impairment of locomotor activity development and motor coordination was found to be attenuated by kaempferol treatment during the neonatal period in rats exposed to cerebral palsy. Neonatal treatment of kaempferol in cerebral palsy rats prevents a substantial reduction in the number of neural precursor cells in the dentate gyrus of the hippocampus, an activated microglia profile, and increased proliferation of microglia in the sub-granular zone and in the granular cell layer. Neonatal treatment with kaempferol increases gene expression of superoxide dismutase and catalase in the hippocampus of rats submitted to the cerebral palsy model. DISCUSSION: Kaempferol attenuates the impact of cerebral palsy on neuromotor behavior development, preventing altered hippocampal microglia activation and mitigating impaired cell proliferation in a neurogenic niche in these rats. Neonatal treatment with kaempferol also increases antioxidant defense gene expression in the hippocampus of rats submitted to the cerebral palsy model.

Rapid Collection of Human Rectal Secretions Using OriCol Devices Is Suitable for Measurement of Mucosal Ig without Blood Contamination.

Measurements of IgG and IgA in human rectal secretions are used to evaluate the Abs elicited by HIV vaccines or the bioaccumulation following immunoprophylaxis at the sites of HIV exposure. To improve sampling methods and tolerability of the procedure, we optimized a balloon device (OriCol) for rectal microbiome sampling requiring 10 second inflation and compared this method to a 5 minute collection using sponges. Lubrication of the device did not interfere with IgG, IgA, or hemoglobin ELISA. Lubricated OriCols inflated to 30 cc minimized hemoglobin contamination (<4.68 ng/ml) compared with collections with two sponge types (Weck-Cel: 267.2 ng/ml, p < 0.0001; and Merocel: 59.38 ng/ml, p = 0.003). Median human serum albumin for OriCols was 14.9 µg/ml, whereas Merocels and Weck-Cels were 28.57 µg/ml (p = 0.0005) and 106.2 µg/ml (p = 0.0002), respectively. Consistent with reduced systemic contamination, the median IgG measured in OriCol-collected rectal secretions (986 ng) was lower than secretions from sponges (Weck-Cel: 8588 ng, p < 0.0001; Merocel: 2509 ng, p = 0.0389). The median IgA yield of samples using the OriCol method (75,253 ng) was comparable to that using Merocel (71,672 ng; p = 0.6942) but significantly higher than Weck-Cel sponges (16,173 ng, p = 0.0336). Median recovery volumes for OriCols were 800 µl, whereas Merocels and Weck-Cels were 615 µl (p = 0.0010) and 655 µl (p = 0.0113), respectively. The balloon device was acceptable among 23 participants, as 85.1% experiencing their first collection ranked it as "seven: acceptable - a lot" or "six: acceptable - somewhat" in a seven-point Likert scale. Therefore, lubricated OriCols inflated to 30 cc allowed for a rapid, well-tolerated, blood-free collection of human rectal secretions.

Dysbiosis and Gut Microbiota Modulation in Systemic Sclerosis.

ABSTRACT: Gastrointestinal (GI) involvement is an early manifestation in systemic sclerosis (SSc), affecting more than 90% of patients, and severe GI disease is a marker of poor prognosis and mortality. Recent studies have hypothesized that alterations of the intestinal microbiota, known as dysbiosis, may represent 1 of the possible environmental factors influencing SSc disease status. In addition, specific microorganisms may be associated with SSc pathogenesis, progression, and GI manifestations. Therapeutic approaches aiming to modulate the intestinal microbiota have emerged, as alternatives to treat GI symptoms, and dietary interventions, probiotic administration, and fecal microbiota transplantation are potential therapies for SSc patients. However, given the complexity and variability of pathogenesis and clinical manifestations in SSc, these therapies need to be combined with additional interventions that target other disease components. Here, we summarize studies addressing intestinal dysbiosis in SSc and discuss the potential of microbiota modulators to treat SSc-related GI disorders.

Determinant factors of sarcopenia in individuals with Parkinson's disease.

OBJECTIVE: Evaluate the occurrence of sarcopenia and determinant factors in individuals with Parkinson's disease (PD) in a city in northeastern Brazil. METHODS: Case series with 77 men and women (adults and older adults) with PD. The risk of sarcopenia was determined using the SARC-F and SARC-CalF screening tools. The diagnosis of sarcopenia was based on the new consensus published by the European Working Group on Sarcopenia in Older People (EWGSOP2). Sarcopenic obesity was diagnosed based on the criteria proposed by Stenholm. Disease stage and severity were determined using the Hoehn and Yahr scale and the Unified Parkinson Disease Rating Scale, respectively. RESULTS: The prevalence of sarcopenia was 19.5% and was associated with age, poor performance on activities of daily living and poor nutritional status. No significant association was found between the SARC-F score and the diagnosis of sarcopenia. The main factors that determined the variation in the parameters for the diagnosis of sarcopenia in the present sample were age, disease severity, body weight, and SARCF score. CONCLUSION: Despite the low prevalence in the present study, sarcopenia progresses with the worsening of the nutritional status and functional capacity of individuals with PD. Further studies are needed on the factors involved in the genesis of sarcopenia. The SARC-F questionnaire is related to parameters for the diagnosis and severity of sarcopenia as well as the severity of PD.

Electrochemical and Spectrophotometric Methods for Polyphenol and Ascorbic Acid Determination in Fruit and Vegetable Extracts.

RESEARCH BACKGROUND: Fresh-cut fruits and vegetables are considered sources of antioxidant compounds. However, their shelf life is limited due to nutritional, quality and safety deterioration. Therefore, in recent decades, various methods have been reported for food processing and preservation, as well as for the determination of antioxidant compounds, due to their many benefits when consumed. The aim of the present work is to compare the performance of electrochemical and spectrophotometric methods in the analysis of the content of polyphenolic compounds and ascorbic acid in extracts from fruits (eggplant), edible roots (carrot) and leaves (rocket, lettuce and chard), and evaluate their capability to detect small changes in the antioxidant content in the eggplant extracts previously irradiated with different UV-C light intensities. EXPERIMENTAL APPROACH: Polyphenolic compounds and ascorbic acid were determined by electrochemical and spectrophotometric methods. An enzymatic biosensor and a nanocomposite sensor were used for polyphenolic compounds and ascorbic acid, respectively, in electrochemical measurements, while Folin-Ciocalteu and Kampfenkel methods were used for spectrophotometric measurements. RESULTS AND CONCLUSION: Results obtained through the different methodologies were comparable and consistent with each other. Both methods allowed determining the content of ascorbic acid and polyphenolic compounds in the fruit and vegetable extracts. Moreover, both techniques enable the detection of the analyte concentration changes in samples exposed to different UV-C intensities and storage days. Finally, it was observed that the antioxidant capacity depends on the type of food, treatment and storage period. NOVELTY AND SCIENTIFIC CONTRIBUTION: Both methodologies were suitable for the quantification of analytes; however, the electrochemical sensors provided higher specificity and selectivity, applicable to different fruit and vegetable matrices, obtaining results with higher precision, in shorter time and with a smaller sample volume, minimizing the economic costs because of the lower consumption of reagents.

Transition from open partial nephrectomy directly to robotic surgery: experience of a single surgeon to achieve "TRIFECTA".

INTRODUCTION: Recent data suggest that robotic platform has become the most accessible minimal invasive surgery even for surgeons without previous training in laparoscopy. Laparoscopic partial nephrectomy (LPN) is a well-stablished procedure, however, with high level of complexity and long learning curve that limit its use. OBJECTIVE: To describe safety, efficiency and learning curve of a single surgeon without previous experience in LPN to reach "TRIFECTA" at robot-assisted partial nephrectomy (RAPN). PATIENTS AND METHODS: This is a retrospective study, with prospective data collection of 101 patients submitted to RAPN by a single surgeon. In order to analyze the learning curve, sample was chronologically divided in two phases: first phase: P1: 50 first patients, second phase: P2: 51 subsequent patients. TRIFECTA was defined as: ischemia time lower than 25 minutes, negative surgical margin and absence of severe complications (Clavien >2). RESULTS: Mean age of patients was 54 years (SD=11.85), median tumor size was 32mm (SD=17) and surgery was performed with zero ischemia time in 33.6% of patients (29.8% at P1 and 40.9% at P2). Demographic data of patients were similar between both groups, except tumor size (P1=27.5mm vs. P2=35.3mm; p=0.02) and body mass index (BMI) (P1=26.6kg/m2 vs. P2=29kg/m2; p=0.03). Rate of bleeding, surgical time, presence of positive margin and peri-operatory surgical complications were similar in both phases. TRIFECTA was higher in P2 in relation to P1 (P1: 58% vs. P2: 87.8%; p=0.002) and median time of hot ischemia was significantly lower at P2 (P1: 17.3 vs. P2: 11.7; p=0.02). At multivariate analysis independent factors related to TRIFECTA included: chronological phase (OR 10.74; 95% IC: 1.63-70.53; p=0.013) and tumor size (OR 0.95; 95% IC: 0.91-0.99; p=0.024). CONCLUSION: RAPN seems to be safe and efficient with good functional and oncological results (TRIFECTA) since the beginning. Experience improvement was related to treatment of larger tumors, higher proportion of patients with zero ischemia and higher rate of TRIFECTA.

Rank-based two-sample tests for paired data with missing values.

Two-sample location problem is one of the most encountered problems in statistical practice. The two most commonly studied subtypes of two-sample location problem involve observations from two populations that are either independent or completely paired, but a third subtype can oftentimes occur in practice when some observations are paired and some are not. Partially paired two-sample problems, also known as paired two-sample problems with missing data, often arise in biomedical fields when it is difficult for some invasive procedures to collect data from an individual at both conditions we are interested in comparing. Existing rank-based two-sample comparison procedures for partially paired data, however, do not make efficient use of all available data. In order to improve the power of testing procedures for this problem, we propose several new rank-based test statistics and study their asymptotic distributions and, when necessary, exact variances. Through extensive numerical studies, we show that the best overall power come from the proposed tests based on weighted linear combinations of the test statistics comparing paired data and the test statistics comparing independent data, using weights inversely proportional to their variances. We illustrate the proposed methods with a real data example from HIV research for prevention.

Genetic characterization of Portuguese allochthonous populations of Pinus nigra using ISSRs and SCoTs and extrapolation of their infraspecific taxonomy.

The Pinus nigra distribution in Portugal is restricted to six allochthonous populations with unknown origin and infraspecific taxonomy. This work intends to evaluate their genetic diversity, structure and relationships, and to infer about their infraspecific taxonomy by comparing molecular patterns produced by inter-simple sequence repeat and Start Codon Targeted markers among Portuguese and foreign samples with known taxonomy and provenance. 127 Portuguese P. nigra individuals were clustered per population. The genetic differentiation was higher within rather than among populations. The pooled molecular data indicated high genetic proximity among the Portuguese and foreign samples of subspecies laricio. However, the separate analysis per marker system demonstrated that two varieties of subspecies laricio (corsicana and calabrica) may have been used in the plantations of the Portuguese P. nigra stands performed in the last century. The genetic characterization and extrapolation of the intraspecific taxonomy of these populations provide useful information for forest management, afforestation and germplasm use.

Migratory and lymphoid-resident dendritic cells cooperate to efficiently prime naive CD4 T cells.

To initiate an adaptive immune response, rare antigen-specific naive CD4(+) T cells must interact with equally rare dendritic cells (DCs) bearing cognate peptide-major histocompatibility complex (MHC) complexes. Lymph nodes (LNs) draining the site of antigen entry are populated by lymphoid-resident DCs as well as DCs that have immigrated from tissues, although the requirement for each population in initiating the T cell response remains unclear. Here, we show that antigen processing and presentation by both lymphoid-resident and migratory DCs was required for clonal selection and expansion of CD4(+) T cells after subcutaneous immunization. Early antigen presentation by lymphoid-resident DCs initiated activation and trapping of antigen-specific T cells in the draining LN, without sufficing for clonal expansion. Migratory DCs, however, interacted with the CD4(+) T cells retained in the LN to induce proliferation. Therefore, distinct DC subsets cooperate to alert and trap the appropriate cell and then license its expansion and differentiation.

A Hh-driven gene network controls specification, pattern and size of the Drosophila simple eyes.

During development, extracellular signaling molecules interact with intracellular gene networks to control the specification, pattern and size of organs. One such signaling molecule is Hedgehog (Hh). Hh is known to act as a morphogen, instructing different fates depending on the distance to its source. However, how Hh, when signaling across a cell field, impacts organ-specific transcriptional networks is still poorly understood. Here, we investigate this issue during the development of the Drosophila ocellar complex. The development of this sensory structure, which is composed of three simple eyes (or ocelli) located at the vertices of a triangular patch of cuticle on the dorsal head, depends on Hh signaling and on the definition of three domains: two areas of eya and so expression--the prospective anterior and posterior ocelli--and the intervening interocellar domain. Our results highlight the role of the homeodomain transcription factor engrailed (en) both as a target and as a transcriptional repressor of hh signaling in the prospective interocellar region. Furthermore, we identify a requirement for the Notch pathway in the establishment of en maintenance in a Hh-independent manner. Therefore, hh signals transiently during the specification of the interocellar domain, with en being required here for hh signaling attenuation. Computational analysis further suggests that this network design confers robustness to signaling noise and constrains phenotypic variation. In summary, using genetics and modeling we have expanded the ocellar gene network to explain how the interaction between the Hh gradient and this gene network results in the generation of stable mutually exclusive gene expression domains. In addition, we discuss some general implications our model may have in some Hh-driven gene networks.

Development, validation, and application of a method for selected avermectin determination in rural waters using high performance liquid chromatography and fluorescence detection.

Avermectins (AVM) are macrocyclic lactones used in livestock and agriculture. A quantitative method of high performance liquid chromatography with fluorescence detection for the determination of eprinomectin, abamectin, doramectin and ivermectin in rural water samples was developed and validated. The method was employed to study samples collected in the Pito Aceso River microbasin, located in the Bom Jardim municipality, Rio de Janeiro State, Brazil. Samples were extracted by solid phase extraction using a polymeric stationary phase, the eluted fraction was re-concentrated under a gentle N2 flow and derivatized to allow AVM determination using liquid chromatography with fluorescence detection. The excitation and emission wavelengths of the derivatives were 365 and 470nm, respectively, and a total chromatographic run of 12min was achieved. Very low limits of quantification (22-58ngL(-1)) were found after re-concentration using N2. Recovery values varied from 85.7% to 119.2% with standard deviations between 1.2% and 10.2%. The validated method was applied in the determination of AVM in 15 water samples collected in the Pito Aceso River microbasin, but most of them were free of AVM or showed only trace levels of these compounds, except for a sample that contained doramectin (9.11µgL(-1)). The method is suitable for routine analysis with satisfactory recovery, sensitivity, and selectivity.

Epidemiological Data and Antimicrobial Resistance of Campylobacter spp. in Portugal from 13 Years of Surveillance.

This study extensively analyzed campylobacteriosis surveillance in Portugal from 2009 to 2021, aiming to investigate demographic shifts, seasonal variations, and antimicrobial resistance (AMR) within Campylobacter isolates. Surveillance network and sentinel laboratory-based system data revealed a substantial under-notification of campylobacteriosis cases, suggesting an underestimated disease burden. Notification rates exhibited a paradigm shift, with a notable prevalence among the pediatric population, particularly in children aged 1-4 years, diverging from European reports. Additionally, an emerging trend of Campylobacter infections in younger adults (15-44 years) was observed. The study unveiled a unique seasonal distribution of cases, defying typical summer peaks seen elsewhere. AMR analysis revealed high resistance to ciprofloxacin and tetracycline, in both C. jejuni (93.7% and 79.2%, respectively) and C. coli (96.5% and 93.2%, respectively), stable throughout the studied period (2013-2021). C. coli exhibited significantly higher resistance to erythromycin, gentamicin, ampicillin and ertapenem compared to C. jejuni (p < 0.001). Multilocus Sequence Typing (MLST) data demonstrated the distribution of resistance markers across diverse sequence types, challenging the notion of a clonal origin for multidrug-resistant isolates. In conclusion, the study highlights the need for enhanced surveillance and raises concerns about alarming AMR levels, recommending the implementation of whole-genome sequencing (WGS)-based surveillance for a deeper comprehension of disease patterns and an evolving AMR landscape.

Frailty and spatialization of older adults in the city of Uberlândia with IVCF-20.

OBJECTIVE: To describe the functional clinical profile of elderly people linked to primary health care, using the Functional Clinical Vulnerability Index (IVCF-20) and to spatialize those with the greatest functional decline by primary health care units in the municipality of Uberlândia, in the state of Minas Gerais (MG), in the year 2022. METHODS: A cross-sectional study with secondary data from the Municipal Health Department of Uberlândia-MG. The variables were compared using Student's t-test, Mann Whitney test, Pearson's chi-square, and multinomial logistic regression to obtain the independent effect of each variable. The significance level adopted was 5% (p < 0.05). The georeferenced database in ArcGIS® was used. RESULTS: 47,182 older adults were evaluated with a mean age of 70.3 years (60 to 113 years), 27,138 of whom were women (57.52%), with a clear predominance of low-risk or robust older adults (69.40%). However, 11.09% are high-risk older adults and 19.52% are at risk of frailty. Older men had independently lower odds of moderate and high risk compared to older women (OR = 0.53; p < 0.001). A high prevalence of polypharmacy was observed, 21.40% of the older adult population, particularly in frail older adults, with a prevalence of 63.08%. There was a greater distribution of frail older adults around the central region of the municipality and in health units with a larger coverage area. The IVCF-20 made it possible to screen frailty in primary health care. CONCLUSION: The instrument is capable of stratifying the risk of older adults in health care networks through primary health care, enabling the application of individualized preventive, promotional, palliative, or rehabilitative interventions, according to the clinical functional stratum of the older adult and the compromised functional domains. Risk stratification and spatial distribution of the frailest older adults can be a good strategy for qualifying health professionals with the aim of maximizing the autonomy and independence of the older adults.

Prognostic value of the geriatric nutritional risk index and other hematological markers on long-term survival in the geriatric population.

OBJECTIVES: The prognostic value of hematological markers has not been extensively explored in the geriatric population, particularly in the presence of the frailty phenotype among hospitalized individuals. Therefore, our study aimed to assess the influence of the frailty phenotype in hospitalized geriatric individuals on hematological markers and their impact on short- and long-term outcomes. METHODS: This is a secondary analysis of a prospective cohort study. This study involved hospitalized individuals who were followed during their hospitalization and for nearly 2 years after discharge. At baseline, Fried's frailty phenotype was assessed, as well as hematological markers, including neutrophil-lymphocyte ratio, monocyte-lymphocyte ratio, neutrophil-monocyte ratio, platelet-lymphocyte ratio, systemic inflammation index, prognostic nutritional index, geriatric nutritional risk index (GNRI), and C-reactive protein-albumin ratio. The phase angle derived from bioelectrical impedance analysis was likewise considered a prognostic biomarker. Our main outcomes were hospital length of stay and mortality during follow-up. RESULTS: Frailty occurred in 43.2% of the population. Individuals with the frailty phenotype exhibited worse hematological markers and lower phase angle values. Low GNRI and elevated C-reactive protein-albumin ratio values were independently associated with mortality (hazard ratio = 6.88, 95% confidence interval 2.0-23.6; hazard ratio = 2.2, 95% confidence interval 1.1-4.4). Only higher values of the systemic inflammation index were independently associated with prolonged hospital stays. CONCLUSION: Hematological markers may serve as a feasible tool for prognostic assessment. Individuals with the frailty phenotype and low GNRI represented a worst-case scenario. Geriatr Gerontol Int 2024; 24: 312-318.

Overweight during development dysregulates cellular metabolism and critical genes that control food intake in the prefrontal cortex.

BACKGROUNDS AND AIMS: Childhood obesity is increasing substantially across the world. The World Obesity Federation (WOF) and World Health Organization (WHO) predicted that in 2030 > 1 billion people will be obese, and by 2035 over 4 billion will reach obesity worldwide. According to WHO, the world soon cannot afford the economic cost of obesity, and we need to act to stop obesity acceleration now. Data in the literature supports that the first 1000 days of life are essential in preventing obesity and related adversities. Therefore, using basic research, the present a study that focuses on the immediate effect of overnutrition and serotonin modulation during the lactation period. METHODS: Using a neonatal overfeeding model, male Wistar rats were divided into four groups based on nutrition or serotonin modulation by pharmacological treatment up to 22 days of life. Cellular and mitochondrial function markers, oxidative stress biomarkers and mRNA levels of hedonic and homeostatic genes were evaluated. RESULTS: Our data showed that overfeeding during lactation decrease NAD/NADH ratio, citrate synthase activity, and increase ROS production. Lipid and protein oxidation were increased in overfed animals, with a decrease in antioxidant defenses, we also observe a differential expression of mRNA levels of homeostatic and hedonic genes. On the contrary, serotonin modulation with selective serotonin reuptake inhibitors treatment reduces harmful effects caused by overnutrition. CONCLUSION: Early effects of overnutrition significantly affect the prefrontal cortex at molecular and cellular level, which could mediate obesity-related neurodegenerative dysfunction.

Is phase angle associated with visceral adiposity and cardiometabolic risk in cardiology outpatients?

BACKGROUND/OBJECTIVES: Phase angle (PhA) serves as a prognostic marker in various clinical scenarios, reflecting oxidative stress and cellular damage. Despite its clinical relevance, its connection with adiposity and cardiovascular risk markers remains underexplored. Hence, our study sought to investigate the relationship between PhA and metabolic, adiposity, and cardiovascular risk parameters among outpatients with cardiology diagnosis. SUBJECTS/METHODS: Adults aged between 26 and 59 years, under the care of a cardiology unit, were included. Ultrasound imaging was used to assess visceral adipose tissue (VAT). Single-frequency bioelectrical impedance analysis (BIA) [50 kHz] was employed to calculate PhA, from BIA's resistance and reactance measurements. Muscle strength, body mass index, waist circumference, and waist-to-height ratio were also evaluated. Framingham's risk score was calculated to estimate the cardiovascular risk events. Metabolic blood samples' results were obtained from medical records. RESULTS: One hundred and five participants were included in our study. Low PhA was observed in 29.5% of our sample. Higher PhA values were independently and inversely associated with both higher VAT and cardiovascular risk (adjusted OR: 0.79 [95% CI 0.69;0.91], OR: 0.74 [95% CI 0.60;0.89], respectively). Lower PhA values (≤5.59) were goodly associated with high VAT (AUC: 0.82 p < 0.001). Lower PhA values (≤5.06) were fairly associated with higher cardiovascular risk (AUC: 0.70 p = 0.003). CONCLUSION: Our study provides evidence that PhA is independently and inversely associated with elevated VAT and cardiovascular risk. These findings underscore the potential of PhA as a valuable complementary marker in assessing cardiometabolic health.

Balancing trade-offs: Enhanced carbon assimilation and productivity with reduced nutritional value in a well-watered C4 pasture under a warmer CO2-enriched atmosphere.

The concentration of atmospheric CO2 and temperature are pivotal components of ecosystem productivity, carbon balance, and food security. In this study, we investigated the impacts of a warmer climate (+2 °C above ambient temperature) and an atmosphere enriched with CO2 (600 ppm) on gas exchange, antioxidant enzymatic system, growth, nutritive value, and digestibility of a well-watered, managed pasture of Megathyrsus maximus, a tropical C4 forage grass, under field conditions. Elevated [CO2] (eC) improved photosynthesis and reduced stomatal conductance, resulting in increased water use efficiency and plant C content. Under eC, stem biomass production increased without a corresponding increase in leaf biomass, leading to a smaller leaf/stem ratio. Additionally, eC had negative impacts on forage nutritive value and digestibility. Elevated temperature (eT) increased photosynthetic gains, as well as stem and leaf biomass production. However, it reduced P and K concentration, forage nutritive value, and digestibility. Under the combined conditions of eC and eT (eCeT), eT completely offset the effects of eC on the leaf/stem ratio. However, eT intensified the effects of eC on photosynthesis, leaf C concentration, biomass accumulation, and nutritive value. This resulted in a forage with 12% more acid detergent fiber content and 28% more lignin. Additionally, there was a decrease of 19% in crude protein leading to a 15% decrease in forage digestibility. These changes could potentially affect animal feeding efficiency and feedback climate change, as ruminants may experience an amplification in methane emissions. Our results highlight the critical significance of conducting multifactorial field studies when evaluating plant responses to climate change variables.