Documentation of quality of life (QOL) of patients after transcatheter aortic valve implantation (TAVI) is a Canadian Cardiovascular Society quality indicator. National results have not been reported to date.We conducted an observational cohort study including all TAVI patients, irrespective of surgical risk, treated between January 2016 and June 2019 as documented in the British Columbia TAVI Registry. QOL was measured at baseline, 30 days, and 1 year, using the Kansas City Cardiomyopathy Questionnaire overall score (KCCQ-OS). We used linear regression modelling to examine factors associated with 30-day changes in QOL, logistic regression modelling to identify predictors of sustaining a poor outcome, and Cox regression modelling to ascertain risk estimates of the effect of QOL on 1-year mortality.The cohort included 1706 patients (742 women [43.5%]); median age 83 years . Median (IQR) baseline KCCQ-OS was 45 , indicating severe impairment. Patients alive at 1 year (91.3%) reported a mean improvement of 24.1  points in the KCCQ-OS at 30 days, which was sustained at 1 year . Older age, lower baseline health status, lower aortic valve gradient, lower hemoglobin, atrial fibrillation, and non-transfemoral access were associated with worse 30-day QOL. At 1 year, 65% of patients had a favorable outcome; additional risk factors for 1-year mortality (8.7%) were male sex, New York Heart Association Class IV, severe pulmonary and renal disease, diabetes, and in-patient status.TAVI is associated with significant early improvement in QOL, which is sustained at 1 year. The inclusion of QOL can support treatment decisions and patient-centred evaluation. Our study was conducted in collaboration with Cardiac Services BC (CSBC), a program of the BC Provincial Health Services Authority  responsible for planning, coordinating, funding, and evaluating cardiac care across the province. The study was approved by the University of British Columbia/Providence Health Care Research Ethics Board (H18-00419).Contribution to this registry is mandatory and is a prerequisite for provincial funding. Data documenting patient demographics, risk factors, procedural and in-hospital factors, and 30-day and 1-year follow-up were collected from the 5 BC TAVI centres. Linkages to administrative databases were conducted to validate site-reported data. The all-cause mortality rate through June 30, 2020 was obtained by linkage to the BC Vital Statistics Death Files. The analytical cohort consisted of BC patients who had a single TAVI (TF and non-TF) between January 1, 2016 and June 30, 2019.,,Patient-reported health status was assessed at baseline (pre-TAVI), 30 days, and 1 year after TAVI using the cardiac-specific 12-item Kansas City Cardiomyopathy Questionnaire (KCCQ) PROM.We report baseline characteristics by sex for the analytic cohort as percentages for categorical variables, and as medians with interquartile ranges (IQRs) for continuous variables. We estimated the mean scores at each time point, the mean change from baseline, and the change between time points, along with 95% confidence intervals (CIs), for the KCCQ-OS and the subscales. Changes in KCCQ scores from baseline to 30 days, and to 1 year, were evaluated using linear mixed modeling to account for the random effect of individual patients.,We employed linear regression modeling to identify predictors of change in KCCQ-OS in the first 30 days after TAVI in patients who were alive at that time point; centred mean difference of baseline score was added as an adjustment factor to account for the regression to the mean effect.P value of < 0.05 was considered statistically significant for all analyses. Parameters with a P value of < 0.10 were retained in the multivariable regression models. The proportional hazard assumption was confirmed for the Cox regression model. Statistical analyses were conducted using SAS  and R, version 4.0.2 .All baseline variables included in The rate of missing KCCQ data was 14.3%, 17.3%, and 34.2% at baseline, 1 month, and 1 year, respectively. To mitigate the risks of bias associated with limiting the study to patients with complete QOL assessment that would contribute to an overestimate of favorable outcomes, , we chose to utilize validated strategies to maximize the data available. We employed a multiple imputation strategy to address missing baseline demographics, clinical factors, and follow-up KCCQ scores. Missing data were imputed 40 times using the multiple imputation in chained equation (MICE), under the assumption of missing at random (MAR). Sensitivity analysis for the MAR assumption is included in P = 0.002), more likely to have had a stroke prior to TAVI , more likely to have diabetes , more likely to have impaired renal function , and more likely to be an inpatient at the time of the procedure . Patients with complete data at all 3 time points, and those with missing KCCQ data, did not differ significantly on their baseline KCCQ scores, except in the symptom subscale  and 1557 (91.3%) were alive at 30 days and 1 year, respectively . The basMedian baseline KCCQ-OS was 45  points, indicating that most patients reported symptoms consistent with New York Heart Association (NYHA) Class III.P < 0.001), which was sustained at the 1-year time point with an adjusted mean change of 25.3 points (P < 0.001); a similar pattern was observed in the 4 subscales . Older age , and atrial fibrillation  were independently associated with deteriorating 30-day KCCQ-OS scores among surviving patients. In addition, the use of a non-TF vascular access approach was associated with worse health status recovery at 30 days  was more significantly associated with improvement in KCCQ-OS for men; however, we did not observe the same association for the female patient group . In addition, patients with preexisting atrial fibrillation , previous stroke , or poor renal function  were 70% more likely to derive a poor outcome 1 year after TAVI, whereas the need for a non-TF vascular access approach was associated with an 80% increase  in the risk of a poor outcome . Additional clinical characteristics that emerged as significant predictors of risk of mortality included per 5-year increase of age , male sex , NYHA Class IV , atrial fibrillation , severe pulmonary disease , diabetes , and severe renal disease . Those who were outpatients at the time of the procedure had a higher likelihood of 1-year survival  Registry reported a 27.6 and 31.9 point mean unadjusted change in KCCQ-OS at 30 days and 1 year, respectively, among patients who had TAVI in the United States between 2011 and 2016.,,,,36Our study further confirms the known impact of comorbid burden on patient-reported outcomes.,,Our study further strengthens the evidence of differences in men's and women's clinical presentation with aortic stenosis and outcomes after TAVI. Compared with men, women are generally older, have fewer comorbidities, and have a smaller body index at the time of their procedure.In 2012, the development of a regional system of care to facilitate access to TAVI in BC aimed to guide and monitor indications in the context of rapidly emerging evidence, provide multidisciplinary mentorship, optimize available health resources, and support excellent outcomes from the successive inception of the new provincial sites.Our study should be interpreted in light of several limitations. We highlighted the degree of missing data, and the overall challenges of collecting PROMs in clinical care. Ongoing efforts will be required by policy and clinical leaders to improve data completeness. We carefully considered the options available to account for missing health status data in our analyses. To this end, the analysis of poor outcome was limited to patients who had completed at least a single QOL measurement; we examined differences between the analytic cohort and patients for whom data were missing, and applied rigorous multiple imputations to develop a representative study cohort. In spite of these efforts, missing data should be considered as a potential source of bias in our findings.,Second, our analyses were limited by the availability of covariates in our multivariable models, and may not have fully captured patients’ risk profiles. For example, frailty, cognitive impairment, and disability are known to have deleterious effects on outcomes after TAVI;Last, although the KCCQ-OS has been validated in people with aortic stenosis, the instrument was developed to capture the experience of heart failure. In our sample, 45% of patients reported NYHA Class II or better, and more than 90% had a left ventricular ejection fraction >35%. Thus, the validity of the measurement may be limited across more complex groups, including people with fewer symptoms associated with heart failure, or other issues, including mild cognitive impairment. There is promising evidence of the availability of an instrument that addresses these limitations while strengthening the Canadian perspective on the assessment of QOL.The goals of treatment of severe aortic stenosis are to extend life and enable patients to maximize their QOL. The indications for TAVI continue to evolve to achieve these patient-centred objectives. Recent advances in the way we care for patients aim at facilitating patients’ rapid recovery, and accelerating their experience of improved QOL as early as 2 weeks after TAVI, without compromising patient safety.


Background: The mortality rate following pancreaticoduodenectomy (PD) has been decreasing over the past few years; nonetheless, the morbidity rate remains elevated. The most common complications after PD are post-operative pancreatic fistula (POPF) and delayed gastric emptying (DGE) syndrome. The issue as to which is the best reconstruction method for the treatment of the pancreatic remnant after PD is still a matter of debate. The aim of this study was to retrospectively analyze the morbidity rate in 100 consecutive PD reconstructed with Wirsung-Pancreato-Gastro-Anastomosis (WPGA), performed by a single surgeon applying a personal modification of the pancreatic reconstruction technique. Methods: During an 8-year period (May 2012 to March 2020), 100 consecutive patients underwent PD reconstructed with WPGA. The series included 57 males and 43 females (M/F 1.32), with a mean age of 68 (range 41–86) years. The 90-day morbidity and mortality were retrospectively analyzed. Additionally, a systematic review was conducted, comparing our technique with the existing literature on the topic. Results: We observed eight cases of clinically relevant POPF (8%), three cases of “primary” DGE (3%) and four patients suffering “secondary” DGE. The surgical morbidity and mortality rate were 26% and 6%, respectively. The median hospital stay was 13.6 days. The systematic review of the literature confirmed the originality of our modified technique for Wirsung-Pancreato-Gastro-Anastomosis. Conclusions: Our modified double-layer WPGA is associated with a very low incidence of POPF and DGE. Also, the technique avoids the risk of acute hemorrhage of the pancreatic parenchyma. Pancreatoduodenectomy (PD) is the only chance of cure for patients diagnosed with a tumor involving the periampullary region. Nowadays, thanks to high quality imaging for pre-operative assessment, advances in surgical techniques and perioperative management, the mortality rate after PD has decreased to 3–5% in dedicated centers. Nonetheless, the morbidity rate after PD remains elevated, even in highly specialized centers (30–50%) ,2,3. TheIn current state-of-the-art pancreatic surgery, the primary goal is to reduce major complications , allowing both early recovery and early discharge of the patient.The accepted surgical procedures for PD in cases of primary or secondary malignant conditions involving the periampullary region are pylorus-preserving PD (PPPD) (or Traverso–Longmire) and the classical Kausch–Whipple PD. Although there are several reconstruction techniques, each involves a different pancreatic anastomosis. It is possible to perform a pancreatojejunostomy (PJA) on a single loop, as described by Child in 1944, a PJA on Roux-en-Y, described by Machado, or a pancreatogastrostomy (PGA) ,6,7,8,9.Standard PGA provides for invagination of the pancreatic stump in the gastric cavity, thus exposing the pancreatic remnant to hemorrhagic complications. The aim of this study was to retrospectively analyze the morbidity rate in 100 consecutive PD reconstructed with WPGA, performed by a single surgeon applying a personal modification of the pancreatic reconstruction technique. In addition, a systematic review of the literature is reported, comparing our technique with the existing literature on the topic.During an 8-year period (May 2012 to March 2020) at the Department of General Surgery “Ospedaliera” at the Polyclinic Hospital in Bari , a single surgeon L.V. performed 212 pancreatic resections, 124 of which were PD. We retrospectively analyzed 100 consecutive PD reconstructed with WPGA over this period of time. The study was designed and conducted respecting the STROBE guidelines for observational studies.The indication for surgery was upfront resectable primary or secondary malignancy (92 patients), 6 cases of Intraductal Papillary Mucinous Neoplasia (IPMN), and 2 of chronic “mass-forming” pancreatitis. The preoperative assessment included CT scan, neoplastic markers (CA 19.9 and CEA), MRI in selected patients, and evaluation of the surgical risk according to the ASA classification . No ASA The primary endpoint was to analyze the incidence of POPF and DGE. According to the 2016 ISGPF classification, any measurable volume of drained fluid on or after POD 3, with amylase levels 3 times the upper normal limit (3 × ULN), was defined as POPF only in cases of clinical relevance. Instead, an increased amylase value in drainage fluid without clinical consequences was defined as a biochemical leak (BL) . According to the ISGPS definition, delayed gastric emptying (DGE) was diagnosed in cases needing insertion of a nasogastric tube (NGT) after POD 3 or intolerance to solid oral intake after POD 7 . SecondaThe operations were performed by the same surgeon, LV, and the reconstruction technique was always a personal, modified technique for WPGA in this series of patients. Compared to the standard PGA technique, the most important modifications adopted since 1998 were the use of an external catheter instead of an internal stent and the “double-layer” anastomosis, where only the gastric seromuscular layer is sectioned and the hole for the duct-to-mucosa anastomosis is created when the “armed” stent is passed through the posterior gastric wall. Additionally, the surgeon modified the type of sutures used for the anastomosis, adopting an absorbable monofilament instead of a non-absorbable material.After pancreatic section, the pancreatic remnant was mobilized 3 cm toward the tail, with accurate dissection from the splenic vein and the surrounding tissues. Before performing the pancreatic anastomosis, the main pancreatic duct was explored to check its patency. The seromuscular incision, performed with a scalpel on the posterior gastric wall, was slightly larger than the greatest diameter of the pancreatic stump A. SutureAn external stent, which is a catheter of 6 to 10 Fr in diameter, almost as large as the duct, was introduced into the Wirsung prior to performing the duct-to-mucosa anastomosis. Then, this catheter was anchored to the Wirsung section surface with a single absorbable stitch. The external stent facilitates the performance of the anastomosis as it avoids occlusion of the duct by the sutures.The duct-to-mucosa anastomosis was performed with interrupted monofilament absorbable suture. This anastomosis was begun at the anterior circumference, placing one or more stitches in each quadrant, depending on the main duct diameter B. The stFinally, the gastric seromuscular layer was sutured to the posterior pancreatic surface. A double suture was performed, anastomosing the main pancreatic duct to the gastric mucosa and anchoring the pancreas to the gastric wall protected by the gastric mucosa itself. In this way, corruption of the pancreatic parenchyma by gastric acid secretions was avoided and intraluminal hemorrhage prevented. The catheter was secured with a Vicryl Rapid stitch to the anterior gastric seromuscular layer. It was successively exteriorized through the anterior abdominal wall under the left costal arch and fixed with an external stitch at the end of the operation C. Two drAfter surgery, the patients usually returned to the surgical ward. The nasogastric tube was removed on POD 1 and the patient started oral intake on POD 3. The external stent was closed as soon as possible  and removed after POD 10, during recovery or after discharge.A five-step search strategy was conducted in Pubmed. Firstly, we searched for sentinel studies. Secondly, we identified keywords. Thirdly, the following complete search strategy was employed: ((pancreas[Title/Abstract] OR pancreatic[Title/Abstract]) AND (stomach[Title/Abstract] OR gastric[Title/Abstract]) AND anastomosis[Title/Abstract]) OR (pancreatogastric[Title/Abstract] OR pancreaticogastric [Title/Abstract]). Fourthly, studies on WPGA as the reconstruction technique after PD were selected. Studies on different procedures, such as PGA, were excluded. Lastly, the references of the included studies were searched to find additional relevant papers. The last search was performed on 14 July 2020. No language restriction was adopted. Four investigators  independently searched for papers, screened titles and abstracts of the retrieved articles, reviewed the full-texts, and selected articles for inclusion.The following information was extracted independently by two investigators  in a piloted form: (1) general information on the study ; (2) details of the surgical technique; (3) complications, including the number of patients with POPF (B-C), DGE, and PPH; (4) global morbidity. For each selected article, the main paper and The series included 57 (57%) males and 43 (43%) females (M/F 1.32), the mean age was 68 (range 41–86) years, and median BMI was 25 (range 18.5–39). Some of the patients had previously been treated in outlying hospitals: 19 patients underwent biliary drainage as a temporary solution to the jaundice: 1 percutaneous transhepatic biliary drainage, 17 biliary endoprosthesis and 1 sphincterotomy without endoprosthesis positioning because of technical problems, which caused acute iatrogenic pancreatitis. One patient was referred to our center after “palliative” GEA had been performed because he was considered unresectable.A Kausch–Whipple PD was performed in 55 cases, while 45 patients underwent a PPPD, according to the indications for PD and extension of the tumor. Standard lymphadenectomy was always associated, and involved the removal of lymphatic groups 5, 6, 8a, 12a, b, 13, 14v and 17, according to the Japanese classification . ExtendeOperative data are reported in Eight patients (8%) developed a clinically relevant pancreatic fistula (5 grade B and 3 grade C) and 8 patients (8%) suffered a biochemical leak . Grade BThree patients (3%) presented “primary” DGE, while in four cases (4%) we observed “secondary” DGE, related to abdominal collections or POPF. All cases were managed conservatively with prokinetics and/or repositioning of NGT for a few days. We observed only one case of prolonged DGE, which was solved with dietary re-education.We observed 32 complications in 26 patients : none of the patients experienced early-onset hemorrhage (in the first 24 h), whereas all cases are classified as late-onset PPH since they occurred more than 24 h postoperatively . Three pAdditionally, one case of biliary fistula (1%) occurred but no cases of enteric fistula.The surgical mortality was 6% (6 patients): three patients died of necrotic hemorrhagic pancreatitis, two patients due to grade C POPF complications and one patient, who underwent segmental venous resection, died of fatal vascular thrombosis.The mean operative time was 224 (range 140–420) minutes and no intraoperative complications occurred. Nine patients (9%) received intraoperative blood transfusions. The reintervention rate was 3%: one patient was operated because of necrotic hemorrhagic pancreatitis complications and two patients because of hemoperitoneum. The median hospital stay was 13.6 days (range 7–55). Two patients were readmitted, one for hemoperitoneum caused by a pseudo-aneurysm of the hepatic artery and one for severe wound infection with wall dehiscence.n = 34), no data of interest (n = 22), PGA without details on the surgical technique (n = 22), PJA (n = 19), overlapping data (n = 1), or patients undergoing surgical palliation (n = 1). Finally, seven articles were included in the systematic review, since they described a Wirsung-Pancreato-Gastro-Anastomosis  ,2,30–50% ,2,Pancreatic reconstruction after PD can be performed by means of several techniques, but choosing between PGA and PJA is the main issue. In 1988, Icard  demonstrThe most significant disadvantage of PGA is the increased risk of pancreatic hemorrhage, due to erosion of the pancreatic remnant by gastric secretions ,9,10. InThe technique presented in this series is a double pancreatic suture. Since the gastric mucosa is interrupted only where the Wirsung-mucosa anastomosis is performed, the pancreatic stump is protected by the gastric mucosa against gastric acid secretions. In theory, the limit of this technique is the impossibility of performing it in cases of a markedly friable pancreatic parenchyma. Notably, when Wirsung cannulation cannot be achieved, WPGA on an external stent cannot be performed. Nonetheless, we succeeded in performing this anastomosis even in cases of a soft pancreatic parenchyma, after Wirsung duct cannulation. The catheter acted as a guide while performing the anastomosis, avoiding pancreatic duct occlusion by the sutures. Importantly, it works as internal perianastomotic drainage so it allows early removal of NGT. Pancreatogastric sutures were applied to anchor the pancreas to the stomach and reduce tension on the anastomosis.Whichever technique is used for pancreatic reconstruction, POPF and DGE are the most common complications after PD, which mostly affect the postoperative course and the length of stay, increasing hospital costs .Several meta-analyses of randomized controlled trials (RCTs) have focused on pancreatic anastomosis, although the issue as to which is the best reconstruction method for the treatment of the pancreatic remnant after PD is still a matter of debate ,28,38,42In our series, we observed 8 patients (8%) with a clinically relevant pancreatic fistula (grade B-C). In the literature, a grade B-C pancreatic fistula rate of 11 to 28.3% is reported ,45,46,47DGE syndrome is another important issue after pancreatic surgery, occurring in 19 to 61% of patients ,48; it oSeven patients presented “isolated” abdominal collections (7%), not related to other abdominal complications . The diagnosis was made by CT scan, performed in four cases because of fever  and in 3 cases for anemia (3% blood collections). All cases were handled conservatively, with no need for reoperation.We observed 8 hemorrhagic complications (8%), all of them classified as late-onset, according to the ISGPS definition . Four paNone of the patients experienced early-onset hemorrhage, defined as PPH raised within the first 24 h postoperatively . SpecifiOnly one patient suffered a biliary leak, probably because in our reconstruction technique the distance between the pancreatic and biliary anastomoses protects the latter from pancreatic complications ,39. Six Roulin et al.  reviewedThe reintervention rate was 3% (3 patients). One patient underwent abdominal toilet for necrotic-hemorrhagic pancreatitis complications but died despite reoperation. Two patients suffered abdominal bleeding and were reoperated for hemorrhagic shock. One of them presented with pancreatic stent dislocation two weeks after surgery.The median hospital stay was 13.6 (7–55) days. Non-complicated patients were discharged after 7–10 days, thanks to postoperative management based on ERAS (enhanced recovery after surgery) protocols, which are strongly recommended after pancreatic surgery ,16. FurtThe limits of our study are represented by its retrospective nature and the small number of patients and complications observed. Also, this is the personal modification of a single experienced surgeon, who started with pancreaticoduodenectomy in 1998. Therefore, we cannot state that our technique for WPGA is widely reproducible or could reduce the rate of complications if adopted by other groups of surgeons.The characteristics and technical details of the Wirsung-Pancreato-Gastro-Anastomoses included in the review are described in Telford and Mason  describeTakao and Shinchi et al. ,34 reporThe techniques reported by the remaining authors (four of seven in our review) involve different pancreatic sutures —see In the reviewed papers, we found that there was often a lack of information about the study inclusion and exclusion criteria , Wirsung diameter , pancreatic parenchyma or other characteristics that could be related to surgical results. Furthermore, Shinchi et al.  did not The difference in defining postoperative complications, as well as the lack of information about patient characteristics, exclusion criteria and follow-up period, could influence data collection, and therefore, a correct interpretation of postoperative outcomes. Therefore, we avoided comparing our results to other WPGA found in the literature. On the other hand, the aim of this review was to confirm the originality of our modified double-layer Wirsung-Pancreato-Gastro-Anastomosis.Nowadays, pancreatic surgery offers satisfactory results in terms of mortality, but morbidity after PD remains elevated, whatever the technique used for treatment of the pancreatic stump. Despite all the guidelines and recommendations, currently there is no general consensus in the literature about the “gold standard” reconstruction technique to reduce the rate of POPF, DGE and surgical morbidity after PD. In this sense, the best technique is the one that reduces the risk of complications, especially pancreatic fistula and abdominal collections, and also the cases of “secondary DGE”.Our modified double-layer WPGA, in which the gastric mucosa reduces the risk of pancreatic stump damage by the gastric acid secretions, is associated with a very low incidence of pancreatic fistula and DGE. Furthermore, this technique reduces the risk of acute hemorrhage of the pancreatic parenchyma as compared with PGA. Nonetheless, the goal to avoid life-threatening pancreatic stump complications, such as necrotic hemorrhagic pancreatitis, which is probably a consequence of “sub-acute” damage, has yet to be achieved.Our results are limited by the retrospective nature of the study, besides the small number of complications observed. Further studies are needed to confirm the safety of our modified technique.


Acne is a common inflammatory disease of sebaceous glands, which brings extensive emotional and psychological distress to patients. Although acupuncture has certain advantages in the treatment of acne, the curative effect is not exact. The purpose of this trial is to evaluate the feasibility, preliminary efficacy, and safety of the “Spleen and Stomach Guiyuan Acupuncture Method” (SSGA) in the treatment of gastrointestinal damp-heat acne.The proposed protocol is planned as a randomized, assessor-blind, conventional-treatment-controlled trial to evaluate the efficacy of SSGA on gastrointestinal damp-heat acne. Seventy six gastrointestinal damp-heat acne patients will be randomly divided into 2 groups and receive SSGA or conventional acupuncture treatment. The entire study period is 12 weeks, including an 8-week treatment period and a 4-week follow-up period. All patients will receive 16 sessions of acupuncture treatment over 8 weeks. The primary outcome is the investigation global assessment (IGA) at week 8, which is an overall assessment of the degree of the inflammatory and non-inflammatory lesion. The secondary outcomes include IGA, the total facial lesion count , the acne-specific quality of life, etc at weeks 8 and 12. The Expectation and Credibility of treatment rating scale will be used to measure the patients’ attitudes to acupuncture after the first treatment. Adverse events will also be recorded.This study is helpful to evaluate the feasibility, preliminary efficacy, and safety of SSGA in the treatment of gastrointestinal damp-heat acne. The results will be used in sample size calculations for subsequent large-scale studies.Trial registration: Chinese Clinical Trial Registry ChiCTR2100047363. Registered on June 13, 2021. It usually occurs on the face, chest, upper back, and neck, including mild, moderate, and severe. There are different inflammatory lesions at different stages, such as acne, blackheads, pustules, papules, and cystic nodules. Acne is the eighth most prevalent disease in the world, affecting 9.4% of the world's population. It can affect 85% of teenagers, of which moderate to severe accounts for about 20%. A Chinese survey revealed that the prevalence rate in the 19-year-old age group was as high as 46.8%. Though acne was mostly seen in adolescents, it was more common in women than men after the age of 23.–11 As a common disease, it has a wide range of potential hazards and related costs. In mild cases, the symptoms are uncomfortable, affect the face, emotional and social psychological distress. In severe cases, they may have emotional and psychological distress, which even causes depression and suicide.Acne is an inflammatory disease of sebaceous glands common in teenagers, These medications generally require a long-term application. Sometimes, the side effects of these drugs are unavoidable, such as the possibility of drug resistance, dryness, peeling, erythema, and skin irritation. Furthermore, the recurrence rate is relatively high after treatment. With the development of medicine in recent years, some non-pharmacological therapies for acne are widely used, such as lasers, light devices including LED device, photodynamic therapy, intense pulsed light, and acupuncture. Acupuncture is widely used to treat various skin diseases. The World Health Organization has recommended acupuncture as an effective treatment for acne. Acupuncture, as a non-pharmacological therapy, has certain advantages in the treatment of acne and has fewer side effects. The existing randomized controlled trials have shown that acupuncture has a certain effect in the treatment of this disease.,15 It can not only improve the skin damage of patients with acne but also improve the quality of life.–20At present, most of the treatments for acne are topical and oral retinoic acid, antibiotics and benzoyl peroxide, oral hormones or contraceptives, and so on.–20 The core of Traditional Chinese Medicine (TCM) is syndrome differentiation and treatment. According to the theory of TCM, acne is located in the skin, closely related to the lung, spleen, stomach, and intestine. The basic pathogenesis is heat toxin steaming the skin. The gastrointestinal damp-heat type is the most common in clinical practice. In line with the theory of TCM and clinical experience, the “Spleen and Stomach Guiyuan Acupuncture Method” (SSGA) selects acupoints according to dialectical classification and is mainly used to treat this syndrome. In addition, we found the SSGA is simple and easy to perform, and the effect is good in clinical practice. However, there is no relevant literature to report. Therefore, this trial intends to observe the clinical efficacy of gastrointestinal damp-heat acne through SSGA.However, the existing clinical studies have shown that the curative effect of acupuncture on acne is not exact. The effective rates of different acupuncture treatments are different. Some are relatively high, and some are relatively low. This is because these clinical studies did not adopt the principle of syndrome differentiation and treatment for the therapy of this disease, but only conventional treatment methods.22.1The proposed study is a prospective, parallel, two-armed randomized controlled trial using a 1:1 allocation ratio with a 4-week follow-up. The study will be conducted at Dongzhimen Hospital of Beijing University of Chinese Medicine and the Third Affiliated Hospital of Henan University of Traditional Chinese Medicine. The outline of the study patient process is in Figure The research protocol was approved by the Medical Ethics Committee of Dongzhimen Hospital of Beijing University of Chinese Medicine (2021DZMEC-087-01) and by the Medical Ethics Committee Third Affiliated Hospital of Henan University of Chinese Medicine. We will obtain the patient's informed consent. Study results will be disseminated in peer-reviewed journals, through public events, and relevant third sector organizations.2.22.2.1Patients with acne will refer to the Chinese Journal of Clinical Dermatology published in April 2017:1.The age of onset is mostly male and female in adolescence.2.The main affected parts are the face, front chest, and back of the neck.3.The skin lesions are in the form of inflammatory papules, blackheads, whiteheads, pustules, cysts, or dark red nodules. In severe cases, there may even be scars, often accompanied by seborrhea, which is chronic.2.2.2With reference to the “Thirteenth Five-Year Plan” textbook “Traditional Chinese Medicine Surgery,” the diagnostic criteria for gastrointestinal damp-heat acne are as follows:1.White or blackhead pimples on the face, chest, and back may be accompanied by light yellow or white lipid plugs; or red needle-like papules accompanied by redness and pain; or red papules accompanied by pustules.2.With oily face; or with dry mouth and bad breath.3.Thick stools, yellow urine.4.The tongue is red, the coating is yellow and greasy, and the pulse is slippery.2.2.3Patients who meet the following conditions will be included:1.13 to 30 years old, male or female patients with acne.2.Patients meet the diagnostic criteria for acne in Western medicine and the gastrointestinal damp-heat type according to the TCM syndrome differentiation criteria3.Investigation Global Assessment (IGA) score is level 3 (moderate) or level 4 (severe).4.Symptoms have been on for more than 6 months.5.Have a certain level of reading comprehension and be able to complete questionnaires independently.6.Patients did not use acne drugs, such as oral, topical, or traditional Chinese medicine, within 4 weeks before entering the group.7.Those who did not participate in other clinical studies within 1 month and were guaranteed not to participate in other clinical studies during this study period.2.2.4Patients who report the following conditions will be excluded:1.Pregnant and lactating women, women who have the desire to give birth in the past 2 months.2.Have a history of mental illness.3.Severe liver and kidney dysfunction.4.Cardiovascular, endocrine, hematopoietic system, immune system, and other primary diseases.5.Drug-induced acne and occupational acne.6.Patients who are unwilling to cooperate with acupuncture treatment.7.In the course of treatment, other acne treatments are combined, such as oral or topical treatment with other drugs, laser therapy, and traditional Chinese medicine masks.2.2.5Seventy six patients will be recruited through hospital outpatient clinics, advertisements on the hospital's social media (WeChat), and leaflets displayed by community service centers. The recruitment will provide a phone number so that potential patients can contact the researchers. The dermatologist will screen patients who meet the inclusion and exclusion criteria and use a digital camera (Canon EOS 200D) to take facial photos. The study assistant will be responsible for providing study information, such as the objectives, interventions, duration, benefits, and risks. Agreed to participate in the study patients will sign informed consent. The confidentiality of information recorded by patients will be protected.2.2.6Patients will be randomly assigned to receive SSGA or conventional acupuncture at a ratio of 1:1. Sealed opaque envelopes will be applied to confirm random hiding. The random sequence number and group assignment information will be sealed in orderly envelopes. With the addition of the patients, these envelopes will be unfolded one by one in order, and the research assistant will record the assigned serial number in the case report form (CRF). The research assistant who does not participate in treatment or evaluation will keep these envelopes.2.2.7Patients, outcome assessors, and statisticians will turn a blind eye to this assignment. However, due to the characteristics of acupuncture, acupuncturists will not be deceived in this trial. Patients will not know their group status and patients in different treatment groups will be treated in different treatment rooms. Besides, they will be separated by a curtain to avoid communication during the trial. For the successful implementation of blinding, all researchers will be trained before the start of the trial.2.3All patients will receive treatment for 8 weeks, twice a week (every Monday and Thursday), 30 minutes each time, a total of 16 sessions. Acupuncturists will be required to hold a Chinese medicine practitioner license and have more than 1 year of clinical experience. The treatment will use sterile disposable steel needles . According to the different acupuncture points and the depth of acupuncture, sterile disposable steel needles of different specifications will be selected. And acupuncture will be artificially stimulated for 10 seconds to achieve “Deqi”. That is, the subject will have special sensations such as soreness, numbness, swelling, and weight. And the surgeon has feelings of tightness under the needle.2.3.1Group A will receive SSGA. The acupuncturist will use sterile disposable steel needles to stimulate left SP9 (Yinlingquan), left SP8 (Diji), left SP7 (Lougu), right ST36 (Zusanli), right ST37 (Shangjuxu), right ST39 (Xiajuxu). After routine disinfection, the acupuncturist will insert disposable steel needles vertically into SP9, SP8, SP7, ST36, ST37, and ST39. The specific selection of acupuncture points, needle specifications and the depth of acupuncture are shown in Table 2.3.2Group B will receive conventional acupuncture treatment. The acupuncturist will use sterile disposable steel needles to stimulate, bilateral LI11 (Quchi), bilateral LI4 (Hegu), bilateral ST44 (Neiting), bilateral GB14 (Yangbai), bilateral SP9 (Yinlingquan), bilateral ST36 (Zusanli). The acupuncturist will insert the disposable steel needles vertically into LI11, LI4, ST44, GB14, SP9, and ST36 after routine disinfection. The specific selection of acupuncture points, needle specifications, and the depth of acupuncture are shown in Table 2.42.4.1,22The primary outcome measurement will be expressed by the change in IGA score from baseline to the end of 8-week treatment. The IGA score is an overall assessment of the degree of inflammatory and non-inflammatory lesions and is divided into 6 levels.Grade 0 (0 points): the skin is smooth and clean, without any inflammatory or non-inflammatory lesions; Grade 1 (1 point): the skin is almost smooth, non-inflammatory lesions are rare, and no more than 1 small inflammatory lesion; Grade 2 (2 points): mild, with some non-inflammatory lesions, and only a few inflammatory lesions ; Grade 3 (3 points): moderate, there are multiple non-inflammatory lesions, and there may be some inflammatory lesions, but no more than 1 small nodular lesion; Grade 4 (4 points): severe, with multiple non-inflammatory and inflammatory lesions, but only a few nodular lesions; Grade 5 (5 points): extremely severe, with multiple non-inflammatory and inflammatory lesions, and multiple nodular cystic lesions.2.4.2,23 inflammatory lesion counts, non-inflammatory lesion counts, and Acne-Specific Quality of Life at 8 and 12 weeks compared with baseline display.The secondary measurements will be measured by changes in IGA, total facial lesion counts ,Total lesion count includes inflammatory lesion counts and non-inflammatory lesion counts. Inflammatory lesions include inflammatory papules, pustules, cysts, and nodules. Non-inflammatory lesions include blackheads and whiteheads. Lesions will be evaluated on the forehead, cheeks, nose, and chin. This scale is mainly used to test the quality of life of patients with skin diseases. Each item is divided into 4 levels according to the specific conditions of the patient, with a score of 0 to 3. The total score of 16 items is the patient's skin disease quality of life score. The total index score is 30 points. The higher the score, the worse the patient's quality of life.Acne-specific quality of life has 14 items in total, covering 3 areas, including self-perception, social function, and emotional function.The Expectation and Credibility of treatment rating scale will be used to measure the patients’ attitudes to acupuncture after the first treatment. The patients will be asked the following question: “Do you think SSGA will be effective in treating the symptoms of ance?”2.4.3The data for this study will be collected by the assistant and filled in CPFs. The independent investigator will enter the relevant information and data of this study into an Excel spreadsheet to protect its confidentiality. No one outside the research group will have access to relevant information. Participants’ research information may not be published outside of the research if there is no written permission from the participants.2.52.5.12 test to compare group A and group B. For secondary outcomes, continuous variables including IGA, TCL, Inflammatory lesion count, Non-inflammatory lesion count, Qol-Acne, and the Expectation and Credibility of acupuncture treatment will be compared at 8 and 12 weeks, using an unpaired Student t test or Wilcoxon rank-sum test (depending on the situation). A P value of <.05 will be considered a limit of statistical significance.Statistical analysis of all data will use IBM SPSS statistical version 24.0 software . For continuous variables, the data will be represented by the mean (standard deviation) or median (interquartile range) according to the normal distribution. Enumerated data will be displayed in percentage. For the primary outcome, we will calculate the IGA score at 8 weeks, and use the χ2.5.2π0 = 76.7%. Combined with clinical experience, the total effective rate of SSGA in treating acne is 98.48%, then the overall rate of expected test results was π = 98.48%. One-sided cutoff of this trial: zα = 1.645, zβ = 1.282, δ=π-π0 = 0.9848-0.767 = 0.2178. Enter the formula to get the estimated sample content value n≈32. Considering that “dropping” may increase the sample size by up to 20%, the sample size was estimated to be 38 cases. Finally, Group A and Group B are according to the random number table method.The total sample size was 76 cases and they are randomly divided into 2 groups. According to the trial design, the sample size estimation method used a completely random design sample rate and the population rate comparison. The sample size is calculated according to the following formula: 2.6To promote patient retention and complete follow-up, total interventions to patients are free of charge. The data relating to this study will be recorded in the CRFs, and then input into the Excel spreadsheet by independent investigators as the first level of control to guarantee the accuracy of the data. The second level of data integrity will include data integrity validation and data monitoring throughout the study carried out regularly. All original forms (including the consent forms and CRFs) will be safely stored in Dongzhimen Hospital of Beijing University of Chinese Medicine. The Research Ethics Committee of Dongzhimen Hospital of Beijing University of Chinese Medicine will audit the trial independently of the investigators every 5 months and will decide whether to end the study early.From baseline to week 12, adverse events and their numbers will be recorded. These adverse events will include adverse events related to acupuncture, such as needle fainting, infection, hematoma, sensation after acupuncture, as well as adverse reactions that cannot be related to acupuncture. For the safety assessment, the trial assistant will record the details of all adverse events in the CRF. Any serious adverse event will be reported to the ethical committees of Dongzhimen Hospital of Beijing University of Chinese Medicine within 24 hours, and the Dermatology Department will compensate for any injuries related to the intervention during the study period. The specific trial intervention process is shown in Figure 3 The general effects of acne are physical sensory symptoms such as skin itching and pain. However, the most serious impact is on the quality of life. This influence is affected by various factors, which makes it difficult to quantitatively analyze. In youth, the self-confidence and self-esteem of acne sufferers are affected. If it can’t be cured, it may bring continuous negative psychological effects on adults, such as shame, embarrassment, depression, anxiety, and even suicide. Therefore, complementary and alternative medicine in modern practice regarding various dermatology is becoming more and more extensive.Acne is a chronic inflammatory skin disease, and its pathogenesis is still not completely clear. Modern studies suggest that its onset is related to the relative increase of androgens, increased secretion of sebum, hair follicles mouth of excessive and Propionibacterium acnes infection and so on. In addition, the immune imbalance, the role of inflammatory factors, and genetic related factors have also been recognized. It is widely used in China and has achieved good results in the treatment of acne, chloasma, eczema, allergic skin diseases, and other skin diseases. However, the existing trials only provide routine treatment for this disease and do not conduct syndrome differentiation.–30 TCM treats acne and adopts the principle of syndrome differentiation and treatment. Starting from the origin, the advantages are significant.,32 Therefore, under the guidance of TCM theory and clinical experience, we have designed a study on acne with the gastrointestinal damp-heat syndrome. This trial is based on the guidance of TCM theory. We aim to evaluate the changes in the count of facial skin lesions and the improvement of quality of life in patients with acne by SSGA. This study is a multi-center clinical randomized trial, which is more in line with clinical practice and closer to the real world. Moreover, the selection of acupoints is within a relatively safe range of the lower limbs, avoiding operations on the local lesions, so the patients’ acceptance is relatively high.As a unique alternative treatment method, acupuncture has the function of “external treatment of internal diseases” and can treat systemic diseases through the conduction of meridians and acupoints.3.1Some limitations of our trial should be recognized. First of all, this trial is only for a certain type of acne, which is relatively limited. But this type of acne is the most common in the clinic, therefore we also cure and observe the majority of acne patients. Second, as a study, the sample size of this trial is relatively small. We will conduct a larger sample size study based on this trial to verify the effectiveness and safety of SSGA again in the future. Last, although this study cannot blind the acupuncturists, the patients will be blind to the group assignment well. Both treatments for the two groups of patients are real acupuncture, but the selected acupoints are different. These patients in both groups will have the feeling of “Deqi” during acupuncture. That is, they will have special sensations such as acidity, numbness, sinking, and swelling. In general, we designed this study and hope that this trial will provide favorable evidence for the treatment of gastrointestinal damp-heat by SSGA.We would like to thank all participants who contributed to this research.Data curation: Hui-Shang Feng, Guang-An Wang.Funding acquisition: Chao-Qun Yan, Kai-Bing Tian.Investigation: Hui-Shang Feng, Ya-Qi Yao.Literature retrieval: Ping-Ping Duan, Chao-Qun Yan.Recruiting patients: Ping-Ping Duan, Hui-Shang Feng, Ning Sun, and Ya-Qi Yao.Software: Ping-Ping Duan, Yuan Chen, Kai-Bing Tian.Supervision: Guang-An Wang.Writing – original draft: Ping-Ping Duan, Chao-Qun Yan, Hui-Shang Feng, Ning Sun.Writing – review & editing: Ping-Ping Duan, Guang-An Wang.


Pectobacterium strains were isolated from soft rot-diseased vegetables. Here, we report their genome sequences and characteristics. Five isolates were found to be Pectobacterium versatile, while the other six were determined to be Pectobacterium brasiliense.Eleven  Pectobacterium and Dickeya species representing the leading disease-causing agents in the field and postharvest  and complete (n = 3) genome sequences.Soft rot disease of plants is a deterioration of plant tissues, resulting in smelly, mushy, and inedible fruits and vegetables. The bacteria that cause soft rot are among the most important plant pathogens , with Petharvest . They emtharvest . Once diAGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GTTACCTTGTTACGACTT-3′), for amplification and an NCBI BLASTn search against the NCBI nonredundant (nr) database identified the isolates as Pectobacterium species  and isolates SR5 to SR8, SR10, and SR11 to be most similar to Pectobacterium brasiliense PcbHPI01 . No plasmids were identified in the isolates; all have type III secretion systems, multiple pectin lyases, cellulases, proteases, iron uptake systems, and flagellar genes. This work provides the basis for further experiments on these agriculturally and economically important plant pathogens.The genome sizes ranged from 4.85 to 5.08 Mbp, the number of contigs ranged from 1 to 71, and the GC content was approximately 52% . The ANIPectobacterium sequences have been deposited in GenBank under the BioProject accession number PRJNA767613. The GenBank and SRA accession numbers for each isolate are listed in The


The composition and function of gut microbiota is crucial for the health of the host and closely related to animal growth performance. Factors that impact microbiota composition can also impact its productivity. Ammonia (NH3), one of the major contaminants in poultry houses, negatively affects poultry performance. However, the influence of ammonia on broiler intestinal microflora, and whether this influence is related to growth performance, has not been reported. Our results indicated that ammonia caused changes to cecal microflora of broilers, and these changes related to growth performance. Understanding the effects of ammonia on the intestinal microflora of broilers will be beneficial in making targeted decisions to minimize the negative effects of ammonia on broilers.Proteobacteria phylum was increased after 35 ppm ammonia exposure. At genus level, ammonia increased the relative abundance of Escherichia–Shigella and decreased the relative abundance of Butyricicoccus, Parasutterella, Lachnospiraceae_UCG-010, Ruminococcaceae_UCG-013 and Ruminococcaceae_UCG-004. Negative correlation between Escherichia–Shigella and growth performance, and positive correlation between bacteria genera  and growth performance was observed. In conclusion, ammonia exposure caused changes in the structure of cecal microflora, and several species were either positively or negatively correlated with growth performance. These findings will help enhance our understanding of the possible mechanism by which ammonia affect the growth of broilers.In order to investigate the influence of ammonia on broiler intestinal microflora and growth performance of broiler chickens, 288 21-day-old male Arbor Acres broilers with a similar weight were randomly divided into four groups with different NH3 levels: 0 ppm, 15 ppm, 25 ppm, and 35 ppm. The growth performance of each group was recorded and analyzed. Additionally, 16s rRNA sequencing was performed on the cecal contents of the 0 ppm group and the 35 ppm group broilers. The results showed the following: a decrease in growth performance in broilers was observed after 35 ppm ammonia exposure for 7 days and 25 ppm ammonia exposure for 14 days. At phylum level, the relative abundance of  Gaseous ammonia NH3), the foremost alkaline gas in poultry houses, is emitted primarily by the degradation of litter that contains unutilized nitrogen  and nega, the for−3) for 30 days (8 h/day); the results showed that ammonia could cause changes in inflammatory markers and beta diversity of intestinal microflora in fattening pigs [The effects of ammonia, a typical inhaled air pollutant, on gut microbiota has been investigated. In one study, six female pigs were exposed to ammonia , during the entire experiment. Additionally, airflow was controlled during the exposures to ensure adequate ventilation, minimize build-up of animal-generated contaminants , and avoid thermal stress. The litter material was removed from the chambers every three days to reduce NH3 volatilization. Broiler grower feed and water was supplied ad libitum. The diet composition according to NRC 1994  was shown in The animal trials were performed as previously described . BrieflyAll birds were weighted (after 12 h fasting) at 7, 14 and 21 days after the start of the trial, and the performance parameters including ADFI (average daily feed intake), FCR, BWG  − (start weight)) and BW (body weight) were measured. At day 21, one bird of each replication of the 0 ppm group and the 35 ppm group; were euthanized by cervical dislocation and the cecal contents were obtained and stored at −80 °C for further analyses.Microbial DNA was extracted from cecal contents using HiPure Stool DNA Kits  according to manufacturer’s protocols, and its concentration and quality were determined by the NanoDrop 2000 UV-vis spectrophotometer  and 1% agarose gel electrophoresis, respectively. The V3-V4 hypervariable regions of 16S rRNA gene was amplified with primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTATCTAAT-3′) by the thermocycler PCR system . PCR product was purified using the AMPure XP Beads  and quantified using ABI StepOnePlus Real-Time PCR System . Purified amplicons were pooled in an equimolar solution and then paired-end sequenced (PE250) on an Illumina platform, according to the standard protocols.t-test in R project Vegan package (version 2.5.3).Sequencing libraries were generated using SMRTbell TM Template Prep Kit , following manufacturer’s recommendation. The library sequencing was performed on the Illumina HiseqTM 2500/4000 by Gene Denovo Biotechnology Co., Ltd. . According to the unique barcode of each sample, pairing sequences were assigned to each sample, and the primer sequencing and barcode were removed. The matching end was merged and read using FLASH. Based on FASTP (version 0.18.0), quality filtering of raw data was performed under specific filtering conditions to obtain high-quality clean tags. All chimeric tags were removed using the UCHIME algorithm. The feature tables and feature sequences were obtained by DADA2. The clean data was clustered into operational taxonomic units (OTUs) of ≥97% similarity using the UPARSE (version 9.2.64) pipeline. According to the SILVA (release 132) classifier, feature abundance was normalized using the relative abundances of each sample. Alpha diversity was used to analyze the complexity of sample species diversity by Chao1, observed species, goods coverage, Simpson and Shannon, which were calculated using QIIME (version 1.9.1). Beta diversity (between sample), such as PCoA and NMDS, was performed in R project Vegan package (version 2.5.3). The abundance statistics of each taxonomy was visualized using Krona (version 2.6). Species comparison among groups was computed by Tukey’s HSD test in R project Vegan package (version 2.5.3). The function prediction of the OTUs was inferred using PICRUSt (version 2.1.4). Analysis of function difference between groups was calculated by Welch’s p < 0.05 was considered statistically significant. The correlation between growth performance and microbial species richness was measured by Spearman’s correlation analysis, which was conducted using the R Vegan package.The data of growth performance were presented as mean ± standard error. The significance of the difference between means was determined by one-way (ANOVA) analysis followed by Tukey’s test, and p < 0.05) in the 25 and 35 ppm groups compared with the control group, and BWG and BW were significantly lower in the 35 ppm group compared with the other groups. At day 14, ADFI, BWG and BW were significantly lower, and F/G was significantly higher in the 35 ppm group compared with the 0, 15 and 25 ppm groups, and BW in the 25 ppm group was significantly lower compared with the 0 and 15 ppm groups. At day 21, BWG and BW were significantly lower in the 25 and 35 ppm groups compared with the 0 and 15 ppm groups, and BWG and BW in the 35 ppm group were significantly lower compared with the 25 ppm group. Moreover, ADFI and F/G were significantly lower and higher separately in the 35 ppm group compared with the other groups.The growth performance of broilers in the different groups was measured by ADFI, F/G, BWG and BW. The results are shown in A total of 1,525,416 clean tags were obtained and clustered into 13,984 OTUs (1075.75 ± 73.48 per sample); the detailed information of sequencing data was shown in p > 0.05).The effects of ammonia on alpha diversity in the cecal contents of broilers is shown in p < 0.05).PCoA  based on Unweighted-UniFrac and NMDS  were used to visualize the beta diversity of cecal microbiota from two groups. Analysis of similarities based on unweighted UniFrac is shown in Firmicutes and Bacteroidetes phyla acted as the predominant taxa in two groups. The Proteobacteria phylum relative abundance in the treatment group was significantly higher (p < 0.05) compared with the control group. At genus level, there were significant differences (p < 0.05) in six microbial species between the treatment group and control group, among which the relative abundance of Escherichia–Shigella was higher (p < 0.05) in the treatment group, and the relative abundance of Butyricicoccus, Parasutterella, Lachnospiraceae_UCG-010, Ruminococcaceae_UCG-013 and Ruminococcaceae_UCG-004 was lower in the treatment group.A taxonomic analysis of the gut microflora in the cecal contents of broiler was shown in p < 0.05).The differences of presumptive biological function of cecal contents microflora between the control group and the treatment group were presented in Escherichia–Shigella was positively correlated with F/G and negatively correlated with ADFI, BWG and BW. Ruminococcaceae-UCG-013 was negatively correlated with F/G and positively correlated with BWG and BW. Lachnospiraceae-UCG-010 was positively correlated with ADFI, BWG and BW, and negatively correlated with F/G. Parasutterella was positively correlated with ADFI, BW and BWG; Ruminococcaceae-UCG-004 and Butyricicoccus were positively correlated with ADFI, BWG and BW.The correlation between growth performance and indicator species was shown in In this study, the effects of ammonia on growth performance and the intestinal microflora of broiler chickens were investigated. Additionally, the correlation between growth performance and intestinal microflora under ammonia exposure has also been analyzed.Ammonia is the most prominent toxic gas in poultry houses. It originates from the breakdown of undigested protein and uric acid in the litter  and adveFirmicutes, Bacteroidetes and Proteobacteria were the most dominant phyla in the cecal of broilers between two groups, which was consistent with previous studies [Proteobacteria was higher in the ammonia group, which is in agreement with the increase of Escherichia–Shigella genus.Our results showed that a variety of microbiota communities lived in the cecal of broilers, and there was a significant difference in gut microflora composition and structure among two groups, indicating that ammonia exposure could induce changes in the symbiotic relationship of microorganisms. Ammonia exposure changed the beta diversity but did not change the alpha diversity, indicating that ammonia changes the species composition but hardly influences the species richness and evenness. The structural response of gut microbiota to ammonia exposure was investigated in this study; results showed that  studies ,27,28. AEscherichia–Shigella genera invade and proliferate within colonocytes and mucosal macrophages [Escherichia–Shigella. Some species of rophages , while trophages . DamagesButyricicoccus, Parasutterella, Lachnospiraceae_UCG-010 and Ruminococcaceae_UCG-013, Ruminococcaceae_UCG-004 was observed in the ammonia group. Butyricicoccus, Ruminococcaceae_UCG-013 and Ruminococcaceae_UCG-004 belong to the same phylogenetic order , which is autochthonous benign microbes primarily inhabit the caecum [Lachnospiraceae benefits gut development and health by degrading plant fiber and producing SCFAs [Simultaneously, the significantly lower relative abundance of e caecum , which ie caecum . In adding SCFAs . These tng SCFAs ,8, whichng SCFAs . All of The composition and function of gut microbiota is crucial for the health of the host and closely related to animal growth performance. The relationship between intestinal flora and growth performance was explored by observational experiments in previous studies and, indeed, there was a significant difference in gut microflora composition and structure between the birds with the highest and lowest feed conversion efficiency ,15,35. Escherichia–Shigella and ADFI, BWG and BW was observed, which is in line with previous studies [In this study, we analyzed the relationship between growth performance and the different genera that ammonia caused. A negative correlation between  studies . Another studies . The hyp studies , and cau studies , thus reButyricicoccus, Parasutterella, Ruminococcaceae_UCG-004, Ruminococcaceae-UCG-013 and Lachnospiraceae-UCG-010 was positively related with growth performance of broilers. Except for the Parasutterella, all of these genera were related to the order Clostridiales, which ferments diverse plant polysaccharides to SCFAs [In this study, the relative abundance of to SCFAs . In lineto SCFAs  found thSCFAs, the major component constitutes of bacterial metabolites, have broad effects on various aspects of host physiology , especiaParasutterella has been defined as a core component of gut microbiota, correlated with various microbial-derived metabolites such as aromatic amino acid, bilirubin, purine and bile acid derivatives [Parasutterella in the treatment group is in keeping with the depleted amino acid, cofactors and vitamin metabolism pathways, together with poor performance.The genus of ivatives . Alterativatives ,46. In tIn conclusion, in this study, we found that ammonia exposure caused changes in the structure of cecal microflora. At the genus level, we found that several species were positively or negatively correlated with growth performance. These results will help enhance our understanding of the possible mechanism by which ammonia affects the growth of broilers.


To identify differentially expressed and clinically significant mRNAs and construct a potential prediction model for metabolic steatohepatitis (MASH).We downloaded four microarray datasets, GSE89632, GSE24807, GSE63067, and GSE48452, from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) analysis and weighted gene co-expression network analysis were performed to screen significant genes. Finally, we constructed a nomogram of six hub genes in predicting MASH and assessed it through receiver operating characteristic (ROC) curve, calibration plot, and decision curve analysis (DCA). In addition, qRT-PCR was used for relative quantitative detection of RNA in QSG-7011 cells to further verify the expression of the selected mRNA in fatty liver cells.Based on common DEGs and brown and yellow modules, seven hub genes were identified, which were NAMPT, PHLDA1, RALGDS, GADD45B, FOSL2, RTP3, and RASD1. After logistic regression analysis, six hub genes were used to establish the nomogram, which were NAMPT, RALGDS, GADD45B, FOSL2, RTP3, and RASD1. The area under the ROC of the nomogram was 0.897. The DCA showed that when the threshold probability of MASH was 0–0.8, the prediction model was valuable to GSE48452. In QSG-7011 fatty liver model cells, the relative expression levels of NAMPT, GADD45B, FOSL2, RTP3, RASD1 and RALGDS were lower than the control group.We identified seven hub genes NAMPT, PHLDA1, RALGDS, GADD45B, FOSL2, RTP3, and RASD1. The nomogram showed good performance in the prediction of MASH and it had clinical utility in distinguishing MASH from simple steatosis. MASH is developed from simple steatosis and can progress to cirrhosis and even liver cancer. A previous study reported that the overall global prevalence of NAFLD diagnosed by imaging was approximately 25.24 and 7–30% of patients with NAFLD had NASH, indicating the overall prevalence of NASH was approximately between 1.5 and 6.45% ,9. The gIn this study, we aimed to screen potential mRNAs for the diagnosis of MASH. Differentially expressed genes (DEGs) between NASH patients and healthy controls were identified in GSE89632, GSE24807, and GSE63067. Then we constructed weighted gene co-expression modules and screened significant genes in modules mostly related to the status of NAFLD. The common genes in DEGs and significant genes in modules were considered as hub genes related to the disease. Based on the decision curve analysis (DCA) and receiver operating characteristic (ROC) curve, we validated the clinical utility of the nomogram of hub genes in predicting MASH.22.1https://www.ncbi.nlm.nih.gov/geo/), up to March 1, 2020. The searches included the keywords  and (organism: Homo sapiens).We conducted dataset searches from the Gene Expression Omnibus (GEO) database of the National Center for Biotechnology Information  study type was expression profiling by array; (ii) samples were from liver tissue; (iii) studies included control and case samples. The search and selection process are shown in Figure S1. We chose datasets with the top three sample sizes for DEGs and chose datasets that included controls, steatosis and NASH samples for weighted gene co-expression network analysis (WGCNA) and validation.The datasets GSE89632, GSE24807, GSE63067, and GSE48452 were downloaded from the GEO database. GSE63067 included two steatosis samples, nine NASH samples, and seven healthy samples . GSE8963GSE63067, GSE24807, and GSE89632 were used to identify DEGs. GSE89632 was analyzed with the weighted gene co-expression network. Finally, GSE48452 was used to construct and validate the prediction nomogram.2.2https://www.ncbi.nlm.nih.gov/geo/geo2r/) was used to compare two groups of samples to identify DEGs. DEGs between NASH samples and healthy controls were analyzed in the datasets GSE63067 and GSE89632 respectively. p-value <0.05 and log FC absolute value >1.2 were used as a filter for the datasets GSE63067 and GSE89632. Bioinformatics analysis was based on the R software 3.6. With the Combat function in the SVA version 3.5 R package, the batch effects in GSE24807 were corrected [p-value <0.05 and log FC absolute value >2 were used as a filter. The common DEGs were listed and the Venn diagram was made.The online analysis platform GEO2R  was set to construct modules (http://metascape.org/gp/index.html#/main/step1).With WGCNA R package, clusters (modules) of highly correlated genes were found and the correlation between modules external sample traits was constructed for GSE89632 . First,  modules . Externa2.4p-value for the disease was <0.05 were considered as significant genes. Hub genes were the intersection of DEGs and significant genes, which were NAMPT, PHLDA1, RALGDS, GADD45B, FOSL2, RTP3, and RASD1. To further observe the relation between hub genes and clinical data, the heatmap of hub genes and samples was drawn with the pheatmap R package.Based on the WGCNA R package, gene significance (GS) and connectivity between genes and genes were calculated. Kwithin was the connectivity of a gene and other genes that were in the same module. GS was the correlation between gene expression and clinical data. Then, genes in the brown and yellow module whose Kwithin was top 5% and GS 2.5GSE48452 was used to construct and validate the prediction model with the rmda, rms, and pROC R package. The data of patients with NASH or simple steatosis were normalized by zero-mean normalization. The logistic regression analysis was performed, and PHLDA1 was little contributed to MASH. Therefore, we constructed a prediction nomogram for MASH which included NAMPT, RALGDS, GADD45B, FOSL2, RTP3, and RASD1, and the predicted value of the nomogram for MASH was obtained. To evaluate the nomogram, the ROC curve, DCA, and calibration plot were performed.2.62 atmosphere. At about 70% confluence, the cells were treated with or without 0.2 mM free fatty acid . After 16 h treatment, the cells were collected for further experiments.The human normal liver cell line QSG-7701 was obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences . It was cultured in RPMI-1640 medium  with 10% fetal bovine serum, and incubated at 37°C in a humidified 5% CO−ΔΔCT method and normalized by β-actin. All operations were repeated thrice.Total RNA was extracted from collected cells using miRNeasy Mini Kit  according to the manufacturer’s instructions. The reverse transcription was performed with Reverse Transcription Kit  and the cDNAs were quantified by real-time PCR by Roche LightCycler96 using QuantiNova SYBR Green PCR Kit . Primers used for qRT-PCR are listed in Table S2. qRT-PCR was carried out with the condition of 2 min for initial denaturation, 45 cycles for denaturation at 95°C for 10 s, annealing and extension at 55°C for 20 s, and melting curves analysis at default procedure. Relative mRNA levels were calculated by the 22.7t-test was performed to compare differences between groups. p < 0.05 was statistically significant.Data were reported as mean ± SD. Student’s Ethics and consent: The ethics approval and consent to participate were not applicable.33.1p-value <0.05, log FC absolute value >1.2); 83 DEGs were screened in GSE63067 ; and 1,643 DEGs were screened in GSE24807 . The common DEGs were presented in a Venn diagram  and steatosis . Yellow module was second related to disease  and steatosis   . Brown mThere were 551 genes in the brown module and 412 genes in the yellow module. GO and KEGG pathway analyses for genes in the two modules were performed. The top five significant GO molecular functions (MFs), biological processes (BPs), and cellular components (CCs), and top 15 KEGG pathways were demonstrated . The enr3.3p-value. The Kwithin of repeated genes were averaged. Screened by Kwithin and GS p-value, brown module and yellow module owned 27 and 20 significant genes, respectively. Intersected by significant genes and DEGs, hub genes, seven in total, were NAMPT, PHLDA1, RALGDS, GADD45B, FOSL2, RTP3, and RASD1 . The expression of NAMPT, RALGDS, GADD45B, FOSL2, RASD1, and RTP3 did not statistically differ between NASH and simple steatosis, while the expression of PHLDA1 was higher in NASH than in simple steatosis .Through the above analysis, we finally kept 19 samples with NASH, 20 samples with simple steatosis, and 18 controls in the dataset GSE89632. The clinical characteristics and the expression of hub genes are shown in 3.5GSE48452 was used to construct a logistic regression model. The model of NAMPT, RALGDS, GADD45B, FOSL2, RTP3, and RASD1 is shown as the nomogram . The calDCA calculated the net benefit without additional clinical information, such as life-years saved or quality of life improved . In Figu3.6The expression of hub genes in QSG-7011 cells with or without FFA was quantified by qRT-PCR, and the results are shown in 4In the study, we used the analysis of DEGs and WGCNA to identify hub genes. Not a single gene, but clusters of highly correlated genes were detected and related to clinical traits with the use of WGCNA . ThroughGEO2R analysis obtained the DEGs between NASH samples and controls in the three datasets. These datasets were from different platforms, and so we used common DEGs to reduce the effect of different platforms. We combined common DEGs and significant genes for disease status in WGCNA to get hub genes that were able to predict NASH and distinguish NASH from steatosis. Finally, seven genes overlapped, which were NAMPT, PHLDA1, RALGDS, GADD45B, FOSL2, RTP3, and RASD1. A prediction model was constructed through logistic regression analysis. Then, we visualized the model and performed the ROC curve and decision curve analyses for the model.Samples with NASH were different from simple steatosis in histology, including steatosis of hepatocytes, lobular inflammation severity, and ballooning intensity. Although there was no significant statistical difference in the expression of hub genes, the decision curve revealed the prediction model had clinical utility, and it had net benefit within certain risk probability. The area under the ROC curve was 0.897, and the curve illustrated that the sensitivity of the model was superior to specificity. However, we did not compare other diagnostic methods for MASH with our model, and whether the model was better than other diagnostic methods still need to be reevaluated .In our study, we identified seven hub genes: NAMPT, PHLDA1, RALGDS, GADD45B, FOSL2, RTP3, and RASD1. These hub genes were considered to have a contribution to the pathogenesis of MASH. Because of the small sample size, PHLDA1 showed little contribution to MASH in regression analysis; therefore, PHLDA1 was excluded and the other six hub genes were made a logistic regression analysis. At the same time, we verified the expression of hub genes in QSG-7701 cells with FFA, and the expression of NAMPT, RALGDS, GADD45B, FOSL2, and RASD1 was consistent with the results of the bioinformatics analysis. However, the relative expression of RTP3 was lower in QSG-7701 cells with FFA than in controls, which was contrary to the WGCNA. The expression of all hub genes between groups was not statistically significant, except FOSL2, possibly because of the small sample size.Z-score normalization of NAMPT is higher, and the points are higher, which indicates that NAMPT plays an important role in MASH. However, studies indicated that the deficiency of NAD played a role in aged NAFLD [NAMPT, nicotinamide phosphoribosyltransferase, or visfatin, promotes nicotinamide to convert to nicotinamide mononucleotide (NMN). NMN finally converts to nicotinamide adenine dinucleotide (NAD), which is a vital coenzyme in cellular redox reactions in all organisms and participates in many signaling pathways . NAMPT ped NAFLD ,25, and ed NAFLD . Therefoed NAFLD  and diabed NAFLD ,28.PHLDA1, pleckstrin homology like domain family A member 1, was a phosphatidylinositol-binding protein and it could suppress AKT . Zhang eRALGDS, Ral guanine nucleotide dissociation stimulator, is an activator of RalA. RalA and RALGDS are important to Ras-induced oncogenic transformation of cells . GADD45BThe study contributed to understanding the molecular mechanism of MASH from the perspective of mRNA and provided potential biomarkers for the prediction of MASH. These potential biomarkers showed good performance in predicting MASH and had clinical utility in distinguishing MASH from simple steatosis. Because the biopsy is affected by the quality of the material taken and the experience of doctors, the results of the biopsy may not fully reflect the condition of the patient. By detecting the expression of hub genes in liver cells, a predicted value is calculated by the model and it can help doctors objectively evaluate the patient’s disease status to a certain extent according to the cut-off value, and provide a reference index for less experienced doctors. Although there is still a long way before clinical application, it provides some new targets for future work.However, the relation between hub genes and MASH or MAFLD has been studied little. It needs further study to provide more precise clinical information about diagnosis and progression. The limitations of our study should be aware of. The samples we used were not large enough. These datasets were not suitable for joint analysis as they were from different platforms. The clinical information of GSE24807 and GSE63067 were not available, which might affect the results. The baseline data of hub genes were not available, and so no comparison with baseline gene expression was made. Our model was from liver tissue, and the specificity for MASH was good. However, the expression of the model in serum needs to be observed for further evaluation.In conclusion, NAMPT, PHLDA1, RALGDS, GADD45B, FOSL2, RTP3, and RASD1 were identified as the hub genes in the progress of MAFLD. The combination of six genes could act as a potential diagnostic model for MASH and have clinical utility in distinguishing MASH from simple steatosis. However, clinical studies with large samples are needed to further research the applicability of the model in the diagnosis for MASH.


An increased proportion of ICU admissions were found in patients from Latin America compared with native Spanish patients when adjusted by age and gender, with no significant differences in in-hospital mortality.The aim of this study was to describe and compare the clinical characteristics of hospitalized patients with COVID-19 pneumonia according to their geographical origin. This is a retrospective case-control study of hospitalized patients with confirmed COVID-19 pneumonia treated at Vall d’Hebron University Hospital (Barcelona) during the first wave of the pandemic. Cases were defined as patients born in Latin America and controls were randomly selected among Spanish patients matched by age and gender. Demographic and clinical variables were collected, including comorbidities, symptoms, vital signs and analytical parameters, intensive care unit admission and outcome at 28 days after admission. Overall, 1080 hospitalized patients were registered: 774 (71.6%) from Spain, 142 (13.1%) from Latin America and the rest from other countries. Patients from Latin America were considered as cases and 558 Spanish patients were randomly selected as controls. Latin American patients had a higher proportion of anosmia, rhinorrhea and odynophagia, as well as higher mean levels of platelets and lower mean levels of ferritin than Spanish patients. No differences were found in oxygen requirement and mortality at 28 days after admission, but there was a higher proportion of ICU admissions (28.2% vs. 20.2%,  At the end of 2019, China first reported a group of cases of severe acute respiratory syndrome by a new virus called SARS-CoV-2. Currently, it is a global pandemic and 172,956,039 cases have been reported and 3,726,466 people have died in total by 7 June 2021 [Many risk factors have been described that predict the evolution throughout the hospitalization of these patients, such as being aged > 65 years, male, a high Charlson score and a respiratory rate upon their admission consistently associated with increased mortality ,8,9,10. The objectives of the study are (1) to describe the main clinical characteristics of a cohort of patients hospitalized with a diagnosis of pneumonia caused by SARS-CoV-2 and (2) to compare clinical variables and outcomes between Spanish natives and Latin American patients.The Vall d’Hebron COVID-19 Prospective Cohort Study includes all consecutive adult patients (≥18 years old) who were hospitalized for COVID-19 at Vall d’Hebron University Hospital, a 1100-bed public tertiary care hospital in Barcelona (Spain). The hospital is the reference center of an area with a total population of 1,664,182  .This is a retrospective case-control study in which all hospitalized patients treated at the hospital during the first wave (from 1 March to 30 April 2020) with laboratory-confirmed COVID-19 and radiologically confirmed pneumonia were included. A laboratory-confirmed case was defined as a patient with a real-time reverse-transcriptase polymerase chain reaction (RT-PCR) SARS-CoV-2-positive result in any respiratory sample . Patients born in Latin American countries were defined as cases; controls were randomly selected from those Caucasian people born in Spain in a 1:4 ratio and matched by age and gender. The main exclusion criterion was the absence of pneumonia in the chest X-ray.Overall, 1080 patients with confirmed COVID-19 pneumonia hospitalized during the study period were registered in the database: 774 (71.6%) from Spain, 142 (13.1%) from Latin America (LA), 43 (3.9%) from other countries and 121 (11.2%) cases without information regarding country of origin. From the 142 patients from LA, all of them fulfilled the inclusion criteria and were considered as cases. On the other hand, 558 Spanish patients were randomly selected as controls. Overall, 700 patients were included in this study.Data were recorded in the Research Electronic Data Capture software . We collected variables including age, gender, country of origin, comorbidities , behavioral factors, such as smoking or alcohol consumption, symptoms, vital signs and analytical parameters at admission, need of intensive care unit (ICU) admission, treatment received and outcome at 28 days after admission .Patients were treated according to the hospital’s clinical protocol available at that time. Tocilizumab was reserved for patients with respiratory failure and interleukin (IL)-6 levels > 40 pg/mL or D-dimer > 1500 ng/mL.p value from the likelihood ratio test of the models with and without the covariate was calculated. In that sense, each case was compared with their corresponding controls. A type I error of 5% was considered. All analyses were carried out with STATA 15.1 .Continuous variables were expressed as mean and standard deviation or medians and interquartile range, as appropriate. Categorical variables were summarized as absolute numbers and percentages. As data are age and gender matched case-control, in order to compare differences, the usual contrast test cannot be used, as it does not take into account the dependency of case and control. A conditional univariable logistic regression was performed and the Procedures were performed in accordance with the ethical standards laid down in the Declaration of Helsinki as revised in 2013, and the study protocol was approved by the Ethical Review Board of the Vall d’Hebron University Hospital . The Ethical Review Board agreed that informed consent was not necessary given the retrospective nature of the study and the anonymization of the information.As mentioned previously, 700 patients were selected for the study. From the 142 patients who were born in LA, their countries of origin were as follows: Ecuador: 23.9% (34/142), 29 (20.4%) from Perú, 19 (13.4%) from Bolivia, 17 (12.0%) from Honduras, 15 (10.7%) from Colombia, 9 (6.3%) from Venezuela, 4 (2.8%) from Dominican Republic, 3 (2.1%) from Brazil, 3 (2.1%) from Chile, 3 (2.1%) from Cuba, 2 (1.4%) from El Salvador, 2 (1.4%) from Paraguay and 2 (1.4%) from Uruguay. From the overall study population (700 patients), the epidemiological and clinical characteristics of patients are summarized in p = 0.0026), rhinorrhea  and odynophagia  than Spanish patients. Regarding analytical parameters at admission, patients from LA had higher mean levels of platelets  and lower mean levels of ferritin  than Spanish patients. When comparing clinical outcomes at 28 days after admission, patients from Latin America showed a higher proportion of ICU admissions  than Spanish patients. However, we did not find any difference between both groups regarding oxygen requirement and mortality.When comparing clinical symptoms at admission between both groups, we observed that patients from LA had a higher proportion of anosmia , when adjusted for age and gender, a degree that is considered pre-obesity , which cRegarding the clinical manifestations, we found a higher proportion of arthromyalgia, anosmia and cacosmia among Latin American patients, which could suggest a different pathophysiological response of the host against the virus. Related to anosmia, it has been hypothesized that SARS-CoV-2 has neurotropic characteristics, being able to invade peripheral nerve terminals and enter the central nervous system through trans-synaptic pathways ,43. It iThe study showed a higher percentage of patients requiring ICU admission among Latin American patients compared with the Spanish population. It was in accordance with the oxygen requirements during admission (higher in the Latin American group), although it did not reach statistical significance. Moreover, although the need for ICU admission was higher, no differences in mortality were observed. These results could be explained by transient worsening in the clinical symptoms (mostly respiratory) due to an increase in systemic inflammation, which are sufficient for ICU admission needs, but not too severe for increasing the risk of death. Genetic factors that have not been studied yet but with a relevant role in pathophysiology might have also contributed ,49.It is important to note that current therapeutic options for COVID-19 pneumonia include the use of corticosteroids and other immunomodulators (such as tocilizumab or baricitinib), drugs that can increase the risk of reactivation of latent infections, including those endemic in geographical areas where migrants come from, such as the strongyloidiasis ,51. ThisThis study has some limitations given its retrospective nature. First, especially during the first wave of the pandemic, COVID-19 management protocol amendments or modifications were periodically performed. However, the clinical protocols for COVID-19 management used in this study were the same between Spanish natives and Latin Americans. Second, because of operational limitations during an outbreak, some patient’s information is missing, but the sample size of the study may have allowed obtaining comprehensive results. Third, we included patients admitted to the hospital due to COVID-19; therefore, even if we detected an increased rate of ICU admissions in LA, this precludes us from reaching conclusions regarding community transmission in this population group.In summary, we observed an increased rate of ICU admissions among hospitalized adult patients with COVID-19 pneumonia from Latin America compared with native Spanish patients. Furthermore, there was a higher rate of arthromyalgia, cacosmia and anosmia observed in patients from Latin America. These differences could be explained by socio-economic factors or still unknown genetic factors, so further investigations are needed. Understanding ethnic disparities can help so as to create health prevention strategies.