INTRODUCTION

Diverticular disease (DD) is a common gastrointestinal disease of unknown etiology. The symptoms of DD are similar with symptoms of irritable bowel syndrome (IBS)[1], e.g., abdominal pain, bloating and altered bowel habits, and are present in 10%-25%of subjects[2]. About 1.5%-4% of patients with DD develop diverticulitis at some time during their lives[3,4].An acute attack of diverticulitis may lead to chronic symptoms called post-diverticulitis IBS, in analogy with post-infectious IBS observed after an acute attack of gastroenteritis[5,6]. The hypothesis behind IBS development is that low-grade inflammation and/or altered intestinal gut microbiota in DD may contribute to visceral hypersensitivity and dysmotility with ensuing symptoms[7,8].

高血压是比较多见的一种慢性疾病,以老年群体为主,合并多种疾病。高血压患者的心肾脑肝等器官均会受到损伤,引起各种脏器的衰竭。患者血流动力学出现变化,血循环受到阻力,左心室出现代偿性肥厚,引起心衰,危急患者的生命安全,需要尽早的采取治疗措施。冠脉造影、心脏彩超是对高血压左室肥厚伴左心衰竭进行诊断的方式,心电图对早期的症状难以检测,冠脉造影费用高,有放射性,所以心脏彩超是常用的诊断方式。

The gut microbiota is discussed as important for the etiology and pathophysiology in a wide range of diseases. Bacterial diversity is higher in lean compared to obese individuals, and in healthy states compared to unhealthy states, and some bacterial groups, e.g.,Enterobacteriaceae, are associated with over-weight and inflammation[9-11]. The family Enterobacteriaceae is commonly found in the gut ecosystem, where Escherichia coli is the most abundant species of the family[9]. Low bacterial diversity and increased levels of Enterobacteriaceae/Escherichia coli have been linked to inflammatory bowel disease (IBD) in humans[12-14]. The findings of abundance of Enterobacteriaceae/Escherichia coli in experimental animal models of intestinal inflammation[15], and the ability of these bacteria to induce colitis[16], have strengthened the hypothesis that these bacteria are of importance in the etiology of IBD.

现有变电站均通过调度数据专网接入调控中心D5000系统服务器。通过调度专用数据网(光端机)分出一个站间带宽不小于2 Mb/s(每秒下载速度不少于300 KB/s)的局域网通道接口给网络五防系统使用，使网络五防服务器与控端五防主站、所辖各变电站五防子站组成局域网络，网络结构图如图4所示。

Only a few studies have been performed regarding microbial composition in DD. Recently, lower amounts of Enterobacteriaceae were found in the colon mucosa of DD patients compared with healthy controls[17], whereas higher amounts of Akkermansia and no difference in the Escherichia coli subgroup were found in feces in another DD cohort[18].

The primary aim of the present study was to compare the level of the large Gram-negative bacterial family Enterobacteriaceae and gut bacterial diversity in colon mucosa between consecutive patients diagnosed with DD and patients with normal endoscopic findings.Secondary aims were to evaluate the influence of demography, socioeconomic status, lifestyle habits,inflammatory parameters and gastrointestinal symptoms on the gut microbiota.

MATERIALS AND METHODS

Study population and study design

All consecutive patients referred to elective colonoscopy at the Department of Endoscopy, Skåne University Hospital, Malmö, were invited to participate in the study. All patients were in a stabile clinical condition,and no one suffered from any acute inflammation,such as diverticulitis. The only exclusion criteria were age of ≤ 18 years and inability to understand the Swedish language. The patients were informed in oral and written communications at the arrival to the Department the day of examination. If they agreed to participate, they had to complete a study questionnaire about demography, socioeconomic status, lifestyle habits, family history and medical history, the Visual Analog Scale for Irritable Bowel Syndrome (VAS-IBS),and a nutrition questionnaire to analyze dietary habits.The colonoscopy was performed according to clinical routines. Four different mucosa biopsies were obtained from the mid part of the colon descendens. Samples were stored at -80 °C until the gut microbiota was analyzed by quantitative polymerase chain reaction(qPCR) and terminal restriction fragment length polymorphism (T-RFLP). Blood samples were collected according to clinical routines and analyzed at the Department of Clinical Chemistry. A study protocol was completed by the physician about clinical findings and histopathological diagnoses. The patients were divided into two groups depending on the colonoscopy finding:patients with DD, and patients without any diverticula who served as controls.

Tissue sampling

The patients were examined by colonoscopy according to clinical routines after prior laxation with Laxabon® (potassium chloride and macrogol;BioPhausia, Stockholm, Sweden). At the end of the colonoscopy, when the clinical examination was completed, four different mucosa biopsies were obtained from intact, inter-diverticular mucosa in the mid part of the colon descendens. This location was chosen since the left colon is the region most often affected by diverticula and is more accessible than the right colon. The biopsies were immediately frozen in liquid nitrogen and kept frozen at -80 °C until analysis.Histopathological examination was performed on separate mucosa samples when IBD had to be excluded or verified.

Questionnaires

Study questionnaire: The questionnaire included questions on age, body mass index (BMI), family history, lifestyle habits, educational achievement,occupation, civil status, circumstances concerning delivery and breast-feeding, place of birth and moving patterns, and medical history. The patients had to answer whether they had been diagnosed with celiac disease, IBD, lactose intolerance, reflux or ulcer. They were asked whether they experienced gastrointestinal symptoms which fulfilled the Rome IV criteria of functional dyspepsia or IBS[19,20]. This questionnaire was in structure and design similar to questionnaires used by other large current population-based and on-going screening projects in Sweden (i.e., LifeGene, EpiHealth,BIG-3, SCAPIS).

VAS-IBS: The VAS-IBS was used to investigate gastrointestinal complaints in the study groups. VAS-IBS is a validated, self-rating questionnaire for estimation of the most common gastrointestinal complaints experienced during the last 2 wk[21]. This questionnaire has also been validated for estimation of symptoms over time[22]. The five items measured in the VAS-IBS address the symptoms of abdominal pain, diarrhea,constipation, bloating and flatulence, and nausea and vomiting. These items were measured on a scale from 0-100, where 0 represented severe problems and 100 represented a complete lack of problems. Whether the patient suffered from symptoms or not, was defined as a score above the median values in healthy subjects[22].

Food questionnaire: The questionnaire included questions about dietary intake each meal in the form of red meat, fish or vegetables, making it possible to estimate dietary patterns. The number of days per week for intake of juice, coffee/tea, milk, sour milk,muesli, berries and fruit, marmalade, bread, cheese,ham and egg at breakfast, or snack were filled in. The participants were asked whether the lunch and dinner were homemade, or whether the participant had a lunch or dinner at a restaurant or a frozen precooked meal.

Microbial analyses

DNA extraction: Three of the four mucosa samples,mean weight 15 ± 0.6 mg, were used for DNA extraction. DNA was isolated and purified by EZ1 Advanced XL (EZ1 DNA Tissue Kit and Bacteria Card;Qiagen, Hilden, Germany)[10,23].

qPCR: The amount of Enterobacteriaceae was estimated using a qPCR assay according to Karlsson et al[10]. Primers used for the qPCR assay have been used and published previously[24,25]. The detection limit was 102 genes/reaction. For standard curves, 10-fold dilution series of the target DNA were made in EB buffer(Qiagen). Number of bacteria was expressed as log10 16S rRNA genes/g feces.

Microbial diversity: T-RFLP was applied to assess the microbial diversity, as previously described[26].Thresholds for internal standard and terminal restriction fragments (T-RFs) were set to 5 and 15 fluorescence units, respectively.

Calculations: Microbial diversity was estimated by calculation of richness (number of T-RFs) and Shannon-Wiener and Simpson’s diversity indices as described by Karlsson et al[10], with the exception that T-RFs within 40-580 base pairs were included in the T-RFLP profile analysis and calculation. The diversity indices take into account both richness and evenness when considering the relative abundance of bacterial groups. Both indices are commonly used to assess microbial diversity[27].Samples below the limit of detection (in qPCR) were replaced by the limit of detection for statistical analysis.

Patient categorization

Depending on presence or absence of diverticula, the included patients were divided into patients with DD or control patients. The control patients either exhibited normal macroscopic endoscopic and microscopic histopathological findings or presence of benign polyps.The group categorization was performed independent of gastrointestinal symptoms. All patients with IBD or malignancy were excluded from the study. The diagnosis of IBD was set when the patients fulfilled the criteria for Crohn’s disease, ulcerative colitis or microscopic colitis, i.e., clinical and endoscopic findings in addition to inflammation at the histopathological examination, in accordance to the diagnoses criteria[28].

Statistical analysis

The statistical calculations were performed using the SPSS software, version 24.0 (Armonk, NY, United States). Non-parametric tests were used because of the low number of participants in each group and the skewed distribution of the values of VAS-IBS.Comparisons of continuous variables between groups were performed by either Mann-Whitney U test or Kruskal-Wallis test. Fisher’s exact test was used for dichotomous variables, and Spearman’s correlation test was used for correlations between parameters. Values are presented in median and interquartile ranges (IQRs)or number and percentage. P ＜ 0.05 was considered statistically significant.

RESULTS

Patient characteristics

In total, 77 patients were invited to participate in the study. Nineteen patients denied to participate and 58 patients were included. Six patients were later excluded since they fulfilled the criteria for IBD, and one because of colon malignancy. Finally, 51 patients were included in the present study, 16 with DD and 35 controls without organic changes visible at the colonoscopy or at the histopathological examination (n = 12), except non-malignant polyps (n = 23). The reasons for referral to colonoscopy were presence of gastrointestinal symptoms which rendered a colonoscopy to exclude IBD, malignancy or DD (n = 17), follow-up after previous resection of polyps (n = 17), rectal bleeding(n = 11), screening for cancer due to heredity (n = 4),or perforation to the urinary tract (n = 2). Only one subject in the DD group had a history of verified acute diverticulitis.

There was an equal sex distribution in the groups.Subjects without DD were slightly older than controls[68 (62-76) years vs 62 (40-74) years, P = 0.072],which may explain that more DD patients than controls had completed primary school as the highest education level. Age differences may also explain the lower degree of physical activity in the DD group. A few patients in both groups had been treated with antibiotics during the last 6 mo (Table 1). The moving patterns did not differ between groups. Sporadic cases of heart and lung diseases were found in both groups (data not shown).

Gastrointestinal symptoms

Altogether, 22 patients (43.1%) fulfilled the Rome IV criteria for IBS. The prevalence of functional dyspepsia,IBS, gastric ulcer, lactose intolerance and reflux was equally distributed between groups. Each symptom item estimated by the VAS-IBS questionnaire was present in about half of all patients examined. Only 4 patients in each group did not have any form of gastrointestinal symptoms (Table 2). There was a wide variety in symptom intensity within each group also. None of the items in VAS-IBS correlated with age (data not shown).

Dietary patterns

All the participants who completed the nutrition questionnaire (n = 42) started the day with a breakfast,which in the vast majority of cases consisted of coffee or tea, together with bread and/or muesli and milk products. Twenty-seven participants had homemade lunch, whereas ten participants had lunch at a restaurant or had precooked meals, and five participants never had any lunch. Thirty-three participants had dinner at home, whereas eight participants had regular dinner at a restaurant or did not have dinner.Those who had homemade lunch suffered from more gastrointestinal symptoms compared with those who did not eat lunch, had lunch at a restaurant or had precooked meals, although bloating and flatulence was the only item that reached statistical significance [52(25-93) vs 88 (70-100), P = 0.024]. The difference could not be related to any differences in socioeconomic factors or smoking or alcohol habits (data not shown)or in age span [66 (50-76) vs 65 (59-72), P = 0.851].When the patients were divided into three groups depending on lunch habits [(1) home-maid lunch; (2)lunch at a restaurant or precooked meals; and (3) no lunch], those who had homemade lunch registered the most severe gastrointestinal symptoms on all the VAS scales, although the differences did not reach statistical significance (data not shown).

Microbiota and inflammatory biomarkers

Patients with DD had significantly higher levels of Enterobacteriaceae than patients without diverticula(P = 0.043; Table 3). Although patients with DD moreoften had lower education and less physical activity, the different subgroups of these parameters did not affect the amount of Enterobacteriaceae, diversity indices of Shannon-Wiener or Simpson, or the number of T-RFs(P = 0.413, P = 0.803, P = 0.770, and P = 0.588,respectively, vs P = 0.684, P = 0.616, P = 0.745, and P= 0.316, respectively). There were no differences in any parameters between controls with and without polyps(data not shown).

Abundance of Enterobacteriaceae/Escherichia coli is associated with IBD, both in animal models and in humans[12,13,15,16]. The gut microbiota generates biologically active small molecules, e.g., amino acids, short-chain fatty acids, sugars and organic acids, which are presumed to affect the health of the host[30]. Basic microbiome metabolism was altered in IBD, with reduced amino acid synthesis and carbohydrate metabolism and increased nutrient uptake. Furthermore, genes involved in pathogenesis processes such as secretion of enterotoxins, walldegrading enzymes and cytokine production were over-represented in Crohn’s disease[13]. This would lead to tissue destruction and bacterial overgrowth,with structural and functional dysbiosis.

Several of the patients had humoral inflammatory parameters above or beneath the reference values,i.e., plasma-C-reactive protein (CRP): ＜ 3 mg/L; blood-leucocytes: 3.5-8.8 × 109/L; blood-thrombocytes 125-340 × 109/L; and plasma-albumin: 36-48 g/L. The level of inflammatory biomarkers did not differ between patients with or without DD (Table 3). Neither did presence nor absence of IBS affect the plasma levels of CRP (P = 0.194) and albumin (P = 0.902), or blood levels of leukocytes (P = 0.912) and thrombocytes(P = 0.509). There was no correlation between any of the inflammatory biomarkers and the level of Enterobacteriaceae or bacterial diversity (data not shown).

Neither the amount of Enterobacteriaceae nor the diversity indices correlated with age, BMI, or any items of the VAS-IBS (data not shown). When calculating differences between patients with and without any of the gastrointestinal symptoms, there were no differences in amount of Enterobacteriaceae or diversity indices (data not shown). Presence of IBS did not affect the amount of Enterobacteriaceae (P = 0.867),Shannon-Wiener index (P = 0.533), Simpson’s index (P= 0.478), or number of T-RFs (P = 0.828).

There were no differences in the amount of Enterobacteriaceae or the diversity indices between those who had a regular vs irregular breakfast intake of coffee/tea, dairy products, or cereals. The gut microbiota parameters examined were not influenced by intake of homemade lunch or dinner, smoking and alcohol habits, intake of probiotics and antibiotics, or movement patterns (data not shown).

DISCUSSION

In the present study examining symptomatic patients with elective colonoscopy, patients with DD had higher amount of Enterobacteriaceae compared with patients without diverticula, whereas the presence of gastrointestinal symptoms or IBS did not affect the amount of Enterobacteriaceae. Patients who had homemade lunch showed more symptoms of bloating and flatulence than those who did not have any lunch or had lunch at a restaurant/precooked meal. None of the studied lifestyle and socioeconomic parameters affected the amount of Enterobacteriaceae or bacterial diversity of the gut.

32 Distrutti E, Monaldi L, Ricci P, Fiorucci S. Gut microbiota role in irritable bowel syndrome: New therapeutic strategies. World J Gastroenterol 2016; 22: 2219-2241 [PMID: 26900286 DOI:10.3748/wjg.v22.i7.2219]

There was an inverse correlation between the amount of Enterobacteriaceae and Simpson’s index(rs = -0.361, P = 0.033) and a tendency to correlation between Enterobacteriaceae and Shannon-Wiener index(rs = -0.299, P = 0.081). The Shannon-Wiener and Simpson’s indices correlated with each other (rs = 0.947,P ＜ 0.001) and number of T-RFs (rs = 0.917, P ＜ 0.001 and rs = 0.772, P ＜ 0.001, respectively).

In the present study of DD, the abundance of Enterobacteriaceae in the colon mucosa at a distance from the diverticula could hypothetically reflect a low-grade inflammation in the bowel wall. The previous publication by Barabra et al[17] suggested chronic low-grade gut mucosa inflammation in DD,through histopathological examination. Such lowgrade inflammation was not reflected in the humoral inflammatory system, confirmed by overall normal CRP and blood cells levels, but may be captured in mucosal biopsies[17,31]. A low-grade inflammation may contribute to pain sensitization and visceral hypersensitivity and symptom development[7,8], which contributes to the increased risk of IBS after acute diverticulitis[6].

It remains unclear whether microbial changes are a cause or a consequence of DD. We do not know whether inflammation is a primary event, leading to weakening of the bowel wall and eventually to development of diverticula, or if inflammation is secondary to the presence of DD distant in the bowel with retention of luminal contents and bacterial overgrowth. Even if the microbial changes are secondary, the dysbiosis may further accelerate the pathologic process and weakening of the bowel wall by mechanisms explained above[13].

Microbial dysbiosis in combination with genetic,environmental, and psychosocial factors are proposed to be involved in the etiology of IBS[20,32]. Escherichia coli was increased in Chinese IBS patients compared with controls, whereas no differences of these feces bacteria were found between IBS patients and healthy controls from other regions[33]. This is in line with our present study, which did not show any correlations between gastrointestinal symptoms or IBS and Enterobacteriaceae.

Gastrointestinal symptoms without visible organic damages are called functional bowel disorders, where IBS is the most common of the disorders, with a prevalence of 10%-15% in the population[20]. A great deal of the present patients suffered from IBS or IBS-like symptoms, whereas some patients experienced gastrointestinal symptoms without fulfilling the Rome IV criteria[20]. Symptomatology is not enough to distinguish between different bowel disorders, as found in the present study. It has previously been shown that patients with IBS have as severe symptoms as those with organic changes, i.e., primary Sjögren´s syndrome and enteric dysmotility[34]. A great symptomatic overlap between DD and IBS is described previously[1], which further underlines that disease classification must be based on organic criteria and not on symptoms solely. Biomarkers for IBS and DD are lacking, but measurements of markers of dysbiosis, inflammatory cells in mucosa, and metabolomes may be able to distinguish IBS from DD in the future. Probiotic therapy is an efficient treatment of IBS[35], whereas the evidence of efficiency in treatment of DD is insufficient.

发放40份教学满意度调查表，回收40份，均有效。实验组的教学满意度高于对照组，差异具有统计学意义（P＜0.05）（表2）。

Since this was a cross-sectional study, we do not know the reason for more symptoms being present in the group with homemade food. The reason may depend on patients with more severe symptoms avoiding visiting a restaurant, to have better control over their food intake.

就这两句话，让大家都沉默了，不约而同地向ICU门口望过去，突然意识到打什么打，现在救活权筝比什么都重要。

The strength in the present pilot study is that we have analyzed mucosal biopsies instead of feces. The mucosa microbiota composition is anticipated to be more reliable than the feces composition. To compare another patient group with similar degree of symptoms seems more appropriate than to compare DD with healthy, non-symptomatic subjects. Further, we have considered food intake and other lifestyle habits affecting microbiota composition. The weakness is the small cohort size. Furthermore, since the patients were enrolled consecutively, there was no matching between cases and controls of, e.g., age, sex or lifestyle habits.In a larger study, some of the demographic parameters and lifestyle habits could have shown statistically significant influence on the gut microbiota. We chose to initially perform this as a pilot trial with a limited amount of patients, as the methodology is very expensive.Since it now has been shown that there are differences in DD according to the gut microbiota, it is important to continue with further studies and more extensive analyses. Since this was a cross-sectional study, we do not know whether the microbial alterations are primary in the development of diverticula or just secondary to DD, with retention of luminal content.

In this pilot study, patients with DD had higher amount of Enterobacteriaceae in the colon mucosa compared to patients without DD. Assessment of gut microbiota may distinguish DD from other patient groups and may be involved in etiology and pathophysiology of the disease. Gastrointestinal symptomatology seems to not be related to the amount of Enterobacteriaceae or to the bacterial diversity.

ARTICLE HIGHLIGHTS

Research background

Diverticular disease (DD) is a common gastrointestinal disease of unknown etiology. The symptoms of DD are similar with symptoms of irritable bowel syndrome (IBS). The gut microbiota is discussed as important for the etiology and pathophysiology in a wide range of diseases. Bacterial diversity is higher in lean compared to obese individuals, and in healthy states compared to unhealthy states, and some bacterial groups, e.g., Enterobacteriaceae, are associated with over-weight and inflammation. The family Enterobacteriaceae is commonly found in the gut ecosystem, where Escherichia coli is the most abundant species of the family. Only a few studies have been performed regarding microbial composition in DD. Recently, lower amounts of Enterobacteriaceae were found in the colon mucosa of DD patients compared with healthy controls, whereas higher amounts of Akkermansia and no difference in the Escherichia coli subgroup were found in feces in another DD cohort. Thus, it is hypothesized that gut microbiota is involved in the etiology and pathophysiology of DD, but the few studies performed so far have shown inconclusive results.

Research motivation

Today, there is no efficient treatment option for DD, neither to prevent disease development nor to reduce the symptoms when the disease has been established, which renders a lot of suffering to the patients. To find out the etiology is crucial to be able to prevent and efficiently treat the disease. New knowledge within this disease field may point out the direction for future research.

Research objectives

16 Garrett WS, Gallini CA, Yatsunenko T, Michaud M, DuBois A,Delaney ML, Punit S, Karlsson M, Bry L, Glickman JN, Gordon JI, Onderdonk AB, Glimcher LH. Enterobacteriaceae act in concert with the gut microbiota to induce spontaneous and maternally transmitted colitis. Cell Host Microbe 2010; 8: 292-300 [PMID:20833380 DOI: 10.1016/j.chom.2010.08.004]

Research methods

All consecutive patients referred to elective colonoscopy at the Department of Endoscopy, Skåne University Hospital, Malmö, were invited to participate in the study. If the patients agreed to participate, they had to complete a study questionnaire about demography, socioeconomic status, lifestyle habits,family history and medical history, the Visual Analog Scale for Irritable Bowel Syndrome (VAS-IBS), and a nutrition questionnaire to analyze dietary habits.The colonoscopy was performed according to clinical routines. Four different mucosa biopsies were obtained from the mid part of the colon descendens.Samples were stored at -80 °C until the gut microbiota was analyzed by quantitative polymerase chain reaction (qPCR) and terminal restriction fragment length polymorphism (T-RFLP). Blood samples were collected according to clinical routines and analyzed at the Department of Clinical Chemistry. A study protocol was completed by the physician about clinical findings and histopathological diagnoses. The patients were divided into two groups depending on the colonoscopy finding: patients with DD, and patients without any diverticula who served as controls. Three of the four mucosa samples,mean weight 15 ± 0.6 mg, were used for DNA extraction. DNA was isolated and purified by EZ1 Advanced XL (EZ1 DNA Tissue Kit and Bacteria Card; Qiagen,Hilden, Germany). The amount of Enterobacteriaceae was estimated using a qPCR assay according to Karlsson et al. Primers used for the qPCR assay have been used and published previously. The detection limit was 102 genes/reaction. For standard curves, 10-fold dilution series of the target DNA were made in EB buffer (Qiagen). Number of bacteria was expressed as log10 16S rRNA genes/g feces. T-RFLP was applied to assess the microbial diversity, as previously described. Thresholds for internal standard and terminal restriction fragments (T-RFs) were set to 5 and 15 fluorescence units, respectively.Microbial diversity was estimated by calculation of richness (number of T-RFs)and Shannon-Wiener and Simpson’s diversity indices as described by Karlsson et al, with the exception that T-RFs within 40-580 base pairs were included in the T-RFLP profile analysis and calculation. The diversity indices take into account both richness and evenness when considering the relative abundance of bacterial groups. Both indices are commonly used to assess microbial diversity. Samples below the limit of detection (in qPCR) were replaced by the limit of detection for statistical analysis.

Research results

Finally, 51 patients were included in the present study, 16 with DD and 35 controls without organic changes visible at the colonoscopy or at the histopathological examination (n = 12), except non-malignant polyps (n = 23).The reasons for referral to colonoscopy were presence of gastrointestinal symptoms which rendered a colonoscopy to exclude inflammatory bowel disease, malignancy or DD (n = 17), follow-up after previous resection of polyps(n = 17), rectal bleeding (n = 11), screening for cancer due to heredity (n = 4), or perforation to the urinary tract (n = 2). Only one subject in the DD group had a history of verified acute diverticulitis. There was an equal sex distribution in the groups. Subjects without DD were slightly older than controls [68 (62-76) years vs 62 (40-74) years, P = 0.072]. Altogether, 22 patients (43.1%) fulfilled the Rome IV criteria for IBS. The prevalence of functional dyspepsia, IBS, gastric ulcer, lactose intolerance and reflux was equally distributed between groups.Each symptom item estimated by the VAS-IBS questionnaire was present in about half of all patients examined. Only 4 patients in each group did not have any form of gastrointestinal symptoms. There was a wide variety in symptom intensity within each group also. None of the items in VAS-IBS correlated with age. Those who had homemade lunch suffered from more gastrointestinal symptoms compared with those who did not eat lunch, had lunch at a restaurant or had precooked meals, although bloating and flatulence was the only item that reached statistical significance [52 (25-93) vs 88 (70-100), P = 0.024]. The difference could not be related to any differences in socioeconomic factors or smoking or alcohol habits or in age span [66 (50-76) vs 65 (59-72), P = 0.851].Patients with DD had significantly higher levels of Enterobacteriaceae than patients without diverticula (P = 0.043). Although patients with DD more often had lower education and less physical activity, the different subgroups of these parameters did not affect the amount of Enterobacteriaceae, diversity indices of Shannon-Wiener or Simpson, or the number of T-RFs (P = 0.413, P = 0.803,P = 0.770, and P = 0.588, respectively, vs P = 0.684, P = 0.616, P = 0.745, and P = 0.316, respectively). There were no differences in any parameters between controls with and without polyps. There was an inverse correlation between the amount of Enterobacteriaceae and Simpson’s index (rs = -0.361, P = 0.033)and a tendency to correlation between Enterobacteriaceae and Shannon-Wiener index (rs = -0.299, P = 0.081). The Shannon-Wiener and Simpson’s indices correlated with each other (rs = 0.947, P ＜ 0.001) and number of T-RFs(rs = 0.917, P ＜ 0.001 and rs = 0.772, P ＜ 0.001, respectively). Several of the patients had humoral inflammatory parameters above or beneath the reference values, i.e. plasma-C-reactive protein (CRP): ＜ 3 mg/L; blood-leucocytes: 3.5-8.8× 109/L; blood-thrombocytes 125-340 × 109/L; and plasma-albumin: 36-48 g/L.The level of inflammatory biomarkers did not differ between patients with or without DD. Neither did presence nor absence of IBS affect the plasma levels of CRP (P = 0.194) and albumin (P = 0.902), or blood levels of leukocytes (P =0.912) and thrombocytes (P = 0.509). There was no correlation between any of the inflammatory biomarkers and the level of Enterobacteriaceae or bacterial diversity. Neither the amount of Enterobacteriaceae nor the diversity indices correlated with age, BMI, or any items of the VAS-IBS. When calculating differences between patients with and without any of the gastrointestinal symptoms, there were no differences in amount of Enterobacteriaceae or diversity indices (data not shown). Presence of IBS did not affect the amount of Enterobacteriaceae (P = 0.867), Shannon-Wiener index (P = 0.533), Simpson’s index (P = 0.478), or number of T-RFs (P = 0.828). There were no differences in the amount of Enterobacteriaceae or the diversity indices between those who had a regular vs irregular breakfast intake of coffee/tea, dairy products, or cereals. The gut microbiota parameters examined were not influenced by intake of homemade lunch or dinner, smoking and alcohol habits, intake of probiotics and antibiotics, or movement patterns. The problems that remain to be solved are whether the difference in gut microbiota composition are primary events in the disease development or secondary to the DD. The causality to DD must still be defined.

Research conclusions

The new finding of the present study is the abundance of Enterobacteriaceae in colon mucosa in DD, and that this abundance was not related to age,BMI, socioeconomic parameters, gastrointestinal symptoms or lifestyle habits. Microbial diversity was not affected by DD or any other parameters measured. The new theory that this study proposes is that the composition of gut microbiota is involved in DD. The summarization of this study is that gut microbiota may be affected in patients with DD. This study is the first study where a clinical cohort of patients is consecutively enrolled during colonoscopy to analyze gut microbiota in colon mucosa, where the only difference between the groups compared is the presence or absence of colon diverticula.Previous studies have enrolled participants in screening programs or analyzed microbiota composition in feces. The authors also studied socioeconomic features and lifestyle habits in the cohort, to be able to adjust for confounders.The new hypotheses proposed are that gut microbiota is involved in DD and that demography, socioeconomic parameters and dietary habits may be of less importance for the microbiota than the presence or absence of colon diverticula.The new methods proposed are the enrolment of consecutive clinical patients in scientific trials, analyses of gut microbiota in mucosa instead of feces,analysis of microbial diversity to get a general reflection of the gut microbiota,analysis of the amount of Enterobacteriaceae or other bacteria by qPCR, and estimation of gastrointestinal symptoms by the VAS-IBS questionnaire. The new phenomenon found were that presence or absence of colon diverticula are more important for gut microbiota than demography, socioeconomic parameters, gastrointestinal symptoms, or lifestyle habits. Another new phenomenon was that patients with homemade lunch had more gastrointestinal symptoms than patients who did not eat lunch or had lunch at a restaurant.The authors confirmed the hypothesis that the amount of Enterobacteriaceae was affected by DD, but failed to confirm the hypothesis that overall bacterial diversity was influenced by colon diverticula. The authors also failed to confirm the hypotheses that demography, socioeconomic parameters, gastrointestinal symptoms and lifestyle habits were associated with gut microbiota composition.The major implication for clinical practice in the future is to consider dysbiosis in patients with DD. Tests to determine gut microbiota are available for clinical use, and should be considered in the management of these patients.

Research perspectives

The experience the authors have learnt from this study is that presence or absence of DD is more important for the gut microbiota composition than demography, socioeconomic parameters, gastrointestinal symptoms, and lifestyle habits. The authors have also learnt from this study that homemade food is not always the best for patients in the management of gastrointestinal symptoms. The authors must further study the importance of gut microbiota in DD. The authors should continue to include patients with DD in experiments to analyze gut microbiota composition to get larger cohorts, and to perform clinical trials to evaluate the effect of probiotics in symptom management of DD. The best method is to analyze gut microbiota in colon mucosa instead of feces. The VAS-IBS is also a useful tool to estimate gastrointestinal symptoms.

REFERENCES

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3 Hjern F, Johansson C, Mellgren A, Baxter NN, Hjern A.Diverticular disease and migration--the influence of acculturation to a Western lifestyle on diverticular disease. Aliment Pharmacol Ther 2006; 23: 797-805 [PMID: 16556182 DOI: 10.1111/j.1365-2036.2006.02805.x]

第二天，川矢果然又来拜师。百里香欣然应允，并设宴款待。川矢大为高兴，对百里香的手艺赞不绝口。拜师酒后，川矢不仅行了三叩九拜大礼，还奉上了一笔银票作见面礼。

4 Shahedi K, Fuller G, Bolus R, Cohen E, Vu M, Shah R, Agarwal N, Kaneshiro M, Atia M, Sheen V, Kurzbard N, van Oijen MG,Yen L, Hodgkins P, Erder MH, Spiegel B. Long-term risk of acute diverticulitis among patients with incidental diverticulosis found during colonoscopy. Clin Gastroenterol Hepatol 2013; 11:1609-1613 [PMID: 23856358 DOI: 10.1016/j.cgh.2013.06.020]

父母的信仰是儿童信念发展的锚定起点(Boyatzis et al., 2006; Ozorak, 1989)，不同信仰的父母有着不同的观念和行为方式。亲子谈话是传递父母的信仰及信念的重要方式(Boyatzis et al., 2006)。因此，父母可能通过亲子谈话传递他们的来生信念，进而影响着儿童对死亡的认知及来生信念。研究二考察儿童来生信念与父母来生信念、死亡相关话题亲子谈话的关系，帮助我们理解儿童来生信念的特点，以及父母来生信念对儿童来生信念的影响过程及机制。

5 Humes DJ, Simpson J, Neal KR, Scholefield JH, Spiller RC.Psychological and colonic factors in painful diverticulosis. Br J Surg 2008; 95: 195-198 [PMID: 17939130 DOI: 10.1002/bjs.5962]

13 Morgan XC, Tickle TL, Sokol H, Gevers D, Devaney KL, Ward DV, Reyes JA, Shah SA, LeLeiko N, Snapper SB, Bousvaros A,Korzenik J, Sands BE, Xavier RJ, Huttenhower C. Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment. Genome Biol 2012; 13: R79 [PMID: 23013615 DOI:10.1186/gb-2012-13-9-r79]

7 Simpson J, Scholefield JH, Spiller RC. Origin of symptoms in diverticular disease. Br J Surg 2003; 90: 899-908 [PMID:12905541 DOI: 10.1002/bjs.4277]

8 Clemens CH, Samsom M, Roelofs J, van Berge Henegouwen GP, Smout AJ. Colorectal visceral perception in diverticular disease. Gut 2004; 53: 717-722 [PMID: 15082591 DOI: 10.1136/gut.2003.018093]

式中,[Sij]为柔度矩阵,αi,i,j=1,2,3为热膨胀系数。均一化接合层的等效热膨胀系数列阵可推导为:

9 Hakansson A, Molin G. Gut microbiota and inflammation.Nutrients 2011; 3: 637-682 [PMID: 22254115 DOI: 10.3390/nu3060637]

基于这些关系表的频繁挖掘方法可以采用直接连接的方法，形成一张大表。但是这种方法会导致性能低下、统计偏斜等问题。故本文利用关系数据库的特点对传统方法进行改进。算法主要包括如下4个步骤。

26 Sand E, Linninge C, Lozinska L, Egecioglu E, Roth B, Molin G, Weström B, Ekblad E, Ohlsson B. Buserelin treatment to rats causes enteric neurodegeneration with moderate effects on CRF-immunoreactive neurons and Enterobacteriaceae in colon, and in acetylcholine-mediated permeability in ileum. BMC Res Notes 2015; 8: 824 [PMID: 26710832 DOI: 10.1186/s13104-015-1800-x]

11 Fåk F, Karlsson CL, Ahrné S, Molin G, Weström B. Effects of a high-fat diet during pregnancy and lactation are modulated by E.coli in rat offspring. Int J Obes (Lond) 2012; 36: 744-751 [PMID:21730967 DOI: 10.1038/ijo.2011.118]

12 Baumgart M, Dogan B, Rishniw M, Weitzman G, Bosworth B,Yantiss R, Orsi RH, Wiedmann M, McDonough P, Kim SG, Berg D,Schukken Y, Scherl E, Simpson KW. Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum. ISME J 2007; 1: 403-418[PMID: 18043660 DOI: 10.1038/ismej.2007.52]

6 Cohen E, Fuller G, Bolus R, Modi R, Vu M, Shahedi K, Shah R, Atia M, Kurzbard N, Sheen V, Agarwal N, Kaneshiro M, Yen L, Hodgkins P, Erder MH, Spiegel B. Increased risk for irritable bowel syndrome after acute diverticulitis. Clin Gastroenterol Hepatol 2013; 11: 1614-1619 [PMID: 23524129 DOI: 10.1016/j.cgh.2013.03.007]

此外还发现，目前的粉丝文化研究中重粉丝形象的整体性特征，轻群体内部的微观性特征，特别是在对粉丝社群成员关系的描述中，大多指向高团结、强凝聚的友好关系，以及小部分追逐权力的竞争关系。但是，冲突关系、群体极化也是粉丝社群内部非常真实的写照，既往研究这部分的关照略显不足，且仅有的相关研究聚焦于不同的偶像团体粉丝社群之间而非同一偶像团体粉丝社群内部。

14 Mondot S, Lepage P. The human gut microbiome and its dysfunctions through the meta-omics prism. Ann N Y Acad Sci 2016; 1372: 9-19 [PMID: 26945826 DOI: 10.1111/nyas.13033]

15 Arthur JC, Perez-Chanona E, Mühlbauer M, Tomkovich S,Uronis JM, Fan TJ, Campbell BJ, Abujamel T, Dogan B, Rogers AB, Rhodes JM, Stintzi A, Simpson KW, Hansen JJ, Keku TO,Fodor AA, Jobin C. Intestinal inflammation targets cancer-inducing activity of the microbiota. Science 2012; 338: 120-123 [PMID:22903521 DOI: 10.1126/science.1224820]

2011年到2014年的时候，在哈蒙德家南面大约150公里处，一系列爆破打通了埃尔华河与赛利希海之间的通道，美国国家公园管理局摧毁了华盛顿州奥林匹克半岛上的两座旧水电站，启动了美国历史上最大的水坝拆除工程。虽然很多人都知道，拆除水坝可以为鲑鱼洄游清除障碍，但很少有人会想到，这一举措同时也可以让更多的漂流木畅通无阻地进入大海。

The primary aim of the present study was to compare the level of the large Gram-negative bacterial family Enterobacteriaceae and gut bacterial diversity in colon mucosa between consecutive patients diagnosed with DD and patients with normal endoscopic findings. Secondary aims were to evaluate the influence of demography, socioeconomic status, lifestyle habits, inflammatory parameters and gastrointestinal symptoms on the gut microbiota. These objectives were possible to realize by the present study design. Further studies according to the same study design, but with larger patient cohorts, are important to perform to confirm the results.

17 Barbara G, Scaioli E, Barbaro MR, Biagi E, Laghi L, Cremon C,Marasco G, Colecchia A, Picone G, Salfi N, Capozzi F, Brigidi P,Festi D. Gut microbiota, metabolome and immune signatures in patients with uncomplicated diverticular disease. Gut 2017; 66:1252-1261 [PMID: 27618836 DOI: 10.1136/gutjnl-2016-312377]

18 Tursi A, Mastromarino P, Capobianco D, Elisei W, Miccheli A,Capuani G, Tomassini A, Campagna G, Picchio M, Giorgetti G,Fabiocchi F, Brandimarte G. Assessment of Fecal Microbiota and Fecal Metabolome in Symptomatic Uncomplicated Diverticular Disease of the Colon. J Clin Gastroenterol 2016; 50 Suppl 1: S9-S12[PMID: 27622378 DOI: 10.1097/MCG.0000000000000626]

19 Stanghellini V, Chan FK, Hasler WL, Malagelada JR, Suzuki H,Tack J, Talley NJ. Gastroduodenal Disorders. Gastroenterology 2016; 150: 1380-1392 [PMID: 27147122 DOI: 10.1053/j.gastro.2016.02.011]

20 Mearin F, Lacy BE, Chang L, Chey WD, Lembo AJ, Simren M, Spiller R. Bowel Disorders. Gastroenterology 2016 [PMID:27144627 DOI: 10.1053/j.gastro.2016.02.031]

21 Bengtsson M, Ohlsson B, Ulander K. Development and psychometric testing of the Visual Analogue Scale for Irritable Bowel Syndrome (VAS-IBS). BMC Gastroenterol 2007; 7: 16[PMID: 17475020 DOI: 10.1186/1471-230X-7-16]

22 Bengtsson M, Persson J, Sjölund K, Ohlsson B. Further validation of the visual analogue scale for irritable bowel syndrome after use in clinical practice. Gastroenterol Nurs 2013; 36: 188-198 [PMID:23732784 DOI: 10.1097/SGA.0b013e3182945881]

23 Stenblom EL, Weström B, Linninge C, Bonn P, Farrell M, Rehfeld JF, Montelius C. Dietary green-plant thylakoids decrease gastric emptying and gut transit, promote changes in the gut microbial flora, but does not cause steatorrhea. Nutr Metab (Lond) 2016; 13:67 [PMID: 27777602 DOI: 10.1186/s12986-016-0128-4]

翠香不耐贮藏。贮期超过3个月，果实硬度就会快速下降。出库的翠香猕猴桃，1周左右变软，之后3～5天果实就会发胀酒化。因此，买回来的翠香应在冰箱内冷藏暂存，分批拿出食用。常温下放显眼处，以免忘记食用导致变坏。

24 Bartosch S, Fite A, Macfarlane GT, McMurdo ME. Characterization of bacterial communities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal microbiota. Appl Environ Microbiol 2004; 70: 3575-3581 [PMID: 15184159 DOI: 10.1128/AEM.70.6.3575-3581.2004]

1.3.4 治疗计划 使用6MV-X射线进行照射，处方剂量是50 Gy，每次2 Gy；剂量体积约束条件：PTV：90%体积高于50 Gy，肾脏：30%体积低于30 Gy，脊髓：0%体积低于45 Gy，肝脏：30%体积低于40 Gy，肺脏：20%体积低于20 Gy，心脏：20%体积低于40 Gy。每例患者做六个治疗计划，都使用5、7、9野等分布野，脊髓的保护区边界是椎管内壁(f2)与脊髓硬脊膜(f1)。

25 Castillo M, Martín-Orúe SM, Manzanilla EG, Badiola I, Martín M, Gasa J. Quantification of total bacteria, enterobacteria and lactobacilli populations in pig digesta by real-time PCR. Vet Microbiol 2006; 114: 165-170 [PMID: 16384658 DOI: 10.1016/j.vetmic.2005.11.055]

10 Karlsson CL, Onnerfält J, Xu J, Molin G, Ahrné S, Thorngren-Jerneck K. The microbiota of the gut in preschool children with normal and excessive body weight. Obesity (Silver Spring) 2012;20: 2257-2261 [PMID: 22546742 DOI: 10.1038/oby.2012.110]

27 Magurran AE. An index of diversity. In measuring biological diversity. Oxford: Blackwell Science Ltd, 2004

青海漫长的地质发展历史，复杂的地质构造运动，尤其是新构造运动和独特的气候条件，形成了大量具有原始性、神秘性、独特性等特点的典型地质遗迹，地质遗迹类型丰富多样。但针对地质遗迹的分类、评价、保护与开发以及科学管理等方面的研究较为薄弱，目前也只是在地质遗迹分类、评价、地质公园建设及生态旅游产品开发研究[8-10]等方面有较为零星的研究。基于此，笔者采用查阅文献、野外勘查、专家访谈与评估等方法，以青海省坎布拉国家地质公园为例，进行景观资源综合评价研究，并对地质公园可持续发展问题进行了探讨，研究结果对于正确认识公园遗迹景观状况，深入进行遗迹景观的保护、开发利用和管理等工作具有参考价值。

28 Walsh AJ, Bryant RV, Travis SP. Current best practice for disease activity assessment in IBD. Nat Rev Gastroenterol Hepatol 2016;13: 567-579 [PMID: 27580684 DOI: 10.1038/nrgastro.2016.128]

29 Tang MS, Poles J, Leung JM, Wolff MJ, Davenport M, Lee SC, Lim YA, Chua KH, Loke P, Cho I. Inferred metagenomic comparison of mucosal and fecal microbiota from individuals undergoing routine screening colonoscopy reveals similar differences observed during active inflammation. Gut Microbes 2015; 6: 48-56 [PMID: 25559083 DOI: 10.1080/19490976.2014.1000080]

30 Martinez KB, Leone V, Chang EB. Microbial metabolites in health and disease: Navigating the unknown in search of function.J Biol Chem 2017; 292: 8553-8559 [PMID: 28389566 DOI:10.1074/jbc.R116.752899]

31 Humes DJ, Simpson J, Smith J, Sutton P, Zaitoun A, Bush D,Bennett A, Scholefield JH, Spiller RC. Visceral hypersensitivity in symptomatic diverticular disease and the role of neuropeptides and low grade inflammation. Neurogastroenterol Motil 2012; 24: 318-e163 [PMID: 22276853 DOI: 10.1111/j.1365-2982.2011.01863.x]

The present result of higher levels of Enterobacteriaceae in mucosa of DD is in opposite to the previous result of Barbara et al[17]. The differences may be explained by the different study design and different composition of the control group. The present study enrolled mainly symptomatic patients examined by colonoscopy to exclude organic diseases or patients with heredity for colon cancer. Barbara et al[17] used asymptomatic or symptomatic patients enrolled to colonoscopy in a screening program to exclude malignancy or as followup after polyp resections. Thus, the control group in Barbara et al[17] consisted of a smaller cohort (n = 14)of asymptomatic subjects, and a lower percentage of symptomatic DD, with sex and age differences between groups. The microbiota composition differed between mucosal biopsies and feces[17]. We decided not to analyze fecal microbiota in our study, since there are greater differences between fecal and mucosal microbiota than between individual subjects, and it is considered more reliable to measure microbiota composition in mucosa than feces[29]. The general composition estimated by microbial diversity may be more important to health than the levels of individual bacterial strains[9,10,14].

33 Zhuang X, Xiong L, Li L, Li M, Chen M. Alterations of gut microbiota in patients with irritable bowel syndrome: A systematic review and meta-analysis. J Gastroenterol Hepatol 2017; 32: 28-38[PMID: 27300149 DOI: 10.1111/jgh.13471]

34 Bengtsson M, Hammar O, Mandl T, Ohlsson B. Evaluation of gastrointestinal symptoms in different patient groups using the visual analogue scale for irritable bowel syndrome (VAS-IBS).BMC Gastroenterol 2011; 11: 122 [PMID: 22073983 DOI: 10.118 6/1471-2393-13-201]

35 Ford AC, Quigley EM, Lacy BE, Lembo AJ, Saito YA, Schiller LR, Soffer EE, Spiegel BM, Moayyedi P. Efficacy of prebiotics,probiotics, and synbiotics in irritable bowel syndrome and chronic idiopathic constipation: systematic review and meta-analysis. Am J Gastroenterol 2014; 109: 1547-1561; quiz 1546, 1562 [PMID:25070051 DOI: 10.1038/ajg.2014.202]