digestive

消化性
  • 文章类型: Journal Article
    为了研究肠道消化酶的活性,肝脏抗氧化酶,免疫酶,在Biofloc模型下,大嘴鲈鱼(Micropterussalmoides)中的糖代谢酶,在300升玻璃罐中进行了一项实验。实验包括一个对照组,以基础饮食喂养,和一个biofloc组,其中添加葡萄糖以保持C/N比为15。每组有三个平行设置,每个鱼缸放养密度为20条鱼。实验持续了60天,采用零水交换水产养殖模式。结果表明,在培养期结束时,初始体重之间没有显着差异,最终重量,WGR,SGR,Biofloc组和大口鲈鱼对照组的SR(p>0.05),而biofloc组较低的FCR和较高的PER是显著的(p<0.05);肠α-淀粉酶,胰蛋白酶,Biofloc组大口鲈鱼的脂肪酶活性显着增加了37.20%,64.11%,和51.69%,分别,与对照组相比(p<0.05);肝脏超氧化物歧化酶和过氧化氢酶活性,Biofloc组大口鲈鱼的总抗氧化能力显着提高了49.26%,46.87%,98.94%(p<0.05),而丙二醛含量显着降低了19.91%(p<0.05);肝脏溶菌酶,碱性磷酸酶,Biofloc组大嘴鲈鱼的酸性磷酸酶活性显着提高了62.66%,41.22%,29.66%,分别(p<0.05);肝脏葡萄糖激酶,丙酮酸激酶,葡萄糖-6-磷酸激酶,丙酮酸激酶,葡萄糖-6-磷酸酶,糖原合成酶活性显著提高46.29%,99.33%,32.54%,和26.89%,分别为(p<0.05)。研究表明,培养大嘴鲈鱼的Biofloc模型不仅可以增强消化酶活性,抗氧化能力,和免疫反应,但也可以促进葡萄糖代谢过程和降低饲养成本。本研究为今后生产大嘴鲈鱼的健康养殖提供数据支持。为优化Biofloc技术培养模式提供了理论参考,对于促进水产养殖健康绿色发展至关重要。
    To investigate the activities of intestinal digestive enzymes, liver antioxidant enzymes, immunological enzymes, and glucometabolic enzymes in largemouth bass (Micropterus salmoides) under the biofloc model, an experiment was conducted in 300-liter glass tanks. The experiment comprised a control group, which was fed a basal diet, and a biofloc group, where glucose was added to maintain a C/N ratio of 15. Each group had three parallel setups, with a stocking density of 20 fish per tank. The experiment ran for 60 days, employing a zero-water exchange aquaculture model. The results showed that at the end of the culture period, there were no significant differences between the initial weight, final weight, WGR, SGR, and SR of the biofloc group and the control group of largemouth bass (p > 0.05), whereas the lower FCR and the higher PER in the biofloc group were significant (p < 0.05); intestinal α-amylase, trypsin, and lipase activities of largemouth bass in the biofloc group were significantly increased by 37.20%, 64.11%, and 51.69%, respectively, compared with the control group (p < 0.05); liver superoxide dismutase and catalase activities, and total antioxidant capacity of largemouth bass in the biofloc group were significantly increased by 49.26%, 46.87%, and 98.94% (p < 0.05), while the malondialdehyde content was significantly reduced by 19.91% (p < 0.05); liver lysozyme, alkaline phosphatase, and acid phosphatase activities of largemouth bass in the biofloc group were significantly increased by 62.66%, 41.22%, and 29.66%, respectively (p < 0.05); liver glucokinase, pyruvate kinase, glucose-6-phosphate kinase, pyruvate kinase, glucose-6-phosphatase, and glycogen synthase activities were significantly increased by 46.29%, 99.33%, 32.54%, and 26.89%, respectively (p < 0.05). The study showed that the biofloc model of culturing largemouth bass can not only enhance digestive enzyme activities, antioxidant capacity, and immune response but can also promote the process of glucose metabolism and reduce feeding costs. This study provides data support for healthy culturing of largemouth bass in future production, provides a theoretical reference for optimizing the biofloc technology culture model, and is crucial for promoting the healthy and green development of aquaculture.
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  • 文章类型: Journal Article
    安全和抗炎的基于植物的天然产物在慢性炎性疾病如骨关节炎或炎性肠病的治疗中日益受到关注。其中,藏红花,一种源自番红花污名的香料,可以具有抗炎特性,因此将是用于治疗此类病症的有希望的治疗剂。然而,藏红花在人类体内的抗炎分子机制尚不清楚。在这项研究中,结合人类血清代谢物和细胞培养物,我们评估了消耗专利藏红花提取物(Safr\'InsideTM)产生的循环代谢物对软骨细胞和结肠上皮细胞对炎症应激反应的影响。使用事后测试进行了参数或非参数方差分析。我们证明了藏红花摄入的含有代谢物的人血清减弱了软骨细胞中IL-1β刺激的PGE2和MMP-13的产生,并限制了ICAM-1,MCP-1,iNOS的增加。IL-1β和TNF-α联合炎症刺激后人上皮细胞中的MMP-3。总之,这些数据为藏红花在细胞水平和慢性炎症性疾病中对软骨细胞和肠上皮细胞有益作用的潜在机制提供了新的发现.
    Safe and anti-inflammatory plant-based natural products present an increasing focus in the treatment of chronic inflammatory diseases such as osteoarthritis or inflammatory bowel diseases. Among them, saffron, a spice derived from the stigma of Crocus sativus, could have anti-inflammatory properties and would be therefore a promising therapeutic agent for the treatment of such conditions. However, the anti-inflammatory molecular mechanisms of saffron in humans are still understudied and unclear. In this study, combining human serum metabolites and cell cultures, we evaluated the effect of circulating metabolites from the consumption of a patented saffron extract (Safr\'InsideTM) on the chondrocytes and colon epithelial cell responses to inflammatory stress. Parametric or non-parametric Analysis of Variance with post hoc tests was performed. We demonstrated that human serum containing metabolites from saffron intake attenuated IL-1β-stimulated production of PGE2 and MMP-13 in chondrocyte cells and limited the increase in ICAM-1, MCP-1, iNOS, and MMP-3 in human epithelial cells following combined IL-1β and TNF-α inflammatory stimulation. Altogether, these data provide new findings into the mechanisms underlying the beneficial effects of saffron on chondrocytes and enterocyte cells at the cellular level and in the context of chronic inflammatory disorders.
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  • 文章类型: Journal Article
    淋巴结基质细胞(LNSC)是免疫系统中经常被忽视的组成部分,但在维持组织稳态和协调免疫反应中起着至关重要的作用。我们对这些细胞在细菌感染情况下的功能的理解仍然有限。我们之前证明了单核细胞增生李斯特菌,兼性细胞内食源性细菌病原体,必须在肠系膜淋巴结(MLN)中尚未确定的细胞类型内复制才能全身扩散。这里,我们证明单核细胞增生李斯特菌可以入侵,逃离空泡,指数复制,并在体外感染的两个LNSC群体的胞质溶胶中诱导I型IFN应答,成纤维细胞网状细胞(FRC)和血液内皮细胞(BEC)。感染的FRC和BEC在体外感染后也产生显著的趋化因子和促炎细胞因子应答。流式细胞术分析证实,在食源性感染小鼠后,GFP+单核细胞增生利斯特氏菌与少量MLN基质细胞相关。使用荧光显微镜,我们发现这些细胞相关的细菌是细胞内的单核细胞增生李斯特菌,在小鼠感染3天的过程中,感染的FRC和BEC的数量发生了变化。这些感染的LNSC群体的离体培养显示出可行的,在琼脂平板上生长的复制细菌。这些结果强调了FRC和BEC在食源性感染期间作为单核细胞增生李斯特菌合适的生长生态位的潜力,并有助于MLN内促进李斯特菌病清除的促炎环境。
    Lymph node stromal cells (LNSCs) are an often overlooked component of the immune system but play a crucial role in maintaining tissue homeostasis and orchestrating immune responses. Our understanding of the functions these cells serve in the context of bacterial infections remains limited. We previously showed that Listeria monocytogenes, a facultative intracellular foodborne bacterial pathogen, must replicate within an as-yet-unidentified cell type in the mesenteric lymph node (MLN) to spread systemically. Here, we show that L. monocytogenes could invade, escape from the vacuole, replicate exponentially, and induce a type I interferon response in the cytosol of 2 LNSC populations infected in vitro, fibroblastic reticular cells (FRCs) and blood endothelial cells (BECs). Infected FRCs and BECs also produced a significant chemokine and proinflammatory cytokine response after in vitro infection. Flow cytometric analysis confirmed that GFP+  L. monocytogenes were associated with a small percentage of MLN stromal cells in vivo following foodborne infection of mice. Using fluorescent microscopy, we showed that these cell-associated bacteria were intracellular L. monocytogenes and that the number of infected FRCs and BECs changed over the course of a 3-day infection in mice. Ex vivo culturing of these infected LNSC populations revealed viable, replicating bacteria that grew on agar plates. These results highlight the unexplored potential of FRCs and BECs to serve as suitable growth niches for L. monocytogenes during foodborne infection and to contribute to the proinflammatory environment within the MLN that promotes clearance of listeriosis.
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  • 文章类型: Journal Article
    背景:最近的证据表明整体饮食质量,根据饮食评分评估,可能在上消化道(UGI)癌症的发展中起作用。然而,现有的饮食评分来自高收入国家,其饮食习惯与UGI癌症负担最高的地区不同,可用数据有限的地方。这项研究旨在调查整体饮食质量与UGI癌症高危地区食管癌和胃癌风险之间的关系。
    方法:我们在2004年至2008年期间从伊朗东北部招募了50045名年龄在40-75岁之间的个体,并每年随访至2020年7月。人口统计数据,饮食,并使用经过验证的问卷收集各种暴露。饮食质量通过计算健康饮食指数(HEI)来评估,替代健康饮食指数(AHEI),替代地中海饮食(AMED),停止高血压(DASH)的饮食方法和世界癌症研究基金-美国癌症研究所(WCRF-AICR)评分。
    结果:在平均12年的随访中,359名参与者发展为食道癌,358人发展为胃癌。调整后,基线饮食评分的每个标准差增加与食管癌风险降低12%和胃癌风险降低17%相关.食管癌与AMED的依从性呈较强的负相关(HRQ4-vs-Q1=0.69(0.49-0.98),P-趋势=0.038)。胃癌与WCRF-AICR呈较强的负相关(HRQ4-vs-Q1=0.58(0.41-0.83),P-趋势=0.004),和DASH(HRC4-vs-C1=0.72(0.54-0.96),P-趋势=0.041)。这些关联在不同的人口亚组之间具有可比性。在该人群中,我们没有观察到HEI和AHEI评分与UGI癌症之间的显着关联。
    结论:尽管个体食物组的消费量不同,在这一高危人群中,坚持现有饮食建议(来自高收入国家)与随后发生食管癌和胃癌的风险较低相关.教育公众拥有健康的饮食习惯可能是预防高危地区UGI癌症的有效策略。
    BACKGROUND: Recent evidence suggests overall diet quality, as assessed by dietary scores, may play a role in the development of upper gastrointestinal (UGI) cancers. However, the existing dietary scores are derived from high-income countries with different dietary habits than regions with the highest burden of UGI cancers, where limited data is available. This study aimed to investigate the association between overall diet quality and risk of esophageal and stomach cancers in a high-risk region for UGI cancers.
    METHODS: We recruited 50045 individuals aged 40-75 between 2004-2008 from northeastern Iran and followed them annually through July 2020. Data on demographics, diet, and various exposures were collected using validated questionnaires. Diet quality was assessed by calculating the Healthy Eating Index (HEI), Alternative Healthy Eating Index (AHEI), Alternative Mediterranean Diet (AMED), Dietary Approaches to Stop Hypertension (DASH), and World Cancer Research Fund-American Institute for Cancer Research (WCRF-AICR) scores.
    RESULTS: During an average 12 years of follow-up, 359 participants developed esophageal cancer and 358 developed stomach cancer. After adjustments, each standard deviation increase in baseline dietary scores was associated with up to 12% reduction in esophageal cancer risk and up to 17% reduction in stomach cancer risk. Esophageal cancer showed stronger inverse associations with adherence to AMED (HRQ4-vs-Q1=0.69 (0.49-0.98), P-trend=0.038). Stomach cancer showed stronger inverse correlation with WCRF-AICR (HRQ4-vs-Q1=0.58 (0.41-0.83), P-trend=0.004), and DASH (HRC4-vs-C1=0.72 (0.54-0.96), P-trend=0.041). These associations were comparable across different population subgroups. We did not observe significant associations between HEI and AHEI scores and UGI cancers in this population.
    CONCLUSIONS: Despite the differences in consuming individual food groups, adherence to the available dietary recommendations (derived from high-income countries) was associated with lower risk for subsequent esophageal and gastric cancers in this high-risk population. Educating the public to have a healthy eating pattern might be an effective strategy towards prevention of UGI cancers in high-risk regions.
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  • 文章类型: Journal Article
    M6A对肿瘤的发生和发展至关重要。m6A调节因子的表达模式在各种肿瘤中不同。分析转录组表达统计以及来自数据库的临床数据以区分患有消化道肿瘤的患者。基于不同的m6A调节剂的表达模式,患者被分为几组。生存分析提示m6A簇中患者预后存在显著差异。结果显示m6A表达模式与能量代谢和核苷酸代谢重叠。功能分析暗示肿瘤细胞中的m6A修饰可能驱动代谢重编程以维持癌细胞的快速增殖。我们的分析强调了m6A风险表征各种代谢特征,并预测消化道肿瘤的化疗敏感性,为m6A调节因子作为预测患者预后的标志物提供证据。
    M6A is essential for tumor occurrence and progression. The expression patterns of m6A regulators differ in various kinds of tumors. Transcriptomic expression statistics together with clinical data from a database were analyzed to distinguish patients with digestive tract tumors. Based on the expression patterns of diverse m6A regulators, patients were divided into several clusters. Survival analysis suggested significant differences in patient prognosis among the m6A clusters. The results showed overlapping of m6A expression patterns with energy metabolism and nucleotide metabolism. Functional analyses imply that m6A modifications in tumor cells probably drive metabolic reprogramming to sustain rapid proliferation of cancer cells. Our analysis highlights the m6A risk characterizes various kinds of metabolic features and predicts chemotherapy sensitivity in digestive tract tumors, providing evidence for m6A regulators as markers to predict patient outcomes.
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  • 文章类型: Journal Article
    消化系统疾病(DDs)是一个全球性的健康问题,具有重大的流行病学和经济影响,鉴于他们的高患病率。
    这项研究调查了1987年至2021年中国与DD相关的死亡率趋势,重点是城乡鸿沟。此外,它旨在确定年龄的具体影响,period,和DDs死亡率队列。
    需要优先考虑和分配更多资源用于未来的疾病管理,以便有效应对城市化和人口老龄化带来的挑战。
    UNASSIGNED: Digestive diseases (DDs) are a global health concern with a substantial epidemiological and economic impact, given their high prevalence.
    UNASSIGNED: This study investigated the trends in mortality related to DDs in China from 1987 to 2021, focusing on the urban-rural divide. Additionally, it aimed to determine the specific impacts of age, period, and cohort on DDs mortality.
    UNASSIGNED: There is a need to prioritize and allocate more resources toward the future management of DDs in order to effectively address the challenges posed by urbanization and aging populations.
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  • 文章类型: Journal Article
    这项研究的目的是通过定量评估恶心等胃肠道症状的患病率,系统分析和总结COVID-19对消化系统的影响,呕吐,腹痛,便秘,腹泻,厌食症.报告COVID-19病例。我们同时调查了其他变量,以确定COVID-19患者中此类症状的相关性,这些症状可能会影响疾病的预后和结局。本系统综述提供了有关该问题的最新文献,因为它需要更多的科学讨论,以便更好地告知医学界和当局,以便可以采取适当措施来控制病毒爆发。
    搜索MEDLINE数据库以识别相关文章。对16项合格研究的数据进行了分析和合成,这些研究专门报道了COVID-19患者的胃肠道症状以及疾病预后。对具有与其他相关独立变量相关的特定胃肠道症状患病率的足够信息的研究进行了荟萃分析。
    从搜索策略来看,我们确定了16篇符合我们资格标准的文章,包括10项横断面研究,2队列研究,1项RCT和3项观察性研究。从这些汇集的研究中,6篇文章专门讨论了COVID-19患者的胃肠道症状,并进行了充分的讨论。在总共3646名患者中,(16.2%-10.1%)患者出现胃肠道症状。最常见的胃肠道症状是腹泻(47%),但最常见的临床表现是发热(77.4%)。在成年患者中,高血压(11.6%)是最常见的合并症.在具有GI症状的16.7%的患者中注意到粪便样品中存在病毒RNA。在抱怨有胃肠道症状的患者中,在很大程度上观察到肝功能异常,ALT水平升高(10.9%)和AST升高(8.8%)的患者。在一项研究中报告了垂直传播的证据(14.2%),该研究强调了病毒传播的程度和模式。据观察,在6项专门报告有胃肠道症状的患者的研究中,绝大多数患者接受抗病毒治疗(68.6%)作为标准疾病管理方案,但最终的疾病结局在这种情况下死亡(8.4%)。出院(45.6%)并不只与一个治疗因素相关,但其他疾病严重程度指标如胸部CT阳性(87.82%)导致疾病预后不良,与有胃肠道症状的非严重COVID-19患者(71.1%)相比,有胃肠道症状的严重患者(28.9%)出现疾病预后不良.
    COVID-19患者中胃肠道症状的存在与可能是直接病毒毒性导致的不良疾病预后呈正相关。医生必须认识到消化症状是COVID-19患者的重要特征。因此,应进行准确和有针对性的胃肠道症状记录和病毒性粪便样本调查,以了解快速演变的疾病症状学.
    UNASSIGNED: The aim of this study is to systematically analyze and summarize the implications of COVID-19 on the digestive system by quantitatively evaluating the prevalence of gastrointestinal symptoms such as nausea, vomiting, abdominal pain, constipation, diarrhea, anorexia. reported in COVID-19 cases. We simultaneously investigated other variables to determine the association of such symptoms in COVID-19 patients which can potentially influence the disease prognosis and outcome. This systematic review presents an updated literature on the issue as it requires more scientific discussion in order to better inform the medical community and authorities so that appropriate measures can be taken to control the virus outbreak.
    UNASSIGNED: MEDLINE database was searched to identify relevant articles. Data was analyzed and synthesized from the 16 eligible studies which exclusively reported GI symptoms in COVID-19 patients along with the disease prognosis. A meta-analysis of studies having adequate information regarding the prevalence of specific GI symptoms in association with other relevant independent variables was performed.
    UNASSIGNED: From the search strategy, we identified 16 articles which fit our eligibility criteria comprising of 10 cross-sectional studies, 2 cohort study, 1 RCT and 3 observational studies. From these pooled studies, 6 articles exclusively talked about COVID-19 patients in which GI symptoms were reported and adequately discussed. In a total of 3646 patients, GI symptoms were documented in (16.2%-10.1%) patients. The most prevalent GI symptom was diarrhea (47%) but the most common clinical manifestation reported was fever (77.4%). Among the adult patients, hypertension (11.6%) was the most frequently reported comorbidity. Presence of viral RNA in stool sample was noted in 16.7% patients with GI symptom. In patients who complained of having GI symptoms, an abnormal liver function was largely observed, with an elevated ALT level in (10.9%) and an elevated AST in (8.8%) of the patients. Evidence of vertical transmission (14.2%) was reported in one study which highlights the extent and mode of viral transmission. It was observed that a great majority of the patients in the 6 studies reporting specifically on patients with GI symptoms were on antiviral therapy (68.6%) as the standard disease management protocol but the eventual disease outcome as in this case died (8.4%), discharged (45.6%) was not linked to just one therapeutic factor but other indicators of disease severity such as positive chest CT findings (87.82%) have led to a poor disease prognosis which was noted in (28.9%) severe patients with GI symptoms compared to (71.1%) non-severe COVID-19 patients with GI symptom.
    UNASSIGNED: Presence of GI symptoms in COVID-19 patients has shown to have a positive association with the poor disease prognosis likely as a result of direct viral toxicity. It is important for the physicians to recognize digestive symptoms as an important characteristic in COVID-19 patients. Hence, precise and targeted documentation of GI symptoms and viral stool sample investigations should be performed in order to understand the rapidly evolving disease symptomology.
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  • 文章类型: Case Reports
    食管吻合口漏是严重的并发症,死亡率高(20-50%)1,尤其是在恶性疾病的术后情况下。它们发生在5-30%的病例中,1通常表现为严重纵隔脓毒症。修复手术已被内窥镜支架的使用所取代,夹子,胶水和真空疗法。后者是最简单和最不容易进一步复杂化。由于内海绵不是市售的,我们介绍了一种自制的床边endo海绵,有明显的成功。
    Oesophageal anastomotic leaks are serious complications with high mortality (20-50%)1 especially in the post-operative setting of malignant disease. They occur in 5-30% of cases,1 usually presenting with severe mediastinal sepsis. Revision surgery has been replaced by the use of endoscopic stents, clips, glue and vacuum therapy. The latter is the simplest and least liable to further complication. As an endo sponge was not commercially available, we introduced a self-made bed-side endo sponge, with evident success.
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  • 文章类型: Journal Article
    我们评估印度西北部胃出口梗阻(GOO)的病因和临床谱的研究显示,恶性原因(54.9%)比良性原因(45.1%)更常见。恶性肿瘤的常见原因是胆囊(37.5%),胃癌(31.8%)和胰腺癌(19.6%);最常见的良性原因是阿片类药物滥用(29%),消化性溃疡(21.6%),摄入腐蚀剂(20.2%)和慢性胰腺炎(12.3%)。
    Our study to evaluate the aetiological and clinical spectrum of gastric outlet obstruction (GOO) in North-west India showed malignant cause (54.9%) was more common than benign (45.1%). Common causes of malignancy were gall bladder (37.5%), gastric (31.8%) and pancreatic carcinoma (19.6%); commonest benign causes were opioid abuse (29%), peptic ulcer disease (21.6%), ingestion of corrosives (20.2%) and chronic pancreatitis (12.3%).
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  • 文章类型: Journal Article
    目的:Roux-en-Y胃旁路术(RYGB)常伴有长期胃肠道(GI)症状。缺乏对这些症状的因果关系的直接机械洞察,但肠道微生物组的变化已被提出发挥作用。通过这项研究,我们旨在调查RYGB之前是否存在与随访期间胃肠道症状相关的微生物易感性,并评估涉及哪些微生物群.
    方法:总共,纳入67例RYGB患者。对手术前和手术后1年获得的粪便样品进行鸟枪宏基因组测序。为了评估胃肠道症状,患者填写胃肠道生活质量指数(GIQLI)问卷,并根据GIQLI总评分和评分变化(手术后与基线比较)分组.使用极随机树预测模型来鉴定与术后胃肠道症状相关的最独特的微生物种类。
    结果:基线和手术后1年样本之间的β多样性差异显着,手术后的微生物组类似于更多的生态失调。鉴定了关于总GIQLI(AUC0.77)或deltaGIQLI评分(AUC0.83)的最具预测性的物种。这些物种中的许多是已知的丁酸产生者或已知支持它们的物种和/或具有抗炎特性的物种,包括肠球菌,普氏粪杆菌,和大反刍动物。
    结论:与高GI评分相关的有益共生肠道菌群与足够的肠道发酵能力相关,这表明这些物种可能对术后胃肠道功能障碍具有保护作用。
    Roux-en-Y gastric bypasses (RYGB) are frequently accompanied by long-term gastrointestinal (GI) symptoms. Direct mechanistic insight into the causation of these symptoms is lacking, but changes in the intestinal microbiome have been proposed to play a role. With this study, we aimed to investigate whether a microbial predisposition exists before RYGB which is associated with GI symptoms during follow-up and to evaluate which microbial groups are involved.
    In total, 67 RYGB patients were included. Shotgun metagenomic sequencing was performed on fecal samples obtained just before and 1 year after surgery. To assess GI symptoms, patients filled out Gastrointestinal Quality of Life Index (GIQLI) questionnaires and were divided into groups based on their total GIQLI score and change in score (postsurgery versus baseline). Extremely randomized tree predictor models were used to identify the most distinctive microbial species associated with postoperative GI symptoms.
    Beta diversity differed significantly between baseline and 1-year post-surgery samples, with the post-surgery microbiome resembling a more dysbiotic profile. The most predictive species regarding total GIQLI (AUC 0.77) or delta GIQLI score (AUC 0.83) were identified. Many of these species are known butyrate producers or species known to support them and/or species with anti-inflammatory properties, including Coprococcus eutactus, Faecalibacterium prausnitzii, and Ruminococcus callidus.
    Beneficial commensal gut microbiota related to a high GI score were associated to adequate intestinal fermentative capacity, suggesting these species might have protective properties against postoperative GI malfunctioning.
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