Bisphenol A (BPA) is an environmental toxin utilized for the production of polycarbonate plastics and epoxy resins. Due to BPA's extensive production and environmental contamination, human exposure is unavoidable. The effects of low-dose of BPA on various body tissues and organs remain controversial. Our study investigated the potential of BPA to induce biochemical, histopathological, and immunohistochemical changes in the coronary artery and myocardium and the potential protective role of L-carnitine (LC). 24 adult Wistar albino male rats were divided equally into a control group, a BPA-treated group , and a BPA plus LC-treated group . BPA-exposed rats demonstrated structural anomalies in the coronary artery tissue including vacuolation of cells in the media and detachment of the endothelium of the intima. Congestion of blood vessels and infiltration by polynuclear cells were observed in the myocardium. There was an enhanced collagen deposition in both tissues indicating fibrosis. Immunohistochemical changes included enhanced eNOS and caspase-3 expression in the coronary artery and myocardium indicating vascular disease and apoptosis, respectively. Oxidative damage was evident in the coronary artery and the myocardium of BPA-treated rats, which was indicated by the reduced level of glutathione (GSH) and elevated malondydehyde (MDA) levels. The coadministration of LC significantly improved BPA-induced structural alterations and oxidative stress. In conclusion, BPA could potentially cause pathologic changes and oxidative damage in the coronary artery and myocardium, which could be improved by LC coadministration. Bisphenol A is one of the greatest volumes of chemicals produced globally, and more than one hundred tons are released into the environment every year . It is uThe health hazards related to BPA are mostly because of the partial polymerization leaving some unbound monomer BPA molecules in the product. Such monomers become released into food or beverages with time, particularly due to the effects of heat, acidic, or basic conditions . It was Bisphenol A acts through several mechanisms. More specifically, it interacts with multiple receptors such as estrogen, aryl hydrocarbon, androgen, thyroid hormone, and glucocorticoid receptors. BPA binds to classical nuclear estrogen receptors, classical and nonclassical membrane-bound ERs, and G protein-coupled receptors 30 . MoreoveThough the endocrine-disrupting effects of BPA have been thoroughly evaluated, there is still some uncertainty regarding its potential to harm the health. Much of such uncertainty is because of the controversies that surround the design and interpretation of results from hypothesis-driven BPA research .Being an endocrine disruptor, BPA can trigger several disorders, including developmental and reproductive system abnormalities, impaired brain and neurologic functions, malignancy, cardiovascular disease (CVD), diabetes, early puberty, obesity, and immune dysregulation .Cardiovascular diseases are the leading cause of mortality globally. Research has found that oxidative damage has a significant role in the development of coronary artery disease (CAD) . A systeμg/kg/d) accelerated atherosclerosis progression in a genetic mouse model prone to endothelial dysfunction and vasculitis [Experimentally, it was shown that low BPA doses could affect estrogen signaling in cultured rodent cardiomyocytes . Also, esculitis .BPA exposure was also associated with atherosclerosis progression in the coronary artery and aorta of rabbits , and othIn addition, Aboul Ezz and colleagues administered BPA to adult male rats by oral route for 6–10 weeks (25 or 10 mg/kg/d) and assessed oxidative damage in the cardiac tissue. They reported enhanced lipid peroxidation, low GSH, and catalase levels. BPA was also associated with a reduction in nitric oxide, which may result in vasoconstriction and reduced blood supply to the cardiac tissue. There was also reduced acetylcholinesterase activity among the BPA-treated rats, which might cause a decrease in heart rate and contractility .2+-handling protein expression, ion channel inhibition/activation, oxidative damage, production of free radicals, and genome/transcriptome modifications [The cardiotoxic effects of BPA could be triggered through its action on estrogenic receptors, modification of cardiac Caications , 23.β-oxidation of fatty acids and has a role in the metabolism of branched-chain amino acids and the stabilization of cell membranes [LC is a nonprotein amino acid, which is formed from lysine and methionine amino acids. It promotes the embranes . Also, sembranes , 26. ComIn the current study, we followed the ethical guidelines according to the ethical norms approved by the Medical Research Ethics Committee, Mansoura University (Code No.: R/96).BPA and LC were purchased from Sigma–Aldrich Inc. The required doses underwent dissolution in corn oil and were given to experimental animals by oral gavage.The study was performed on 24 male adult Wistar albino rats weighing about 200–250 g. Rats were housed and maintained under standard environmental conditions at a temperature of 22°C ± 2°C, 12 h of light/dark cycle, 41%–55% relative humidity, and provided with food and water throughout the experimental period. All animal work was carried out in accordance with the guidelines for the use of animals in research established by Mansoura University, Egypt. Also, the animals underwent handling according to the Guide for the Care and Use of Laboratory Animals .The study duration was 4 weeks. The rats were randomly divided into three groups (8 rats each) as follows: Group 1: untreated rats and served as controls (received corn oil), Group 2 (BPA 40 mg/kg/d): rats were treated with 40 mg/kg/d BPA dissolved in corn oil and given by oral gavage. This dose was chosen following the Chapel Hill BPA expert panel consensus statement, which defined the “low BPA dose” dose below the LOAEL (50 mg/kg/d) in animal models , and GroAt the end of the experimental work, rats were intraperitoneally injected with pentobarbital (50 mg/kg). Their hearts were then dissected. The left ventricles were cut perpendicular to the long axis into rings of 1   –2 mm width and utilized for the measurement of tissue GSH and MDA, and histopathological and immunohistochemical examination.μm thicknesses, and deparaffinized using 100% xylene, followed by rehydration with 100% and then 70% ethyl alcohol. Transverse sections underwent staining with hematoxylin and eosin (HE) for visualization of general tissue morphology and Masson trichrome to detect fibrotic areas. Stained sections were examined under light microscopy [The myocardial and coronary artery tissues of the sacrificed rats were dissected immediately, and part of it was fixed in 10% neutral buffered formalin for histopathologic examination. The paraffin-embedded sections were prepared to be 5 croscopy , 31.μm thickness. They were collected on poly-L-lysine coated slides, dried in a thermostat at 37°C for 24 h to achieve an appropriate adhesion of the biologic material to the surface of the slide, and then stained with the appropriate antibodies (anti-eNOS and anticaspase-3). Immunohistochemistry examination by eNOS was performed according to Felaco et al. [For the immunohistochemical study, sections were cut at 3 o et al. , while eo et al. .g for 20 minutes, and the resultant supernatant was used for oxidative activity analysis. GSH and MDA concentrations were measured in the tissue homogenates utilizing commercially available kits.The myocardial and coronary artery specimens from the sacrificed rats were homogenized in phosphate buffer solution  at 1 ml volume/g tissue wet weight ratio of 4 : 1. Homogenates underwent centrifugation at 13,000 × The data were analyzed using IBM SPSS Statistics for Windows, v 22.0. Armonk, NY: IBM Corp. Qualitative data were presented as numbers and percentages. Quantitative data were presented as means and standard deviations for parametric data following testing normality by the Kolmogorov –Smirnov test. The significance of a result was judged at the (0.05) level. The one-way ANOVA test was utilized to compare more than 2 independent groups with the post hoc Tukey test to detect pair-wise comparison.There were no reported mortalities among the experimental animals. In each group, the same area of each section was selected for hematoxylin and eosin, Masson trichome, eNOS, and caspase-3 stainings. The current study revealed the following findings.To investigate BPA's effects on the coronary artery, sections were stained using HE and examined using a light microscope. Unlike the control rats, which demonstrated the normal histological structure of the artery, sections from the BPA group show vacuolation of cells in the media and detachment of the endothelium of the intima of the artery. In contrast, sections taken from rats that received the BPA + LC demonstrated a near-normal structure to control rats . RegardiP < 0.001) (P < 0.001). However, LC coadministration was associated with a significant reduction in collagen deposition between muscle fibers (P < 0.001). Moreover, as shown in P < 0.001).The Masson Trichrome stain stains the collagen-rich fibrotic areas in a bluish color. As demonstrated in < 0.001) . On the e fibers  (P < 0.0P < 0.001) (P < 0.001). In contrast, the BPA + LC group showed a decrease in caspase-3 expression in comparison to the BPA group (P < 0.001). The quantitative analysis of caspase-3 expression area percentage of the heart demonstrated that BPA enhanced caspase-3 expression in comparison with control rats (240.69 ± 4.84% versus 182.92 ± 6.44%) and that coadministration of LC and BPA was associated with a significant reduction in caspase-3 expression in comparison with the BPA group (204.47 ± 10.59% versus 240.69 ± 4.84%) (P < 0.001)  . The apoPA group  (P < 0.0< 0.001) .P < 0.001). Conversely, coadministration of LC caused a significant reduction in eNOS expression in the coronary artery (P < 0.001) (P < 0.001). On the other hand, there was a decreased expression of the stain in the BPA + LC group (P < 0.001). The quantitative analysis of the eNOS expression area % of the heart (P < 0.001).In our study, the endothelial function was assessed by staining coronary artery sections with the eNOS immune stain . BPA cau< 0.001) . Regardihe heart  revealedP < 0.001) between BPA-treated rats and control rats (approximately 3-fold for MDA and 2.5-fold for GSH). In the group of BPA + LC, the MDA demonstrated a statistically significant decrease (P < 0.001) of the mean MDA level (20.75 nmol/g versus 32.82 nmol/g) while the GSH demonstrated a statistically significant rise (P < 0.001) of mean GSH level (1.22 mmol/g versus 0.82 mmol/g)  .BPA is a pollutant that induces different health problems including metabolic disorders, hormonal-based tumors, and cardiovascular diseases . AccordiThe current study demonstrated significant histopathological and immunohistochemical changes in the coronary arteries and the hearts of the rats. Starting with coronary artery changes, the current BPA-associated histopathological alterations in the coronary artery, which included vacuolation of cells in the media and detachment of the endothelium of the intima of the artery, and increased collagen fibers in the arterial wall, are suggestive of coronary artery vasculitis and atherosclerosis. These changes can be elucidated by the influence of BPA on the endothelial cells, resulting in elevated levels of surface adhesion molecules that increase permeability and induce immune cell infiltration . BPA couα and IL-1β can reduce the left ventricular function and cause loss of cardiomyocytes via apoptosis. These cytokines also have the ability to induce neurohumoral activation and cause oxidative damage, resulting in the initiation of the p38-MAP kinase and nuclear factor-κB. Collectively, this induces cardiomyocytolysis and reduces Ca+2 uptake by the sarcoplasmic reticulum, and therefore impairs cardiac inotropy [On the other hand, the myocardium of BPA-exposed rats showed congestion of the blood vessels and infiltration by polynuclear cells in the myocardium. These findings are suggestive of the inflammation linked to BPA administration. Proinflammatory cytokines such as TNF-inotropy . The coninotropy , who repIn addition, the myocardial changes were suggestive of increased fibrosis as indicated by a significant increase in collagen fibers. Similar findings in the myocardium have been reported previously after oral administration of BPA (50 mg/kg) to rats, where the myocardium of BPA-exposed rats showed a significantly increased collagen fiber deposition in comparison to control rats . Also, Bβ, NF-α, and interferon-γ [In our work, the apoptosis caused by BPA was evaluated by the immunohistochemical study of the proapoptotic marker caspase-3. BPA caused apoptosis in the coronary artery and the cardiac tissue as indicated by enhanced caspase-3 expressions. The immunohistochemistry examination of caspase-3 in cardiac slices revealed that caspase-3 expression was minimal in the control group, while it is significantly elevated in the BPA group and decreased in the BPA + LC group. Indeed, treatment of rats with LC improved the apoptotic effects of BPA, as evidenced by down-regulatingcaspase-3 expressions. Myocyte apoptosis is associated with high concentrations of proinflammatory cytokines including IL-1rferon-γ .Due to the importance of eNOS activity to keep the vascular dilatation, eNOS expression was investigated in this study. Nitric oxide (NO) is produced by the endothelial nitric oxide synthase (eNOS) enzyme by converting the L-arginine to NO in the vasculature . The eNO2+/calmodulin-dependent mechanism [Our findings revealed an increased expression of eNOS in BPA-exposed rats, which was in agreement with Klint et al. . It shouechanism , and theechanism .At the biochemical level, BPA-induced oxidative stress was investigated. The biochemical analysis showed the occurrence of oxidative damage in BPA-treated rats that was indicated by the reduced level of the antioxidant GSH and the elevated level of the lipid peroxidation marker (MDA). The heart is significantly sensitive to free radicals due to its high oxidative metabolism and due to its few antioxidant defenses. ROS are cytotoxic agents, which cause oxidative stress by targeting biomolecules like membrane lipids and cellular DNA . TherefoThe increased evidence of BPA-related negative effects on health has encouraged researchers to search for a drug or natural substance that protects against these effects, especially for those at high exposure risk . This stExposure to BPA has been reported to be linked to CVD. However, few studies have focused on its association with coronary artery pathology. This study concluded that BPA exposure can result in biochemical and pathologic alterations in the myocardium and coronary artery. The results suggest that oxidative stress, fibrosis, and apoptosis play important roles in BPA-related toxicity. Importantly, the harmful alterations caused by BPA could be partially improved by the coadministration of LC. However, more studies are required to evaluate the effects of prolonged exposure to different BPA doses on the cardiovascular system. Also, it is recommended to regularly administer LC to help reduce the deleterious effects of exposure to BPA-containing products.


Hence, cell wall compartments were maintained. Consequently, there was a reduction in browning symptoms in fruits by inhibiting polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) activities. Thus, the fruit skin browning index showed almost no symptoms. The lipid peroxidation process and ionic permeability declined as well. The result suggests that, by applying GA/PVP-SA 2 mM as an edible coating, fruit tissue breakdown can be minimized, and the shelf life of peach can be extended up to 10 days without symptoms of tissue breakdown.Peaches are grown in many Egyptian orchards for local and global fresh market sales. The interior fruit tissue breakdown (IFTB), often resulting in decayed peaches, is a severe problem during marketing. Therefore, to minimize FTB of peaches, in this study, gum arabic (GA) and polyvinylpyrrolidone (PVP) were mixed with different concentrations of salicylic acid (SA)  and were applied as edible coating to extend the shelf life of peach fruits. Mature peaches were selected and harvested when peaches reached total soluble solid content (SSC: 8.5%) and fruit firmness of about 47 N. Fruits were coated and stored at room temperature (26 ± 1 °C and air humidity 51 ± 1%) for 10 days during two seasons: 2020 and 2021. Fruit coated with GA/PVP-SA 2 mM showed a significant ( Prunus persica L. cv, ‘Early Sweet’), which belongs to the family Rosaceae, is one of the most popular fruits in the world and, in particular, in the Egyptian market due to its nutritive value and characteristic flavor [The peach fruit  has permitted GA as a safe additive coating in food industries [Gum arabic (GA) is one of the most common polysaccharides and is naturally extracted from the bark of dustries . Moreovedustries ,16. Strudustries . The chadustries , stabilidustries , and micdustries . GA is wdustries ,22,23. Pdustries . PVP, widustries . PVP hasdustries ,27. The dustries . Acceptadustries . Food apdustries .Salicylic acid  is an endogenous growth regulator and a signal molecule that is critical for the induction of resistance to biotic and abiotic stress. In plants, it exists as a free phenolic acid and as conjugated forms that are constructed by hydroxylation, glycosylation, or methylation of the aromatic ring . EarlierBotrytis cinerea in strawberry fruit. It is also recognized that SA has fungicidal properties on the brown rot of sweet cherry fruit, which is caused by Monilinia fructicola [Regarding public doubts about the undesirable effects of synthetic fungicides on human health and the environment, there is continuing research into new substitutes for the application of synthetic chemicals. One of the alternatives might be the use of SA, which has revealed antifungal properties on some fruits and other plants . For insucticola .There are no available data in the literature concerning the use of a biopolymer blend of GA/PVP supplemented with salicylic (SA) acid as an edible coating. Thus, the present study aimed to evaluate the ability of GA/PVP/SA edible coating formulations in minimizing the incidences of tissue breakdown in peach fruits’ ‘Early Sweet‘ during the trading period.−1. Meanwhile, the peaks at 3422, 2927, 1654, 1429, 1030, and 776 cm−1 were noted for GA. The IR spectrum of PVP shows the characteristic peaks observed at 3450, 1656, 1435, 1289, and 845 cm−1. Moreover, the characteristic bands at 3451, 2926, 1294, and 776 cm−1 were found in the GA/PVP blend (−1 wavenumbers.VP blend . The FT-GA/PVP blends as edible coatings possessed significant effects on weight loss of peach fruits compared with control uncoated fruits . The GA/GA/PVP blends had a significant impact on the hue angle (color appearance parameter) of peach fruits compared with control uncoated fruits . ThroughResults indicated that GA/PVP coating blends have a significant impact on peach fruit firmness compared with control uncoated fruits . The fru−1), while the control fruits displayed a progressive decline in phenolic contents on the 10th day , respectively.During 10 days of storage at 25 °C, the contents of TPs and TFs of uncoated fruits were significantly lower than those of GA/PVP treatments . Noticea−1 mg protein−1), while the highest activities of PPO and PAL were recorded in uncoated fruits , throughout the shelf-life period (days) for ‘Early sweet’ peach fruit. A significant interaction at p ≤ 0.001 was observed when coating treatment of GA/PVP–SA and storage periods (days) were considered as a factor. Noticeably, all the activities of CWDEs increased gradually up to the sixth day of the experiment. Moreover, both LOX and PG enzymes continued to increase in activity until the 10th day of postharvest life; however, the CEL activity decreased. As presented in −1 mg protein−1), CEL (17.85 U min−1 mg protein−1), and PG (2.86 U min−1 mg protein−1) on the 10th day. Conversely, the GA/PVP–SA 2 mM treatment presents the highest significant reduction in CWDEs, i.e., LOX (0.99 U min−1 mg protein−1), CEL (9.64 U min−1 mg protein−1), and PG (0.43 U min−1 mg protein−1), at the end of the experiment.p < 0.001 between postharvest time (days) and GA/PVP–SA treatments. Perceptibly, both cell membrane termination and MDA and EL% increased gradually over all treatments up to the 10th day of the postharvest period, contrasting with the initial rates at harvest time. Moreover, the control treatment showed more rapid MDA accumulation and ion permeability percentage during the postharvest period compared to other coating treatments. The coating treatment of GA/PVP–SA 2 mM is recognized as the most valuable one in minimizing the alternations in cell membrane parameters, where it recorded the values of 0.32 μM g−1 FW and 13.24% for MDA and EL%, respectively, on the 10th day of storage time.Experimental data of MDA and EL% as indicators of membrane disruption are presented in this study. −1 h−1 for both gasses on the second day of the shelf-life period. Consequently, it minimizes both ethylene and carbon dioxide production until up to the end of the storage time . It can be concluded from −1), and the stretching (a shoulder at 3063 cm−1) and bending (1297 cm−1) vibrations of C–H bonds of aromatic rings, C=O bonds (1659 cm−1), and C=C bonds of benzene rings . Overall, the FT-IR spectrum of SA in this study agreed to a large extent with that obtained by [The IR spectrum of salicylic acid  reveals ained by .−1 is ascribed to OH groups of the carbohydrate structure. Meanwhile, the major IR bands observed at 2927 cm−1 were assigned to the vibrational modes of C–H groups. Peaks present in the spectra at 1654 and 1429 cm−1 correspond to the occurrence of the carboxylic groups. It is well known that carboxylic acids display a characteristic OH in-plane bending band at 1430 cm−1 [−1 wavenumber may be due to the symmetrical stretching of uronic acid carboxylates in the structure of GA. The peaks found between 800 cm−1 and 1200 cm−1 represented C–C, C–O, and C–O–C stretching and C-OH and C–H bending modes of the polymer backbone. Bands detected in the spectra at 776 cm−1 may be assigned to the 1–4 linkage of galactose and 1–6 linkage of mannose [−1 in the FT-IR curve of GA may be assigned to arabinogalactan. In the present work, the bands found between 700 and 500 cm−1 were attributed to the pyranose rings. Our results concerning the FT-IR spectrum of GA were in accordance with those obtained by [For GA, the broad peak observed at 3422 cm430 cm−1 . Therefo mannose . The peaained by .−1 are assigned to hydroxyl stretching, and the peaks at 1435 and 845 cm−1 correspond to the CH2 scissoring vibrations and CH2 bending, respectively. The peaks at 1656 and 1289 cm−1 are assigned to C=O stretching and C–N stretching. Our results concerning the FT-IR spectrum of PVP were in accordance with those obtained in [For PVP, the peaks observed at 3450 cmained in .−1, while the peak at 776 cm−1 in this blend confirmed the presence of 1–4 linkage of galactose and 1–6 linkage of mannose related to GA. The FT-IR spectrum of GA/PVP–SA 2 mM has prominent bands at 3240, 1660, 1612, and 1483 cm−1 wavenumbers due to the presence of SA in the polymer blend. Meanwhile, the peak found at 1296 cm−1 might be related to the pyrrolidone structure of PVP. The board peak of hydroxyl groups related to GA shifts to the wavenumber of 3528 cm−1. Moreover, the shifted peak at 760 cm−1 related to the 1–4 linkage of galactose and 1–6 linkage of mannose might support the presence of GA in GA/PVP–SA 2 mM treatment.In the GA/PVP blend, the C–N bending vibration from the PVP pyrrolidone structure was shifted to be at 1294 cmAloe gels as a coating source were able to diminish water loss of palm and peach fruits compared with uncoated fruits. Polysaccharide biodegradable coatings can decrease water loss of the fruit tissues by forming a physical barrier around the fruit surface [Coating polymers are generally made of fats, proteins, and polysaccharides that inhibit water loss ,43 and p surface . Overall surface . It is t surface . PVP is In addition, using PVP increases coating performance to maintain peach fruit quality and decline the water evaporation. Thus, the use of pure PVP is restricted. To overcome this difficulty, PVP is fabricated with GA in this study to obtain a blend possessing acceptable properties, i.e., forming a physical barrier and preserving a high amount of water .The presence of phenolic ingredients increases cellular immunity due to their biological properties, such as antioxidant capacity and protection against fungi and bacteria . GA/PVP–Enzymatic browning is a common phenomenon that can usually be detected in different fruits, which unfavorably affects the nutritional value and other quality attributes. This happens when the phenolic ingredients are oxidized by PPO to their quinone derivatives and, additionally, are oxidized to form melanin pigment accountable for the browning reactions . In the The impact of SA on phenolic contents and the activities of browning enzymes depend on some critical factors, such as type of stress, SA concentration, availability, plant variety, postharvest conditions, and so on. In this respect, postharvest treatment of peach fruit with SA at 2 mM recorded lower PPO activity, accompanied by higher activities of antioxidant enzymes during shelf-life storage . MoreoveSkin browning is a common problem for extending the postharvest life of peach fruit, which is most sensitive to mechanical injury. Browning is chiefly caused by the enzymatic oxidation of endogenous phenols into quinones . SBI weiAloe vera gel under shelf-life storage over 30 days had a rate of diminished hue angle that was much slighter than the uncoated fruits. It could be suggested that GA/PVP blends can prevent color rapid deterioration [The acceptable color and appearance of peach fruit is the critical factor for its friendly marketing. The hue angle of vegetables and fruits was mostly affected by the coating treatment. Moreover, coated peach fruits’ color should stay parallel to a fresh one by combating any deterioration of color during storage. In this regard, the little rate of decrease in hue angle of peach fruits coated with GA/PVP blends in comparison to uncoated fruits reflects the effectiveness of these polymer composites in avoiding color rapid deteriorations. Our results agreed with , who shoioration . The proAloe gels had no effect on the level of firmness in peach fruit stored at 20 °C for six days; its values gradually decreased in coated and uncoated fruits alike, with no significance [A. vera coating film decreasing the firmness loss of peach fruits stored at 4 °C has been recently reported [During the ripening process, fruit firmness decreased gradually due to the induction of the activities of CWDEs. In this study, GA/PVP formulations could preserve peach fruit firmness, especially GA/PVP–SA 2 mM. The literature data revealed a discrepancy in the impact of different edible coatings on peach fruit firmness, validating the proper role of SA in the present study. For instance, it was found that ificance . In contreported .The present data elucidated the efficient role of GA/PVP biopolymer coatings in diminishing the activities of PG, CEL, and LOX . Thus, tThe plant cell wall is a complex reticulate structure, which consists of structural proteins, pectin, cellulose, and hemicellulose . Pectin The hydrolysis of pectin is catalyzed by related enzymes comprising pectin methylesterases, PG, pectate-lyase, and β-galactosidase, of which PG has been proposed to act as a vital role . MoreoveCellulases (E.C. 3.2.1.4) hydrolyze β-1,4 linkages of cellulose, cellobiose, and cellodextrin. Generally, they are multienzyme complexes having endo-1,4-β-glucanase, β-glucosidase, and cellobiohydrolase activity . In fruiIn this study, the GA/PVP biopolymer coating efficiently diminished the activities of PG and CEL. These desired effects progressively increased in the presence of salicylic acid in the polymer composites. Inhibition of fruit CWDEs and preservation of firmness using edible coating formulations have been noted by several researchers. In this respect, Dave et al.  found th3DkLOX by SA, concomitant with the preservation of fruit firmness, inhibition of weight loss, and ethylene production during persimmon fruit storage. Moreover, the inhibitory effect of nitric oxide on LOX and ethylene biosynthesis in the shelf life of peach fruit has been previously reported [LOX in delaying peach fruit ripening and preserving firmness.Lipoxygenase  is an enzyme that commonly exists in plant tissues, which activates the oxidation of polyunsaturated fatty acids to form corresponding hydroperoxides. The fatty acid hydroperoxides formed by the activity of LOX are possibly harmful to membrane function by initiating increased rigidity . It alsoreported . The higreported , the earreported . The resMDA is the product of lipid peroxidation, and its accumulation is indicative of cell membrane degradation. It was previously described that the increased amount of MDA is principally due to the increased activity of LOX . Our resThe usage of biodegradable coatings alone or incorporated with bioactive additives in fruit postharvest technology significantly reduces the accumulation rate of MDA, which preserves the functions of cellular membranes and, thus, also reduces cell permeability rates . This cop  <  0.05) lower compared to uncoated fruits after the second day until the end of the storage period. Polymer coatings acted as a barrier film, providing a different internal atmosphere and a selective membrane for permeation of ethylene in and out of the fruit, as well as diminished production of ethylene by the fruit tissues [The respiratory performance of GA/PVP/SA-coated peach fruits  presente tissues . Moreove tissues .The influence of SA on ethylene production was studied on different fruits, i.e., tomato , apple , and SelPrunus persica) ‘Early Sweet’ cv. Trees were planted in a commercial orchard in Meet-Gamer province, Egypt, in clay soil that is 11 years old. All trees were grafted on ‘Nemaguard’ rootstock at 4 × 4 m2 plantation distance. The trees were also pruned in an open vase shape under drip irrigation. The ranch administration program was connected by the proposals of the Egyptian Agricultural Ministry. Fruits were selected from the shaded side of trees in the same uniform at full maturation in May [The present study was conducted throughout the season (2020 and 2021) on peach fruit (n in May . At harvGum arabic (GA) powder was supplied by El-Gomhoria Company, Cairo city, Egypt. It was prepared by adding 60 g GA to 500 mL distilled water. Thereafter, the solution was stirred by hot magnetic stirring at 55 °C for 60 min. Then, the solution was cooled at room temperature and filtered to remove impurities using a fine piece of cloth . To prep−1 at a resolution of 4 cm−1 [For identification of the functional groups and chemical bonding in pure SA, GA, PVP, and the dry powders of blend solutions of GA/PVP and GA/PVP-SA 2 mM, 1 mg of each sample was mixed with 300 mg of fine potassium bromide (KBr). The thin pellets were prepared by pressing with the hydraulic pellet press and were then subjected to Fourier transform infrared spectrophotometer  in the range of 500–4000 cmf 4 cm−1 ,42.Peach fruit samples (15 fruit per replicate) were weighed at harvest time up to the end of the experiment time interval (2 days). Weight loss % was computed based on the initial value at harvest time .As for fruit skin, the browning parameter was determined by browning spots visually during shelf-life duration. The browning scale was identified in five categories . The catFruit firmness was measured as the necessary force to penetrate the tissue using the fruit texture Effegi penetrometer  .−1 fresh weight (FW) as gallic acid equivalents (GAE) [−1 FW catechin equivalent (CE) [TPs content of peach fruit samples was evaluated using Folin–Ciocalteu reagent and gallic acid as a standard phenolic compound. TPs content was spectrophotometrically estimated at 750 nm. Data were represented as mg 100 gts (GAE) . Meanwhient (CE) .A gram of fruit sample in time was homogenized with 5 mL of Tris-HCl buffer . Then, the mixture was centrifuged at 4 °C  and the resultant supernatant was kept at −20 °C until further steps.EC: 1.14.18.1) was assessed according to the procedure of [−1 mg protein−1.Polyphenol oxidase (PPO) (edure of  by mixinEC: 4.3.1.24) activity was assessed spectrophotometrically at 290 nm according to the steps described by [−1 boric acid buffer (pH 8.8). The activity of PAL was lastly expressed as units U min−1 mg protein−1.Phenylalanine ammonia-lyase (PAL)  containing EDTA (20 mM), cysteine-HCL (0.02 M), and Triton X-100 (0.05%). Then, centrifugation was carried out at 15,000× essments . The supessments .D-galacturonic acid after assessing the absorbance at 540 nm. PG activity was expressed as U min−1 mg protein−1.The assay of polygalacturonase (PG) activity was evaluated according to the scheme designated by . Volumes−1 mg protein−1.Assay of cellulase (CEL): the activity of CEL was evaluated according to the method described by . The clew/w)). The mixture was then centrifuged for 20 min at 17,000 rpm at 4 °C. The resultant supernatant (0.1 mL) was mixed with 2.8 mL of sodium phosphate buffer , and then 0.1 mL of linoleic acid sodium salt (0.005 M) was added. The activity of LOX was observed according to the resultant rise in absorbance at 234 nm due to hydroperoxide formation. The activity of LOX was expressed as U min−1 mg protein−1.Lipoxygenase  activity was evaluated relating to the scheme . LipoxygTwo equidistant readings were taken in the equatorial region of each fruit. The results were expressed in Newtons (N) , and totw/v) meta-phosphoric acid and 0.2 mL of 2% (w/v) ethanolic butylated hydroxytoluene (BHT). The resultant mixture was centrifuged at 15,000× g rpm for 15 min. Then, 1.0 mL of the resultant supernatant was added to 0.1 mL of 2% (w/v) BHT, 0.5 mL of 1% (w/v) of thiobarbituric acid (TBA) in 0.05 M NaOH, and 0.5 mL of 20% (v/v) hydrochloric acid. The tube mixture was incubated at 95 °C for 30 min. After cooling, the resultant chromogen was extracted by adding 0.8 mL of n-butanol, centrifugation was performed to separate the organic phase, and the absorbance of thiobarbituric acid reactive substances (TBARS) was read at 532 nm. The compound of 1,1,3,3-tetra-ethoxy-propane (Sigma) was used to prepare the calibration curves, and finally, micromoles of MDA per gram FW for the examined samples were estimated. The electrolyte leakage percentage rate (EL%) was evaluated by a conductivity ion meter. Data were exhibited as a percentage [Malondialdehyde (MDA) was determined according to the method in . One grarcentage .Ethylene and respiration of peach were estimated on a fruit sample of 6 peaches after a 2-day interlude. Fruits were incubated and saved in 1000 mL glass jars sealed for a one-hour interval of the shelf-life term in time for all further analyses. Gas samples only from each of the jars around the fruit were extracted, and both ethylene and carbon dioxide were examined by gas chromatography techniques (GC) . However, ethylene was monitored by GC-6000 Vega Series  and carbon dioxide was measured by GC PBI-Dansensor Checkmate-9900 .p < 0.05 using Co-Stat software package Ver. 6.303 . To forecast the characteristics of the treated fruits during the shelf life, a linear regression technique was performed, and the criterion of R2 > 0.9 was considered as a proper judge for the fitness of the model [The parameters of water loss %, fruit skin color hue angle, and skin spot browning index were analyzed using one-way analysis using a complete block design when GA/PVP–SA treatments were considered as a factor. However, the distractive parameters were analyzed using two-way analysis using a factorial experiment in a completely randomized block design for chemical parameters when storage period (days) and GA/PVP–SA treatments were investigated as factors. The means of all examined treatments were compared using Duncan’s multiple range test at he model .The outcomes obtained from this research show that the peach fruit coated with GA/PVP mixed with SA at a concentration of 2 mM gives a significant effect on diminishing fruit tissue breakdown during postharvest life. This result can be summarized into three points: First, the treatment inhibits the activities of CWDEs. Second, it decreases the lipid peroxidation process and ionic penetrability rate. Third, the coating treatment decreases the activities of browning enzymes, and therefore, the brown incidence color on the fruits decreases during the shelf-life period. It can be said that GA/PVP–SA 2 mM treatment can retard the deterioration of peach fruit during shelf life. Future studies for the development of information on the impact of GA/PVP–SA treatment to increase fruit storability under shelf-life conditions are needed.


Mental Health Literacy (MHL) has become a focus of research in recent decades, as a prerequisite for early identification and intervention for mental health problems. Although several instruments have been developed for assessing MHL, there is a need for brief and psychometrically sound measures to capture important aspects of MHL in large and diverse adult samples. The present study aimed to: (1) provide a revised and shorter version of a previously validated questionnaire for assessing MHL; and (2) examine the psychometric properties of the MHLq-SVa in student samples from six different countries .The study involved 2180 senior school and undergraduate students, aged between 17 and 25 years old, from China, India, Indonesia, Portugal, Thailand, and the United States. Participants responded to the Mental Health Literacy Questionnaire for young adults (MHLq-ya), in their native language, following its translation and adaptation for each culture. The MHLq-ya comprises 29 items, organized into four dimensions: Knowledge of mental health problems; Erroneous beliefs/stereotypes; First-aid skills and help-seeking behavior; Self-help strategies. Confirmatory factor analyses and internal consistency analyses were performed on the combined data.Data from the different countries supported a shorter version of the questionnaire (MHLq-SVa), composed of 16 items that fit with previously defined dimensions. Internal consistency and between-factor correlations further supported the adequacy of the instrument’s psychometric properties.The study provided preliminary support for the construct validity and reliability of the MHLq-SVa as a measure for assessing MHL in young adults from six different countries and languages. Future studies are needed to further validate the measure and undertake multicultural comparisons of MHL in diverse samples from around the globe. According to the Global Burden of Disease Study, in 2019 approximately 792 million people presented mental health problems, a number that rises to 970 million people if substance use is included  have repThe increasing prevalence of mental health problems globally suggests the need to adopt preventative measures, as early detection of signs and symptoms increases the probability of success in terms of treatment outcomes –13. One Research has examined the role of variables that potentially influence MHL levels, namely gender , 18, proSeveral instruments have been developed to assess MHL , soGiven the increasing need to assess MHL among young adults and given time constraints when undertaking such assessments, the current study aimed to expand the analysis of the psychometric properties of the MHLq-YA to provide a revised and shorter version. In addition, we examined the psychometric properties of this short version in student samples from six different countries .The study involved 2180 senior school and undergraduate students, aged between 17 and 25 years old, from six different countries – China, India, Indonesia, Portugal, Thailand, and the United States.The sample from China included 496 undergraduates , aged from 17 to 22 years . The sample from India included 284 undergraduate students , aged between 18 and 25 years . The Indonesian sample included 197 undergraduate students , aged between 18 and 23 years . The Portuguese sample included 382 undergraduate students , aged between 17 and 25 years . The Thai sample included 385 undergraduate students , aged between 18 and 24 years . The United States sample included 436 undergraduate students aged between 18 and 25 years .Prior to data collection, the MHLq-YA was translated into the predominant language of each country, following the guidelines for translating and adapting items : (1) traParticipants’ recruitment occurred through in person contact with students  and through online dissemination .The study followed ethical guidelines in each country with all participants providing written informed consent. The sociodemographic and MHLq-YA forms were self-administered using online platforms , in-person paper-and pencil questionnaires , or both . Data collection occurred both prior to  and during  the COVID-19 pandemic.The protocol included a sociodemographic section, comprising self-reported questions about gender and age, and the MHLq-YA.The original version of MHLq-YA  includedConsidering the preliminary evidence from the MHLq-YA study , first wFor the calculation of total score, items from “erroneous beliefs/stereotypes” dimension were reverse-scored. Internal consistency was analysed in all samples through Cronbach’s Alpha and McDonald’s Omega , as wellThe construct validity of the MHLq-YA was tested by performing a CFA with the Portuguese sample. Considering the previous results of the exploratory study of psychometric properties , local aThe revised version of MHLq-YA, now named Mental Health Literacy questionnaire- Short Version for adults (MHLq-SVa), fit well to the Portuguese data (X2(95) = 153.17 × 2/gl = 1.62 ; CFI = 0.95; NNFI = 0.95 RMSEA = 0.040). At the level of local adjustment, the factorial weights were globally high, ranging between 0.50 and 0.97 . The standardized loadings revealed some concerns in two countries, regarding items MHLq13 (Indonesia), MHLq15, and MHLq27 (India).The quadri-dimensional model showed similar goodness-of-fit indices in US (X2 (95) = 206.547 × 2/gl = 2.17; CFI = 0.97; NNFI = 0.96; RMSEA = 0.052), China (X2 (95) = 186.190 × 2/gl = 1.96; CFI = 0.95; NNFI = 0.94; RMSEA = 0.044), Thailand (X2 (95) = 180.768 × 2/gl = 1.90; CFI = 0.95; NNFI = 0.94; RMSEA = 0.048), India (X2 (95) = 257.482 × 2/gl = 2.71; CFI = 0.88; NNFI = 0.85; RMSEA = 0.072) and Indonesia (X2 (95) = 183.044 × 2/gl = 1.92; CFI = 0.95; NNFI = 0.94; RMSEA = 0.051). The model also showed a good local adjustment in these five countries .Following the preliminary study of the psychometric properties of the MHLq-YA , where sThis study has two major strengths. First, it allowed for the development of a shorter version of a questionnaire for assessing MHL in young adults, which will be easier to administer, less time consuming to score, and less burdensome for participants. Second, this was the first time that this questionnaire was tested in different cultures, from distinct regions , in languages spoken in countries with large populations, facilitating future research focused on multicultural comparisons of MHL. There are also limitations. The sample sizes of each country varied significantly, potentially compromising comparative analyses, such as measurement invariance. Data collection procedures (paper-pencil vs. online) and timing (pre and during COVID-19 pandemic) differed between countries. The fact that some data were collected during the pandemic could have influenced participants’ responses, since mental health awareness is thought to have increased during this period e.g., ,44). The. The44])Future studies should address and overcome the limitations stated above, but also contribute to the strengthening and applicability of this instrument. First, considering the concerns regarding the factor loadings of some items, as well as the non-significant correlation between Factor 2 and Factors 1 and 3 in India, new data collection and analyses should be developed in India and Indonesia, in order to explore the need to revise item translation or make cultural adjustments. Second, other instruments should be considered, not only to control social desirability, but also to further study the MHL construct by means of examining concurrent validity . Third,Preliminary validation of the MHLq-SVa suggests that it is a valid and reliable measure for assessing MHL in young adults from six different countries and languages . Future studies are needed to test measurement invariance and other relevant psychometric properties, allowing multicultural comparisons of MHL.


Metagenomic analysis showed that Bacteroidetes (53.9%) and Firmicutes (37.1%) were the dominant bacterial phyla in yak rumen across dietary treatments. In GSG samples, Actinobacteriota, Succinivibrionaceae_UCG-002, and Ruminococcus albus were the most abundant, while Bacteroides was significantly more abundant in NSG samples (p < 0.05) than that in GSG. The known fiber-degrading fungus, Neocallimastix, was significantly more abundant in NSG and SG samples, while Cyllamyces were more prevalent in NSG rumen than in the SG rumen. These findings imply that a diverse consortium of microbes may cooperate in response to fluctuating nutrient availability, with depletion of known rumen taxa under nutrient deficiency. Archaeal community composition showed less variation between treatments than bacterial and fungal communities. Additionally, Orpinomyces was significantly positively correlated with acetate levels, both of which are prevalent in GSG compared with other groups. Correlation analysis between microbial taxa and VFA production or between specific rumen microbes further illustrated a collective response to nutrient availability by gut microbiota and rumen VFA metabolism. PICRUSt and FUNGuild functional prediction analysis indicated fluctuation response of the function of microbial communities among groups. These results provide a framework for understanding how microbiota participate in seasonal adaptations to forage availability in high-altitude ruminants, and form a basis for future development of probiotic supplements to enhance nutrient utilization in livestock.Yak is the only ruminant on the Qinghai-Tibetan Plateau that grazes year-round. Although previous research has shown that yak rumen microbiota fluctuates in robust patterns with seasonal foraging, it remains unclear whether these dynamic shifts are driven by changes in environment or nutrient availability. The study examines the response of yak rumen microbiota  to simulated seasonal diets, excluding the contribution of environmental factors. A total of 18 adult male yaks were randomly divided into three groups, including a nutrition stress group , a grazing simulation group , and a supplementation group . Volatile fatty acids (VFAs) profiling showed that ruminal acetate, propionate and total VFA contents were significantly higher ( Bos grunniens), referred to in Chinese as the “boat of the plateau,” is the only large mammal endemic to the Qinghai-Tibet Plateau, and yak husbandry is tightly interwoven in the lifestyle of the Tibetan people, providing indispensable goods, such as dairy, meat, textiles, and fuel  by simulating grazing conditions in winter pasture, warm season pasture, and winter pasture with supplementation. To this end, high throughput metagenomic rRNA gene sequencing was used to characterize rumen community composition and diversity under different grazing conditions. Then, to test the hypothesis that specific microbes are enriched under different grazing conditions, leading to differences in rumen fermentation, we characterized the volatile fatty acid (VFA) profiles of all rumen samples and conducted correlation analysis between microbial taxa (ASVs) and different VFAs. This study extends our understanding of the role of rumen microbes in metabolic adaptation to extreme seasonal changes in forage availability.ad libitum access to water. The remaining feed was collected and weighed before morning feeding to calculate the actual feed intake.Procedures of the current study were approved by the Animal Welfare and Ethics Committee of Lanzhou University . The study was conducted in August 2021, at the Yak Breeding Cooperative in Nimu County, Lhasa City .The experiment selected 18 male yaks  and randomly divided into three different diet groups shown in . The expOn the last day (day 35) of the experiment, a stomach tube  was used to collect rumen fluid from each yak before morning feeding. The initial 50 ml of fluid were discarded to avoid contamination by saliva. Then, 40 ml of rumen fluid was taken and stored in a sterile enzyme-free cryopreservation tube with liquid nitrogen freezer and transferred immediately to the laboratory and stored in a –80°C refrigerator. The collected samples were mainly used for the determination of volatile fatty acids (VFAs) and microbial community analysis.g at 4°C for 15 min, metaphosphoric acid solution(0.2 ml) containing 2-ethyl butyric acid as internal standard was added to supernatant(1 ml). After mixing well and standing for 30 min at 4°C. The mixed solution was centrifuged at 3850×g at 4°C for 15 min, and the supernatant was collected and filtered (0.22 μm) for VFAs determination using a gas chromatograph  with an AT-FFAP type capillary column (30 m × 0.32 mm × 0.5 μm) and a flame ionization detector, with the temperature regime as: 90°C for 1 min, increased to 120°C at 10°C/min for 1 min, then increased from 120 to 150°C at 10°C/min, and maintained at 150°C for 3 min. The injection hole temperature was 250°C, and the auxiliary chamber temperature was 250°C.Volatile fatty acids (VFAs) were determined by modified method from The collected feed samples were dried in a forced-air oven at 65°C for 48 h. After crushed and then passed through a 1 mm sieve, feed samples were weighed and dried at 105°C to a constant weight to thus subjected to dry matter (DM) and crude protein (CP) determination by the Kjeldahl method , while nRumen fluid stored at −80°C was subjected to genomic DNA extraction using TIANamp Stool DNA Kit . DNA spectrophotometer  was used for DNA yield and purity screening. Normalized genomic DNA was applied to perform barcode PCR with a set of primers . Phusion® High-Fidelity PCR Master Mix (New England Biolabs) was used in the PCR reactions, cycling conditions were as follows: 1. denaturation step at 98°C, 1 min; 2. 30 cycles at 98°C for 10s, 50°C for 30 s, 72°C for 30 s; and 3. a final 5 min extension at 72°C. The PCR products were thus subjected to agarose gel electrophoresis for separation and visualization. The final products were sequenced on an Illumina NovaSeq platform and 250 bp paired-end reads were generated.Paired-end reads were assigned to each sample by barcodes identification. After cutting off the barcodes and primer sequences, Paired-end reads were merged and spliced by FLASH v 1.2.11 . QualityAlpha and beta diversity were calculated from different indices in QIIME2 (Version QIIME2-202006). Principal Coordinate Analysis (PCoA) was used to obtain principal coordinates and visualize differences of rumen samples in complex multi-dimensional data. The results were plotted using ade4 and ggplot2 packages in R. LEfSe analysis was performed through the Huttenhower Lab Galaxy Serverp values were used to separate means and statistical significance was accepted at p < 0.05.The association between rumen microbiota and VFA through Spearman’s correlation analysis. One-way ANOVA by SAS version 9.2 was used to compare rumen VFAs. Non-parametric factorial Kruskal–Wallis sum-rank tests were performed to test for differences among groups at the bacterial phylum and genus levels, and Dunn’s test was performed to separate means where significance was found. Tukey-adjusted n = 6 animals per treatment; p < 0.05) in GSG than in NSG or SG, while the concentrations of rumen butyrate and valerate, as well as the acetate: propionate ratio showed no difference among the three experimental groups. These results suggested that metabolic processes in yak rumen indeed varied with nutrient intake, leading us to hypothesize that rumen microbiota contributed to this process.In order to better understand the differences in microbial-associated metabolic processes in yak rumen under varying nutrient availability, volatile fatty acid (VFA) composition was determined in yak rumen fluid of animals treated with simulated nutrient deficiency (NSG), simulated warm season grazing (GSG), or supplemented cold season grazing (SG) , while NSG had intermediate richness between the other treatments (p > 0.05) , 12  and 3  bacterial taxa significantly associated with NSG, GSG and SG yak rumen, respectively  was significantly higher in the GSG rumen (1611) than in the SG rumen (1360) (eatments . Shannon > 0.05) . Princip > 0.05) , with GSectively .Bacteroidetes (53.97%) and Firmicutes (37.16%) were the dominant phyla in all three treatment groups  comprising the next most abundant taxa. These four phyla with highest relative abundance accounted for >90% of all bacterial ASVs. At the genus level, the predominant genera included Rikenellaceae_RC9_gut_group (24.25%), uncultured_bacterium_f_F082 (8.01%), and Prevotella_1 (5.12%) in all samples  but lower abundance of Firmicutes . Yak rumen from GSG also had the highest abundance of Actinobacteriota among all the groups . At the genus level, the relative abundance of Bacteroides in NSG was higher than that in the GSG rumen , while Lachnospiraceae_ND3007_group was significantly enriched in SG  and Succinivibrionaceae_UCG-002 was significantly higher in GSG rumen  compared with other groups (p < 0.05) species level ASVs such as Fibrobacter succinogenes, Butyrivibrio fibrisolvens, and Ruminococcus albus. The relative abundance of Fibrobacter succinogenes and Butyrivibrio fibrisolvens in SG was significantly higher  than that in NSG, while GSG rumen had the higher abundance of Ruminococcus albus than that in SG group   . These cBased on the observed differences in bacterial communities between treatments, metagenomic sequencing of the fungal ITS hypervariable region was conducted to examine changes in rumen fungi, which are well known to contribute to cellulose degradation in ruminants. In total, 92,749 cleaned reads were obtained for each sample after splicing and filtering, with a total of 1,441 ASVs detected in the three groups combined after denoising. Among these fungal ASVs, 213 were common to all three groups, while 395 were unique to NSG rumen, 383 were unique to SG, and 200 were exclusively detected in GSG .p < 0.01) , 17 , and 26  fungal taxonomic groups that were significant indicator taxa in the NSG, GSG, and SG rumen, respectively  and species richness (Chao 1) in yak rumen indicated that both indexes were lower in GSG than in the NSG and SG rumen ( < 0.01) ,B. PCoA  < 0.01) . Subsequectively .Neocallimastigomycota (81.29%) and Ascomycota (12.91%) were the most abundant phyla in all rumen samples . At the genus level, predominant genera included Piromyces (20.13%), Orpinomyces (19.01%), and Neocallimastix (16.56%) in all samples. Less prevalent but significantly abundant genera included Caecomyces, Cyllamyces, Aspergillus, Anaeromyces, and Penicillium, accounting for 12.00, 10.26, 7.16, 1.37, and 1.28% of fungal ASVs, respectively  and Neocallimastix was lower . Yak rumen in the SG rumen had the highest abundance of Anaeromyces among the three groups , while Cyllamyces was enriched in NSG compared to SG , and Buwchfawromyces was more abundant in NSG than in the GSG rumen (p < 0.05)  . In summIn addition to bacterial and fungal contribution to rumen metabolism, we hypothesized that rumen archaea may also vary under different nutrient conditions. To test this possibility, metagenomic sequencing of the archaeal was conducted in rumen of yaks from each treatment group, which resulted in 1,699,212 clean reads, after quality control and filtering, from all samples combined. On average, 83,546 valid sequences were obtained for each sample, which contained 1,997 total ASVs. There were 164 ASVs shared by all three groups, while much larger proportions were unique to each grazing simulation, including 545 ASVs exclusive to NSG rumen, 428 ASVs unique to SG rumen, and 481 ASVs only found in GSG rumen .p < 0.05), although species richness (Chao 1) was not significantly different among the three groups  and Aenigmarchaeota (0.02%). At the genus level, Methanobrevibacter (81.03%) and Methanosphaera (2.47%) were predominant across samples (Methanosphaera in NSG was significantly lower than in GSG (p < 0.05), but did not differ from that in SG  indicated that SG (4.8) had significantly higher diversity than in GSG (4.4) and NSG (3.9) . In addition, fungal function prediction using FUNGuild software showed that pathotrophs, symbiotrophs, and saprotrophs were the major components. Plant_Pathogen and Endophyte-Plant_Pathogen were significantly enriched in SG than that in NSG (p < 0.05), and Animal_Pathogen-Endophyte-Plant_Pathogen-Wood_Saprotroph was significantly more abundant in SG than that in GSG (p < 0.05). Moreover, the relative abundance of Plant_Pathogen-Soil_Saprotroph-Wood_Saprotroph was significantly higher in GSG than that in NSG (p < 0.05)  .p.251.o5 was negatively correlated with acetate and propionate concentrations ; Lachnospiraceae_ND3007_group showed a positive correlation with valerate and isovalerate concentrations , and Christensenellaceae_R-7_group was negatively correlated with butyrate, valerate and isovalerate concentrations . In addition, the Ruminococcaceae_NK4A214_group bacterial ASV was significantly negatively correlated with all VFAs except isovalerate . Among fungal ASVs, the relative abundance of Orpinomyces was significantly positively correlated with acetate concentration , while Piromyces was negatively correlated with acetate and isobutyrate concentrations . At the genus level, Penicillium and Candida were positively correlated with valerate and acetate concentrations, respectively .Based on the above detection of microbial taxa significantly associated with different grazing simulation treatments, we next conducted Spearman’s correlation analysis to determine whether and which yak rumen microbes shared an association with significant VFAs . This anDeep_Sea_Euryarchaeotic_Group (DSEG) was positively correlated with Rikenellaceae_RC9_gut_group  and Cyllamyces . Methanosphaera ASV counts were positively correlated with Succinivibrionaceae_UCG-002, Quinella, Orpinomyces, and Aspergillus ; Methanosphaera was negatively correlated with Neocallimastix and Cyllamyces . Rikenellaceae_RC9_gut_group, Prevotella, and F082 were negatively correlated with Bacteroides and Buwchfawromyces, Papillibacter and Methanobrevibacter, Bacteroides, and Cyllamyces, respectively . There were also significant positive correlations between Lachnospiraceae_ND3007_group and Penicillium, between Orpinomyces and Succinivibrionaceae_UCG-002, between Papillibacter and Anaeromyces, and between Cyllamyces and Buwchfawromyces. By contrast, negative correlations in ASV counts were identified between Piromyces and Methanobrevibacter, between Cyllamyces and Aspergillus, between Succinivibrionaceae_UCG-002 and Neocallimastix, and between Quinella and Cyllamyces .Analysis of mutual correlations among high-abundance rumen microbiota  at the genus level  showed tYak is the only ruminant on the Qinghai Tibetan plateau that utilizes pasture year-round. Previous work has shown that yak rumen microbiota is robust to seasonal changes in forage availability, potentially enhancing the efficiency of fiber degradation during winter months . HoweverVolatile fatty acids reportedly participate in maintaining rumen homeostasis by providing an ideal acidic environment for the rumen microbiota , and VFAR. flavefaciens and F. succinogenes are both known to produce large amounts of succinate through fiber degradation, which is increased by conversion to propionate . At the species level, R. albus was more abundant in GSG rumen than that in SG rumen, while F. succinogenes, B. fibrisolvens, and R. flavefaciens were more abundant in SG samples than NSG. Together with the higher Firmicutes levels in NSG than GSG, enrichment with these known fiber-degrading bacteria  was significantly positively correlated with Lachnospiraceae_ND3007_group (Firmicutes), although further experiments and functional analysis based on metagenomic study are necessary to determine whether these clades can directly cooperate to utilize different components of the plant fiber.Yak rumen contain a complex network of symbiotic microorganisms that can synergistically ferment plant fibers, providing nutrients for themselves and the host . In thisThis study comprehensively explores the response of yak rumen microbiota to simulated modes of seasonal feeding to exclude the impact of environmental factors. Our results show that different microbes in yak rumen gain dominance under (simulated) seasonal changes in nutrient availability, with relatively high nutrient availability promoting enrichment for known fiber degrading bacteria, and nutrient stress selecting for bacteria and fungi that are competitive under low nutrient availability and enhance the efficiency of host nutrient utilization. This study expands the scope of our understanding of microbial alteration and interactions under fluctuating nutrient conditions. Our findings provide a framework for future studies examining precise nutrient interventions or cold season probiotic treatments to enhance yak rumen function in nutrient utilization.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: NCBI SRA database with accession number PRJNA863131.XH and RL conceived the study and designed the experiments. XY and XF performed DNA extraction and drafted the manuscript. XY, HJ, QZ, Basangwangdui, SD, and QZ coordinated in animal experiment and sample collection. XY, XF, and XH analyzed the data and contributed to data interpretation. All authors contributed to the article and approved the submitted version.This work was supported by National Key R&D Program of China (2021YFD1200904), Special Item of Regional Collaborative Innovation in Tibet Autonomous Region  and the Open Project Program of State Key Laboratory of Barley and Yak Germplasm Resources and Genetic Improvement (XZNKY-2021-C-014-K06) and National Modern Agricultural Technology System (CARS-37).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cognitive testing and EP symptoms  were assessed at baseline and follow-up (mean follow-up time 12 months). Mild EP symptoms were present at treatment onset in 40% of the participants. EP symptoms were related with lower performance in neurocognitive testing at baseline and at follow-up, especially among those with nonaffective psychotic disorder, and especially in tasks requiring speed of processing. No associations between EP symptoms and social cognition were detected. In linear regression models, when positive and negative symptom levels and chlorpromazine equivalents were accounted for, baseline EP symptoms were associated with worse baseline global neurocognition and visuomotor performance. Baseline EP symptoms also longitudinally predicted global, verbal, and visuomotor cognition. However, there were no cross-sectional associations between EP symptoms and cognitive performance at follow-up. In sum, we found both cross-sectional and longitudinal associations between EP symptoms and neurocognitive task performance in the early course of psychosis. Those without EP symptoms at the start of treatment had higher baseline and follow-up neurocognitive performance. Even mild EP symptoms may represent early markers of long-term neurocognitive impairment.Extrapyramidal (EP) symptoms such as tremor, rigidity, and bradykinesia are common side effects of most antipsychotics, and may associate with impaired performance in neurocognitive testing. We studied EP symptoms in first-episode psychosis (FEP;  Most antipsychotic medications have a potential to induce EP symptoms as a side-effect4. The first generation, but also the second generation antipsychotics, are known to induce EP symptoms5. It has also been reported that motor impairments associate with clinical features of psychotic illness, such as negative symptoms2, although not all studies have found such associations7. Motor impairments may also index disease severity8 and predict a worse outcome of psychosis11.Extrapyramidal (EP) symptoms are caused by disordered dopaminergic regulation of movement, and are prototypically seen as parkinsonian symptoms, such as tremor, rigidity, and slowed movement. Mild EP symptoms are common already among individuals experiencing their first psychotic episode12. Similarly to EP symptoms, cognitive deficits associate with negative symptoms13, a more severe clinical picture, and worse functional outcome15. While cognitive performance may be influenced by antipsychotics16, the association is complex17.Cognitive impairment is a common feature of psychotic disorders9. In individuals with schizophrenia, severe EP symptoms have been associated with worse neurocognitive performance when controlling for severity of psychopathology, both at a composite score level and in several cognitive domains such as verbal memory, processing speed, and working memory18. In another study, motor impairment predicted a cognitive factor in people with schizophrenia when age, gender, and education were controlled for19. In individuals with schizophrenia or schizoaffective disorder, some cognitive tests correlated with psychomotor abnormalities20. Neurocognitive performance has been reported to associate with EP symptoms also among outpatients with schizophrenia at baseline and 6-month follow-up, when controlling for anxiety and depression21. Furthermore, a recent study among people with schizophrenia found that neurocognition, but not social cognition, was directly correlated with EP symptoms5. Studies on the association between EP symptoms and cognition at the beginning of psychotic disorders have been scarce. Among persons with first-episode psychosis (FEP), EP signs longitudinally associated with deficits in memory, executive functioning, and attention in 6-month follow-up assessments22. However, not all studies have found connections between motor and cognitive impairments in FEP6.Motor impairments have been found to be linked to greater cognitive impairment in psychotic disorders9, and could thus be an intrinsic feature and a possible endophenotype of psychotic disorders, even marking liability to schizophrenia23. EP symptoms have been described in neuroleptic-naïve patients, unaffected first-degree relatives, and high psychosis risk individuals24. In one study, motor impairment predicted the cognitive performance not just among people with schizophrenia, but also among their unaffected first-degree relatives19. Cognitive deficits are similarly evident also in groups with psychosis risk symptoms and among unaffected relatives25. Abnormal motor performance associated with impaired cognition across multiple domains in individuals with high psychosis risk26.EP symptoms do not seem to merely reflect medication side effects but may represent a neurobiological mechanism related to the etiology of psychosis. It has been proposed that in addition to being antipsychotic drug induced, motor impairments in psychotic disorders may be spontaneous27, which measures motor effects such as tremor and stiffness. We investigated how EP symptoms associated with performance in cognitive testing during the first year after the onset of FEP. We took into account the daily dose equivalents of antipsychotic medications and the severity of psychosis symptoms, since antipsychotic medication may be partially or wholly responsible for these associations.As cognitive deficits are among the symptoms that severely interfere with daily functioning in psychosis, it is important to understand factors that may influence cognitive performance. Motor symptoms have been a part of the psychosis concept since Kraepelin, however, here we investigated the implications of mild EP symptoms instead of more severe abnormal psychomotor behaviors. We assessed EP symptoms with the Simpson-Angus Scale5. EP symptoms such as psychomotor retardation are associated to prolonged reaction times resulting in poor performance in cognitive tasks dependent of motor activity18. Therefore, we sought to determine whether current EP symptoms associated with deficits in motor and speed-reliant tasks, with the main hypothesis that even mild EP symptoms could impair performance in these, but not other types of tasks.A possible mechanism explaining the associations between EP symptoms and cognition is that performance in cognitive tasks is affected by motor disturbances caused by the EP symptoms22, we tested a secondary hypothesis of whether EP symptoms act as a prognostic marker for cognitive symptoms by testing for longitudinal associations between baseline EP symptoms and cognitive performance a year later. Investigating cognitive correlates of minor motor abnormalities in FEP in a follow-up setting separates this work from many previous studies.Additionally, based on previous results on the predictive value of EP symptoms on disease severityp = 0.238) or gender (p = 0.140). FEP participants from the two sites did not differ in age (p = 0.891), gender (p = 0.213), or the symptom sum scores at either time point (p ≥ 0.085). In the controls, there was a difference between the sites in the gender distribution (p = 0.001) but not in age. Participants attending or not attending follow-up did not differ in terms of baseline cognition (g factor p = 0.090), EP symptoms (p = 0.321), or negative symptoms (p = 0.371), but those attending follow-up had lower baseline positive symptoms (p = 0.023).The participants with cognitive data included 256 persons: 113 with FEP (71 from the Helsinki site and 42 from the Turku site), as well as 143 control participants (62 from Helsinki and 81 from Turku). Social cognition data were available for 66 FEP participants and 62 controls from Helsinki. Of the FEP group, 82% were diagnosed with nonaffective psychotic disorder (ICD diagnosis codes F20–29) and 18% with affective psychotic disorder (psychotic depression or bipolar disorder). The most common antipsychotics among patients were risperidone (36%), olanzapine (34%), and quetiapine (20%) at baseline, and olanzapine (17%), aripiprazole (14%), risperidone (13%), and quetiapine (11%) at follow-up. Supplementary Table p < 0.001). Participants with FEP from the two sites did not differ in terms of baseline or follow-up cognitive factors.Cognitive results can be seen in Table g factor = −0.34, p = 0.006).Rank-order correlations between measures in the FEP group are shown in Supplementary Table g factor = −0.23, p = 0.017). At follow-up, neurocognitive factors had significant negative correlations with both positive and negative symptom levels.As for the associations between cognition and symptoms, at baseline, negative symptoms correlated negatively with all cognitive factors .Single EP symptom scores are presented in Supplementary Table 2p > 0.05). Although baseline EP symptoms did not differ between diagnostic groups, at follow-up EP symptoms were more common among participants with nonaffective psychosis (Χ2p = 0.021).EP symptoms were not associated with age or gender in the FEP group Table . EP sympp = 0.074; Table The presence of baseline EP symptoms was unrelated to symptom severity. Negative symptom levels tended to be higher in patients with any baseline EP symptoms (mean 2.2) than in those without them mean 1.7) but the difference did not meet statistical significance (.7 but thp = 0.001), but not with positive symptoms.At follow-up, having EP symptoms was associated with more severe negative symptoms . Of the patients with follow-up EP symptom data, out of the 60 patients still using antipsychotics, 43% presented with EP symptoms, compared to 24% of the 17 not using antipsychotics (Χ2p = 0.139).Follow-up EP symptoms and CPZE were not associated either (As can be seen in Table p ≥ 0.240). In the nonaffective group, baseline EP symptoms were associated with lower baseline g factor (p = 0.008) and verbal (p = 0.012) and visuomotor performance (p = 0.006) but not social cognition (p = 0.126), as well as with the follow-up g factor (p = 0.009), verbal (p = 0.009), and visuomotor (p = 0.013) performance.These associations were not significant among those diagnosed with affective psychosis , but not with follow-up general neurocognition (p = 0.066) or verbal performance (p = 0.110). None of these associations reached statistical significance if the FEP group was divided into diagnostic groups.In addition, in the whole FEP group, having EP symptoms at follow-up was associated with lower follow-up visuomotor performance , β = −0.2, p = 0.037; model R2 = 0.08, adj. R2 = 0.04). Looking at the two neurocognitive domains separately, with the same predictors, baseline EP symptoms were associated with lower baseline visuomotor performance , β = −0.2, p = .021). However, baseline EP symptoms did not significantly associate with verbal performance , β = −0.2, p = 0.096) or social cognition , β = −0.1, p = 0.561).In linear regression models, when controlling for baseline positive and negative symptoms and CPZE (which were not significant predictors), having baseline EP symptoms was associated with a lower baseline g factor a year later , β = −0.3, p = 0.013; model R2 = 0.19, adj. R2 = 0.14), again controlling for the same baseline variables. Using the two neurocognitive factors, baseline EP symptoms predicted both follow-up Verbal , β = −0.3, p = 0.007) and follow-up Visuomotor performance , β = −0.3, p = 0.017).In regression models predicting follow-up neurocognition with baseline EP symptoms, having EP symptoms at illness onset predicted a lower g score , β = −0.1, p = 0.659) or Verbal or Visuomotor domains.Finally, follow-up EP symptoms were not a significant predictor of follow-up We investigated extrapyramidal signs as predictors of performance in cognitive testing in the early stages of psychotic illness: soon after entering to treatment and again 9–18 months later (mean follow-up time one year). Gait, elbow rigidity, and tremor scores of the Simpson-Angus Scale were used for assessment of EP symptoms. Forty percent of the individuals with a recent FEP had mild levels of EP symptoms and none had more severe EP symptoms. Before controlling for confounding factors, having EP symptoms at baseline associated cross-sectionally with baseline neurocognitive impairment, as well as longitudinally with follow-up neurocognitive impairment. A year later, cross-sectional associations between EP symptoms and visuomotor neurocognition  were found. No associations between EP symptoms and social cognition were detected.g factor level, and both the verbal and visuomotor domains separately. There were no cross-sectional associations between EP symptoms and cognition at follow-up when controlling for symptom severity and medication.In regression models, we found that soon after the onset of psychosis, EP symptoms associated with neurocognitive impairment. The association was not explained by severity of psychopathology or antipsychotic dose. At the neurocognitive domain level, baseline EP symptoms predicted baseline visuomotor performance, but the association between EP symptoms and verbal performance was not statistically significant when positive and negative symptoms and antipsychotic medication were controlled for. Looking at longitudinal associations, baseline EP symptoms predicted follow-up neurocognition at the composite 18, who found that the association between EP symptoms and cognition could be explained by motor speed, and concluded that EP symptoms affect performance on cognitive tasks rather than core neurocognitive abilities per se. Another study found that processing speed largely explained the cognitive decline in people with first-episode schizophrenia28, highlighting the importance of processing speed on cognition. In addition, the role of deficits in executive functioning, such as initiation and planning, is crucial in the neurocognitive testing situation, and can be linked to response inhibition or delays.We had hypothesized that motoric slowing and stiffness would specifically affect processing speed but not necessarily the other cognitive domains. The tasks with speed limits , where slowing down clearly affects performance, were indeed significantly associated with EP symptoms on the single task level  method has its limitations, because DDDs have not been developed for the purpose of determining dose equivalence of dopamine receptor blockade33.To our knowledge, only a few studies37. In the current study, anticholinergic medication was not controlled for, as such medication is very rarely used in Finland. On the other hand, some antipsychotics have anticholinergic effects which may affect cognition. In addition, although other medications, such as benzodiazepines, are not known to induce EP symptoms, they may affect cognitive performance.Whereas our interest was in the milder level of EP symptoms, previous works have typically studied the effect of more severe EP symptoms, for example, the use of anticholinergic medication to treat iatrogenic EP symptoms. Anticholinergic medication has been reported to associate with long-term cognitive impairmentMild EP symptoms were common among young adults with FEP, and not merely as side-effects of antipsychotic medication, but also as a psychosis symptom dimension. Even mild EP symptoms predicted poorer neurocognitive performance at illness onset, and independently from symptom severity or antipsychotic dose. EP symptoms may slow the person down and cause motor coordination difficulties in cognitive testing, but they might also reflect a more ubiquitous state of dopaminergic modulation affecting subcortical cognitive processing. Studying cognitive domains unaffected by psychomotor slowing could further elucidate the nature of these associations.22 or clinical outcome38, irrespective for antipsychotic treatment. This is possibly related to dopamine system dysregulation9, or a disturbance in the cortical-striatal-thalamocortical neuronal network39. Further studies are needed to verify whether EP symptoms at treatment onset predict impaired neurocognitive outcome.The associations between EP symptoms at illness onset and poorer neurocognitive performance a year later also cannot be explained by the effect of EP symptoms during the testing situation. Some earlier FEP studies have found that EP symptoms at illness onset may be markers of later neurocognitive impairment40. The participants were recruited from hospitals and outpatient clinics in 2010–2017 and in both sites interviewed with the Brief Psychiatric Rating Scale, Expanded version 4.0 (BPRS)41 as soon as possible after they had commenced treatment and were able to provide informed consent, as judged by the treating personnel. Of the FEP participants in Turku, 32 persons were interviewed with the Positive and Negative Syndrome Scale (PANSS)42 instead. Both sites used the Structured Clinical Interview for the DSM-IV, Research Version (SCID-I/P)43, with trained research staff conducting the interviews. Diagnoses were set by a senior psychiatrist based on SCID as well as medical records from received mental health treatment. As a criterion for inclusion in Helsinki, psychosis was defined as a score ≥ 4 (moderate or higher) in BPRS unusual thought content (delusions) or hallucinations. In Turku, the inclusion criterion was a psychotic disorder as defined by the Structured Interview for Prodromal Syndromes 5.0 Presence of Psychotic Symptoms criteria44, complemented by medical records. Exclusion criteria at both sites were substance-induced psychoses and psychotic disorders due to a general medical condition.Participants were recruited from two geographically distinct Finnish sites, Helsinki and Turku, both including young adults with first psychiatric treatment contact for affective and non-affective psychosisIn addition, age- and gender-matched control participants from the same catchment areas were recruited through the Finnish Population Information System. The exclusion criteria were psychotic disorder, conditions preventing MRI, and chronic neurological or endocrinological diseases, but other mental health problems were allowed. Controls were assessed with the same measures as the FEP group.29. The controls were assessed at baseline and again one year later. The Turku Early Psychosis Study included baseline and 9–12 month assessments for FEP and control participants45. In the whole FEP group used here, the follow-time varied between 272–539 days, mean ± SD 369 ± 47 days.The Helsinki Early Psychosis Study protocol included one meeting as soon as possible after the treatment had started (baseline) and follow-ups after two and twelve months. The cognitive testing was performed both at two months  and twelve months46.The participants gave written informed consent to participation. The study protocols were approved by the Ethics Committees of the Hospital Districts of Helsinki and Uusimaa and Southwest Finland, and by the institutional review boards of the Finnish Institute for Health and Welfare and the University of Helsinki. The study was carried out in accordance with the sixth version of the Declaration of Helsinki47 and the Wechsler Memory Scale, Third Edition 48, as well as the Trail Making Test49, and the Verbal Fluency test (semantic and phonemic)50.Cognitive testing was administered by a psychologist at two time points to both FEP and control participants. In the present study, we use data for those tasks which were in use at both sites. These include measures from the Wechsler Adult Intelligence Scale, Third Edition g factor model was estimated with Mplus 8.151. In addition, we used a confirmatory two-dimensional factor model separating the correlated Verbal and Visuomotor factors, which has been presented in our previous work40. Supplementary Table To summarize baseline neurocognitive performance, a one-dimensional For the follow-up cognition, factor scores were calculated using the baseline models with the same parameters, instead of estimating the models again. All the same tasks were used except for Vocabulary, which was not included at follow-up. The model descriptors of baseline and follow-up neurocognitive models are presented in Supplementary Table 52. We have previously obtained a one-dimensional factor solution of the Hinting task53, taking into account the varying difficulty level and relevance of the task items, and the factor scores were used in the analyses as a measure of social cognition.In addition, but only at the Helsinki site as part of the first testing, the theory of mind domain of social cognition was assessed with the Hinting taskThe cognitive factor scores were transformed into age- and gender-corrected residuals based on the performance of controls at baseline, to be used in all analyses.27 in each study phase. On this scale, ratings from 0 to 4 are used, with 0 indicating “normal” motor functions, 1 indicating mild impairment, and higher scores increasingly severe impairment. Raters were trained research staff who also conducted clinical interviews. In Helsinki, five items were used: gait, arm dropping, elbow rigidity, leg pendulousness, and tremor. In Turku, items gait, elbow rigidity, and tremor were included. Here, we used the three items common to both sites: gait, elbow rigidity, and tremor. We divided the participants into those having 0 points on all three items and those having any EP symptoms. Supplementary Table EP symptoms were evaluated using shortened versions of the Simpson-Angus Scale40, we used symptom equivalents for the FEP subgroup interviewed with PANSS instead of BPRS. We considered PANSS item P3 Hallucinatory behavior to correspond to BPRS item 10 Hallucinations, P1 Delusions in PANSS to correspond to item 11 Unusual thought content in BPRS, PANSS P2 Conceptual disorganization to correspond to BPRS item 15 Conceptual disorganization, and PANSS N1 Blunted Affect to correspond to BPRS item 16 Blunted affect.Similarly as in our previous work combining FEP participants from the two sitesAt both time points, positive psychotic symptoms were calculated as the sum of hallucinations, unusual thought content, and conceptual disorganization item scores, while blunted affect was used to assess negative symptoms.33 are reported.Information on medication was collected from interviews and medical records. The DDD based CPZE54. The limit for statistical significance was p < 0.05. The age- and gender-corrected factor score residuals for the composite g factor, the two-dimensional neurocognitive factors, and the social cognition factor were used in the analyses. We also looked at neurocognitive correlates of EP symptoms on a single task score level.For statistical analyses we employed IBM SPSS Statistics for Windows, version 272 or Mann-Whitney U tests. Spearman rank-order correlations (r) were used to examine the associations between continuous variables of interest, such as cognitive factor scores, symptom levels, and CPZE levels. To adjust for multiple comparisons in Supplementary Table 55.The participant groups were compared with Pearson’s ΧU test, to see whether any associations would replicate , and then longitudinally between baseline EP symptoms and follow-up cognition . These associations were investigated also separately in the two diagnostic groups. Common language effect sizes (CL) were calculated from Mann-Whitney values as U/n1 × n2.Cross-sectional associations between EP symptoms and cognition at both time points were calculated separately with the Mann-Whitney 2 and adjusted R2 values. In addition, standardized β values are provided to allow comparability between models.Linear regression models were estimated in the FEP group, controlling for antipsychotic medication (CPZE), positive symptoms, and negative symptoms. The models predicted (1) baseline cognition with baseline predictors  (2) follow-up cognition with the same baseline predictors, and (3) follow-up cognition with follow-up predictors . For these regression models, we report unstandardized B coefficients with 95% confidence intervals (CI) as well as RLindgren Supplement


Bile acid (BA) metabolism may be influenced by gut dysbiosis and alterations of intestinal epithelium in patients with Crohn's disease (CD). Here, we aimed at investigating the alterations of serum BA profile in CD patients and analyzing the correlation between BAs and CD disease activity.  A total of 62 CD patients (29 active and 33 remission) and 33 healthy volunteers (HVs) were enrolled in this retrospective study. Serum BA profiles were measured by liquid chromatography-tandem mass spectrometry.  Levels of primary BAs components, including cholic acid (CA) and chenodeoxycholic acid (CDCA), showed no significant difference when compared with HVs. Secondary BAs (SBA) were significantly decreased in CD patients compared with HVs. Importantly, the deoxycholic acid (DCA) and glycodeoxycholic acid (GDCA) levels were significantly lower in CD active than in CD remission patients. The DCA/(DCA + CA) ratio was also decreased in CD active patients than in CD remission patients while the LCA/(LCA + CDCA) ratio showed no difference between them. Principal component analysis also indicated a clear separation among the three groups, with a total variance of 93.43%. The correlation analysis showed that the SBA, DCA, GDCA levels, and DCA/(DCA + CA) ratio had an inverse relationship with Crohn's Disease Activity Index.  The BA profile exhibits significant alterations in CD patients. The SBA, DCA, GDCA levels, and DCA/(DCA + CA) ratio were significantly decreased in CD active patients. The DCA/(DCA + CA) ratio had an inverse correlation with CD disease activity. Crohn's disease (CD) represents a relapsing inflammatory bowel disease (IBD) featuring chronic abdominal pain, diarrhea, obstruction, and perianal disease . While pα-hydroxylase (CYP7A1) catalyzes the transformation of cholesterol to primary BAs, including cholic acid (CA) and chenodeoxycholic acid (CDCA) [α-dehydroxylate of the gut microbiota, involving deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), and lithocholic acid (LCA) [In the liver, cholesterol 7d (CDCA) . Primaryd (CDCA) , 7. The id (LCA) .Clostridium subcluster XIVa, serving for 7α-dehydroxylation, is positively correlated with a low DCA/(DCA + CA) ratio [Firmicutes ruminococcaceae with the SBA levels (such as DCA and LCA) in ulcerative colitis patients [Several previous studies analyzed the BA concentration and gut microbiota composition to conduct targeted metabolomics investigations in IBD patients. For example, Duboc et al. revealed a significantly increased ratio of conjugated BAs, paralleled by decreased levels of SBAs in fecal samples of active IBD patients . MoreoveA) ratio . In addipatients . AltogetOther studies have shown that due to decreased diversity of microbiota, SBAs could be reduced in CD patients , 11–13. n = 33) comprised clinically healthy subjects from the Medical Examination Center of the Zhongda Hospital and had no prior history of liver dysfunction or antibiotics taken over the past 3 months. The study was approved by the Ethics Committee of Zhongda Hospital (2021ZDSYLL297-P01).A total of 62 CD patients were consecutively enrolled in this study. All the subjects were hospitalized in the Department of Gastroenterology, Zhongda Hospital of Southeast University, from August 2020 to August 2021. The CD diagnostic criteria were corroborated with clinical, imaging, endoscopic, and histological manifestations based on the European Crohn's and Colitis Organisation consensus . The excSerum samples were collected from all subjects after overnight fasting. BA levels were measured by liquid chromatography-tandem mass spectrometry. Other laboratory data, such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were obtained and documented at the clinical laboratory of the Zhongda Hospital. Data collected from the CD cohort includes age, gender, disease duration, site of disease, and medications. Furthermore, the CDAI score was calculated to differentiate CD patients from active to remission period and the CDAI score <150 was considered as the remission period .t-test, expressed as mean ± standard deviation (SD). Otherwise, the Mann–Whitney U test was employed for comparisons of nonparametric data, expressed as median (interquartile range). The correlation was assessed by the Pearson's correlation coefficient or nonparametric Spearman's rank correlation coefficient. Metabolomic analysis was performed by heatmaps and principal component analysis (PCA) to assess the separation between groups using R software (version 3.6). Variables were mean-centered and divided by the SD before statistical analysis in the heatmap. In all statistical tests, P < 0.05 was considered as statistically significant.Statistical analysis was performed with SPSS Statistics 23.0 and GraphPad Prism 8.4.0 software. The analysis involved tests of normality and homogeneity of variance. The differences between groups were assayed for parametric by one sample P > 0.05) was documented. About 3% of CD active patients, but no CD remission patients were administered with corticosteroid therapy. 72% of CD active and 85% of CD remission patients were treated with biological agents, such as anti-tumor necrosis factor α (anti-TNF α) or anti-α4β7 integrin antibodies, but there was no significant difference in the exposure to biologics between CD active and remission patients. CD active patients had significantly higher CRP and ESR levels than CD remission patients (P < 0.001), and CDAI scores could differentiate them from active to remission period.In this retrospective study, we included 62 CD patients and 33 controls out of which 29 were CD active patients  and 33 CD remission patients . Healthy volunteers (HVs) involved 20 males and 13 females, with an average age of 46.0 ± 10.9 years . No signP < 0.01; P < 0.05; Concentrations of total and individual BAs were detected in different groups . GeneralIn order to further explore the metabolic differences within these samples, PCA analysis was conducted. Our analysis indicates a clear separation among the CD active, CD remission, and HVs when involving all identified BAs . The varα-dehydroxylation [Having delineated that gut microbiota contributes to the transformation from primary to SBAs via 7xylation , we sougrs = −0.336, P = 0.008), shown in rs = −0.453, P < 0.001) was also documented [As several BAs were significantly altered between CD active and remission patients, we analyzed the correlation of them with CDAI scores . SBAs wecumented .Our investigations revealed that significant alterations of the serum BA profile in CD patients. SBAs, such as DCA and GDCA, were significantly lower in CD active than in CD remission patients. Furthermore, the DCA/(DCA + CA) ratio was also decreased in CD active patients. The heatmap substantiated these findings and the PCA analysis revealed a clear separation within these groups. In addition, the DCA/(DCA + CA) ratio had an inverse correlation with the clinical disease activity of CD patients.α therapy. Yang et al. investigated the association of decreased fecal BAs and gut microbiota dysregulation and the mechanism of inflammatory responses through BA-activated receptors in patients with ulcerative colitis [Previous studies had reported that the alterations of both serum and fecal BA concentrations in IBD patients. Duboc et al. found that low SBAs could be associated with the impaired gut microbiota . However colitis . Howeverα-dehydroxylate of Clostridium subcluster XIVα and represents the conversion from primary to SBAs [Our study revealed a significant decrease in SBAs in CD patients. SBAs are considered to play an anti-inflammatory role in IBD patients , 19. The to SBAs . Here, wrs = −0.336). Furthermore, the DCA and GDCA levels showed an inverse correlation with CD activity . The DCA/(DCA + CA) ratio was also negatively correlated with CDAI scores (rs = −0.453). Overall, it seems likely that increased inflammatory responses tend to decrease SBA levels in CD patients with higher CDAI scores.However, the association between the BA profile and disease activity still remains elusive. Given the alterations of several BAs among the CD active, CD remission, and healthy population, we evaluated the correlation of these indicators and disease activity. SBAs had a negative relationship with CDAI scores  ratio may be a potential biochemical signature for delineation of the intestinal inflammation in CD patients.