Leading physicians from each EP lab were contacted electronically; participation was voluntary.n = 30); 50 and 45 % of active centres participated in the last 2 instalments of the registry. A mean of 508 ± 270 standard and complex catheter ablation procedures were reported annually for 2015–2016 by all responding centres. The most frequently performed ablation targets atrial fibrillation (PVI) arrhythmia accounting for 36 % of all procedures (mean = 164 ± 85). The number of full time physicians ranges between 1 and 7 per centre, (mean = 4). The mean wait time to see an electrophysiologist for an initial non-urgent consult is 23 weeks. The wait time between an EP consult and ablation date is 17.8 weeks for simple ablation, and 30.1 weeks for AF ablation. On average centres have 2 (range: 1–4) rooms equipped for ablations; each centre uses the EP lab an average of 7 shifts per week. While diagnostic studies and radiofrequency ablations are performed in all centres, point-by-point cryoablation is available in 85 % centres; 38 % of the respondents use circular ablation techniques.All Canadian EP centres were identified (This initiative provides contemporary data on invasive electrophysiology lab practices. The EP registry provides activity benchmarks on national trends and practices.The online version contains supplementary material available at 10.1186/s12913-021-06441-0. Characterization and reporting of resources in health care is vital for means allocation, designing new facilities, and policy making. Interventional cardiac electrophysiology (EP) is a rapidly evolving field. Ablation technologies to treat cardiac dysrhythmias became widely available and affordable. Over the past decades, the number of centres in Canada that perform catheter ablations of arrhythmias and other invasive EP procedures has significantly augmented. An increasing number of certified electrophysiology specialists are entering the field as indications for complex ablations are growing. We established the first Canadian nationwide cardiac ablation registry in 2011, to understand the variability in the conduct of EP procedures and resource allocation across the country. The purpose of this publication is to provide a 6-year update on national practices of invasive cardiac EP.) and with funding support from International Winter Arrhythmia School (IWAS). Since the registry’s inception in 2011, we continuously identified and added EP centres to our database through the CHRS, IWAS symposium and industry liaisons. There is an EP lab in most major university-affiliated hospitals; and 120 practicing interventional cardiac electrophysiologists, performing standard and complex ablations that serve Canada’s 37 million population [The “EP Ablation Registry” survey was developed for this project by an independent steering committee in collaboration with the ablation committee of Canadian Heart Rhythm Society  was from all university affiliated hospitals that perform EP studies and catheter ablations. Six centres (43 %) perform EP procedures on pediatric (< 18 years) population. There was a slight increase in the average amount of EP enabled rooms since 2011 (mean 1.9 up from 1.6). On average, the equipped rooms are used specifically for ablation activity at 75 % capacity in 2015–2016, while in 2011–2012 they were available for ablations only at 70 % capacity (room was at 100 % if used for ablation 5 days/week). Four of the 14 (29 %) centres use the operating room for cardiac rhythm management device implantation, for an average of 3 days per week.Diagnostic EP studies and radiofrequency ablations were consistently performed in all participating centres since 2011; ablations using Contact Force catheters have been performed in all centres since 2015. Radiofrequency ablation remains the predominant technique; while the popularity of point-by-point cryoablation and balloon ablation have grown in the last 6 years. Please refer to Table The survey collected data on imaging and electroanatomical mapping systems. CARTO and intracardiac echo were the most widely utilized in 86 % centres, increasing from 78 % of centres in 2011, followed by EnSite NavX-Velocity and EnSite NavX Precision utilized in 71 and 57 % of centres respectively. Only one centre reported the current use of Rhythmia™ and one centre has continued to use LocaLisa since 2011. Two centres in Canada clinically use Robotic Magnetic Navigation systems .In total, there were 57 full-time physicians  who performed ablations in 2015–2016. 43 % of the responded programs employ 22 part-time physicians, with an average of 1.6 part timers per centre (range 0–12). The number of full-time and part-time EP staff have been increasing .There was a trend in more part-time electrophysiologists from 24 to 28 % of total EPs. Ten of the fourteen (71 %) hospitals employ 35 EP fellows, with an average of 2.5 trainees per centre. The ratio of teaching staff (full-time and pro-rated part-time) to trainees is approximately 2:1. We observed a trend of a decreased ratio between staff members and trainees over the polled time.In 12 of 14 centres, there is an average of 4.6 nurses trained for EP procedures (range: 0–10); and an average of 3 permanent EP (range: 0–7). Seven (50 %) centres employ EP technicians, with an average of 1.5 technicians per centre (range: 0–6). By 2016 non-3D mapping/recording systems became operated predominantly by EP nurses and industry reps, they are the primary operators in 50 and 29 % of the centres respectively, EP techs operated 21 % of the systems in 2016. Table In total, 7106 standard and complex catheter ablation procedures were reported annually for 2015–2016 by all responding centres, an average of 508 ± 270 per centre; this has significantly increased from 2011 to 2012, when the national average obtained by our survey was 421 ± 202 ablations. This is consistent with the increased number of rooms that are solely equipped for ablations, which have increased  versus mean = 1.6; (1–3)), dedicated to ablations 7 days versus 5 days per week. Centres are steering away from doing implants in the operating rooms towards ablation labs ; part-time physicians were assumed to perform one half of the ablation volume of full-timers. Figure On average, each EP specialist was found to perform 117 ± 70 ablations annually in 2015–2016, similar to 113 ± 42 procedures in 2011–2012 , 32 % for the years 2013-4 and 30 % in 2011-2. Paroxysmal AF decreased from accounting for the majority (93 %) of all AF ablations in 2012 to 55 % of the cases in 2016, contrary to persistent AF which increased from 29 to 47 %. Chronic success remains consistently > 70 % for paroxysmal AF procedures, and varies between 60 and 90 % for persistent AF.Substrate-specific data were available from eight centres from 2016, seven centres from 2015; six centres in 2014, five centres in 2013, thirteen centres in 2012, and seven centres in 2011. This comprised a total of 18,864 ablation procedures that were performed over the 6 years (2011–2016), and were included in the following substrate specific analysis. The most frequently performed ablation was targeting AF (PVI) arrhythmia accounting for 36 % of all procedures in 2015-16 (The second common procedure was the ablation of atrioventricular node reentry tachycardia (AVNRT) accounting for 18 % of all procedures in 2015–2016 (n = 1489), which has decreased from 2011 when this dysrhythmia comprised 23 % of all ablated substrates. The total number of cavotricuspid isthmus (CTI) ablations in 2015-16 was 1473 averaging 98 ± 71 ablation procedures per centre (range 37–327), comprising 17 % of total volume of ablations. Ablation of Atrioventricular Junction and ventricular tachyarrhythmias were the most rarely performed ablations. In 2015–2016 they accounted for 7 and 8 % of ablations respectively, and 8 and 7 % in 2013 and 2014. Table Major complications from ablation of accessory pathways (AVRT), AVNRT, and CTI include pericarditis, transient PR prolongation, tamponade, pericardial effusion, stroke, indication for a permanent pacemaker, vascular complications requiring intervention, and death. Minor complications were defined as vascular, hematuria, arteriovenous fistula, pseudoaneurysm, and pain during procedure. The following are self-reported procedural complication and success rates for the 2015–2016 survey. The rate of both major and minor post-procedural complications for the aforementioned substrates were reported as < 10 % in 6 of 7 centres that provided complication rate data, and < 1 % in 1 of the centres. Two centres reported pulmonary embolisms post procedures. The chronic success rate, defined as ‘no recurrence or palpitations were documented other than target arrhythmia’ was greater than 90 % in most (80 %) of reporting centres. Partial success, defined as ‘recurrence with episodes of lesser frequency and severity, requiring the patient to be on the same medications’, occurred with < 10 % frequency in 2 centres, greater than 90 % frequency in 2 centres, and 10–20 % in the remaining centre.Ablation of AF most commonly leads to minor complications that requires a hospital stay of > 24 h and other complications that don’t require intervention: vascular complications, hematuria, arteriovenous fistula, pseudoaneurysm, and groin pain. Six of 7 centres that reported complications in 2015–2016 had a < 10 % minor complication rate, and one centre reported a < 1 % incidence of minor complications. Major complications of AF: vascular complications requiring intervention more specifically pericarditis, tamponade, pericardial effusion, stroke, need for permanent PM, atrio-esophageal fistula, death, occurred with the same frequency. Chronic success of AF as well as CTI/MAT/FAT ablations were defined as ‘no recurrence or documentation other than target arrhythmia’, it was > 80 % in 2 centres, 71–80 % in 1 centre, > 90 % in 1 centre, and 1 centre reported a 61–70 % rate for 2015–2016. Partial success was < 10 % in 2 centres, 21–30 %, 71–80 %, and > 90 % in 1 centre each.Ablation of Ventricular Tachycardia had similar major complication rates. Five out of seven centres reported < 10 % minor complication rates, 1 centre reported a < 1 % rate, and in one centre 10–20 % of procedures yielded minor vascular complications of AV fistula and pseudoaneurysm not requiring intervention.In 75 % of polled centres reported that patients are discharged > 24 h post AF ablation procedure, and in 25 % centres patients are discharged same day. Those undergoing RVOT ablations 50 % were discharged same day, and 50 % >24 h post ablation. In AVRT/AVNRT/CTI ablations 75 % of patients are discharged same day, and 25 % in > 24 h.Paroxysmal AF was more common than persistent and permanent AF. Paroxysmal AF made up 55 % of annual AF cases in 2016, and made up the majority (93 %) of annual AF cases in 2013. Chronic success, defined as ‘12 months free from documented AF of 30 seconds or more’, was achieved after about 70 % of ablations of paroxysmal AF, ranging from 69 to 71 % between 2013 and 2016. Chronic success was achieved for a range from 60 to 90 % of non-paroxysmal AF ablations. Cases had to be redone between 15 and 22 % of paroxysmal AF, and between 17 and 28 % of non-paroxysmal AF ablations. Table In 2016, PVI procedures were done under general anesthesia ‘rarely’ in 50 % of the centres, ‘most of the time’ in 33 % of the centres, and used for ‘all’ the cases in 17 % of the centres. Conversely, 50 % use non-general anesthesia most of the time, 38 % use it rarely, and 13 % don’t use it at all. During the years 2014 and 2013, 25 % of the centres used general anesthesia in half of atrial fibrillation ablation cases; 63 % of the centres performed most of the cases under general anesthesia and 13 % of the centres rarely used general anesthesia. Meanwhile, 25 % of the cases used non-general anesthesia in half and most of the cases, and 50 % of the centres only rarely used non-general anesthesia.Finally, the questionnaire evaluated the type of imaging that was done prior to AF ablation; five centres provided the data for the year 2016. Pre-procedural MRI or CT was done in 76–90 % of the cases by 2 centres, and > 90 % in 3 centres. Pre-procedural TEE was done in > 90 % of the cases in 3 centres, 76–90 % of the cases in 1 centre, and done 1–25 % of the time in the remaining centre. In 2015, 40 % centres conducted pre-procedural CT or MRI imaging on 76–90 % of the cases, 60 % on > 90 % of the AF ablation cases. In both and 2013 and 2014, 75 % centres conducted the pre-procedural CT or MRI imaging, while 13 % did an MRI or CT on 76–90 % of the cases and 13 % of centres do an MRI or CT on less than 25 % of the AF procedures.This paper presents the trends of types of activities performed and facilities available in Canadian EP practice by reporting results of the 3 administered surveys encompassing data from the years 2011 to 2016. The response rates are comparable with other interventional EP surveys’ response rates including Canada, United States, and Germany . CappatoOverall, the average number of ablation procedures has steadily augmented, from a mean of 421 ± 202 cardiac ablations in 2011–2012 to 508 ± 270 in 2015–2016 per reporting Canadian centre. This growth of procedural volumes is above the population growth in Canada and likely to reflect increased availability of procedural care and increased awareness of the referring community of treatments available. The gains in the amount of procedures were also likely due to improved efficiencies of technology and workflow. The volume of ablations per Canadian centre has surpassed the volumes reported in the latest 3 European registries; in Spain, the mean procedures per centre was 156 ± 126 in 2017 [The growing trends in the number of full and part-time physicians are in line with other reports. The increasing amount of part-time staff in Canada represents community-based cardiologists/electrophysiologists getting access to tertiary based EP labs, and expanding access to community based operators.The annual ablation volume per operator  has consistently surpassed the guidelines put forth by the American College of Cardiology, which recommends practicing EP specialists to perform more than 20–50 ablations annually to maintain their competency level . This isThe number of trainees has decreased . There is a trend of lesser EP training popularity as EP workforce became more saturated in North America. A training and practice paper by Krahn et al. stated that three fourths of 413 EP fellows who have trained in Canada came from outside the country. Although most foreign trainees return to their country of origin, repatriation is not universal [The survey allows to establish the number of trainees feasible to accommodate per centre. European Heart Rhythm Association requirements state that an electrophysiological training centre should have at least 200 ablation procedures per year; 93 % of the polled centres in Canada performed > 200 ablations per year, whereas 69 % of the German centres reported a > 200 annual ablations volume. A comparison with the American Heart Association, American College of Cardiology, and Heart Rhythm Society advanced training statement revealed that requirements suggested for the United States are similar to those established in Europe . TherefoThe American College of Cardiology, American Heart Association, and Heart Rhythm Society Advanced joint training statement provides a recommendation of 160 catheter ablations as the minimum procedural volume of interventional procedures to achieve and demonstrate competence in clinical cardiac electrophysiology, for the 2-year fellowship training . In refePulmonary vein isolation for AF has remained the most frequent ablation target in Canada since 2011, with its absolute rate further increasing. In the year 2016 alone, PVI for AF accounted for 39 % of all annual procedures. This frequency is similar to European registries’ reports of AF being the most targeted substrate, accounting for 47 % of ablations done in Germany in 2015 [The survey has evolved since its original installment to encompass more detailed information on lab infrastructure; we have also started collecting data regarding wait times to see an EP specialist and get a cardiac ablation procedure, see The participation rate does not provide a full representation of all Canadian EP centres; as not all centres responded. The retrospective and self-reported nature of the data may limit analysis. Our aim for future surveys is to include prospective data, and increase participation rate.The EP Ablation Registry continues to systematically collect data pertaining to ablation procedures nationwide, the reported data provides a unique opportunity to illustrate trends in Canadian EP practices over last 6 years. The registry demonstrated that the Canadian EP procedural intensity of practice and training is comparable to international standards. This information may provide guidance for making decisions related to staff and trainees capacity for the EP community and for health policy decision makers, as cardiac electrophysiology continues to augment in Canada and worldwide.Additional file 1:Additional file 2:Additional file 3:Figure S1 Supplement. Waitlist A: wait times to see an electrophysiologist for an initial non-urgent consult.Additional file 4:Figure S2 Supplement. Waitlist B: wait times between EP consult and date of ablation procedure.Additional file 5:


An increased consumption of fresh fruit and vegetables is associated with health benefits ,2. Inclun = 133), a “Food and Nutrition Knowledge” validated questionnaire [n = 6), and semi-structured interviews (n = 6). All interviewed gardeners understood the purpose and signed the informed consent.An UG in Setúbal, Portugal, was selected for this study. All data was collected in October, 2018 and included: demographic data from the gardeners (ionnaire  with 20 n = 133) 11,999€per year.The UG had 138 plots with 133 in use. Most of the gardeners were men (59.9%) and under 65 years of age (74.4%). 30,5% had an income under (Our results meet those of recent studies that enhance the importance of UG in feeding populations, especially those with diminished food security and low access to fresh quality products . Increas


Transarterial chemoembolization (TACE) and radiofrequency ablation (RFA) are effective treatment methods for unresectable hepatocellular carcinoma (HCC). However, there is still a lack of clinical research on whether early sequential RFA, compared with late combination therapy, can improve the long-term efficacy of initial TACE treatment.This retrospective study investigated a cohort of patients who underwent combination therapy using TACE and RFA (TACE followed by RFA) from January 2010 to January 2020 at our medical centre. A total of 96 patients underwent TACE combined with early RFA , which was called TACE + eRFA. Thirty-four patients received 1–2 palliative TACE treatments first and then underwent TACE treatment combined with late RFA (TACE + lRFA). All patients continued to receive palliative TACE treatments after intrahepatic lesion progression until reaching intolerance. The overall survival (OS) rate, time to tumour progression (TTP), tumour response rate and major complication rates were compared between the two groups.P = 0.013), median TTP , objective response rate (ORR)  and disease control rate (DCR) (94.8% vs 73.5% P = 0.002) between the two groups. Multivariable analysis revealed that the Barcelona Clinic Liver Cancer stage was an independent risk factor for OS. Meanwhile, multivariable analysis revealed that TACE + eRFA was associated with an enhanced TTP.There were significant differences in the median OS (46 months vs 33 months; Early sequential RFA treatment in patients with early-intermediate HCC can improve local tumour control and clinical outcomes while reducing the frequency of TACE treatment. In clinical practice, in HCC patients initially treated with TACE, it is recommended to combine RFA as soon as possible to obtain long-term survival. Cancer is the first or second cause of premature death (30–69 years old) in 134 countries worldwide . HCC exhRadiofrequency ablation (RFA) is the first-line ablation technique applied through an electrode tip inserted into the target lesion that induces coagulation necrosis and is a widely accepted treatment option for patients with early-stage HCC . CompareIn recent years, transarterial chemoembolization (TACE) has been widely used as a palliative therapy in the treatment of HCC patients who are unsuitable for radical therapies . TACE caRecently, studies have indicated that RFA combined with TACE may improve the local control of tumours with a diameter of over 3 cm , 21. HowThis was a retrospective study performed in accordance with local and national laws and abiding by the guidelines of the Helsinki Declaration. Approval for this study was obtained from The Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. The need for informed consent was waived by The Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology.P > 0.05). Prior to undergoing the initial TACE treatment, all patients were informed of the benefits and risks of the combination therapy. TACE + eRFA was defined as early sequential RFA treatment  following initial TACE. There were patients who were unwilling to take the risk, could not afford the cost of two treatments, or recognized the efficacy of TACE treatment alone but refused to undergo the initial TACE combination therapy. In these patients, following 1–2 palliative TACE treatments, TACE + lRFA was performed on the target lesion.From January 2010 to January 2020, 436 consecutive patients underwent combination therapy using TACE and RFA (TACE followed by RFA) at our medical centre. Prior to these patients undergoing initial TACE therapy, the treatment strategy was recommended by the multidisciplinary tumour board. Considering the patient’s age, any serious comorbidities , the patient’s compliance, the tumour location , the lack of donor livers and the limited efficacy of TACE and RFA alone, some patients were not deemed suitable for liver resection or liver transplantation; these patients were recommended for combination therapy (TACE followed by RFA). The time of RFA following TACE was dependent on the disappearance of symptoms postembolization and the recovery of liver function. In this study, the mean interval between TACE and RFA in the TACE combined with early RFA (TACE + eRFA) group was 6.3 days (1–14 days), and the median interval was 5 days. In the TACE combined with late RFA (TACE + lRFA) group, the mean interval between TACE and RFA was 6.1 days (2–13 days), and the median interval was 5.5 days. There was no significant difference in the interval between the two groups (The diagnosis of HCC depended on the diagnostic criteria of the European Association for the Study of the Liver (EASL) and the American Association for the Study of Liver Disease (AASLD) , 10. A t9/L); (3) other malignancies or liver metastases; and (4) incomplete clinical data  any previous treatment for HCC; (2) renal failure, cardiac failure or haemorrhagic risk  and OS rate were compared between the TACE + eRFA and TACE + lRFA groups. The time of combined treatment to disease progression  was defined as the TTP. OS was defined as the interval between the first TACE procedure and either death or the last follow-up. The modified Response Evaluation Criteria in Solid Tumors (mRECIST) was used to evaluate the treatment response at approximately 1–1.5 months after the combined treatment . A complLaboratory tests and contrast-enhanced CT or magnetic resonance imaging (MRI) examinations were performed one month after combined treatment. Imaging (contrast-enhanced CT or MRI) and laboratory examinations were performed every 2–3 months for patients. Once local progression or intrahepatic metastasis occurred, palliative TACE treatment was given until it was deemed intolerable by the patient. Follow-up continued until patient death or the end point of this study.P value of < 0.05 was considered statistically significant. Quantitative data are presented as the mean ± standard deviation, and discrete variables are presented as proportions. Quantitative data were analysed by Student’s t-test, while categorical data were analysed by the chi squared test. The Kaplan–Meier method was used to evaluate differences in the TTP and OS rate between the two groups. Univariate analyses were implemented with the log-rank test. Variables with a P value of < 0.10 were entered into the multivariate analysis, which was performed using a Cox proportional hazard regression model.All analyses were performed using SPSS 24.0 software , and a From January 2010 to January 2020, a total of 436 patients received combination therapy as a treatment option, and 306 patients were excluded since they did not meet the study requirements, as highlighted in Fig. P = 0.000). In addition, the disease control rate was 94.8% in the TACE + eRFA group and 73.5% in the TACE + lRFA group (P = 0.002). Hence, compared with patients in the TACE + lRFA group, those in the TACE + eRFA group benefitted from an enhanced tumour response.The objective tumour regression rate was 89.6% in the TACE + eRFA group and 61.8% in the TACE + lRFA group (P = 0.84).Within the TACE + eRFA group, three patients had severe complications, for an incidence of 2.1%. Two patients developed a biloma following TACE, and one patient suffered from an intestinal perforation after RFA. There were two cases of severe complications in the TACE + lRFA group, for an incidence of 2.9%. A subcapsular haematoma in the liver was observed on CT in two patients. There was no significant difference in the incidence of major complications between the two groups (P = 0.013) .The median OS was 46 months 95%CI: 38.0–54.0) in the TACE + eRFA group and 33 months 95%CI: 27.4–38.6) in the TACE + lRFA group (7.4–38.6 8.0–54.0 P < 0.001)  in the TACE + eRFA group and 14 months (95% CI 7.2.0–20.9) in the TACE + lRFA group (The combination of TACE and RFA has several theoretical advantages over RFA or TACE alone , 26. FirPresently, there is a lack of relevant literature on the timing of combination therapy. Most studies recommend RFA treatment within two weeks post-TACE –27 to beIn this study, due to various reasons, some patients failed to undergo combination therapy within two weeks; they underwent 1–2 TACE procedures before treatment and continued to receive palliative TACE treatments after progression. Our results demonstrate that early sequential RFA treatment Fig.  providesSimilar to other studies , 32, 34,However, the sample size was small, and the retrospective, non-randomized experimental design was the main flaw of the study. Consequently, a prospective, randomized controlled trial is necessary to verify our results. In addition, due to the limited number of samples, no stratified analysis was conducted.In essence, early sequential RFA treatment in patients with early- and mid-stage HCC can improve local tumour control and improve clinical outcomes, such as OS and TTP, while reducing the frequency of TACE treatment. In clinical practice, for HCC patients initially treated with TACE, it is recommended to combine RFA as soon as possible to improve the long-term survival of individual HCC patients.


Suction drainage is commonly applied after total knee arthroplasty (TKA) and unicompartmental knee arthroplasty (UKA) to reduce hematoma, swelling and to favor surgical wound healing. However, its efficacy remains controversial; thus, the purpose of this study is to evaluate drainage efficiency in the management of postoperative bleeding in TKA and UKA.The cohort comprised 134 clinical records of patients affected by knee osteoarthritis (OA) who underwent either TKA or UKA. All the patients were subdivided into 2 groups: the first one with drainage and the second one without drainage (respectively 61 and 73 patients). For each group, hemoglobin levels in the preoperative, first, second and third postoperative day were collected. Postoperative complications such as swelling, bleeding from the surgical wound or the need for blood transfusion, were also recorded.p = 0.715), second (p = 0.203) and third post-operative day (p = 0.467) between the two groups. Moreover, no significant correlation between knee swelling or transfusion rate and the drainage was observed (p = 0.703 and p = 0.662 respectively). Besides, a significant correlation was found between bleeding from the surgical wound and the absence of drainage (p = 0.006).Our results did not show any significant difference of hemoglobin levels in the first (The study demonstrates how the routine use of suction drainage does not provide substantial benefits in the postoperative blood loss management after TKA or UKA.NCT04508101, 09/08/2020, Retrospectively registeredClinicalTrials.gov III Knee arthroplasty is the most common treatment for osteoarthritis (OA) and other joint disorders , 2. SpecThis retrospective, single center study was conducted at Istituto Ortopedico Galeazzi, Milan, Italy. Approval by the Ethics Committee of Ospedale San Raffaele, Milan, Italy  was obtained on July 2020 and the study was registered in the ClinicalTrials.gov Registry. We analyzed 134 clinical records of patients who underwent either TKA or UKA in the period between March 2019 and March 2020. Patients were subdivided into 2 groups: the first one with drainage (D) and the second one without drainage (ND) (respectively 61 and 73 patients). All the patients underwent physical examination before surgery. Plain radiographs were performed to confirm the diagnosis and to assess OA grade. In case of clinical suspect of unicompartmental OA, magnetic resonance imaging (MRI) was prescribed to confirm the diagnosis and to assess the integrity of anterior and posterior cruciate ligament and lateral meniscus to ensure a correct treatment choice. We included patients affected by OA with either TKA or UKA indication. Patients showing contraindications to tranexamic acid (TXA), such as allergy/hypersensitivity, intrinsic risk for thrombosis or thromboembolism and hereditary thrombophilia [Patients included in the present study followed FT pathway. The surgery was performed by the same surgical team, specialized in knee surgery, in regional anesthesia. The patients were placed in supine position with 90 degrees’ knee flexion. In patients undergoing UKA a tourniquet was positioned at the proximal extremity of the thigh, whereas tourniquet was not applied in TKA patients. Among the 134 prostheses, a posterior stabilizing prosthesis was placed in 90 patients and a medial unicompartmental prosthesis was placed in 44 patients. For all the patients, we performed a midline skin incision and medial parapatellar approach. The prostheses were cemented following the conventional technique, using antibiotic-treated cement. Two 500 mg/5 ml TXA vials were administered systemically, and one vial locally via drainage conduct or through intraarticular injection where the drainage was not present. After surgery, standard deep vein thrombosis prophylaxis was administered for 45 days using nadroparin calcium 0,4 ml or 0,6 ml by weight (respectively < 80 kg and > 80 kg). A strict pain control was performed using analgesics and anti-inflammatory drugs to ease an early mobilization according to FT pathway. All patients underwent a rehabilitation program, which included early active and passive knee mobilization, and isometric muscle reinforcement, aiming to a faster recovery and a consequent reduction in hospitalization . Patientp-values were calculated at each time interval  for each group. Fisher’s exact test was applied to evaluate differences in proportions of categorical variables  between study groups. Moreover, regression analysis and linear models were adopted to assess the influence of more than two variables on the same parameter. Additionally, we also performed the abovementioned tests considering patients undergoing TKA and UKA separately. Statistical significance was set at p < 0.05.The statistical analysis has been performed using R software v3.6.1 . Shapiro Wilk test was used to assess the normal distribution of continuous variables. According to data distribution, parametric (unpaired Student’s t test) or non-parametric test (Wilcoxon’s test) were used to evaluate differences among the study groups. Specifically, the average of hemoglobin values and p = 0.266), t1 (p = 0.715), t2 (p = 0.203) and t3 (p = 0.467), showing a similar downward trend at each time interval (Table p < 0.001 and p = 0.038). Moreover, there was no significant difference in knee swelling average between ND (3.6%) and D (5.6%) cases, as well as in transfusion rate (14.5 and 17.1% respectively). Indeed, double-cross tables showed no association between the presence of drainage and knee swelling (p = 0.703), or transfusion rate (p = 0.662). Furthermore, we noticed that wound bleeding was mainly present in the group without drainage (13.2% vs 1.4%), showing a significant association (p = 0.006) (Table p = 0.020). Moreover, transfusion rates were significantly more frequent in TKA  and this was confirmed  by a logistic regression analysis considering the same variables reported above, where also “sex” (p = 0.010) and the initial hemoglobin values (p < 0.001) demonstrated significant influence on these events. The influence of patient gender on transfusion probability was not observed in the univariate analysis . Concerning knee swelling, we found no association neither with drainage (p = 0.795) nor with the other variables.Both groups were similar and uniform in term of sex, affected side and surgical procedure. Specifically, among 134 patients (90 TKA and 44 UKA), we performed 45 TKA and 17 UKA in D group, and 45 TKA and 27 UKA in ND group. Patients’ characteristics are summarized in Table p = 0.287), t1 (p = 0.592), t2 (p = 0.888) and t3 (p = 0.669) was found (Table p = 0.999), and in transfusion rate . Moreover, wound bleeding was mainly reported in ND group (13.5% vs 2.0%), but this difference was not significant in the univariate analysis (p = 0.060) (Table p = 0.075), and that the need for transfusion was higher among males  and it was reduced with higher initial hemoglobin values (p < 0.001). Drainage has no influence on these events.Considering only patients who underwent TKA, no statistically significant difference between the two groups at t0 p = 0.87, t1 (pp = 0.604), t1 (p = 0.212), t2 (p = 0.336) and t3 (p = 0.394) (Table p = 0.380). The need for transfusion was instead comparable between the two groups without statistical significance . Wound bleeding was instead observed only in ND group, without statistical significance  (Table Patients who underwent UKA showed a comparable downward trend in hemoglobin values, showing no statistical significance at t0 (t1 p = 0.12, t2 (pt2 p = 0.36 and t3In the present study emerged that drainage employment in TKA and UKA does not provide significant differences in the postoperative bleeding control. The strength of this study was in the choice to include both TKA and UKA in the analysis, considering the two procedures either as variables and separate study groups.Knee arthroplasty is considered one of the most effective surgical procedures for OA in the orthopedic clinical practice . RecentlAlthough our results are in line with the most recent medical literature, several limitations should be addressed. An extended observation would have likely provided additional information about the length of stay. However, the aim of this study was solely to assess the influence of suction drainage on postoperative blood management rather than on hospitalization length. It should be noticed that tourniquet was used only in UKA as established by our internal protocol, and so it can be assumed as part of the procedure and not as a confounding variable. Furthermore, including two different procedures could lead to bias. Nevertheless, both the studied groups were characterized by a homogeneous distribution of TKA and UKA cases, and the two procedures were also analyzed as different populations.Given our results and considering the current literature, we believe that the employment of suction drainage does not provide substantial benefits in the postoperative blood loss management in tourniquet-free TKA and in UKA performed with tourniquet.


Risk of preeclampsia varies by month of delivery. We tested whether this seasonal patterning may be mediated through maternal vitamin D concentration using antenatal exposure to UV‐B radiation as an instrumental variable.2 and was associated with reduced risk of preeclampsia with evidence of a dose‐response relationship . Associations were demonstrated for UV‐B exposure in all 3 trimesters.Scottish maternity records were linked to antenatal UV‐B exposure derived from satellites between 2000 and 2010. Logistic regression analyses were used to explore the association between UV‐B and preeclampsia, adjusting for the potential confounding effects of month of conception, child's sex, gestation, parity, and mean monthly temperature. Of the 522 896 eligible singleton deliveries, 8689 (1.66%) mothers developed preeclampsia. Total antenatal UV‐B exposure ranged from 43.18 to 101.11 kJ/mThe seasonal patterning of preeclampsia may be mediated through low maternal vitamin D concentration in winter resulting from low UV‐B radiation. Interventional studies are required to determine whether vitamin D supplements or UV‐B–emitting light boxes can reduce the seasonal patterning of preeclampsia. A population‐wide study of deliveries in NorwayIt is plausible that vitamin D may play a role in this seasonal patterning. Case‐control studies have found significantly lower circulating 25‐hydroxy vitamin D (25(OH)D) concentrations in women with preeclampsia and eclampsia, compared with healthy, normotensive pregnant women.Vitamin D is produced in response to UV‐B exposure. The amount of UV‐B reaching the earth's surface is highest in summer months and, in winter, is usually too low to stimulate vitamin D production in high‐latitude countries. UV levels are strongly correlated with season, but superimposed on this annual cycle there is an 11‐year solar cycle over which changes in the sun's oscillatory magnetic field result in variations in the number of sunspots and, therefore, solar radiation. As a result, UV levels in a given season vary between years; therefore, adjusting for month of conception can disentangle associations with UV from associations with other seasonally patterned environmental and behavioral phenomena.Our study aim was to explore whether antenatal UV‐B exposure was associated with preeclampsia incidence, independent of month of conception, among all singleton children born in Scotland over an 11‐year period.https://www.isdscotland.org/Products‐and‐Services/eDRIS/Use‐of‐the‐National‐Safe‐Haven/.The data sets analyzed during the current study are available in the National Services Scotland National Safe Haven, All methods were carried out in accordance with the EU General Data Protection Regulation. All experimental protocols were approved by National Health Service Scotland's Public Benefit and Privacy Panel for Health and Social Care on December 5, 2019 (Reference 1617‐0001). Informed consent waived by National Health Service Scotland's Public Benefit and Privacy Panel for Health and Social Care.International Classification of Diseases, Ninth Revision (ICD‐9) codes 642.4 or 642.5, or International Classification of Diseases, Tenth Revision (ICD‐10) codes O14.0, O14.1, or O14.9. Since the early 1990s, gestational age at delivery has been confirmed by ultrasound conducted in the first half of pregnancy in >95% of pregnant women in the United Kingdom. If there is more than 7 days' difference between gestational age calculated from ultrasound and from last menstrual period, the former is used.We linked routine maternity records on singleton infants delivered in Scotland between 2000 and 2010 inclusive to environmental databases on UV solar irradiance and temperature. The Scottish Morbidity Record 02 records the child's sex and date of birth, gestation at delivery, whether it was a multiple delivery, and any maternal diseases. Preeclampsia was defined as ftp://apollo.eorc.jaxa.jp/pub/JASMES/Global_05km/). The Great Britain points were then interpolated, via inverse weighted distance, to raster images. The rasters were projected to Ordinance Survey Great Britain 1936/British National Grid. Finally, we took the Northing and Easting centroids of each postcode unit in Scotland (Ordnance Survey codepoint ver 2017.4.0) and used these to extract the UV‐B values for only those grid squares covering residential areas in Scotland. Scotland‐wide mean daily values were then calculated for each month resulting in a mean monthly time series of UV‐B irradiance values measured in W/m2. These were converted to kJ/m2 by multiplying by 86 400 (seconds in the day) and dividing by 1000 to be consistent with other epidemiologic studies. We linked these data to pregnancy records by month and year of conception. UV‐B exposure was calculated over each trimester of pregnancy and total exposure over the whole of pregnancy. These were then categorized into overall and trimester‐specific quintiles  for UV‐B (280–315 nm) was available at daily resolution from 2000 to 2010 inclusive. We downloaded UV‐B values, between January 1, 2000, and December 31, 2010, for each latitude and longitude within the Great Britain bounding box  from the publicly accessible Japan Aerospace Exploration Agency FTP site (Mean monthly air temperatures (°C) for 2000 to 2010 were downloaded from the Centre for Environmental Data Analysis website. These data are derived from the Met Office historical weather observations, which are then spatially interpolated to produce a regular 5‐km grid. The interpolation process takes account of latitude and longitude, altitude and terrain shape, coastal influence, and urban land use.Since UV data were available for only 2000 to 2010 inclusive, children conceived before 2000 or born after 2010 were excluded as they did not have UV exposure data for the whole of their intrauterine period. We excluded from the study children who were born <24 or >44 weeks' gestation, or had birthweight <400 or >6500 g. These exclusions were made because values outside these ranges are indicative of pregnancy complications that may introduce confounding. We tested whether there was evidence of any association between UV‐B exposure and preeclampsia. A series of multivariate logistic regression analyses were performed of the associations between trimester‐specific and overall UV‐B and preeclampsia. The models were adjusted sequentially for month of conception, then child's sex, estimated gestation and parity, then mean monthly temperature. Month of conception was modeled as a dummy variable. All analyses were undertaken using Stata version 14.2  in December to 19.23 kJ/m2  in June. Distribution of conceptions within a month did not vary across months. Therefore, use of a chronologically centered midmonth mean did not introduce bias, although it may have reduced statistical power.Overall, 528 175 singleton children were born in Scotland between 2000 and 2010. Of these, 657 were ineligible for inclusion: 35 were born at <24 weeks' gestation; 21 were born at >44 weeks' gestation, 252 had missing data on gestation; 74 had birthweight <400 g; and 275 had birthweight >6500 g. The remaining 527 518 were eligible for inclusion. A further 2 had to be excluded because the mother's age was missing, 25 because the child's sex was missing, and 4595 because parity was missing. Therefore, the final study population comprised 522 896 deliveries. Over the 11‐year study period (2000–2010), mean monthly exposure to UV‐B ranged from 0.51 kJ/mThe mother developed preeclampsia in 8689 (1.66%) pregnancies, ranging from 1.55% for October conceptions to 1.75% for February conceptions . The incAmong 522 896 pregnancies in Scotland over an 11‐year period, exposure to UV‐B was inversely associated with the risk of preeclampsia, with some evidence of a dose relationship. This was independent of month of conception and, therefore, other seasonally patterned phenomena such as diet, physical activity and smoking, as well as the child's sex, gestation, parity, and temperature.Most vitamin D is produced in the skin through UV‐B exposure. Both season and weather affect the amount of UV‐B reaching the earth's surface. Therefore, in high‐latitude countries, such as Scotland, the amount of UV‐B in winter months is generally insufficient for vitamin D production because of a lower zenith angle and higher cloud cover resulting in a greater amount of atmosphere to penetrate. Vitamin D insufficiency, defined as circulating 25(OH)D concentrations <40 nmol/L, is twice as likely in Scottish residents than people resident in other parts of Britain.Our findings support our hypothesis that the higher risk of preeclampsia during pregnancies resulting from summer conceptions may be attributable to lower levels of exposure to UV‐B, in the second half of pregnancy, likely mediated via lower levels of maternal vitamin D. Our findings are consistent with a study of solar radiation in an Australian cohort, which found that pregnancy hypertension was positively correlated with solar radiation 1 month after conception but negatively correlated with solar radiation 7 months after conception.This study covered the entire Scottish population nonselectively. We used an existing maternity database that is not collected primarily for research use. However, it is subjected to regular quality‐assurance checks in which a sample of digital records are validated against clinical records, and completeness and accuracy have been consistently high for the variables used in this study.As well as UV, other environmental exposures such as temperature exhibit seasonal patterning. Furthermore, lifestyle behaviors such as diet and physical activity vary by season. Therefore, we adjusted for outdoor temperature and month of conception to control for other seasonally patterned exposures for which we did not have data. Adjustment for child's sex was a strength because boys have a different seasonal patterning of conception from girls, with male conceptions peaking in autumn and female conceptions in spring.As with all observational studies, association does not necessarily infer causation since residual confounding is possible. Mendelian randomization provides evidence that low vitamin D concentrations are causally linked to higher blood pressure and hypertension.We calculated mean daily levels of UV‐B for each month over Scotland as a whole. Greater granularity would have been preferable; however, Scotland measures 729 km (453 miles) from the northernmost to the southernmost settlement, which equates to a relatively small range of latitudes (from 54.4° North to 60.5° North). Our calculation of UV exposure assumed that women resident in Scotland remained in Scotland during their pregnancy. No data were available on changes in UV exposure attributable to holidays and trips abroad. A limitation of Scottish Morbidity Record 02 is that it does not record maternal lifestyle data such as physical activity or weight gain during pregnancy. Similarly, we had no data on individual differences in exposure to UV attributable to the proportion of time spent outdoors, choice of clothing, or use of sunblock.We observed an inverse relationship between antenatal UV‐B exposure and risk of preeclampsia. This lends support to the National Institute for Health and Care Excellence guideline that all pregnant women should take 10 µg of vitamin D supplements daily (400 IU/day), and National Health Service guidance on safe sun exposure to produce vitamin D.Dr Pell had the original concept. Drs Pell and Mackay obtained maternity data; and Drs Clemens, Cherrie, and Dibben obtained environmental data. Dr Hastie undertook the statistical analyses. Drs Hastie, Pell, Mackay, Clemens, Cherrie, Dibben, Smith, Stock, and Megaw interpreted the results. Drs Hastie and Pell drafted the manuscript. Drs Hastie, Pell, Mackay, Clemens, Cherrie, Dibben, Smith, Stock, and Megaw revised the manuscript and approved the final version.This work was supported by Health Data Research UK (Edin‐1 to Hastie). Provision of the UV data was funded by a joint Natural Environment Research Council, Medical Research Council, and Chief Scientist Office project grant (NE/P010911/1); Tommy's Charity; and Health Data Research UK funding (Edin‐1). Dr Stock is funded by Wellcome Trust Clinical Career Development Fellowship (209560/Z/17/Z). Funders played no role in conducting the research or writing the paper.None.


Puerariae lobatae radix (Ge‐Gen in Chinese) and Puerariae thomsonii radix (Fen‐Ge) are widely used as medicine and health products, particularly in Chinese medicine. Puerarin and daidzein are the primary bioactive compounds in Puerariae radix. These isoflavones have been used to treat cardiovascular and cerebrovascular diseases, hypertension, diabetes, and osteoporosis. The content of puerarin in Ge‐Gen is about six times higher than that in Fen‐Ge, so its use has a higher pharmacological effect. It is therefore of great importance to effectively distinguish between these two species. However, because their basal plants, P. lobata (Willd.) Ohwi and P. thomsonii Benth., possess an extremely similar appearance, and detecting the level of chemical constituents is just a rough distinction, it is necessary to develop more efficient identification approaches. Here the complete chloroplast genomes of P. lobata and P. thomsonii were deciphered, including sequencing, assembly, comparative analysis, and molecular marker development. The results showed that they are 153,393 and 153,442 bp in length, respectively; both contain 124 annotated genes, including eight encoding rRNA, 29 encoding tRNA, and 87 encoding proteins. Phylogenetic analysis showed that they form a clade, indicating that they originate from the same ancestor. After obtaining 10 intergenic/intronic regions with a genetic distance greater than 0.5 cm, primers were designed to amplify regions of high variability in P. lobata and P. thomsonii. Finally, a 60‐bp differential base fragment, located in the intron of rpl16, was developed as a molecular marker to efficiently distinguish between these two species. Puerariae lobatae (Ge‐Gen) is about six times higher than that in Puerariae thomsonii (Fen‐Ge), but these two plants are visually indistinguishable. By deciphering the complete chloroplast genome of these two plants, the authors developed a molecular marker to efficiently distinguish between them.The content of the medicinal isoflavone puerarin in the root of  CPchloroplastIRreverse complementary regionsLSClong single copy regionP. lobataPueraria lobata (Willd.) OhwiP. thomsoniiPueraria thomsonii Benth.SSCshort single copy regionSSRssimple sequence repeatsPuerariae lobatae radix, known as Ge‐Gen in Chinese, is a popular traditional Chinese herbal medicine that was first described in the Shennong Herbal Classic. The genus Pueraria DC. belongs to the Fabaceae family and comprises about 20 species worldwide. It is mainly distributed in East Asian countries, among which China is the distribution center, accounting for 11 species. The dried roots of Ge‐Gen have been widely used for the treatment of influenza, muscle stiffness, and other sicknesses [Pueraria species: Pueraria lobata (Willd.) Ohwi and Pueraria thomsonii Benth., whose roots were considered the main source of Ge‐Gen because of the wide distribution and its traditional Chinese herbal medicine value, as recorded in the Chinese Pharmacopoeia, 2000 edition [ edition , while d edition .P. lobata are both higher than in P. thomsonii, especially the main bioactive compounds puerarin and daidzein [P. lobata is much better than P. thomsonii. As a result, for accuracy of medication, since 2005 the roots of P. lobata and P. thomsonii have been separately used as Ge‐Gen and Fen‐Ge in the Chinese Pharmacopoeia; The Chinese Pharmacopoeia (2010 edition) specifies the lower limit of puerarin in P. thomsonii and P. lobata are 0.3% and 2.4%, respectively. However, the morphological characteristics of these two species are too similar to distinguish. They are both perennial woody lianas, up to 8 meters in length. They have thick and massive roots; woody stems at the base; pinnate compound leaves growing with three leaflets; ovate‐shaped accumbent stipules; campanulate calyx, covered with yellowish‐brown pubescent; purple corolla, obovate petals; flat pod, covered with brown bristles. The main difference between these two species is that the leaflets of P. thomsonii are rhomboid‐ovate or broadly ovate, the apexes are acute or apiculate, the bases are truncate, the lateral leaflets are slightly smaller and more oblique, and the petals are circular. While the shape of dried roots of the two plants is extremely similar, it is more difficult to identify the processed slices. Moreover, their morphological characteristics are susceptible to environmental conditions, which increases the difficulty for identification [P. lobata and P. thomsonii and their common counterfeits using minor differences. Universal molecular markers, such as internal transcribed spacer (ITS), rbcL and psbA, are widely used for identifying species easily and accurately [According to previous studies, the contents of isoflavones and polysaccharides in daidzein , 6, 7. Tfication . Althougopoeia 20 edition curately , 12, butP. lobata and P. thomsonii have not been thoroughly studied. The complete CP genome of P. thomsonii has been reported [Pueraria DC. represented by P. lobata and P. thomsonii will have great development prospects.The chloroplast (CP) is an organelle of great importance in green plants, which plays a crucial role in photosynthesis, carbon fixation, translation, and biosynthesis . Accordireported , and we P. thomsonii and P. lobata were sequenced, assembled, and annotated. Afterwards, a comparative genomic analysis between the two CP genomes was conducted with the aim of finding highly variable regions and establish the phylogenetic position of the Pueraria DC. in the Fabaceae family. Moreover, a specific molecular marker from the CP genomes of the two Pueraria species was developed and verified. In all, our study aimed to ensure safety in the use of medicinal plants, identify species, and conserve wild Pueraria species.In our study, the genomes of P. lobata and P. thomsonii were collected from Sancha Town, Enshi City, Enshi Prefecture, Hubei Province , China. Samples of P. lobata were collected from the Beijing Medicinal Plant Garden  for reference. Leaves were stored at −80 °C after rinsing. Total DNA was extracted using a plant genomic DNA kit . DNA quality was assessed using the Nanodrop spectrophotometer 2000 , and the integrity was evaluated by 1.0% agarose gel electrophoresis. The sequencing libraries were prepared by using the TruSeq DNA Sample Prep Kit . DNA was fragmented into ~500 bp long fragments randomly for paired‐end library construction [trimmomatic (v. 0.32) [spades (v. 3.10.1) [clc genomics workbench (v. 7), and novoplasty (v. 4.3) [dnastar (v. 11.0) [Fresh leaf materials of v. 0.32) . spades  3.10.1) , clc gen(v. 4.3)  were use(v. 4.3) . The con(v. 4.3) . All thev. 11.0) .ogdraw (v. 1.3.1) [emboss (v. 6.3.1) [P. thomsonii and P. lobata was performed using the mVISTA program [Dual Organellar GenoMe Annotator (DOGMA) , the CP . 1.3.1) . The per. 6.3.1) . Compara program  in Shuff program .reputer [The size and location of long repeat sequences, including forward, palindromic, reverse, and complement repeats in the CP genomes of two species were identified by setting the parameter of the Hamming Distance to 3 (sequence identity ≥90%) and the Minimal Repeat Size to 30 through reputer . Simple reputer .P. lobata, P. thomsonii, and other 42 of Leguminosae, as well as two outgroup species, Nicotiana tabacum and Arabidopsis thaliana. The CP genome sequences were downloaded from the NCBI GenBank (https://www.ncbi.nlm.nih.gov/)  were obtained and then globally matched using Alignment using the Fast Fourier Transform (MAFFT) program [N. tabacum and A. thaliana as outgroup species, phylogenetic relationships were analyzed using the ML method and the Tamura‐Nei model in MEGA6 [In this study, the phylogenetic tree was conducted using the maximum likelihood (ML) method: a total of 46 CP genomes were used for the phylogenetic analysis, including /) Table . After s program . Next, uin MEGA6 . The BOOIn order to analyze sequence diversity and selective pressure, sequences of 124 annotated genes and 146 intergenic/intronic regions were extracted from the two CP genomes, and then aligned by the Clustalw2 (v. 2.0.12) program  with theP. lobata and P. thomsonii, plant samples of P. lobata and P. thomsonii were collected from some other regions  agarose gel for 20 min at 120 volts. Finally, the products were sent to the company for sequencing. The primers are listed in Table The molecular marker was selected based on the alignment and comparison of mVISTA similarities and divergence analysis results. To amplify these regions, primers were designed using Primer Premier 5 . In ordens Table . The proP. lobata and P. thomsonii both display circular molecules with the typical quadripartite structure, including a long single copy region (LSC), a short single copy region (SSC), and a pair of reverse complementary regions (IRa and IRb). The CP genome of P. lobata from Hubei Province was 153,393 bp in length; while that of P. thomsonii was 153,442 bp; both of the total GC contents were 35.41%  and the adjacent genes of the CP genomes of ail Fig. . Genes oP. lobata, among which 20 genes had a single intron, two genes had two introns; while in P. thomsonii there were 22 genes with introns, among which 20 genes had one intron, and two genes had two introns . According to previous studies, the abundance of RPL16 increased in tolerant or sensitive seedlings under dehydration; [rpl16 is very likely related to the dehydration response, as well as photosensitivity, to some extent. As for the intron, it was reported that most of the genes expressed at higher levels tend to possess shorter introns. Natural selection has driven introns to smaller sizes in highly expressed genes to reduce the cost of transcription, and small introns may have evolved to fit more genes into smaller regions [P. lobata may be an ancestral characteristic of the highly expressed gene rpl16, intron length in rpl16 may have decreased as evolved. Taken together, it is quite possible that P. lobata has stronger dehydration resistance and photosensitive activity than P. thomsonii.Through the global comparative analysis and the genetic distance analysis, the diversity and variety of the CP genomes of dration; . Consequ regions , 53. ThuP. lobata and P. thomsonii collected from different regions as reference, the YF‐60 marker developed in this study was verified by PCR amplification and the 1% agarose gel electrophoresis. The novel molecular marker developed in this study could effectively distinguish the two Pueraria DC. species from different geographical regions. In addition, apart from the published P. thomsonii CP genome P. lobata CP genome in NCBI when conducting nucleotide blast (https://www.ncbi.nlm.nih.gov/nucleotide/MT818508.1). Therefore, we further compared and verified the CP genome of P. lobata, and found no difference from P. thomsonii in the YF‐60 marker region where it should have. This result just indicated the phenomenon that P. thomsonii is easily mistaken as P. lobata, and further supported the availability and application value of our developed molecular marker YF‐60. In all, the developed molecular marker in this study will be beneficial to lay a theoretical foundation for identification of species of Pueraria DC. and promote available utilization and protection of wild Pueraria DC. resources.For the sake of further confirmation of the accuracy, with the total genomic DNA extracted from leaves of The authors declare no conflicts of interest.BW, MH, and CL conceptualized the project. MH and BW administrated the project. JL and MY did the formal analyzing. JL, MY and MJ analyzed and interpreted the data. MY and JL applied the software. JL and MY wrote the original draft. BW supervised the work. JL validated the work. JL, MH, YL, and BW reviewed and edited the article. All authors read and agreed on the published version of the article.Fig. S1. Circular Gene map of the complete CP genome of Puerariae thomsonii. Genes drawn inside the circle are the transcribed clockwise, and those on the outside are transcribed counter‐clockwise. The dark gray area in the inner circle corresponds to the GC content, whereas the light gray refers to AT content. LSC: large single copy region, SSC: small single copy region, IRa, IRb: copies of inverted repeat regions.Click here for additional data file.Table S1. CP genome information for phylogenetic tree construction.Click here for additional data file.Table S2. Sample information.Click here for additional data file.Table S3. Annotated genes of CP genome.Click here for additional data file.Table S4. Different genes in different regions in Pueraria lobata.Click here for additional data file.Table S5. The lengths of introns for the splitting genes in Pueraria lobata.Click here for additional data file.Table S6. The lengths of introns for the splitting genes in Puerariae thomsonii.Click here for additional data file.Table S7. k2p genetic distance of protein‐coding region of Pueraria lobata and Puerariae thomsonii ≥ 0.1.Click here for additional data file.Table S8. k2p genetic distance of intergenic/intronic region of Pueraria lobata and Puerariae thomsonii ≥ 0.5.Click here for additional data file.Table S9. SSR statistics of CP genome of Pueraria lobata and Puerariae thomsonii.Click here for additional data file.Table S10. SSR distribution in Pueraria lobata CP genome.Click here for additional data file.Table S11. SSR distribution in Puerariae thomsonii CP genome.Click here for additional data file.


Ischemic stroke (IS) in young patients may differ in etiology and prognosis from later‐life IS, which is much more common. A number of single‐center and population‐based cohorts of affected individuals have been published, but information on the long‐term prognosis of these patients is limited.IS patients (≤55 years), discharged over a 10‐year period, were evaluated and prospectively followed. Subgroups were evaluated for type of stroke, antecedent risk factors (RF), long‐term outcomes, and occupational status over several years of follow‐up.n = 118) and younger  age groups. Traditional RF—hypertension, diabetes mellitus, hyperlipidemia—were significantly associated with IS, and increased with age. The distribution and type of IS were similar in both groups, except for an increase in small vessel IS among the older subgroup (p = .003).178 IS individuals from 2001–2010 were divided into older , a similar proportion of patients in both subgroups had a recurrent IS, but no significant differences were found in most disability indices. Approximately one third of patients suffered from moderate to severe disability, and were unable to return to their prior work.Of the evaluable patients at 5.1 ± 2.5 years of follow‐up  ischemic stoke patients discharged over a 10 year period were prospectively evaluated and followed. Of 178 evaluable patients, raditional risk factors such as‐ hypertension, diabetes mellitus, hyperlipidemia were significantly associated with IS, and increased with age. The distribution and type of ischemic stroke were similar in both groups except for an increase in small vessel IS among the older subgroup (P = 0.003), and disability increased with the number of strokes. Our study has the following potential limitations: it included young IS cohorts from only two departments of our hospital. Full volume diagnostic studies were not completed by all patients. For statistical analysis, we did not separate patients with first and recurrent event on admission. Functional status on the day of discharge was not observed, and ethnic differences were not determined. The review of the follow‐up period, and the obtainment of information about a patient's functional or occupational status was done by telephone, and therapeutic follow‐up management was not included.1The incidence of ischemic stroke (IS) has increased globally by 25% among adults under age 60 Hankey, . IS statWe therefore conducted a prospective study to evaluate and follow‐up adults younger than 55 years of age, following hospital discharge for acute IS. Analysis focused on risk factors (RF), etiology, stroke subtypes, and long‐term clinical outcomes. We tested the hypothesis that younger patients differ in their RF, IS characteristics, and outcomes, and aimed to compare IS patients from a younger subgroup (aged 18–45 years) with those from an older one (aged 46–55 years).2All surviving patients aged 18–55 years, with a discharge diagnosis of IS between 2001 and 2010, from two departments of medicine out of four at an academic medical center, were invited for ambulatory evaluation and follow‐up. Older patients and individuals with active cancer, intra‐cerebral hemorrhage, or other neurological diseases, were excluded.Chart review was done for each patient, and was complemented by a full medical history, physical examination, routine and ancillary diagnostic work‐up focusing on RF for IS. The latter included echocardiography, 24‐h ECG (Holter) monitoring, cervical artery Doppler study, and evaluation for hypercoagulability. RF were categorized as "traditional" (modifiable and non‐modifiable), and "additional"  RF.Neuroimaging was reviewed and evaluated for stroke subtypes according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification: large artery atherosclerosis, small vessel occlusion, cardioembolism, and other determined and undetermined etiologies. Arterial territory of IS was classified as hemispheric , posterior fossa (including cerebellum and brain stem), multiple infarcts, and negative imaging.Long‐term patient follow‐up included recurrent stroke and other cardiovascular events, epileptic seizures or death, and were based on information obtained from hospital charts, and contacting the patient's primary physician and/or death registries. Disabilities were evaluated through structured telephone interview with the patient (or caregiver when the patient was unable to communicate). The interview determined functional status by completing detailed and validated questionnaires, including the modified Rankin scale (mRS), measuring the degree of disability/dependence after stroke; the Barthel index (BI), reflecting current actual performance activities of daily living (ADL) and finally, the London Handicap Scale (LHS), providing a descriptive profile of disadvantages experienced in the domains of mobility, physical independence, orientation, social functioning, economic self‐sufficiency, and occupational status (OS).The study was IRB approved. No therapeutic interventions were initiated by the authors. However, a new diagnosis with potential therapeutic implications was communicated to the patient's primary physician.χ2 test and Fisher's Exact test to compare categorical variables across groups, as well as by Student's t test to compare means. Stepwise logistic regression model, using forward selection analysis, was used to determine the correlation between variables such as disability scores and age. p values < .05 were considered significant.Statistical analysis was done using SAS 9.1.3 software (SPSS Inc.). Parameters of patients of different age groups (18–45 and 46–55) were compared by Pearson's 3n = 118). Women constituted 54% of all study individuals, with gender distribution similar in the two subgroups. Intriguingly, 17/178 patients (10%) had a family history of stroke, and 83/178 (47%) reported a previous stroke/transient ischemic attack (TIA). These variables were not significantly different between the two cohorts . Approximately one third of these individuals were 18–45 years old . This younger subgroup had a median age of 40 versus 51 in the older cohort (3.1p = .0003), and there were more subjects with four or more RF (p = .002) than in the younger subgroup. Traditional RF such as hypertension, diabetes, and dyslipidemia, as well as a history of previous atherosclerotic cardiovascular disease (ASCVD), were significantly more prevalent among individuals in the older versus the younger cohort (p = .002). In particular, coronary artery disease (CAD), atrial fibrillation (AF), carotid artery stenosis (CAS), and peripheral vascular disease (PVD), were all more common in the older subgroup, but only CAD reached a level of statistical significance.RF for study participants are presented in Table p = .002), dyslipidemia , and ASCVD . In contrast, the younger 18–45 age cohort had a significantly lower prevalence of these traditional RF, but hormonal factors  and antiphospholipid syndrome  emerged as more prevalent in this group. Only a few patients had no identifiable RF; 12% in the younger, and 5% in the older subgroup, but this was not statistically significant.Multivariate analysis showed a persistent strong correlation of IS in the older 46–55 age subgroup, with hypertension , with more diabetes , hypertension , smoking , and alcohol abuse . Men also had significantly more CAD , CAS , and PVD  (Appendix 1). On multivariate analysis, the three most prominent RF in men remained diabetes ; ASCVD , and alcohol (p = .007). In contrast, women had more valvular heart diseases , and autoimmune diseases . As expected, hormonal factors in women retained their significance on multivariate analysis. Obesity, arrhythmia, and other RF showed no significant difference between genders.Compared with females, male patients  had significantly more RF per individual . Despite careful evaluation, the type of stroke could not be determined in 25% of younger, and 9% of older individuals (p = .007). No significant differences between genders were found, including on multivariate analysis (Appendix 1).The relative proportion of types of IS as per TOAST classification is listed in Table 3.3p = .02) and male . On multivariate analysis, the absence of hypertension  or valvular disease  was found to be predictive of negative brain imaging.According to clinical presentation or neuroimaging  the vascular territories of the stroke were not significantly different between the two age subgroups Table , or betw3.4p = NS) (Table p = .02), alcohol abuse (p < .001), and older age were identified as potential predictors of mortality through multivariate analysis. As many as 32% of the patients had a recurrent stroke over the follow‐up period, but recurrence rate was not significantly different between the two age and gender subgroups, but was still correlated with the number of RF , and 46–55 vs. 18–45‐year‐old patients , had a recurrent cardiovascular event (Table Over the follow‐up period of 5.1 ± 2.5 years (similar in both age subgroups), 13/178 (7%) patients died; 3 (5%) in the younger and 10 (9%) in the older groups (3.5Twenty‐seven patients (15%) were alive, but were lost to follow‐up or declined to respond despite their initial consent. Disability follow‐up was obtained in 138 patients, at a mean time interval of 5 years. These patients constituted approximately 80% of the original cohort in each of the age subgroups.p ≤ .05). Indeed, this is reflected by the greater proportion of individuals in the older subgroup classified as "severe disability" (best seen in the 38% vs. 23% unable to work in the OS), but the difference did not reach statistical significance. Multivariate analysis identified four significant factors predictive of severe disability and inability to resume work: hypertension , recurrent stroke (by mRS) , CAD , and large artery disease (by TOAST classification) . The same RF correlated with the inability to resume work in the gender subgroup as well (Appendix 1).We used four different validated instruments  to evaluate long‐term functional outcome and disability. Results are presented in Table 4By definition, “stroke in the young” used to be an event occurring at or before age 44. However, today, due to the increasing span of life, and variability in the cut of upper age border (45–55 years) between statistic groups in the literature, the age period of "stroke in the young" is divided into “early adult life” before age 44, and "midlife” between ages 45 and 55  increased with age and predominated. Most "classic" modifiable RF were already well represented in the younger (18–45 years) and male cohort; thus, comparisons of the age/gender subgroups were frequently found to lack statistical significance. The accumulation of traditional RF in males and with aging, as shown in a Danish group of 40,102 patients, may explain the male predominance among older patients as seen in the Helsinki study, but this proportion has been shown to equalize in patients over age 55 , estrogen use, or an underlying autoimmune disease. This observation is consistent with other published studies . Surprisingly, however, we also found an association with autoimmune diseases (p = .02), which has not been previously reported  patients died, the major cause of death being of vascular origin (54%). A recent long‐term follow‐up study in young IS patients demonstrated a 5‐year cumulative mortality ranging from 5.8% to 11%, 10‐year cumulative mortality from 12% to 17%, and 26.8% after 20 years. The cause of death in 55% of cases was of vascular origin, and in 28%, it was felt to be preventable  of our young patients, after IS, recovered by mRS and BI without disability. Moderate‐to‐severe disability was the same in the gender and age groups. We did not check the level of disability of the patients on the day of discharge, but other Israeli studies found no significant differences between the younger and older patients by mRS  study of 2019 showed stroke as the top‐ranked age‐related cause of disability‐adjusted life‐years (DALYs) worldwide , but did not correlate with gender, and were comparable to the results reported by Varona et al. . Our mul5Younger patients with IS are predisposed by the same traditional vascular stroke RF as older individuals, and these progressively increase with age. We found no significant age‐ and gender‐related differences in terms of RF, etiologies, stroke subtype, and long‐term clinical outcome. The risk of long‐term disability worsens with time due to recurrent vascular events. These data emphasize the need for a strategy for early identification of the already well‐known stroke RF, and to institute an aggressive therapeutic management in all patient groups as a primary and secondary prevention of recurrent stroke.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.


To confirm the critical factors affecting seizure susceptibility in acute pentylenetetrazole (PTZ) mouse epilepsy models and evaluate the prior literature for these factors.Serial cohorts of wild‐type mice administered intraperitoneal (IP)‐PTZ were aggregated and analyzed by multivariate logistic regression for the effect of sex, age, background strain, dose, and physiologic stress  on seizure response. We assessed the reporting of these factors in a comprehensive literature review over the last 10 years (2010–2020).We conducted aggregated analysis of pooled data of 307 mice  with median age of 10 weeks (range: 6–49 weeks) with acute PTZ injection (dose range 40–65 mg/kg). Significance in multivariate analysis was found between seizures and increased PTZ dose (odds ratio (OR) 1.149, 95% confidence interval (CI) 1.102–1.205), older age , physiologic stress , and mixed background strain . Literature review identified 97 papers using acute PTZ‐seizure models. Age, housing, sex, and background were omitted by 61% (59/97), 51% (49/97), 18% (17/97), and 8% (8/97) papers, respectively. Only 17% of publications specified all four factors (16/97).Our analysis and literature review demonstrate a critical gap in standardization of acute PTZ‐induced seizure paradigm in mice. We recommend that future studies specify and control for age, background strain, sex, and housing conditions of experimental animals. A receptor antagonist to induce seizures, and is employed in both acute and chronic models of epilepsy.Rigor and reproducibility in preclinical epilepsy models is essential for understanding epilepsy and expediting antiseizure drug discovery.Acute PTZ model offers many benefits as a seizure paradigm, including its ease of administration, ability to evoke a full range of seizure types, and utility in drug screening.To better understand the influences of age, background strain, sex, and physiologic stress on acute PTZ‐induced seizures, we evaluated aggregated seizure response data from several serial cohorts of IP‐injected mice in the Experimental Neurophysiology Core at Boston Children’s Hospital. We then systematically evaluated the literature from the past 10 years to assess if these critical factors were specified and controlled for in acute PTZ paradigm protocols across several studies. Our study and literature review together outline the important factors necessary to specify and control for in acute PTZ‐induced seizure paradigms and serve as a framework for future studies using acute PTZ administration to model epilepsy in mice.All mouse procedures were performed in accordance with the Guide for the Humane Use and Care of Laboratory Animals, and the studies included were approved by the Animal Care and Use Committee of Boston Children’s Hospital. All mice were housed in a 12‐h light–dark cycle, climate‐ and humidity‐controlled room, with access to food and water ad libitum.Littermates and sexes were divided evenly across experimental groups. Cohorts of wild‐type control mice were included in this analysis, some of which have previously been published.PTZ  was dissolved fresh before each experiment in 0.9% saline at 10 mg/mL. All mice were injected during the light phase of the light–dark cycle. Mice were weighed prior to dosing and injected IP (volume PTZ (mL) = weight (kg) × dose (mg/kg)/10 mg/mL). Post‐injection, mice were placed in individual transparent cages and monitored for seizure over a 10‐min period. After a 10‐min observation period, mice were killed. Our primary endpoint was GTCS, as behaviorally indicated by loss of balance and continuous, uncontrollable jerking (Racine score of 5). A GTCS ended when the animal regained balance or righted itself and ceased jerking  or died. Start and finish time of GTCS and time of death were recorded.We analyzed aggregated data from 22 both published and unpublished cohorts from the Experimental Neurophysiology Core at Boston Children’s Hospital over a 7‐year period (2013–2020) using the same standardized method for PTZ‐induced seizures. Because these cohorts were collected and measured in comparable ways and between‐cohort heterogeneity is negligible, aggregated analysis of the pooled data was performed.p‐value of less than 0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism 8 and RStudio software. All factors in analysis were compared to seizure response. Univariate comparisons were performed by Pearson’s chi‐squared tests for categorical data , Mann‐Whitney U tests for continuous, non‐parametric data (age and dose) and Spearman correlation. Fisher’s exact test and chi‐square test of trend were performed on subgroup analysis. Multivariate comparisons were performed by multivariate logistic regression analysis. A To identify studies that employed acute PTZ as a seizure paradigm, we performed a PubMed search for the 10 years (January 2010–May 2020) using the following search criteria: (pentylenetetrazole[Title/Abstract] OR PTZ[Title/Abstract]) AND mouse[Title/Abstract] AND (seizure[Title/Abstract] OR epilepsy [Title/Abstract]) NOT kindling[Title/Abstract]. Papers were excluded post hoc if they were review articles, kindling models (repetitive injections of PTZ) otherwise not omitted, PTZ paradigms in zebrafish or rat models only, and/or evaluations of PTZ as a treatment only. Our search yielded 130 results, and 97 papers were included in our analysis.Per reviewed manuscript, we recorded the age, background strain, sex, and housing of experimental mice, and route of PTZ administration, dose, and primary endpoint(s). We systematically evaluated if each of these critical factors were included in the study cohort. A factor was considered unspecified (uns.) if there was no mention of it or there was insufficient detail to replicate the study. Primary endpoints were grouped into categories for simplicity: (1) binomial seizure endpoint , (2) seizure feature , (3) behavioral scoring, (4) EEG characterization, (5) seizure threshold calculation, (6) other, or (7) undefined/unclear. Ages were grouped as previously explained.We evaluated the contributions of age , background strain , sex, physiologic stress , and dose (40–65 mg/kg) to seizure response  in an acute IP PTZ model. In total, our aggregated analysis of pooled data includes 307 mice from 22 cohorts , 220 C57BL/6J background, 87 mixed background; 202 males, 105 females; 255 unstressed , 52 stressed (surgery and/or single‐housed); 160 given 40 mg/kg PTZ, 28 given 48–50 mg/kg PTZ, 63 given 55–57 mg/kg, and 56 given 65 mg/kg Table .p < 0.0001), age (p < 0.027), and physiologic stress (p < 0.0001), but not background strain (p = 0.4) or sex (p = 0.69).Results of our aggregated analysis are summarized in Tables p < 0.0001, OR 1.149, 95% CI 1.102–1.205), older age , physiologic stress , and mixed background   or Cohort 19 (50% (12/24) males and 38% (8/21) females with seizures; p > 0.57) (Table p = 0.003). Collectively, our data confirm age, stress, and background strain are important factors influencing PTZ‐induced seizure susceptibility in wild‐type mice.We performed subgroup analysis for sex and stress. To evaluate sex differences, we analyzed a subgroup of mice where the age, background, stress, and dose were identical and fixed. No significant difference in seizure susceptibility in male and female mice was found within either Cohort 16 (29% (7/24) males and 31% (8/26) females with seizures; 7) Table . To eval7) Table . We idenOur data highlight the importance of accounting for baseline characteristics in PTZ‐induced seizure mouse models of epilepsy. We undertook a comprehensive literature review to evaluate prior studies using acute PTZ‐induced seizures in mouse models. We identified 97 publications over the past 10 years 2010–2020). Our findings are summarized in Table 010–2020.Primary seizure endpoints were utilized in most of the prior literature. Binomial endpoints  were used in 41% (40/97) of studies, with 20% using GTCS (19/97), 22% using a different seizure type (21/97), and 17% using death (16/97). Other endpoints reported were seizure features , seizure rating scale , EEG features , and seizure threshold . Seizure threshold was employed by almost all IV papers . In the studies utilizing the IP route, the most common primary endpoints were either binomial seizure/no seizure  or seizure scoring scales . Forty‐nine percent (47/97) of papers included more than one primary seizure endpoint.We assessed the included publications for specification of age, background, sex, and housing conditions of the experimental mice Fig. . Only 17The purpose of this study was two‐fold: (1) to evaluate the influences of age, background strain, sex, and physiologic stress on seizure susceptibility in an acute PTZ epilepsy mouse model through aggregated analysis, and (2) to critically examine the way the field has accounted for these factors over the last 10 years. In our study, we identified dose, age, and stress as critical factors with significant influences on seizure susceptibility in an acute PTZ paradigm. In our literature review, we found that acute PTZ models are inconsistent across studies. Due to varying endpoints and most studies not explicitly stating confounding factors, it is difficult to compare or replicate results and evaluate potential treatments. Together, our aggregated analysis and comprehensive literature review demonstrate a critical gap in standardizing the acute PTZ‐induced seizure paradigm to study epilepsy. It is likely our findings on age, background strain, sex, physiologic stress, and dose will generalize to other ages, strains, and PTZ routes of administration (IV and SubQ) not employed in our study, but more research is necessary to evaluate the role of factors we do not use in our labs.The American Epilepsy Society (AES) and International League Against Epilepsy (ILAE) Translational Task Force have begun a systematic review of animal research in epilepsy.The most striking finding of our study is the influence of physiologic stress on seizure susceptibility, which we represented through single‐housing and EEG implantation surgeries. Mice exposed to physiologic stress before a PTZ seizure challenge seized twice as often as control mice. Although our study was underpowered to directly address the effect of social isolation on seizures, our findings are consistent with other acute seizure paradigms in rodent models. Social isolation increased seizure susceptibility after pilocarpine‐induced seizures.A limitation of our study is that most of the animals were socially isolated before PTZ administration due to EEG electrode implantation surgery that involved acute exposure to anesthesia. The influence of EEG electrode implantation on seizure susceptibility in mice is poorly understood. Per lab protocol, implanted mice were post‐operatively administered analgesics and allowed to heal for at least a week before any further experiments could be conductedIn addition, we identified age as a significant factor in seizure susceptibility in both our univariate and multivariate analyses. Our findings on age are limited in scope, as 72% of our mice fall in the young adult category, and our age range is restricted to 6–49 weeks. Even with this restricted age range, we still found a significant influence of age on seizure susceptibility. At the same time, age was the most commonly omitted critical factor in the literature, with many papers omitting age all together and others only specifying the mice as “adult.” Our results provide evidence that even small differences in age result in variability in seizure susceptibility. All studies should report the age range of mice used and restrict the range of ages as much as possible. The underlying mechanisms of seizure susceptibility across ages are not well‐defined, and further research is needed in this area.We did not identify sex as a significant contributor to seizure susceptibility in wild‐type mice in an acute PTZ paradigm. Most studies did specify the sex of their experimental mice in their methods, and of these studies, a majority used only males. While we did not find sex differences in our study, it would be hasty to generalize these findings to all possible experimental designs, and, therefore, the sex breakdown of an experimental cohort is essential in any acute PTZ methods section. Sex differences are well‐documented in aging and maturation of mice.Other studies have shown that background strain influences seizure presentation and susceptibility.The cohorts included in our study used the most common acute PTZ method of administration (IP), primary endpoint , and background strain (C57BL/6J), making our findings useful for a majority of the field. We show that dose, age, background strain, and physiologic stress all influence seizure susceptibility. Physiologic stressors of surgery and housing conditions influence PTZ‐induced seizures, but the mechanisms of these effects remain to be determined. As proposed by the AES‐ILAE report on common data elements, our data highlight the importance of including age, background strain, sex, and housing to improve the rigor and reproducibility of preclinical models of epilepsy.M.S. reports grant support from Novartis, Roche, Pfizer, Biogen, Ipsen, LAM Therapeutics, Astellas, Bridgebio and Quadrant Biosciences. He has served on Scientific Advisory Boards for Sage, Roche, Celgene, Aeovian, Regenxbio and Takeda. None of the other authors has any conflict of interest to disclose.We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.Table S1. Cohorts of mice used for multivariate analysis of variables related to PTZ‐induced seizures.Table S2. Summary of literature review for intravenous and subcutaneous routes of PTZ‐induced seizures in mice.Click here for additional data file.Table S3. Full literature review separated by route of PTZ administration.Click here for additional data file.


To evaluate the effects of the concomitant use of proton pump inhibitors (PPIs) and/or antibiotics (Abs) on oncological outcomes in patients with advanced urothelial carcinoma.We retrospectively evaluated 155 patients with advanced urothelial carcinoma who were treated with immune checkpoint inhibitors (ICIs) between August 2015 and April 2021. The concomitant use of PPI or Abs was defined as any PPI or Abs administered within 30 days before ICI initiation and during ICI therapy. The primary outcomes were the effect of PPI and/or Abs use on the objective response rate (ORR) and immune‐related adverse events (irAEs). The secondary outcomes were the effects of PPI and/or Abs use on progression‐free survival (PFS) and overall survival (OS) after ICI therapy analyzed using the inverse probability of treatment weighting‐adjusted Cox regression analysis.Of the 155 patients enrolled in the study, 99 (64%) were PPI users and 56 (36%) Abs users. PPI users were associated with a significantly poorer ORR than non‐PPI users , whereas Abs use was not significantly associated with changes in ORR. The rate of irAEs was not significantly associated with the use of PPIs or Abs. Multivariate inverse probability of treatment weighting‐adjusted Cox regression analysis revealed significantly poorer PFS and OS in PPI users than in non‐PPI users, whereas Abs use was not associated with poorer outcomes.The concomitant use of PPI may adversely affect oncological outcomes in patients with locally advanced or metastatic urothelial carcinoma treated with ICI therapy. Written consent was not obtained in exchange for public disclosure of study information (opt‐out approach).2.2We retrospectively evaluated consecutive 160 patients with locally advanced (cT4 or pN+) or metastatic UC treated with ICI therapy between August 2015 and April 2021 at two academic centers and three general hospitals. We verified our retrospective data before analysis. Five patients who were treated within 3 months or who had no imaging evaluation were excluded. Thus, 155 patients with advanced UC were included. The concomitant use of PPI or Abs was defined as any PPI or Abs administered within 30 days before ICI initiation and during ICI therapy. A single dose of first‐generation cephem antibiotics  were administrated to the majority of patients for peri‐procedural purposes  as a standard of care. The following variables were recorded and evaluated: age, sex, Eastern Cooperative Oncology Group performance status (ECOG‐PS), estimated glomerular filtration rate (eGFR), clinical stage, progression‐free survival (PFS), and overall survival (OS). Tumor stage and grade were stratified based on the eight editions of the TNM classification.2.32, grade >1 hearing loss, grade >1 neuropathy, and/or New York Heart Association (NYHA) Class III heart failure. In addition, we defined the marginal criteria as being ECOG‐PS 1, eGFR 50–60 ml/min/1.73 m2, NYHA Class II heart failure, and age >75–80 years. Patients with two or more marginal factors (such as age ECOG‐PS 1 and eGFR 55 ml/min/1.73 m2) were classified as cisplatin ineligible.Patients received either gemcitabine plus cisplatin; gemcitabine plus carboplatin; or methotrexate, vinblastine, doxorubicin, and cisplatin.2.4We divided the patients into two groups based on the concomitant use of PPI or Abs. The primary outcomes were the effects of PPI and/or Abs use on the objective response rates (ORRs) and immune‐related adverse events (irAEs). The secondary outcomes were the effects of PPI and/or Abs use on PFS and OS after ICI therapy.2.5t test or the Mann–Whitney U test. Fisher's exact test or the χ2 test was used to compare categorical variables. Quantitative variables were expressed as means with standard deviations or medians with interquartile ranges. The rate of OS from ICI therapy until death was estimated using the log‐rank test. The effects of PPI use on PFS and OS after ICI therapy were determined using multivariable Cox regression analyses via the inverse probability of treatment weighting (IPTW) method. The hazard ratio (HR) with a 95% confidence interval (95% CI) was calculated after controlling for potential confounders, including patient age, sex, ICI therapy treatment line, ECOG‐PS at initiation of ICI therapy, tumor type, PPI use, Abs use, and exposure to radiotherapy.We performed statistical analyses using BellCurve for Excel version 3.10 , GraphPad Prism version 7.00 , and R: 4.0.2, A Language and Environment for Statistical Computing . Intergroup differences were examined using Student's 33.1n = 79, 80%) used PPIs before ICI therapy, suggesting a long‐term continuous medication. There were no significant differences in baseline characteristics between the non‐PPI users and PPI users. The proportions of patients with concomitant use of Abs were 38% and 51% in non‐PPI users and PPI users, respectively. Of the PPI users (n = 99), 79 (80%) patients had been using PPI continuously for 3 months before the initiation of ICI therapy.Table 3.2p = 0.005). ORR was not significantly different between the Abs users (21%) and non‐Abs users (33%) . The rate of irAEs was not significantly different between the PPI users and non‐PPI users (p = 0.639)  compared that in PPI users (20%)  Figure .3.33.3.1p = 0.002)     Figure . The OS ) Figure . The PFS) Figure . The OS ) Figure .3.3.2p = 0.004) or patients using either PPI or Abs   or single users   or single users   than in patients using neither  Figure . The rat) Figure . The PFS) Figure . The OS ) Figure .3.3.3p = 0.041, Figure p = 0.036, Figure p = 0.534, Figure p = 0.847, Figure Multivariable Cox regression analyses for PFS and OS was shown in Table 4This study investigated the prognostic impact of the concomitant use of PPI and/or Abs on oncological outcomes in patients with advanced UC treated with ICI. ORR was significantly lower in patients who used PPIs, whereas Abs use did not affect ORR. The background‐adjusted PFS and OS after ICI therapy were significantly shorter in patients who used PPI, which was consistent with the results of a post‐hoc analysis of clinical trials.Lactobacillus and Streptococcus species.Reports concerning the effects of dysbiosis‐inducing drugs are conflicting. Recent studies have suggested that exposure to corticosteroids, antibiotics, and PPI leads to progressively poorer outcomes after ICI therapy.The accumulation of dysbiosis‐inducing drugs was strongly associated with poor PFS and OS after ICI therapy. We found that patients who used both PPI and Abs exhibited significantly poor ORR, PFS, and OS after ICI therapy. A similar observation was reported in a recent study that included 1229 patients with non–small‐cell lung cancer (70%), melanoma (14.7%), renal cell carcinoma (9.2%), and others (6%).The use of corticosteroids is associated with irAEs in patients treated with ICI therapy. A previous study suggested that the use of corticosteroids reduces the efficacy of ICI therapy via reduction of CD8 + T cell proliferation.There is a lack of studies investigating the effects of the concomitant use of PPI or Abs and ICIs on irAEs. As many reports suggested a positive association between the efficacy and immune‐related AEs,Several limitations in this study should be acknowledged. First, because of the retrospective study design, we could not control for selection bias and other unmeasurable confounders. Second, the statistical analysis might be underpowered because of the small sample size. Analyses under a single population are a problem for generalization. Third, lack of data on gut microbiota and corticosteroid use are robust limitations of this study. Forth, we could not address the duration of PPI/Abs treatment and the type of Abs. Nonetheless, this study demonstrates the effect of concomitant PPI and/or Abs use on prognosis in patients with advanced UC treated with ICI therapy. Thus, unnecessary PPI or Abs should be avoided for patients with advanced UC who require ICI therapy. Further prospective studies are required to determine the role of concomitant use of PPI and Abs in the reduced antitumor immune responses via gut dysbiosis.5The concomitant use of PPI may adversely affect oncological outcomes in patients with locally advanced or metastatic UC treated with ICI therapy.This study was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI .The authors have no conflict of interest.Yoshiharu Okuyama is responsible for data collection. Shingo Hatakeyama is responsible for project development, manuscript editing, data analysis, and data collection.Kazuyuki Numakura, Takuma Narita, Toshikazu Tanaka, Yuki Miura,Daichi Sasaki, Daisuke Noro, Noriko Tokui, Teppei Okamoto, Hayato Yamamoto, Shintaro Narita, Takahiro Yoneyama, Yasuhiro Hashimoto are involved in data collection. Tomonori Habuchi and Chikara Ohyama are involved in project development and critical review.Figure S1. Visual abstractA schematic summary of present study was shown.Click here for additional data file.