Structure-based design of non-hypertrophic apelin receptor modulator.

Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the ß-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.

Snapshot of the cannabinoid receptor 1-arrestin complex unravels the biased signaling mechanism.

Cannabis activates the cannabinoid receptor 1 (CB1), which elicits analgesic and emotion regulation benefits, along with adverse effects, via Gi and ß-arrestin signaling pathways. However, the lack of understanding of the mechanism of ß-arrestin-1 (ßarr1) coupling and signaling bias has hindered drug development targeting CB1. Here, we present the high-resolution cryo-electron microscopy structure of CB1-ßarr1 complex bound to the synthetic cannabinoid MDMB-Fubinaca (FUB), revealing notable differences in the transducer pocket and ligand-binding site compared with the Gi protein complex. ßarr1 occupies a wider transducer pocket promoting substantial outward movement of the TM6 and distinctive twin toggle switch rearrangements, whereas FUB adopts a different pose, inserting more deeply than the Gi-coupled state, suggesting the allosteric correlation between the orthosteric binding pocket and the partner protein site. Taken together, our findings unravel the molecular mechanism of signaling bias toward CB1, facilitating the development of CB1 agonists.

Structural insights into the human D1 and D2 dopamine receptor signaling complexes.

The D1- and D2-dopamine receptors (D1R and D2R), which signal through Gs and Gi, respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson's disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-Gs and D2R-Gi signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson's disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system.

Conformational transitions and activation of the adhesion receptor CD97.

Adhesion G protein-coupled receptors (aGPCRs) are evolutionarily ancient receptors involved in a variety of physiological and pathophysiological processes. Modulators of aGPCR, particularly antagonists, hold therapeutic promise for diseases like cancer and immune and neurological disorders. Hindered by the inactive state structural information, our understanding of antagonist development and aGPCR activation faces challenges. Here, we report the cryo-electron microscopy structures of human CD97, a prototypical aGPCR that plays crucial roles in immune system, in its inactive apo and G13-bound fully active states. Compared with other family GPCRs, CD97 adopts a compact inactive conformation with a constrained ligand pocket. Activation induces significant conformational changes for both extracellular and intracellular sides, creating larger cavities for Stachel sequence binding and G13 engagement. Integrated with functional and metadynamics analyses, our study provides significant mechanistic insights into the activation and signaling of aGPCRs, paving the way for future drug discovery efforts.

GPCRs steer Gi and Gs selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

Serotonin (or 5-hydroxytryptamine, 5-HT) is an important neurotransmitter that activates 12 different G protein-coupled receptors (GPCRs) through selective coupling of Gs, Gi, or Gq proteins. The structural basis for G protein subtype selectivity by these GPCRs remains elusive. Here, we report the structures of the serotonin receptors 5-HT4, 5-HT6, and 5-HT7 with Gs, and 5-HT4 with Gi1. The structures reveal that transmembrane helices TM5 and TM6 alternate lengths as a macro-switch to determine receptor's selectivity for Gs and Gi, respectively. We find that the macro-switch by the TM5-TM6 length is shared by class A GPCR-G protein structures. Furthermore, we discover specific residues within TM5 and TM6 that function as micro-switches to form specific interactions with Gs or Gi. Together, these results present a common mechanism of Gs versus Gi protein coupling selectivity or promiscuity by class A GPCRs and extend the basis of ligand recognition at serotonin receptors.

Structures of the human dopamine D3 receptor-Gi complexes.

The dopamine system, including five dopamine receptors (D1R-D5R), plays essential roles in the central nervous system (CNS), and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders. Here, we report two cryo-EM structures of human D3R in complex with an inhibitory G protein and bound to the D3R-selective agonists PD128907 and pramipexole, the latter of which is used to treat patients with Parkinson's disease. The structures reveal agonist binding modes distinct from the antagonist-bound D3R structure and conformational signatures for ligand-induced receptor activation. Mutagenesis and homology modeling illuminate determinants of ligand specificity across dopamine receptors and the mechanisms for Gi protein coupling. Collectively our work reveals the basis of agonist binding and ligand-induced receptor activation and provides structural templates for designing specific ligands to treat CNS diseases targeting the dopaminergic system.

Molecular Basis for Hormone Recognition and Activation of Corticotropin-Releasing Factor Receptors.

Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.

Structural basis of GABAB receptor-Gi protein coupling.

G-protein-coupled receptors (GPCRs) have central roles in intercellular communication1,2. Structural studies have revealed how GPCRs can activate G proteins. However, whether this mechanism is conserved among all classes of GPCR remains unknown. Here we report the structure of the class-C heterodimeric GABAB receptor, which is activated by the inhibitory transmitter GABA, in its active form complexed with Gi1 protein. We found that a single G protein interacts with the GB2 subunit of the GABAB receptor at a site that mainly involves intracellular loop 2 on the side of the transmembrane domain. This is in contrast to the G protein binding in a central cavity, as has been observed with other classes of GPCR. This binding mode results from the active form of the transmembrane domain of this GABAB receptor being different from that of other GPCRs, as it shows no outside movement of transmembrane helix 6. Our work also provides details of the inter- and intra-subunit changes that link agonist binding to G-protein activation in this heterodimeric complex.

Structures of the glucocorticoid-bound adhesion receptor GPR97-Go complex.

Adhesion G-protein-coupled receptors (GPCRs) are a major family of GPCRs, but limited knowledge of their ligand regulation or structure is available1-3. Here we report that glucocorticoid stress hormones activate adhesion G-protein-coupled receptor G3 (ADGRG3; also known as GPR97)4-6, a prototypical adhesion GPCR. The cryo-electron microscopy structures of GPR97-Go complexes bound to the anti-inflammatory drug beclomethasone or the steroid hormone cortisol revealed that glucocorticoids bind to a pocket within the transmembrane domain. The steroidal core of glucocorticoids is packed against the 'toggle switch' residue W6.53, which senses the binding of a ligand and induces activation of the receptor. Active GPR97 uses a quaternary core and HLY motif to fasten the seven-transmembrane bundle and to mediate G protein coupling. The cytoplasmic side of GPR97 has an open cavity, where all three intracellular loops interact with the Go protein, contributing to the high basal activity of GRP97. Palmitoylation at the cytosolic tail of the Go protein was found to be essential for efficient engagement with GPR97 but is not observed in other solved GPCR complex structures. Our work provides a structural basis for ligand binding to the seven-transmembrane domain of an adhesion GPCR and subsequent G protein coupling.

Structural insights into the lipid and ligand regulation of serotonin receptors.

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest subtype family of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1E and other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor-G-protein complexes: 5-HT1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1D bound to 5-HT; and 5-HT1E in complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein-5-HT1A interface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules-particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.

Structural basis of GPBAR activation and bile acid recognition.

The G-protein-coupled bile acid receptor (GPBAR) conveys the cross-membrane signalling of a vast variety of bile acids and is a signalling hub in the liver-bile acid-microbiota-metabolism axis1-3. Here we report the cryo-electron microscopy structures of GPBAR-Gs complexes stabilized by either the high-affinity P3954 or the semisynthesized bile acid derivative INT-7771,3 at 3 Å resolution. These structures revealed a large oval pocket that contains several polar groups positioned to accommodate the amphipathic cholic core of bile acids, a fingerprint of key residues to recognize diverse bile acids in the orthosteric site, a putative second bile acid-binding site with allosteric properties and structural features that contribute to bias properties. Moreover, GPBAR undertakes an atypical mode of activation and G protein coupling that features a different set of key residues connecting the ligand-binding pocket to the Gs-coupling site, and a specific interaction motif that is localized in intracellular loop 3. Overall, our study not only reveals unique structural features of GPBAR that are involved in bile acid recognition and allosteric effects, but also suggests the presence of distinct connecting mechanisms between the ligand-binding pocket and the G-protein-binding site in the G-protein-coupled receptor superfamily.

Structural basis and molecular mechanism of biased GPBAR signaling in regulating NSCLC cell growth via YAP activity.

The G protein-coupled bile acid receptor (GPBAR) is the membrane receptor for bile acids and a driving force of the liver-bile acid-microbiota-organ axis to regulate metabolism and other pathophysiological processes. Although GPBAR is an important therapeutic target for a spectrum of metabolic and neurodegenerative diseases, its activation has also been found to be linked to carcinogenesis, leading to potential side effects. Here, via functional screening, we found that two specific GPBAR agonists, R399 and INT-777, demonstrated strikingly different regulatory effects on the growth and apoptosis of non-small cell lung cancer (NSCLC) cells both in vitro and in vivo. Further mechanistic investigation showed that R399-induced GPBAR activation displayed an obvious bias for ß-arrestin 1 signaling, thus promoting YAP signaling activation to stimulate cell proliferation. Conversely, INT-777 preferentially activated GPBAR-Gs signaling, thus inactivating YAP to inhibit cell proliferation and induce apoptosis. Phosphorylation of GPBAR by GRK2 at S310/S321/S323/S324 sites contributed to R399-induced GPBAR-ß-arrestin 1 association. The cryoelectron microscopy (cryo-EM) structure of the R399-bound GPBAR-Gs complex enabled us to identify key interaction residues and pivotal conformational changes in GPBAR responsible for the arrestin signaling bias and cancer cell proliferation. In summary, we demonstrate that different agonists can regulate distinct functions of cell growth and apoptosis through biased GPBAR signaling and control of YAP activity in a NSCLC cell model. The delineated mechanism and structural basis may facilitate the rational design of GPBAR-targeting drugs with both metabolic and anticancer benefits.

The gut microbiome modulates the susceptibility to traumatic stress in a sex-dependent manner.

Post-traumatic stress disorder (PTSD), a psychological condition triggered by exposure to extreme or chronic stressful events, exhibits a sex bias in incidence and clinical manifestations. Emerging research implicates the gut microbiome in the pathogenesis of PTSD and its roles in stress susceptibility. However, it is unclear whether differential gut microbiota contribute to PTSD susceptibility in male and female rats. Here, we utilized the single prolonged stress animal model and employed unsupervised machine learning to classify stressed animals into stress-susceptible subgroups and stress-resilient subgroups. Subsequently, using 16S V3-V4 rDNA sequencing, we investigated the differential gut microbiota alterations between susceptible and resilient individuals in male and female rats. Our findings revealed distinct changes in gut microbiota composition between the sexes at different taxonomic levels. Furthermore, the abundance of Parabacteroides was lower in rats that underwent SPS modeling compared to the control group. In addition, the abundance of Tenericutes in the stress-susceptible subgroup was higher than that in the control group and stress-resilient subgroup, suggesting that Tenericutes may be able to characterize stress susceptibility. What is particularly interesting here is that Cyanobacteria may be particularly associated with anti-anxiety effects in male rats. This study underscores sex-specific variations in gut microbiota composition in response to stress and sex differences should be taken into account when using macrobiotics for neuropsychiatric treatment, highlighting potential targets for PTSD therapeutic interventions.

Gut microbiota intervention alleviates pulmonary inflammation in broilers exposed to fine particulate matter from broiler house.

The gut microbiota of poultry is influenced by a variety of factors, including feed, drinking water, airborne dust, and footpads, among others. Gut microbiota can affect the immune reaction and inflammation in the lungs. To investigate the effect of gut microbiota variation on lung inflammation induced by PM2.5 (fine particulate matter) in broilers, 36 Arbor Acres (AA) broilers were randomly assigned to three groups: control group (CON), PM2.5 exposure group (PM), and PM2.5 exposure plus oral antibiotics group (PMA). We used non-absorbable antibiotics (ABX: neomycin and amikacin) to modify the microbiota composition in the PMA group. The intervention was conducted from the 18th to the 28th day of age. Broilers in the PM and PMA groups were exposed to PM by a systemic exposure method from 21 to 28 days old, and the concentration of PM2.5 was controlled at 2 mg/m3. At 28 days old, the lung injury score, relative mRNA expression of inflammatory factors, T-cell differentiation, and dendritic cell function were significantly increased in the PM group compared to the CON group, and those of the PMA group were significantly decreased compared to the PM group. There were significant differences in both α and ß diversity of cecal microbiota among these three groups. Numerous bacterial genera showed significant differences in relative abundance among the three groups. In conclusion, gut microbiota could affect PM2.5-induced lung inflammation in broilers by adjusting the capacity of antigen-presenting cells to activate T-cell differentiation. IMPORTANCE: Gut microbes can influence the development of lung inflammation, and fine particulate matter collected from broiler houses can lead to lung inflammation in broilers. In this study, we explored the effect of gut microbes modified by intestinal non-absorbable antibiotics on particulate matter-induced lung inflammation. The results showed that modification in the composition of gut microbiota could alleviate lung inflammation by attenuating the ability of dendritic cells to stimulate T-cell differentiation, which provides a new way to protect lung health in poultry farms.

Identification and mechanism of G protein-biased ligands for chemokine receptor CCR1.

Biased signaling of G protein-coupled receptors describes an ability of different ligands that preferentially activate an alternative downstream signaling pathway. In this work, we identified and characterized different N-terminal truncations of endogenous chemokine CCL15 as balanced or biased agonists targeting CCR1, and presented three cryogenic-electron microscopy structures of the CCR1-Gi complex in the ligand-free form or bound to different CCL15 truncations with a resolution of 2.6-2.9 Å, illustrating the structural basis of natural biased signaling that initiates an inflammation response. Complemented with pharmacological and computational studies, these structures revealed it was the conformational change of Tyr291 (Y2917.43) in CCR1 that triggered its polar network rearrangement in the orthosteric binding pocket and allosterically regulated the activation of ß-arrestin signaling. Our structure of CCL15-bound CCR1 also exhibited a critical site for ligand binding distinct from many other chemokine-receptor complexes, providing new insights into the mode of chemokine recognition.

Contributing Factors Affecting the Length of Hospital Stay among Febrile Patients with Omicron Reported in Suzhou.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has had global attention with regard to the urgent challenging threat to global public health. Currently, the novel Omicron variant is showing rapid transmission across the world, which appears to be more contagious than the previous variants of COVID-19. Early recognition of disease is critical for patients' prognosis. Fever is the most common symptom. We evaluated the clinical characteristics of febrile patients with COVID-19 reported in Suzhou and explored the predictors for a longer duration of hospitalization in febrile patients. METHODS: This retrospective study was carried out in 146 Omicron variant infected patients confirmed by nucleic acid tests in the Affiliated Infectious Hospital of Soochow University between February 13, 2022 and March 2, 2022. Data of febrile and afebrile laboratory-confirmed patients on hospital admission in Suzhou were collected and compared. According to the median length of stay (LOS), febrile cases were divided into short and long LOS groups. Then the predictive factors for a prolonged duration of hospitalization were analyzed using logistic regression methods. Receiver Operating Characteristic (ROC) Curve analysis was used to analyze the effectiveness of the risk factors for prolonged duration of hospitalization in febrile COVID-19 patients. RESULTS: Of the 146 discharged patients in our study, 112 patients (76.7%) caught a fever. Compared to afebrile Omicron patients, febrile patients showed a significantly longer duration of hospitalization (15.00 (5.80) vs. 13.00 (6.00), p = 0.002). Taking the median LOS (15 days) as the dividing point, 64 febrile cases were assigned to the short LOS group and the rest to the long LOS group. The long LOS group had a longer virus shedding duration than the short LOS group (18.42 ± 2.86 vs. 11.94 ± 2.50 days, p < 0.001). Compared to short LOS febrile patients, long LOS patients were older (44.88 ± 21.36 vs. 30.89 ± 17.95 years, p < 0.001) and showed a higher proportion of greater than 60 years old (33.3% vs. 9.4%, p = 0.002; Supplemental Table S2). Febrile patients with long LOS also showed a higher proportion of hypertension (25% vs. 6.3%, p = 0.005) and higher levels of cTnI (5.00 (3.00) vs. 4.00 (2.00) µg/L, p = 0.025). The multivariate analysis indicated that virus shedding duration (OR 2.369, 95% CI 1.684 - 3.333, p < 0.001) was the independent risk factor associated with long-term hospital stay in febrile patients with Omicron. Furthermore, ROC Curve analysis revealed that the area under the curve (AUC) for virus shedding duration to diagnose prolonged duration of hospitalization in febrile COVID-19 patients was 0.951 (95% CI 0.913 - 0.989). The cutoff point was set at 14.5 days. CONCLUSIONS: More than half of the non-severe patients exposed to the new Omicron variant had symptoms of fever. In total, 42.86% of the febrile patients were discharged within 15 days since hospital admission. Febrile Omicron cases took a longer duration of hospitalization compared to afebrile patients, and virus shedding duration (OR 2.369, 95% CI 1.684 - 3.333, p < 0.001) was probably a predictive factor for long-term hospital stays.

Automated machine learning for the identification of asymptomatic COVID-19 carriers based on chest CT images.

BACKGROUND: Asymptomatic COVID-19 carriers with normal chest computed tomography (CT) scans have perpetuated the ongoing pandemic of this disease. This retrospective study aimed to use automated machine learning (AutoML) to develop a prediction model based on CT characteristics for the identification of asymptomatic carriers. METHODS: Asymptomatic carriers were from Yangzhou Third People's Hospital from August 1st, 2020, to March 31st, 2021, and the control group included a healthy population from a nonepizootic area with two negative RT‒PCR results within 48 h. All CT images were preprocessed using MATLAB. Model development and validation were conducted in R with the H2O package. The models were built based on six algorithms, e.g., random forest and deep neural network (DNN), and a training set (n = 691). The models were improved by automatically adjusting hyperparameters for an internal validation set (n = 306). The performance of the obtained models was evaluated based on a dataset from Suzhou (n = 178) using the area under the curve (AUC), accuracy, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and F1 score. RESULTS: A total of 1,175 images were preprocessed with high stability. Six models were developed, and the performance of the DNN model ranked first, with an AUC value of 0.898 for the test set. The sensitivity, specificity, PPV, NPV, F1 score and accuracy of the DNN model were 0.820, 0.854, 0.849, 0.826, 0.834 and 0.837, respectively. A plot of a local interpretable model-agnostic explanation demonstrated how different variables worked in identifying asymptomatic carriers. CONCLUSIONS: Our study demonstrates that AutoML models based on CT images can be used to identify asymptomatic carriers. The most promising model for clinical implementation is the DNN-algorithm-based model.

Effectiveness of Mobile Health Platform-Based Continuity of Care in Osteoporosis Prevention and Treatment.

Objective: To assess the effectiveness of using mobile health platforms for continuous care in preventing and treating osteoporosis. Methods: 114 patients with osteoporosis admitted to Nantong First People's Hospital from March 2021 to June 2022 were recruited and assigned equally via random number table method to receive either routine care (namely education on osteoporosis disease, dietary guidance, exercise guidance, activity guidance, medication supervision, fall prevention, psychological care, and secondary health education at the time of discharge) (routine group) or mobile health platform-based continuity of care (continuity group), with 57 patients in each group. Outcome measures included treatment compliance, disease knowledge of osteoporosis (diet, exercise, risk factors), quality of life level, and care satisfaction. Results: All eligible patients were followed up for one year after discharge from the hospital. Patients with continuity of care showed higher treatment compliance and disease knowledge of diet, exercise, and risk factors than those with routine care (P = .004). Continuity of care was associated with significantly higher MOS 36-item short-form health survey (SF-36) scores (The SF-36 is a self-administered questionnaire containing 36 items that survey overall health status) and nursing satisfaction in patients versus routine care (P = .004). Conclusion: Mobile health platform-based continuity of care effectively enhances post-discharge compliance and knowledge of osteoporosis in patients with osteoporosis, thereby improving post-discharge quality of life and satisfaction with care. Multi-center studies involving diverse healthcare settings and patient populations would provide more robust evidence. Moreover, these findings highlight the potential benefits of incorporating mobile health platforms into the care continuum for osteoporosis patients. Also, by utilizing mobile health platforms, healthcare providers can extend their reach beyond hospital settings and provide continuous care and support to patients, potentially reducing the burden on healthcare systems and improving overall population health outcomes.

Separation and Detection of Catechins and Epicatechins in Shanxi Aged Vinegar Using Solid-Phase Extraction and Hydrophobic Deep Eutectic Solvents Combined with HPLC.

This research presents a new, eco-friendly, and swift method combining solid-phase extraction and hydrophobic deep eutectic solvents (DES) with high-performance liquid chromatography (SPE-DES-HPLC) for extracting and quantifying catechin and epicatechin in Shanxi aged vinegar (SAV). The parameters, such as the elution solvent type, the XAD-2 macroporous resin dosage, the DES ratio, the DES volume, the adsorption time, and the desorption time, were optimized via a one-way experiment. A central composite design using the Box-Behnken methodology was employed to investigate the effects of various factors, including 17 experimental runs and the construction of three-dimensional response surface plots to identify the optimal conditions. The results show that the optimal conditions were an HDES (tetraethylammonium chloride and octanoic acid) ratio of 1:3, an XAD-2 macroporous resin dosage of 188 mg, and an adsorption time of 11 min. Under these optimal conditions, the coefficients of determination of the method were greater than or equal to 0.9917, the precision was less than 5%, and the recoveries ranged from 98.8% to 118.8%. The environmentally friendly nature of the analytical process and sample preparation was assessed via the Analytical Eco-Scale and AGREE, demonstrating that this method is a practical and eco-friendly alternative to conventional determination techniques. In summary, this innovative approach offers a solid foundation for the assessment of flavanol compounds present in SAV samples.

The exposure in ovo to glyphosate on the integrity of intestinal epithelial tight junctions of chicks.

Glyphosate is an ingredient widely used in various commercial formulations, including Roundup®. This study focused on tight junctions and the expression of inflammatory genes in the small intestine of chicks. On the sixth day of embryonic development, the eggs were randomly assigned to three groups: the control group (CON, n = 60), the glyphosate group (GLYP, n = 60), which received 10 mg of active glyphosate/kg egg mass, and the Roundup®-based glyphosate group also received 10 mg of glyphosate. The results indicated that the chicks exposed to glyphosate or Roundup® exhibited signs of oxidative stress. Additionally, histopathological alterations in the small intestine tissues included villi fusion, complete fusion of some intestinal villi, a reduced number of goblet cells, and necrosis of some submucosal epithelial cells in chicks. Genes related to the small intestine (ZO-1, ZO-2, Claudin-1, Claudin-3, JAM2, and Occludin), as well as the levels of pro-inflammatory cytokines (IFNγ, IL-1ß, and IL-6), exhibited significant changes in the groups exposed to glyphosate or Roundup® compared to the control group. In conclusion, the toxicity of pure glyphosate or Roundup® likely disrupts the small intestine of chicks by modulating the expression of genes associated with tight junctions in the small intestine.