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+{"text": "The enteric subgroup F adenoviruses type 40 (Ad40) and 41 (Ad41) are the second most important cause of acute infantile gastroenteritis after rotaviruses. Repeated community outbreaks have been associated with antigenic changes among the Ad40 and Ad41 strains due to host immune pressure. Therefore large field epidemiological surveys and studies on the genetic variations in different isolates of Ad40 and Ad41 are important for disease control programs, the design of efficient diagnostic kits and vaccines against subgroup F adenoviruses. A novel method using sodium dodecyl sulphate SDS/EDTA-pretreated chromatography paper strips was evaluated for the collection, storage and shipping of Ad40/41 contaminated stool samples.This study shows that adenoviral DNA can be successfully detected in the filter strips by PCR after four months storage at -20\u00b0C, 4\u00b0C, room temperature (20\u201325\u00b0C) and 37\u00b0C. Furthermore no adenoviral infectivity was observed upon contact with the SDS/EDTA-pretreated strips.Collecting, storing and transporting adenovirus type 40 and 41 positive stool samples on SDS/EDTA-pretreated chromatography filter strips is a convenient, biosafe and cost effective method for studying new genome variants and monitoring spread of enteric adenovirus strains during outbreaks. Enteric adenoviruses (EAds) are considered to be the second most important causative agent of acute infantile gastroenteritis after rotaviruses. The fastidious subgroup F adenoviruses type 40 (Ad40) and 41 (Ad41) account for the majority of cases of severe acute diarrhea in children less than 2 years of age -5. The cPrevious studies have demonstrated the application of different filter papers for the collection and storage of blood , saliva This study describes the use of SDS/EDTA-pretreated filter paper strips in collection, transportation and storage of adenovirus type 40/41 positive stool samples for subsequent genetic analysis.In the current study we describe the use of chromatography paper strips for the collection, transportation, and storage of EAds type 40/41 positive stool samples. In order to inactivate the adenoviruses and other microorganisms upon contact with the strips, the latter were pretreated with SDS, a surfactant with protein denaturising ability. This can allow safe transportation of the strips without extensive biohazard precautions. To protect the viral DNA from degradation by deoxyribonucleases (DNases) the chromatography strips were also preincubated with EDTA, and Tris-HCl. EDTA chelates magnesium ions, a necessary co-factor for most nucleases and the weak organic base Tris-HCl ensures the proper action of the chelating agent in binding the divalent cations.6 adenoviral particles per ml was serially diluted 1:8 (dilution a), 1:80 (dilution b), 1:800 (dilution c), 1:8000 (dilution d) and 1:80,000 (dilution e). The SDS/EDTA-pretreated filter paper strips were infected with each stool dilution and stored at -20\u00b0C, 4\u00b0C, room temperature (20 to 25\u00b0C), and 37\u00b0C. The presence of adenoviral DNA on the chromatography filter strips was detected by PCR amplification of a 301 bp fragment of the adenoviral hexone gene after storage for 7 days, 14 days, 56 days and 120 days  after three passages of the infected cell line. The eluate of the untreated chromatography strips loaded with adenovirus type 1 caused cytopathic effect in the HeLa cell line. It can be concluded that the SDS/EDTA-pretreated strips can be used for the collection and shipping of adenovirus positive stool samples from remote areas to reference laboratories in a biosafe way.To be sure that the EAd40/41 contaminated filter strips are not infectious, we carried out a biosafety test to find out if any adenovirus could survive onto the SDS/EDTA-pretreated strips. Previously we showed that pathogenic bacteria such as A strips . The bioWe conclude that the use SDS/EDTA-pretreated filter strips for retrieval and subsequent analysis of adenoviral DNA from EAds type 40/41 positive stool specimens is a feasible method for sample collection. The described filter paper strips facilitate collection, transport and storage of adenoviral positive stools because they are biosafe, cost effective and require minimal storage space. This study shows that adenoviral DNA can remain stable for at least 4 months at 37\u00b0C temperature conditions making this method especially attractive for field research or population screening in tropical countries where freezers are not available.2) Whatman grade 17 chr pure cellulose chromatography paper with thickness of 0.92 mm and a flow rate of 190 mm/30 min  was used. Strips of 80 mm \u00d7 4 mm were cut from the chromatography paper and soaked for two minutes in a solution of 2% (w/v) sodium dodecyl sulphate (SDS), 10 mM EDTA and 60 mM TrisHCl. The chromatography paper strips were left to dry overnight at room temperature. Disposable gloves were used during the preparation of the filter paper strips.Highly absorbent  was used for this study. The undiluted feces sample contained approximately 2.6 \u00d7 106 particles per ml of stool, as calculated from a standard curve supplied with the antigen enzyme immunoassay kit. The stool sample was diluted in 1 ml (dilution 1:8) DNase/RNase free water (Sigma) and the following dilutions were used: 1:8 (dilution a), 1:80 (dilution b), 1:800 (dilution c), and 1:8000 (dilution d), and 1:80000 (dilution e). The pretreated chromatography strips were infected with 100 \u03bcl of the different dilutions of stool sample and were left to air dry overnight at room temperature. After complete drying, the infected strips were stored under four different temperature conditions: -20\u00b0C, 4\u00b0C, room temperature (20 to 25\u00b0C) and 37\u00b0C.A diarrhea stool sample that was positive for adenovirus type 40/41 hexon antigen by the Premier Adenoclone2) was used for the DNA extraction performed at the following storage time intervals: 7, 14, 56 and 120 days. The filter paper was inserted into an Eppendorf tube with 500 \u03bcl of Dnase/Rnase free water (Sigma) and thoroughly squeezed out. An aliquot of 200 \u03bcl of the squeezed eluate was used for DNA extraction using the QIAamp DNA Blood Mini Kit  according to the manufacturer's instructions. A set of degenerate consensus primers (forward primer 5'-GCCSCARTGGKCWTACATGCACATC-3' and (reverse primer 5'-CAGCACSCCICGRATGTCAAA-3') were used to amplify a 301 bp fragment of the adenoviral hexone gene [2, and 1 unit Taq polymerase . The PCR was conducted in a Geneamp PCR System 9600 thermal cycler (Applied Biosystems). The thermocycling conditions consisted of denaturation at 94\u00b0C for 3 min, followed by 35 cycles of 30 s at 94\u00b0C, 30 s at 55\u00b0C and 1 min at 72\u00b0C and 5 min of final elongation at 72\u00b0C. PCR products were visualized using polyacrylamide gel electrophoresis and ethidium bromide staining.Half of the strip (160 mmone gene . The PCRin vivo. Therefore adenovirus type 1 was used for the biosafety experiments. The SDS/EDTA-pretreated filter stips were first infected with 100 \u03bcl of the HeLa cell cultured adenovirus type 1 (106 TCID 50/ml) and allowed to dry at room temperature for 60 min. The strips were then placed into an eppendorf tube containing 500 \u03bcl Dulbecco's Modified Eadle Medium (DMEM)  supplemented with 200 mM L-glutamine . The strips were thoroughly squeezed in the medium and the eluate was dialyzed using 3,500-Da Slide-A-Lyzer dialysis cassettes  to remove the cytotoxic SDS. The dialyzed eluate was inoculated on a confluent monolayer of HeLa cells and was incubated at 37\u00b0C in a humified incubator with a 5% CO2 environment. Untreated strips infected with adenovirus type 1 and noninfected SDS/EDTA-pretreated strips were used as positive and negative controls respectively. The presence of cytopathic effect indicated the presence of live replicating virus on the strip. Cytopathic effects were monitored up to the third passage of the tissue culture supernatant.A biosafety experiment was performed to check if adenoviral particles are still infectious after contact with the SDS/EDTS-pretreated chromatography paper strips. Since Ad40 and Ad41 grow poorly in cell culture it is difficult to detect these viruses Eads \u2013 enteric adenovirusesEDTA \u2013 ethylenediamine tetra-acetic acidSDS \u2013 sodium dodecyl sulphateThe author(s) declare that they have no competing interests.KZ conducted the study, carried out the experiments and wrote the manuscript. PM carried out the biosafety experiments. MR developed the filter paper strip sampling method. MVR supervised the study and revised the manuscript. All authors read and approved the manuscript."}
+{"text": "Eligible patients had normal organ functions and Eastern Cooperative Oncology Group performance status \u2a7d2. QOL was measured with European Organisation for Research and Treatment of Cancer QLQ-C30 and LC13 questionnaires. There were no significant differences in median progression-free survival , median overall survival , or tumour response rates . Toxicity was mainly haematologic. In the EG arm granulocytopenia occurred more frequently, leading to more febrile neutropenia. Also, elevation of serum transaminases, mucositis, fever, and decline in LVEF were more common in the EG arm. In the CG arm, more patients experienced elevated serum creatinine levels, sensory neuropathy, nausea, and vomiting. Global QOL was not different in both arms. Progression-free survival, overall survival, response rate, and QOL were not different between both arms; however, overall toxicity was more severe in the EG arm.The purpose of our study was to compare progression-free survival and quality of life (QOL) after cisplatin\u2013gemcitabine (CG) or epirubicin\u2013gemcitabine (EG) in chemotherapy-naive patients with unresectable non-small-cell lung cancer. Patients ( Platinum-based chemotherapy is the standard treatment for patients with advanced non-small-cell lung cancer (NSCLC) who have a good performance status . A meta-In spite of the fact that cisplatin-containing regimens are currently considered the treatment of choice for advanced NSCLC, cisplatin has several disadvantages such as nephro-, neuro-, and ototoxicity. Therefore, nonplatinum-containing regimens have been studied to find less toxic therapies with similar efficacy. Phase III studies have reported similar response rates and overall survival in cisplatin and noncisplatin-based regimens. However, in the majority of these trials the nonplatinum regimens had a more favourable toxicity profile  was observed in 7% of patients. The tumour response rate was 49% and the median survival time was 42 weeks  or epirubicin. Epirubicin in combination with gemcitabine (EG) was administered as an outpatient regimen, the cisplatin\u2013gemcitabine (CG) combination was given as a short inpatient regimen as is often done in European countries. The American Society of Clinical Oncology guidelines (published in 1997) recommended two to eight cycles of platinum-based treatment in advanced NSCLC. We chose a maximum of five cycles. Progression-free survival was the primary end point of the study. Overall survival, response rate, toxicity, and QOL were secondary end points.9\u2009l\u22121, neutrophils \u2a7e1.5 \u00d7 109\u2009l\u22121, platelets \u2a7e100 \u00d7 109\u2009l\u22121, haemoglobin \u2a7e6.2\u2009mmol\u2009l\u22121), normal renal (serum creatinine \u2a7d120\u2009\u03bcmol\u2009l\u22121 or creatinine clearance \u2a7e60\u2009ml\u2009min\u22121) and liver function , and serum aspartate aminotransferase (ASAT) less than three times the upper limit of normal) were required. Patients were excluded if they had active infections, second primary malignancies , uncorrected hypercalcaemia, or an LVEF \u2a7d45% measured by multiple gated acquisition (MUGA) scan. Local medical ethics committees of all hospitals approved the protocol. All patients gave informed consent before study entry.Patients were included if they had histological or cytological diagnosis of unresectable stage III or stage IV NSCLC and at least one measurable or evaluable tumour lesion on physical examination, chest X-ray, or chest CT. No prior chemotherapy was allowed and radiotherapy was permitted as long as no more than 25% of the bone marrow was irradiated. Radiotherapy should have been completed at least 4 weeks before inclusion, and patients should have recovered from any toxic side effect. The irradiated area was excluded from tumour measurements. All patients had to have a performance status \u2a7d2 according to the Eastern Cooperative Oncology Group (ECOG) scale and a life expectancy of at least 12 weeks. An adequate bone marrow reserve (leucocytes \u2a7e3.0 \u00d7 10\u22122) was administered in 250\u2009ml 0.9% NaCl by a 30\u2009min infusion on days 1 (before cisplatin or epirubicin) and 8. Cisplatin 80\u2009mg\u2009m\u22122 (in 1000\u2009ml 0.9% NaCl) was administered intravenously during 3\u2009h after prehydration with 0.9% NaCl on day 2 of each 21-day treatment cycle. Epirubicin 100\u2009mg\u2009m\u22122 (in 50\u2009ml 0.9% NaCl) was administered as an intravenous bolus injection within 5\u2009min on day 1 of each 21-day cycle. For prehydration, patients in the CG arm were admitted to hospital for 2 days. Epirubicin\u2013gemcitabine was administered as an outpatient regimen. Anti-emetics consisted of ondansetron 8\u2009mg and dexamethasone 8\u2009mg twice a day on days 1, 2, and 8. The treatment consisted of a maximum of five cycles and was stopped earlier in case of tumour progression, intolerable toxicity, or patient's wish. No treatment with G-CSF was foreseen. In case of Hb<5.0\u2009mmol\u2009l\u22121 or symptomatic anaemia in combination with Hb<6.0\u2009mmol\u2009l, patients were treated with red blood cell transfusion. Platelet transfusion was given in the event of platelets <10 \u00d7 109\u2009l\u22121 or persistent bleeding in combination with platelets <20 \u00d7 109\u2009l\u22121.Eligible patients were randomised by telephone to receive either cisplatin or epirubicin both with gemcitabine. Gemcitabine (1125\u2009mg\u2009m9\u2009l\u22121 and/or platelets <100 \u00d7 109\u2009l\u22121) or in case of persistent common toxicity criteria (CTC) grade 2 or more nonhaematologic toxicity . The dose of cisplatin or epirubicin for subsequent cycles was reduced to 75% in case of a nadir of neutrophils below 0.5 \u00d7 109\u2009l\u22121 exceeding 7 days, a nadir of platelets below 25 \u00d7 109\u2009l\u22121, thrombocytopenia associated with bleeding, febrile neutropenia, or CTC grade 3 nonhaematologic toxicity (except nausea and vomiting). The cisplatin dose was reduced by 50% for a calculated creatinine clearance between 50 and 70\u2009ml\u2009min\u22121, and in case of a creatinine clearance less than 50\u2009ml\u2009min\u22121 cisplatin was not administered  by the planned dose (mg\u2009m\u22122\u2009week\u22121) for the number of cycles each patient received.Drug administration was postponed to a maximum of 2 weeks if there was no haematologic recovery on day 22  criteria . After tAfter discontinuation of treatment, patients were evaluated every 6 weeks with physical examination, laboratory tests, chest X-ray or CT-scan of the chest, and additional imaging tests on clinical indication to assess tumour progression.At the start of treatment, after three cycles of chemotherapy, and 6 weeks after the end of treatment, QOL was measured with the European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30, supplemented by a 13-item lung-cancer-specific questionnaire module, the EORTC QLQ-LC13. This validated questionnaire was filled in at home and is composed of a core QOL questionnaire covering general aspects of health-related QOL and disease- and treatment-specific symptoms  or EG (n=121). Patient characteristics were not significantly different between both treatment arms . In both arms, two patients died due to septicaemia . In the CG arm, 56% of patients had one or more red blood cell transfusions during treatment, compared to 51% of patients in the EG arm (P=0.433). At the time of study, erythropoietin was not routinely administered. The number of red blood cell transfusions per patient was also not different between both treatment arms.Haematologic toxicity is shown in P=0.001). Patients in the EG arm also had more often short-lasting fever (in the absence of neutropenia). Nausea, vomiting, and fatigue frequently occurred in both arms, but more often in the CG arm. Significantly more patients in the EG arm had a grade 1 (16% in the CG arm vs 42% in the EG arm) and grade 2 (7% in the CG arm vs 13% in the EG arm) decline in LVEF . Clinically evident cardiac failure was not observed during follow-up. In this trial, 28 elderly patients (\u2a7e70 years) were included. In these patients, grade 3 or 4 thrombocytopenia occurred more frequently compared to younger patients, in 78 vs 46% of patients, respectively (P=0.029). No differences in other toxicities were found when comparing them to younger patients.The worst nonhaematologic toxicity per patient is listed in The median (range) number of cycles per patient was 4 (0\u20135) for both arms. The maximum of five cycles was completed in 58 (49%) patients in the CG arm and in 49 (41%) patients in the EG arm .P=0.121) (n=1), death from other causes (n=4), discontinuation of treatment at patient's request after one cycle (n=4), toxicity (n=2), and for other reasons (n=2). Three patients did not receive chemotherapy at all and were, therefore, not evaluable for response. For the analysis, these patients were considered as nonresponders. The response rates for patients with stage III vs stage IV disease were 55%  and 40%  in the CG arm, and 44%  and 30%  in the EG arm, respectively. All these differences were not statistically significant. The tumour response rate in patients with a performance status of 0 or 1 was 43% compared to 36% in patients with a performance status of 2 (P=0.443). Elderly patients (\u2a7e70 years) had a similar tumour response rate compared to younger patients. However, patients with liver metastases (n=20) had a lower response rate (15%) compared to patients without these metastases (44%) (P=0.013). A logistic regression model with potential prognostic factors  showed that the presence of liver metastases was a significant independent negative prognostic factor for tumour response.The overall response rate was 46%  for the CG arm and 36%  for the EG arm (n=77) was 28 weeks .A total of 38 patients in the CG arm (32%) and 39 patients in the EG arm (32%) received second-line chemotherapy, consisting of docetaxel alone or docetaxel combined with carboplatin or irinotecan. A partial response to second-line therapy was observed in 20% of patients, without a difference between CG and EG. In all, 24% of responders and 14% of nonresponders on first-line therapy achieved a partial response to second-line therapy; this difference was also not significant. Median survival after start of second-line chemotherapy  weeks for the CG and EG arm, respectively (P=0.247). Progression-free survival (\u00b1s.e.) at 6 months follow-up was 49% (\u00b15%) vs 40% (\u00b15%) for the CG and EG arm, respectively (P=0.134)  weeks in the EG arm, which was not different between both arms (P=0.143). The 1-year survival rate (\u00b1s.e.) was 45% (\u00b15%) vs 35% (\u00b15%) in the CG and EG arm, respectively (P=0.123)  A Cox regression model was used to find independent prognostic factors for progression-free and overall survivals. The factors disease stage, histology, sex, age, performance status, weight loss, and liver metastases were used in this model. Performance status \u2a7d1, absence of liver metastases, and weight loss <5% were associated with prolonged progression-free and improved overall survivals. Stage III disease was, compared to stage IV disease, associated with longer progression-free survival.The questionnaires filled in at the patient's home at the start of treatment, after three cycles of chemotherapy, and 6 weeks after the end of treatment were returned to the datacenter by 70, 47, and 45% of patients, respectively. Between both treatment arms, no significant differences were found in global health status and functional scales , at all three moments of measurement  included in this trial toxicity (except thrombocytopenia), the tumour response rate, progression-free and overall survivals were not different compared to younger patients. However, in contrast to the fact that 35\u201343% of all lung cancers arise beyond the age of 70 years . HoweverIn conclusion, we found no differences in efficacy and global QOL between cisplatin and epirubicin, both in combination with gemcitabine as first-line treatment for advanced NSCLC. However, the observed differences in toxicity profile are in favour of cisplatin. Therefore, a platinum-based combination regimen remains the recommended treatment for advanced NSCLC."}
+{"text": "Addition of irinotecan to docetaxel does not improve response rate, and increases gastrointestinal toxicity.Response rate and toxicity of second-line therapy with docetaxel (75\u2009mg\u2009m Prior radiotherapy was allowed as long as the irradiated area did not contain the sole measurable or evaluable lesion. Exclusion criteria were active infection, second primary malignancies , symptomatic brain metastases, inflammatory bowel diseases, symptomatic peripheral neuropathy \u2a7egrade 2 according to the Common Toxicity Criteria (CTC) of the National Cancer Institute (version 2.0), serious cardiac diseases, contraindications for use of corticosteroids, pregnancy, breast-feeding, or reproductive potential without implementing adequate contraceptive measures. Local medical ethics committees of all hospitals approved the protocol. All patients gave informed consent.Inclusion criteria for enrolment in the trial were age \u2a7e18 years, stage IIIb\u2013IV NSCLC, failure or relapse after first-line chemotherapy, at least one measurable or evaluable tumour lesion, performance status \u2a7d2 according to the Eastern Cooperative Oncology Group scale, life expectancy of \u2a7e3 months, adequate bone marrow reserve (neutrophils \u2a7e1.5 \u00d7 10\u22122 on day 1 (D arm) or docetaxel 60\u2009mg\u2009m\u22122 and irinotecan 200\u2009mg\u2009m\u22122 both on day 1 followed by lenogastrim 150\u2009\u03bcg\u2009m\u22122\u2009day\u22121 on days 2\u201312 (DI arm). Docetaxel (in 250\u2009ml 0.9% NaCl) was administered as a 1-h intravenous infusion in both treatment arms. Irinotecan  was administered after docetaxel as a 90-min infusion. Lenogastrim ampoules contained 263\u2009\u03bcg recombinant human granulocyte colony-stimulating factor dissolved in 1\u2009ml solvent for subcutaneous injection. Treatment was repeated every 3 weeks for a maximum of five cycles and halted in case of tumour progression, intolerable toxicity or patient's wish. To prevent hypersensitivity reactions caused by docetaxel dexamethason 8\u2009mg was given twice a day during 3 subsequent days starting the day before infusion. Antiemetics consisted of ondansetron 8\u2009mg twice a day on days 1\u20133. In case of diarrhoea, patients were treated with loperamide . When the diarrhoea persisted for more than 48\u2009h or occurred in combination with neutropenia, fever or dehydration, patients were hospitalised and treated with antibiotics.Patients were randomised by block randomisation to receive either docetaxel 75\u2009mg\u2009m9\u2009l\u22121 and/or platelets <100 \u00d7 109\u2009l\u22121). In case of nadir values of neutrophils <0.5 \u00d7 109\u2009l\u22121 or platelets <50 \u00d7 109\u2009l\u22121 exceeding 7 days, febrile neutropenia, or thrombocytopenia associated with bleeding, the dose of docetaxel for subsequent cycles was reduced to 55\u2009mg\u2009m\u22122 in the D arm and to 45\u2009mg\u2009m\u22122 in the DI arm. The dose of irinotecan was reduced to 150\u2009mg\u2009m\u22122 in these cases. In the event of grade 3\u20134 nonhaematologic toxicity (except nausea and vomiting) or grade 2 neuropathy, the doses of docetaxel and irinotecan were reduced with 25% for subsequent cycles. Treatment was stopped if the same severity of toxicity occurred at the reduced dose level treatment or in case of grade 3\u20134 neuropathy. In case of grade 3\u20134 diarrhoea lasting more than 2 weeks despite appropriate therapy, no further irinotecan was administered.Drug administration was postponed  if there was no haematologic recovery on day 22  or DI (n=52). Patient characteristics were not significantly different between both treatment arms . In this arm, six patients were hospitalised for serious diarrhoea compared to one patient in the D arm (P=0.05). Nail changes and arthralgia were only observed in the D arm, where the higher docetaxel dose was administered. Additionally, significantly more patients in the D arm had myalgia (P<0.05).The worst nonhaematologic toxicity per patient is listed in P>0.05). The reasons for treatment discontinuation for both arms were not different. Main reasons for treatment discontinuation were progressive disease and toxicity. Seven patients died due to disease progression while on protocol therapy. Three patients died due to toxicity of treatment, due to paralytic ileus after one cycle , bowel perforation after two cycles , and myocardial infarction after one cycle . Doses of docetaxel (D arm), docetaxel (DI arm), and irinotecan had to be reduced in 4, 2, and 7% of the cycles, respectively. In both arms, drug administration was postponed up to 2 weeks in 2% of the cycles.A total of 206 and 162 cycles were administered in the D and DI arm, respectively. The median (range) number of cycles per patient was 4 (1\u20135) in the D arm, and 3 (1\u20135) in the DI arm. The maximum of five cycles was completed in 25 (45%) patients in the D arm and in 17 (33%) patients in the DI arm  for the D arm and 10%  for the DI arm , discontinuation of treatment at patients request , and discontinuation for toxicity . These patients were considered nonresponders.At the end of the first stage, one response was observed in the D arm e DI arm . Accordivs 15  weeks for the D and DI arm, respectively (P=0.42)  weeks in the DI arm, which was not different between both arms (P=0.69). The 1-year survival rate (\u00b1s.e.) was 26% (\u00b16%) vs 30% (\u00b17%) in the D and DI arm, respectively (P=0.49)  (P=0.42) . The med(P=0.49) .According to the statistical design of this trial, the docetaxel arm, with a response rate of 16%, ranked superior compared to the docetaxel\u2013irinotecan arm, with a response rate of 10%. Using this design, smaller number of patients are required compared to the usual randomised trial design in which sample size calculations are based on statistical significance. The conclusion of this trial \u2013 that addition of irinotecan to docetaxel as second-line chemotherapy in NSCLC does not improve response rate \u2013 is solely based on the ranking of response rate. Although our trial was not powered to detect differences in survival, the efficacy of docetaxel as single agent and docetaxel combined with irinotecan seemed not different in terms of progression-free survival and overall survival.\u03b2-glucuronidase activity from intestinal microflora (Both arms showed a different toxicity profile. Leukopenia, nail changes, myalgia, and arthralgia occurred more frequently in the D arm, while thrombocytopenia and diarrhoea were more common in the DI arm. Toxicities especially occurring in the D arm might probably be related to the higher dose level of docetaxel used in this arm. Nevertheless, toxicity in the docetaxel arm was acceptable. As in other trials, diarrhoea frequently occurred after treatment with docetaxel and irinotecan (The response rate for docetaxel monotherapy found in this trial is comparable to results of other phase II trials, which found a response rate between 16\u201322% (Two other single agents were recently investigated in a second-line setting in NSCLC patients. Gefitinib, an orally active EGFR tyrosine kinase inhibitor, was studied in a randomised phase II trial (Whether combinations are superior to single agents in second-line setting is presently unclear. Although this trial demonstrated no clear benefit using docetaxel with irinotecan, other regimens using the combination of these drugs with filgrastrim support or with celecoxib are currently under investigation (In conclusion, addition of irinotecan to docetaxel as second-line chemotherapy does not improve response rate, and increases gastrointestinal toxicity in patients with stage IIIb or IV NSCLC."}
+{"text": "The link between natural disasters and subsequent fungal infections in disaster-affected persons has been increasingly recognized. Fungal respiratory conditions associated with disasters include coccidioidomycosis, and fungi are among several organisms that can cause near-drowning pneumonia. Wound contamination with organic matter can lead to post-disaster skin and soft tissue fungal infections, notably mucormycosis. The role of climate change in the environmental growth, distribution, and dispersal mechanisms of pathogenic fungi is not fully understood; however, ongoing climate change could lead to increased disaster-associated fungal infections. Fungal infections are an often-overlooked clinical and public health issue, and increased awareness by health care providers, public health professionals, and community members regarding disaster-associated fungal infections is needed. The potential for adverse health events after natural disasters is well recognized and comprises various challenges for public health  are well defined, but others (such as Aspergillus and other molds) are thought to be ubiquitous. The abundance and distribution  of environmental fungi probably depend on climatic or environmental factors such as ambient temperature and moisture , and in 4 (10.5%) of 38 wound isolates from 23 ambulatory patients  is hypothesized to be the most common fungal pathogen associated with near-drowning, although this finding has not been studied specifically in the context of disasters    in 1 tourist , which resolved without antifungal treatment (Syncephalastrum was detected in various clinical specimens from 8 persons whose self-reported exposures to mold ranged from none to heavy, but none had evidence of invasive infection (Few data clearly demonstrate that indoor mold exposures increase the risk for invasive infection in post-disaster settings. Flooding lasted for weeks after Hurricanes Katrina and Rita made landfall on the US Gulf Coast in August and September 2005, respectively, leading to visible mold growth in 46% of 112 inspected homes (Aspergillus fumigatus, A. flavus, and basidiomycetous fungi; culture plates exposed inside the refuge showed a similar fungal profile, suggesting that the indoor environment may have played a role in the patients\u2019 infections (After the 2011 Great East Japan Earthquake and subsequent tsunami, a medical relief team observed unexplained chronic cough among a group of previously healthy persons living in a temporary refuge (Cryptococcus gattii, to expand into areas that are currently more temperate (Climate change could be affecting the ecology of pathogenic fungi in ways that are not yet fully understood; even minor or gradual changes in temperature, moisture, and wind patterns might affect fungal growth, distribution, and dispersal (Huppert and Sparks suggest that global climate change is contributing to greater frequency and severity of extreme weather events and that current patterns of population growth, urbanization, and human activity create conditions that render many communities increasingly vulnerable to these hazards (Disasters are complex events that can result in a wide range of health effects, although infectious disease outbreaks as an immediate consequence of disasters are uncommon. Health care providers should be aware of the possibility for cases or clusters of community-acquired or health care\u2013associated fungal infections among disaster survivors because these infections often appear clinically similar to bacterial infections and can be associated with serious illness and death. These infections can occur in persons who do not have the typical immunocompromising risk factors for fungal infection but who have experienced near-drowning, trauma, or other unusual exposure to the environment, such as a dust storm. A fungal infection should be considered early if a patient has a persistent or progressive infection that is not responding to initial antibacterial treatment, particularly because rapid diagnosis and administration of appropriate antifungal therapy can improve patient outcomes. Prompt restoration of disaster-affected aspects of the local health care infrastructure may help facilitate earlier diagnosis and treatment and possibly reduce the risk for infection associated with the use of contaminated medical equipment or substandard care. Strategies to reduce disaster-associated fungal infections should be considered within the broader context of comprehensive and sustainable risk reduction methods to prevent disaster-related injury and illness."}
+{"text": "Neurotrophin receptors were initially identified in neural cells. They were recently detected in some cancers in association with invasiveness, but the function of these tyrosine kinase receptors was not previously investigated in colorectal cancer (CRC) cells.NTR) receptors at the plasma membrane, whereas TrkA and TrkC, two other high affinity receptors for NGF and NT-3, respectively, were undetectable. We demonstrate that BDNF induced cell proliferation and had an anti-apoptotic effect mediated through TrkB, as assessed by K252a, a Trk pharmacologic inhibitor. It suppressed both cell proliferation and survival of CRC cells that do not express TrkA nor TrkC. In parallel to the increase of BDNF secretion, sortilin, a protein acting as a neurotrophin transporter as well as a co-receptor for p75NTR, was increased in the cytoplasm of primary and metastatic CRC cells, which suggests that sortilin could regulate neurotrophin transport in these cells. However, pro-BDNF, also detected in CRC cells, was co-expressed with p75NTR at the cell membrane and co-localized with sortilin. In contrast to BDNF, exogenous pro-BDNF induced CRC apoptosis, which suggests that a counterbalance mechanism is involved in the control of CRC cell survival, through sortilin as the co-receptor for p75NTR, the high affinity receptor for pro-neurotrophins. Likewise, we show that BDNF and TrkB transcripts (and not p75NTR) are overexpressed in the patients' tumors by comparison with their adjacent normal tissues, notably in advanced stages of CRC.We report herein that human CRC cell lines synthesize the neural growth factor Brain-derived neurotrophic factor (BDNF) under stress conditions (serum starvation). In parallel, CRC cells expressed high- (TrkB) and low-affinity (p75Taken together, these results highlight that BDNF and TrkB are essential for CRC cell growth and survival in vitro and in tumors. This autocrine loop could be of major importance to define new targeted therapies. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is known to play a critical role in the modulation of cell survival, differentiation and apoptosis in the nervous system NTR), as well as a death domain receptor belonging to the tumor necrosis factor (TNF) receptor family, a common receptor for all neurotrophins nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). Neurotrophins are synthesized as precursors (proneurotrophins) that are proteolytically cleaved to mature neurotrophins. Pro-BDNF cleavage can occur either intracellularly by the action of furin or proconvertase, or extracellularly by the action of plasmin, matrix metalloproteinase 7 (MMP-7) or MMP-9 NTR, whereas mature BDNF has greater affinities for TrkB.BDNF signals through two types of cell surface receptors: the high-affinity tropomyosin-related kinase (Trk) receptor B (TrkB), a tyrosine kinase receptor and the low affinity receptor . Written informed consent was obtained by all subjects for this study. Tumor tissues were obtained from 20 patients, who underwent surgical removal of CRC in Limoges University Hospital between January 2006 and February 2007. Four adenocarcinoma patients per stage  was used to isolate total RNA from the cell lines as described in the manufacturer's instructions. The amount of RNA extracted was quantified by measuring the absorbance at 260 nm using the Nanodrop spectrophotometer ND-1000 (Labtech). The purity of the RNA was checked by the ratio DO260/DO280 nm between 1.83 and 2.00. The absence of RNA degradation was confirmed by electrophoresis on a 1.5% agarose gel containing ethidium bromide. Extraction of RNA from patients' tissues was performed as described http://www.wi.mit.edu). Designed primer pairs are listed, along with their expected product size and annealing temperatures in Primers were designed using Primer 3 .After extraction of PCR products with Rapid PCR Purification Systems (MARLIGEN bioscience), according to the manufacturer's instructions, PCR products were directly sequenced using the BigDye\u00ae Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) in a GeneAmp\u00ae PCRSystem 2700 thermocycler (Applied Biosystems). The fragments were purified by isopropanol precipitation. The sequencing gel was run on an automated laser fluorescent DNA sequencer ABI Prism\u00ae 3130\u00d7l Genetic Analyser (Applied Biosystems) and homologies were checked, using Finch TV, after blasting with the BDNF, TrkB145, TrkB95, p75NTR (1 \u00b5g/ml), goat anti-sortilin (1 \u00b5g/ml) and rabbit anti-phospho-Akt  Abs from Santa Cruz Biotechnology, rabbit anti-TrkA (1\u22361000), rabbit anti-TrkC (1\u22361000) and mouse anti-Akt (1\u22362000) Abs from Cell Signaling Technology. Subconfluent cell lines cultures were lysed (5 minutes on ice) in the culture flasks by 1\u00d7 Cell lysis buffer  containing 1 mM PMSF (Sigma-Aldrich). The suspension was sonicated for 1 min (a pulsation of 40 Hz every 20 sec) to degrade chromosomal DNA and centrifuged at 14,000 g for 10 minutes. Supernatants were collected and protein concentration was determined using Bradford protein concentration assay (Sigma). SDS-PAGE was performed and the proteins were electro-blotted onto PVDF membranes (BioTrace\u2122). After one hour of incubation at room temperature in blocking solution (5% non-fat dry milk in PBS), the membranes were exposed to the specific primary Abs in blocking solution overnight at 4\u00b0C. Then, membranes were washed thrice for 5 min with TBS/0.1% Tween-20 and the immunoreactions were detected by horseradish peroxidase-conjugated secondary Ab to mouse, rabbit, or goat Ig (Dakocytomation) diluted at 1\u22362000 in blocking solution for 1 h at room temperature. After washing, visualization of immunocomplexes was accomplished using the Immobilon Western Chemiluminescent HRP Substrate (Millipore). Protein-loading control was performed with anti-Actin Ab . Western blots were scanned using a bio-imaging system . Densitometric analyses were performed using an IMAGEJ software program . Protein expression was determined in relative units in reference to actin expression.Mouse anti-BDNF (1 \u00b5g/ml) and mouse anti-TrkB (1 \u00b5g/ml) antibodies (Abs) were purchased from R&D Systems, rabbit anti-P75NTR  and goat anti-sortilin Ab . Cells were washed 3 times in PBS, and incubated for 2 h at room temperature with Alexa Fluor-conjugated secondary Abs (Invitrogen) diluted 1\u22365000 in PBS. After 3 washes in PBS, nuclei were stained for 5 min with DAPI. After intensive washes, coverslips were inverted on slides and mounted with Dako Fluorescent Mounting Medium (Dakocytomation). Negative controls were cells incubated with irrelevant normal rabbit, mouse, or goat IgG (Sigma). Pictures were taken using a confocal microscope ; surface plots of fluorescence data were generated with IMAGEJ software program.Cells grown on 12-mm coverslips were fixed with 4% PFA at room temperature for 30 min, and permeabilized or not with 0.1% Triton X-100. Nonspecific binding was blocked by 30 min incubation with PBS-2% BSA at room temperature. Coverslips were then incubated overnight at 4\u00b0C in blocking solution with the primary Ab. The following Abs were used: rabbit anti-BDNF Ab , rabbit anti-pro-BDNF Ab , mouse anti-TrkB Ab , rabbit anti-p75After a 24-h, 48-h and 72-h culture, the samples of supernatant from cell lines were collected and stored at \u221280\u00b0C until the day of assay. Immunoassays Systems (Promega), specific for BDNF were performed according to the manufacturer's instructions. Results were expressed as mean \u00b1 SEM (pg/mL). At least three independent experiments were performed for each experimental condition, each with measurements in triplicate.5 per well) were incubated overnight on twelve-well plates before treatment, then cultured for 24-h in serum-free medium with or without exogenous BDNF, K252a or both BDNF and K252a added simultaneously. Proliferation values were measured using a BD LSRFortessa flow cytometry (BD Biosciences). Experiments were performed in triplicate, and three sets of 50,000 cells were collected for every condition. The data were acquired and analyzed by the BD FACSDiva 6.0 software (BD Biosciences). Each experiment was repeated at least thrice.Cell proliferation was measured using the Click-it EdU Alexa Fluor 488 Flow cytometry assay (Invitrogen), according to the manufacturer's instructions. Briefly, cells  according to the manufacturer's instructions. Absorbance values were measured at 405\u2013490 nm dual wavelengths. The absorbance obtained in controls was normalized to a value of 1, as previously described P values<0.05 were considered significant. Mean and SEM values were obtained from at least 3 independent experiments.Statistical significance was determined by a one-way analysis of variances (ANOVA) with statview 5.0 software (Abacus Concepts). NTR expressions were detected in CRC cell lines under basal (10% FCS) culture conditions, both at mRNA (NTR transcripts were predominant in a primary (SW480) and a metastasis (SW620) line  line  (two cel20) line  except frivation . TrkB annscripts  as well nscripts  were notSince CRC expressed TrkB receptors, we searched for an endogenous production of BDNF, a TrkB ligand. BDNF mRNA was detected in all studied cell lines under basal (10% FCS) conditions, predominantly in the two primary CRC lines (WiDr and SW480). Interestingly, following serum starvation for 24 to 72 h, BDNF mRNA levels , as wellThe finding that endogenous BDNF is secreted under serum starvation conditions led us to search for the expression of its high affinity receptor. In basal (FCS-containing) cultures , the higTo determine the function of this ligand-receptor system in the proliferation and survival of WiDr, SW480, SW620 and COLO 205 CRC cells, proliferation and apoptosis assays were performed with exogenous BDNF either in FCS-free or in 10% FCS-containing cultures. After a 24-h culture in serum-free medium, exogenous BDNF significantly increased the proliferation of all studied cell lines, in contrast to the absence of effect in the presence of 10% FCS , 3. SincTo define the signal transduction pathway induced by BDNF/TrkB activation, we searched for Akt phosphorylation in two CRC cell lines following BDNF treatment. Indeed, western blotting revealed that the exposure of WiDr or SW480 to BDNF after a 16-hour serum deprivation, induced Akt phosphorylation (Ser 473) after 5 minutes, reached maximum at 30 minutes (7 to 8-fold increase) and yet detected after 24 hours (3 to 4-fold increase) .We therefore determined apoptotic levels of the four cell lines in the presence of a neutralizing anti-BDNF mAb Altogether, these data suggest that endogenous BDNF is implicated in CRC cell survival in serum-free cultures via TrkB through an autocrine loop. This led to study the role of sortilin in BDNF traffic.The primers used in the study of sortilin transcripts recognized the intracellular part of the protein (sortilin IC involved in neurotrophin trafficking) and the extracellular part (sortilin EC involved in its receptor properties). Sortilin transcript  and protNTR, a death domain receptor, as evidenced in neurons and B lymphocytes but not yet in CRC cells. We thus examined the functional effect of exogenous pro-BDNF on the four CRC cell lines maintained in serum-free medium. Apoptotic ratios significantly increased in all CRC lines, especially in the two primary lines  tissues  were studied as controls. Patients' tumor and adjacent non-tumor tissues were compared. BDNF and its receptors TrkB and p75 tissues . Noteworterparts . These pThe present data provide evidence that endogenous BDNF is an essential autocrine factor able to rescue human CRC cell death under stressed culture conditions through TrkB, its tyrosine kinase receptor. High BDNF expression was reported in neuroblastoma BDNF is synthesized by neurons as pro-BDNF that is cleaved by matrix metalloproteases (MMP), especially MMP-7 and MMP-9 NTR signals independently of Trk, it requires sortilin as a coreceptor, then binds pro-BDNF and induces apoptosis NTR-sortilin complex bound to pro-BDNF. Therefore, the role of sortilin seems to be a complex balance between these two opposite functions. Shedding of the luminal domain of sortilin as described in HT29 cells That BDNF is secreted in association with sortilin, especially under stress conditions, was not previously described in CRC cells. Sortilin was initially known to regulate neurotrophin traffic in human neuronal cells NTR acts as a tumor suppressor in neuroblastoma cell in vivo NTR receptor in tumor was consistent with these findings. Our results suggest that BDNF and its receptors may have a crucial function in facilitating tumorigenesis and progression of CRC tumors.Previous reports have shown that the BDNF/TrkB pathway promotes tumorigenesis, invasiveness, angiogenesis and drug resistance, contributing significantly to the aggressive phenotype of these poor prognosis tumors. Thus, an evaluation of BDNF/TrkB expression in patients with CRC disease may be helpful for a better prediction of the prognosis and treatment outcome. In the present study, we showed that BDNF and TrkB (both forms) were overexpressed in tumor tissue in comparison either to each corresponding non-tumor tissue from the same patient or to the control tissues with benign disease. In addition, it was reported that the low affinity receptor p75vitro and in vivo. We also speculate that sortilin, as a transport protein as well as a potential pro-BDNF receptor, appears to be the key actor of this autocrine loop. Future therapeutic strategies involving BDNF/TrkB and sortilin could be developed from such results to improve targeted treatment of CRC patients.Taken together, all these findings strongly support that autocrine BDNF/TrkB signaling contributes to tumor cell survival in CRC in"}
+{"text": "Prescription opioid use in the United States has become widespread , and stuCDC used Truven Health\u2019s MarketScan Commercial Claims and Encounters and Medicaid data from 2008\u20132012 to assess outpatient pharmacy prescription drug claims for opioid-containing medications among reproductive-aged women (15\u201344 years). The Commercial Claims and Encounters data represent a convenience sample of employed persons with private employer\u2013sponsored insurance and their dependents. The Medicaid data are an annual sample of Medicaid recipients in 10\u201313 states across the United States. Both data sources include person-level information  and claim-level data . This analysis was restricted to women continuously enrolled (\u2265365 days) for the year under study in a private insurance or Medicaid plan that included prescription drug coverage. Outpatient pharmacy prescription medication claims were searched for opioid-containing medications using national drug codes; pure opioid antagonists , medications that block the effects of opioids, were excluded when not combined with an opioid . For each woman, the prescription claims data were analyzed to determine whether she had ever filled a prescription for an opioid medication from an outpatient pharmacy during a given calendar year. The annual proportion of reproductive-aged women with outpatient prescription claims for an opioid during 2008\u20132012 was examined by health care coverage type and specific opioid medication, age group, U.S. geographic region (only available for privately insured women), and race/ethnicity (only available for Medicaid-enrolled women). The average proportion of reproductive-aged women who filled a prescription for an opioid from an outpatient pharmacy each year was also estimated. Chi-square tests were used to determine whether significant differences existed between the frequency of opioid claims among privately insured and Medicaid-enrolled women.There were approximately 4.4\u20136.6 million privately insured and 0.4\u20130.8 million Medicaid-enrolled reproductive-aged women in the study sample each year during 2008\u20132012. Of these, on average 27.7% of privately insured and 39.4% of Medicaid-enrolled women filled a prescription for an opioid from an outpatient pharmacy each year (p<0.001). Opioid prescription claims were highest in 2009 with 29.1% of privately insured women and 41.4% of Medicaid-enrolled women filling a prescription for an opioid. During 2008\u20132012, opioid prescription claims were consistently higher among Medicaid-enrolled women when compared with privately insured women . In 2012The most commonly prescribed opioids during 2008\u20132012 were hydrocodone (reported by an average of 17.5% of privately insured and 25.0% of Medicaid-enrolled women each year), codeine (6.9% and 9.4%), and oxycodone (5.5% and 13.0%) . PrivateSignificant regional and racial/ethnic differences were observed. Among privately insured women, opioid prescription claims were highest among those residing in the South. Among Medicaid-enrolled women, opioid prescription claims were highest among non-Hispanic whites (p<0.001) .Opioid-containing medications are widely prescribed among reproductive-aged women with either private insurance or Medicaid, with approximately one fourth of privately insured and over one third of Medicaid-enrolled women filling a prescription for an opioid each year during 2008\u20132012. In addition, CDC found an average of three opioids prescribed for every four privately insured women and nearly two opioid prescriptions for every one Medicaid-enrolled woman per year. This is a significant public health concern given evidence of adverse pregnancy outcomes with opioid exposure, the likelihood of exposures occurring among unrecognized or unintended pregnancies, and health care provider concerns about using other pain medications during early pregnancy ,5.This analysis presents data among all reproductive-aged women; however, previous studies from slightly earlier time periods have reported data among women who were actually pregnant. In general, more reproductive-aged women filled a prescription for an opioid in this study compared with what has been found among pregnant women. In a study of approximately 534,000 pregnant women with private insurance during 2005\u20132011, 14.4% filled a prescription for an opioid during pregnancy . In a stThe consistently higher frequency of opioid prescribing to Medicaid-enrolled women is of concern because approximately 50% of U.S. births occur to Medicaid-enrolled women . Other sGeographic region was available for the privately insured claims data and showed opioid prescription rates were highest among reproductive-aged women residing in the South and lowest in the Northeast. Race/ethnicity was available for Medicaid data, and indicated opioid prescriptions were nearly 1.5 times higher among non-Hispanic white reproductive-aged women than among non-Hispanic black or Hispanic women. Other reports of opioid prescribing patterns have shown similar geographic trends, with the South having the greatest number of prescription opioid claims ,7, and wAlthough there appeared to be a decline in the frequency of opioids prescribed to both privately insured and Medicaid-enrolled women of reproductive age from 2009 to 2012, any conclusions about changes over time must be interpreted with caution. The apparent decline might indicate improvements in opioid prescribing practices; however, given the potential changes in the composition of the sample used for the privately insured claims data and in the states included in the Medicaid sample each year, this conclusion cannot be drawn from these data. At least one study has noted a decrease in opioid prescriptions among pregnant women, with a decline from 14.9% in 2005 to 12.9% in 2011 , althougThe findings in this report are subject to at least four limitations. First, Truven Health\u2019s MarketScan Commercial Claims and Encounters and Medicaid data are samples and might not be generalizable to the entire U.S. population. Although the privately insured claims data represent approximately 4\u20136 million insured persons, it is likely that they are only representative of persons with employer-based private insurance. The 10\u201313 states that contribute their data to the Medicaid sample each year do so anonymously. Therefore, changes over time in the Medicaid data might reflect changes in the sample of states, instead of broader changes among the Medicaid-enrolled population. It is also likely that certain women will be included in multiple years of either the Commercial Claims and Encounters or Medicaid data. Second, pregnant women were not identified in this analysis; the study population was based on female sex and age 15\u201344 years alone. Whether opioid prescriptions were limited to infertile or contracepting women was not ascertained. Third, these analyses likely underestimate opioid use, because the data only represent outpatient pharmacy claims. No information on inpatient opioid use, opioids obtained without a prescription, or opioids paid for out-of-pocket was available. Finally, although these data represent opioids dispensed by outpatient pharmacies, there was no verification that women actually took the medications.What is already known on this topic?Opioid use among women of reproductive age is a concern because opioid medications have been linked to birth defects and other adverse pregnancy outcomes. Given the high rate (approximately 50%) of unintended pregnancies in the United States, opioid use among reproductive-aged women can result in many early pregnancy exposures.What is added by this report?During 2008\u20132012, more than one fourth of privately insured and more than one third of Medicaid-enrolled reproductive-aged women (15\u201344 years) filled a prescription for an opioid from an outpatient pharmacy each year. Prescription rates were consistently higher among Medicaid-enrolled women when compared with privately insured women.What are the implications for public health practice?More targeted interventions and communications strategies are needed to reduce unnecessary prescribing and use of opioid-containing medications, particularly among women who might become pregnant.This analysis used a large database to estimate the proportion of privately insured and Medicaid-enrolled reproductive-aged women who filled a prescription for an opioid from an outpatient pharmacy. Many women need to take opioid-containing medications to appropriately manage their health conditions; however, in some instances safer alternative treatments are available and use of opioids is unnecessary. Having a better understanding of prescription opioid use just before and during early pregnancy can help inform targeted interventions to reduce unnecessary prescribing of opioids and provide evidence-based information to health care providers and women about the risks of prenatal opioid exposure."}
+{"text": "Trypanosoma cruzi is the agent of Chagas disease, transmitted by hematophagous triatomine vectors. Establishing transmission cycles is key to understand the epidemiology of the disease, but integrative assessments of ecological interactions shaping parasite transmission are still limited. Current approaches also lack sensitivity to assess the full extent of this ecological diversity. Here we developed a metabarcoding approach based on next-generation sequencing to identify triatomine gut microbiome, vertebrate feeding hosts, and parasite diversity and their potential interactions. We detected a dynamic microbiome in Triatoma dimidiata, including 23 bacterial orders, which differed according to blood sources. Fourteen vertebrate species served as blood sources, corresponding to domestic, synantropic and sylvatic species, although four  accounted for over 50% of blood sources. Importantly, bugs fed on multiple hosts, with up to 11 hosts identified per bug, indicating very frequent host-switching. A high clonal diversity of T. cruzi was detected, with up to 20 haplotypes per bug. This analysis provided much greater sensitivity to detect multiple blood meals and multiclonal infections with T. cruzi, which should be taken into account to develop transmission networks, and characterize the risk for human infection, eventually leading to a better control of disease transmission. Trypanosoma cruzi is the agent of Chagas disease, a major vector-borne parasitic disease in the Americas. T. cruzi parasite transmission from triatomine vectors to vertebrate hosts occurs via the insect\u2019s feces through a complex succession of events, making transmission from vector to vertebrate host rather unlikely1. The parasite is nonetheless able to infect a very large variety of mammalian hosts covering many orders, including Marsupialia, Rodentia, Lagomorpha, Chiroptera, Carnivora and Primata. The identification of the host species involved in transmission cycles is key to understand the epidemiology of the disease and the risk for human transmission. The identification of triatomine host feeding patterns and their variations7 is starting to provide useful information to assess the role of different hosts species as parasite reservoirs or for contributing to increasing the risk for human infection. Separate or overlapping transmission cycles among species communities may also be evidenced10. Nonetheless, host feeding sources and feeding behavior of triatomines remain poorly understood as current techniques based on direct sequencing of PCR products can only identify the dominant sequence/host in each sample. The addition of a cloning step prior to sequencing allowed detecting up to 3\u20135 host species in some bugs6, suggesting that multiple blood meals may be more frequent than previously acknowledged. Such multiple blood meals are key to evaluate the potential for parasite transmission between hosts species.T. cruzi into seven discrete typing units (DTUs TcI to TcVI and Tcbat)12, the geographic distribution of the different DTUs as well as their association with transmission cycles restricted to specific habitats and hosts is beginning to be challenged15. Indeed, while it was believed that TcI DTU was largely predominant in Mexico and Central America (over 95% of the strains)16, recent studies have documented the presence of non-TcI parasite strains in triatomines from different regions in Mexico and Central America at high frequencies17. Similarly in the southern US, initial work reported only TcI and TcIV DTUs18, but recent studies also indicate the presence of additional non-TcI DTUs at important frequencies20. Together, these observations indicate clearly that T. cruzi genotype distribution in many countries and regions needs to be re-examined16. Also, potential parasite genetic differentiation and adaptation to specific hosts should be examined to establish the epidemiologic relevance of T. cruzi genotypes in the transmission of Chagas disease. As for the identification of blood feeding source mentioned above, current PCR genotyping methods are also limited to detecting the dominant genotype in biological samples, and the multiclonality of infections is mostly overlooked. Indeed, the genetic analysis of multiple parasite clones isolated from single hosts revealed concurrent infections with up to 10 parasite genotypes per opossum individual21. More recently, the analysis of sequence variation in GP63 through deep sequencing also revealed a large number of gene variants present in Chagasic patients22. The multiclonality of infection in humans is also evidenced in longitudinal studies of patients, a large proportion of which present changes in the dominant DTU identified before and after drug treatment, indicating that infection with multiple DTUs and heterogeneous drug response may be occurring23. Multiclonal infections may thus be the norm rather than the exception, and the interactions among parasite genotypes within vectors and hosts are still mostly unknown.Similarly, while parasite genetic diversity has been known for a long time and has led to the division of 25. For example, the development of Trypanosoma brucei, the agent of African trypanosomiasis, in its tsetse fly vector, is directly influenced by a microbiome-regulated gut immune barrier26. Gut microbiome similarly modulates dengue virus infection in Aedes aegypti mosquitos28, and microbiome manipulation may be used to control virus transmission29. Triatomines harbor a diverse gut microbiome that is beginning to be identified31, but its influence on T. cruzi transmission remains unclear. In Triatoma infestans, infection by T. cruzi induces the overexpression of antimicrobial peptides modulating the microbiome, and this inhibition of the bacterial microflora is important for parasite establishment in the vector32. Nonetheless, our understanding of triatomine microbiome is still too limited to further assess its role in vectorial capacity and T. cruzi transmission, or to take full advantage of its potential manipulation in novel parasite control strategies such as para-transgenesis as suggested before34.Parasite development and the vectorial capacity of vectors may also be affected by the bacteria present in the insect gutT. cruzi transmission. Such interactions may however be critical for parasite transmission dynamics and Chagas disease epidemiology, and may lead to novel disease control approaches. Our objective was thus to develop an integrative and highly sensitive molecular approach using metabarcoding based on next-generation sequencing for the identification of triatomine genotype, gut microbiome, vertebrate feeding hosts, and parasite genetic diversity and their potential interactions.Finally, while some of the aspects mentioned above have begun to be evaluated in an isolated manner, there has been no or little effort to provide an integrative assessment of the ecological interactions that combine to shape Triatoma dimidiata were used in this study. They were collected during entomological surveillance following a pilot vector control intervention during years 2013\u20132015, in the villages of Bokoba, Sudzal and Teya35, located in the Yucatan peninsula, Mexico, about 15\u201320\u2009km apart36, as well as in the sylvatic habitat surrounding these villages (up to 8\u2009km from the villages) using the previously reported primers ITS2_200F (5\u2032-TCGYATCTAGGCATTGTCTG-3\u2032) and ITS2_200R (5\u2032-CTCGCAGCTACTAAGGGAATCC-3\u2032) and PCR conditions41.We used triatomine Internal Transcribed Spacer (ITS)-2 nuclear marker for 42.To identify the microbiome composition, a 140\u2009bp fragment of the bacterial 16\u2009S rRNA gene was PCR amplified (35 cycles) using primers E786F (5\u2032-GATTAGATACCCTGGTAG-3\u2032) and U926R (5\u2032-CCGTCAATTCCTTTRAGTTT-3\u2032) as described before43.For the identification of vertebrate blood meals, a 215\u2009bp fragment of the 12\u2009S rRNA gene was amplified (35 cycles) with the primers L1085 (5\u2032-CCCAAACTGGGATTAGATACCC-3\u2032 and H1259 (5\u2032-GTTTGCTGAAGATGGCGGTA-3\u2032) as described beforeT. cruzi parasites in bugs was detected by PCR amplification (35 cycles) of parasite satellite DNA with primers TcZ45. Parasite genotyping was performed by multiplex PCR (40 cycles) using a mixture of three primers able to amplify T. cruzi DTUs TcI and TcBat (350\u2009bp), TcII, TcV and TcVI (300\u2009bp), and TcIV (380\u2013400\u2009bp), based on the mini-exon gene46. DNA samples purified from reference strains from the relevant T. cruzi DTUs were used as controls.The presence of All PCR reactions were run using Thermo Scientific\u2122 DreamTaq\u2122 DNA polymerase. Positive and negative PCR controls were included in each batch of PCR reactions. A DNA extraction control using only water was also PCR amplified with each for the DNA marker used to detect potential contamination during DNA extraction. All PCR products were separated in 2% agarose gels, stained with ethidium bromide and visualized under UV light, to ensure the presence of bands of the expected size.T. cruzi parasite markers were pooled for each bug, and cleaned using Promega Wizard\u00ae SV Gel and PCR Clean-Up System. Following end-repair and indexing, libraries were prepared and sequenced on a MiSeq (Illumina) or Ion Torrent (Life Technologies) platforms. From 100,000 to 900,000 reads were obtained from each bug after quality and size filtering. Sequence data were deposited in the SRA database under accession number SRR6337087 to SRR6337099. Reads were aligned to reference sequences for each of the target markers in Geneious 9.1 using the Geneious algorithm, corresponding to a depth ranging from 130 to 340,000 reads per marker  haplotype networks48 of ITS-2 sequences were constructed in PopArt 1.7. New ITS-2 haplotype sequences were deposited in Genbank database under accession numbers: MF767417 to MF767433.Sequences for bacterial 16\u2009S gene and vertebrate 12\u2009S rRNA gene were screened for chimeras using UChime50 with update 5, release 11 of the sequence database. Taxonomic identification of bacteria was made at a threshold of >96% sequence identity, and the frequency of sequences for each taxonomic unit was considered as a proxy of its abundance in triatomine gut. Rarefaction curves were elaborated, at the individual and group level, to estimate species richness of our sampling. For blood meal sources, 12\u2009S rRNA sequences were analyzed by MEGABLAST against the entire \u201cnr\u201d GenBank database (version of March 2017). Sequence match with >96% identity was used for species or genus identification, with an E value of at least 6.09E-77, and sequences representing less than 0.5% of a blood meal were discarded from the analysis. Rarefaction curves were also constructed. Sequences identified from the same species were further aligned to detect sequence haplotype variants as an indicator of feeding on different hosts of the same species. A feeding network and parasite transmission pathways was constructed using Cytoscape 3.5, to visualize the frequency of the respective feeding sources as well as possible pathways for parasite transmission among species when multiple blood meals were detected, since feeding sources can be used as evidence of vector-host contact. Nodes of the network represent the various species detected as feeding sources, and edges link hosts that were found in the same individual gut content. Network statistics were calculated as implemented in Cytoscape, and included the average number of neighbors, network density, network centralization, average clustering coefficient, and neighborhood connectivity distribution.For microbiome composition, bacterial 16\u2009S rRNA sequences were analyzed using a Bayesian classifier from the Ribosomal Database ProjectT. dimidiata. Due to large differences between TcI and non-TcI sequences, these were also analyzed separately. T. cruzi mini-exon sequences were deposited in Genbank database under accession numbers: MF770768 to MF770822. To assess if parasites haplotypes were consistently associated within bugs, or rather circulated independently from one another, we determined which haplotypes are shared between bugs, and which are unique. This allowed identifying sets of haplotypes that can be transmitted independently from each others among vectors and hosts. Assuming that no parasite selection occurs in triatomines and a good sensitivity of our method allowing to detect rare haplotypes, this is likely to reflect independent infection events of the bugs, allowing to estimate the possible number of infection events of bugs .Parasite sequences from the mini-exon gene were aligned with sequences from reference strains covering all DTUs (USAOPOSSUM (TcI), Tu18 (TcII), AF1Cl7 (TcII), M6531 (TcIII), M6241 (TcIII), MT4167 (TcIV), CanIII (TcIV), SC43 (TcV), MN (TcV), CL Brener (TcVI) and VSC (TcVI)), and TCS haplotype networks were constructed using PopArt, taking into account the observed frequency of the different haplotypes within bugs, to visualize parasite diversity infecting i/N).ln(ni/N)) as well as Margalef Richness index ((S-1)/ln N) were calculated, with ni representing the number of individuals of species/taxa i, N the total number of individuals, and S the total number of species/taxa. Indices were compared using Student\u2019s t test, and linear regression was used to assess the potential associations among biodiversity indicators. Frequency data were compared by \u03c72 or Fisher\u2019s Exact tests.To further describe microbiome, blood meal sources and parasite genotype sequence diversity, Shannon diversity index  were analyzed, corresponding to an average of 18,627 sequences per bug (N\u2009=\u200914). Four bugs presented a single ITS-2 sequence, and eight presented two sequence haplotypes at a frequency of about 50% each, suggesting that these corresponded to heterozygote bugs. Interestingly, one bug presented four haplotypes, at frequencies of 46, 39, 9 and 6%, respectively. Two of these four sequences were novel and not found in other bugs, thus were not a contamination from other samples, but we could not exclude that they may represent chimeric sequences. Overall, these data confirmed the extensive concerted evolution of this multi-copy gene, which mostly behaves as a single copy sequence. It also confirmed the filtering out of errors/artefacts throughout our amplification, sequencing and analysis process.38, and the haplotype of most bugs corresponded to known ITS-2 haplotypes .Comparison of ITS-2 haplotypes with reference haplotypes confirmed that all analyzed bugs belonged to the previously described Group 3 of this species complexpes Fig.\u00a0. Eleven T. dimidiata microbiome  and Bacillus, Staphylococcus and Anoxybacillus . Importantly, Wolbachia was not detected in any of the samples, indicating that it might be absent from this insect species.About 500,000 partial 16\u2009S rRNA sequences (110\u2009bp long) were analyzed, corresponding to an average of 37,654 sequences per bug (N\u2009=\u200913). A total of 23 bacterial orders were identified with high confidence, and rarefaction curve indicated that most of the richness had been identified through our sampling , with females presenting a larger diversity of orders in their microbiome , and Shannon Index tended to be higher in females . Oceanospirillales, Pseudomonadales, Xanthomonadales, Flavobacteriales, Caulobacterales, Burkholderiales and Actinomycetales were more abundant in females, while Bacillales, Bacteroidales, Enterobacteriales, Neisseriales and Rhodobacterales were more abundant in males. More strikingly, microbiome composition differed strongly based on the dominant blood meal source of the bugs .Interestingly, the composition of bacterial orders was significantly different between males and females , cow (Bos taurus), cat (Felis spp.), mouse (Mus musculus), rat (Rattus spp.), pig (Sus scrofa), turkey  and chicken , in addition to humans (Homo sapiens) (Supplementary Table\u00a0Coendou spp.), squirrel (Sciurus spp.), fruit bat (Artibeus spp.), kinkajou (Potos flavus), and dove (Zenaida spp.) . Shannon diversity index for the blood sources of female and male bugs was also similar , as well as the Margalef richness index .About 218,000 partial vertebrate 12\u2009S rRNA sequences (about 170\u2009bp long) were analyzed for the identification of blood sources of triatomines, corresponding to an average of 15,573 sequences per bug (N\u2009=\u200914). MEGABLAST analysis revealed a diverse range of at least 14 host species, including the expected domestic and synantropic species such as dog  blood meal, accounting for 50\u2013100% of the sequences, while lower frequency sequences may correspond to older or partial blood meals. Thus, multiple host species were detected in all bugs except one, with up to 7 identified host species in a single bug Fig.\u00a0. Within T. cruzi parasites by T. dimidiata among vertebrate host species, a feeding and transmission network was constructed  and strong clustering (average clustering coefficient: 0.85) with a medium centralization  , indicating that highly connected nodes predominate in shaping the network. Indeed, the network shows the limited role of birds as feeding sources, while a mixture of domestic and sylvatic mammalian species contribute to potential parasite transmission pathways . This allowed the detection of an important haplotype diversity within each individual triatomine, ranging from 2 to 20 parasite haplotypes, with frequencies ranging from 0.1% to 65%  from % Tables\u00a0. For a f% Tables\u00a0. For TcI% Tables\u00a0. While tT. cruzi parasites with the number of infection events, feeding sources and gut microbiome diversity in individual T. dimidiata bugs . Unexpectedly, we could not detect an association between parasite diversity and blood meal species diversity (P\u2009=\u20090.96), possibly because of our small sample size combined with the fact that only recent blood meals can be detected, while bugs remain infected with T. cruzi for their lifespan. Also, parasite diversity tended to increase with microbiome diversity, but this did not reached statistical significance .Shannon diversity index was then used to compare the diversity of ugs Fig.\u00a0. As expeT. cruzi transmission and the risk for human infection in many settings. However, most of these approaches have limited sensitivity to assess the full diversity of triatomine feeding hosts and parasite genotypes, or lack integration. We developed here a metabarcoding strategy based on next-generation sequencing to successfully identify a wide range of ecological associations of T. dimidiata in the Yucatan peninsula, Mexico. Analysis of triatomine ITS-2 sequences confirmed that our study focused on ITS-2 Group 3 of T. dimidiata species complex. This group is clearly differentiated from the other ITS-2 group38, with a different ecological niche at the regional level55, although it appears to have a comparable epidemiological role as other sub-species from the dimidiata complex57. The lack of ITS-2 sequence variants in most bugs also confirmed the strong concerted evolution of this multi-copy marker, as well as the reliability of our methodological approach.Molecular approaches are providing very relevant information on T. dimidiata gut microbiome for the first time. We identified that the most common bacterial orders present are similar to those found in the microbiome of other insect vectors such as Aedes, as well as other triatomine species. However, there were also some differences among T. dimidiata and other triatomines. For example, Clostridiales and Rhodocyclales are more frequent in Triatoma infestans, Rhodnius prolixus, and Panstrongylus megistus but marginally present in T. dimidiata51. Enterobateriales predominate in Rhodnius prolixus52, while Bacillales are more abundant in T. dimidiata. Rhizobiales, Burkholderiales, Sphingomonadales, Pseudomonadales, Caulobacterales, Lactobacilales and Xanthomonadales are also relatively abundant in T. dimidiata, but have not been reported in other triatomines51, although they have been observed in mosquitoes such as Aedes spp., Culex spp.60 or ants61. Remarkably, Wolbachia was not detected in T. dimidiata, in agreement with its absence from triatomine species such as Triatoma infestans, Triatoma vitticeps, Dipetalogaster maximus, Panstrongylus maximus30, Triatoma brasiliensis and Triatoma pseudomaculata31, and Rhodnius remains the only triatomine genus in which Wolbachia has been detected in both natural and laboratory populations62.We also obtained extensive information on T. dimidiata microbiome appeared as very dynamic, with differences between males and females, which may reflect differences in nutrient needs. Blood feeding sources may also affect microbiome composition, as suggested by our observation of differing microbiomes among bugs with different dominant blood sources, and further studies are needed to clarify this point. In mosquitoes, the microbiome has been found to be similarly very dynamic, showing seasonal variations64, as well as changes due to the environment, diet, and aging65, although a constant core microbiome could be identified. Further studies should help clarify the variations in T. dimidiata microbiome composition according to its ecological conditions.T. dimidiata68, with the exception of some of the sylvatic hosts such as the kinkajou, porcupine, squirrel and fruit bat. However, the most striking observation was that all bugs except one had blood from more than one host, with up to 11 hosts detected in a single bug. This is considerably higher that what has been observed with previous molecular approaches, which at best can detect 3\u20135 hosts/bug6, and most studies report unmixed/single blood meals7. This highlights the high sensitivity of our deep sequencing approach. We were thus able to identify not only the dominant blood meal, likely corresponding to the most recent/abundant meal, but also the remaining older or partial blood meals present in much lower amount. The identification of blood from different hosts in a bug represents key evidence of host-switching, which appears to be very frequent for T. dimidiata. Feeding frequency of triatomines has been a long sought parameter, as it contributes to transmission dynamics69. Estimates from field studies suggested that it was around a blood meal every 1.7\u20137 days for T. infestans70. Our data suggest about one  blood meal every week, up to one every 3 days, which is very consistent with these previous studies. We thus estimated that T. dimidiata could feed on up to 115 hosts per year, which may provide many opportunities for parasite circulation among hosts and individuals and/or species. Such a large number of feeding hosts is also considerably higher than for other vector species such as mosquitoes, which at best feed on a handful of hosts during their lifespan69, and this is likely to strongly affect parasite transmission dynamics71. In addition, the mixture of blood meals on domestic and sylvatic hosts confirmed the strong overlap of domestic and sylvatic transmission cycles, which may reflect a high mobility of both vectors and hosts between these habitats. This is in agreement with the intrusive behavior of T. dimidiata in this region77.Blood feeding was observed on at least 14 host species, most of which had been reported before as feeding sources for T. cruzi transmission network as it evidences chains of vector-host contacts. Dogs emerged as a key host for parasite transmission to humans, as observed in other regions80, and also in agreement with a relatively high prevalence of infection of this host82. This observation strengthens the rationale for controlling T. cruzi infection in dogs as part of an integral control intervention. This may be achieved by insecticide treatment85 or a vaccine87. Mice also seem to play an important role in parasite transmission, although their infection rate by T. cruzi may be highly variable in the region89. On the other hand, our network indicated that rats and cats played a limited role in parasite transmission to humans, in spite of a significant prevalence of T. cruzi infection observed in previous studies90. Nonetheless, rodent control has been successfully tested as part of a Chagas disease control intervention in Guatemala91. Cows rather than pigs are other domestic animals that appear to contribute to the circulation of T. cruzi in our setting, although limited information is available about T. cruzi infection in these hosts92. Importantly, several sylvatic host species including porcupine, squirrel and fruit bat, reported here for the first time as important blood sources for T. dimidiata, were also strongly connected to potential parasite transmission to humans and other domestic hosts, emphasizing the direct links between sylvatic and domestic hosts of T. cruzi. Importantly, fruit bats have previously been found to be infected by T. cruzi (about 3% for Artibeus spp.) in the southern part of the Yucatan peninsula89.Information on multiple blood meals was used to construct potential Didelphis opossums, which have been reported with a very high T. cruzi infection rate in the region (12\u201354%)93. This may be due to the limited number of bugs analyzed, but infection through other routes such as oral or through anal gland may also play a role for these hosts. PCR primer bias may also occur, although we successfully amplified control opossum DNA from Yucatan with our primers, and opossum has been previously reported using the same primers6. Analysis of additional populations of T. dimidiata, and of the seasonal variations in feeding profiles using our approach should provide additional information to refine feeding and transmission networks, and of the potential source of parasites infecting humans.Unexpectedly, no blood meals were detected from T. cruzi genetic diversity revealed a very high level of parasite diversity in T. dimidiata, with up to 20 parasite haplotypes per bug, based on the analysis of the mini-exon marker. While this diversity may be due to sequence variants in paralogous copies within clones/genomes of this repeated sequence, variations in haplotype frequencies, their apparent independent circulation among bugs, and the presence of several well established parasite DTUs all support extensive multiclonality of infection. While most studies focus on identifying the main dominant genotype circulating in vectors and overlook this multiclonality96, our data emphasize the very high sensitivity of our approach, which provided detailed information on the composition of parasite populations infecting bugs, not only at the level of sequence haplotype, but also on their relative frequency. Indeed, we were able to detect even very low frequency haplotypes, representing as little as 0.1% of the parasite haplotypes present in T. dimidiata. Some of this diversity may be due to contamination among samples following incorrect indexing, which would tend to minimize differences among bugs, or may also represent chimeric sequences and/or sequencing artefacts. However, we also detected rare parasite haplotypes in a single bug (for example H18-TcI (0.2%) is unique to Tey015, or H41-NoTcI (0.1%) is unique to Tey016), which are more likely to be true sequence variants.Analysis of T. cruzi in T. dimidiata is in agreement with the extensive host switching behavior described above, as bugs feeding on a large variety of hosts may likely get infected with a large variety of parasites. Using Shannon diversity index as an indicator of parasite diversity, we could confirm the significant correlation between parasite genetic diversity with the number of infection event of bugs. However, we could not detect any correlation between blood sources diversity and parasite diversity. As mentioned above, this may be due to small sample size, combined with the fact that blood meals only reflect recent feeding host, while T. cruzi infection lasts for the entire lifespan of the bugs. On the other hand, parasite diversity may be associated with microbiome diversity, and future studies should help establish the critical interactions between triatomine microbiome and T. cruzi development51.A high clonal diversity of T. dimidiata for the first time in the region, and possibly additional non-TcI DTUs are also present since our analysis revealed a large number of haplotype variant within the closely related TcV and TcVI DTUs. Although the reference sequences from TcII, TcV and TcVI DTUs are clearly separated, we observed many intermediate sequences much harder to classify. Further studies using additional markers should help refine parasite genetic structure, and the population dynamics resulting from interactions among parasite genotypes. Overall, our results are in agreement with the large diversity of T. cruzi strains and DTUs observed in T. dimidiata in Veracruz, Mexico13, and Guatemala17, as well as in vertebrate hosts from the Yucatan89, even though these studies only documented the dominant genotypes as mentioned above. Our observations raise the questions of the nature of the interactions among parasite haplotypes within vectors, which may play an important role in shaping parasite transmission and the epidemiology of T. cruzi infection98. In any case, it is clear that future studies should take into account the multiclonality of T. cruzi infections to further understand parasite transmission networks and the epidemiology of Chagas disease.TcII DTU was detected in T. dimidiata bugs in an integrative manner. Importantly, our analysis provided much greater sensitivity than conventional molecular approaches to detect multiple blood meals in bugs as well as multiclonal infections with T. cruzi. Moreover, our observations confirm the presence of a very dynamic triatomine microbiome, which may be modulated by blood sources and may interact with T. cruzi parasite. This extensive diversity of feeding sources and parasite infections allowed documenting transmission network. Expanding these studies to additional T. dimidiata populations and at different times of the year will provide unprecedented data to characterize T. cruzi parasite transmission and the risk for human infection, and which ultimately will lead to a better control of disease transmission.In conclusion, we described here a powerful new approach based on metabarcoding through next generation sequencing to provide detailed information on ecological associations of Supplementary materials"}
+{"text": "The synthesis and crystal structure of a potentially redox non-innocent penta\u00adfluoro\u00adphenyl-substituted pyridine di\u00adimine ligand system are reported. 21H9F10N3, represents a potential redox non-innocent pyridine di\u00adimine ligand system. It consists of a central pyridine ring with two penta\u00adfluoro\u00adphenyl substituted imine groups in positions 2 and 6. The whole mol\u00adecule is generated by mirror symmetry, the mirror bis\u00adecting the N and para-C atom of the pyridine ring. The perfluoro\u00adphenyl ring is inclined to the pyridine ring by 73.67\u2005(8)\u00b0. In the crystal, mol\u00adecules stack along the a axis, but there are no significant inter\u00admolecular inter\u00adactions present.The title compound, C Thus, only half of the mol\u00adecule is present in the asymmetric unit \u00b0. The imine nitro\u00adgen atom, N2, is oriented in an anti-conformation with respect to the pyridine nitro\u00adgen, N1. This orientation is in contrast with the mol\u00adecule acting as a tridentate ligand coordinating to the chromium ion in complex tri\u00adchloro\u00ad\u00adeth\u00adyl)pyridine-N,N\u2032,N\u2032\u2032)chromium(III) aceto\u00adnitrile monosolvate chromium(III) aceto\u00adnitrile monosolvate  in toluene (100\u2005ml) was refluxed for 30\u2005h during which time water was removed by a Dean\u2013Stark apparatus. The crude yellow product was washed with cold methanol and filtered producing a pure off-white solid . Colorless block-like crystals were obtained by vapor diffusion of hexa\u00adnes into a saturated di\u00adchloro\u00admethane solution of the title compound. Spectroscopic data: 1H NMR : \u03b4 8.6\u20137.8 , 2.5 , and MS (ESI): m/z 494 [C21H9F10N3]H+.A mixture of 2,6-di\u00adacetyl\u00adpyridine , 2,3,4,5,6-penta\u00adfluoro\u00adaniline  and Uiso(H) = 1.5Ueq(C-meth\u00adyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table\u00a0110.1107/S2056989017008040/su5374sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017008040/su5374Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017008040/su5374Isup3.cmlSupporting information file. DOI: 1553189CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:"}
+{"text": "The fluorinated base layer with a nano-textured surface prepared by a selective fluorination etching process acted as growth seeds in the subsequent a-TiOx deposition. A nanorod-like microstructure was achievable from the resulting a-TiOx film due to the self-assembled deposition. Compared to the a-TiOx film directly deposited onto the untreated base layer, the rate constant of this fluorinate-free a-TiOx film surface for decomposing methylene blue (MB) solution that was employed to assess the film\u2019s photocatalytic activity was markedly increased from 0.0076 min\u22121 to 0.0267 min\u22121 as a mechanism for the marked increase in the specific surface area.The photocatalytic activity of an amorphous titanium oxide (a-TiO The deposition pressure, power, and gas flow rate of TTIP/O2 were controlled at 40 Pa, 100 W, and 120/20 sccm, respectively. A detailed system setup and deposition parameters have been described elsewhere [x film was achieved by pre-irradiating a UV light for 5 h through an anodic alumina membrane mask and then etching in the diluted hydrofluoric (HF) solution for a different time. The selective fluorination etching (hereafter abbreviated to SFE) has been addressed elsewhere [x film using PECVD under the same deposition condition was then coated onto the base layer with surface nano-textures to realize the a-TiOx film surface free of the fluorine ions. In addition, another set of the a-TiOx film crystallized into anatase structures was prepared by post-annealing the un-treated a-TiOx film at 500 \u00b0C for 30 min under oxygen ambient  as a comparison.A 100-nm-thick hydro-generated a-TiOlsewhere . A 100-nx films with and without the SFE treatment as well as prepared using the two-step deposition was measured using a surface profile system . Surface roughness was measured using atomic force microscopy  with the tapping mode. The surface and cross-section morphologies were observed by a field emission scanning electron microscope  operated at 3 kV. Fourier transform infrared spectrometry  and X-ray photoelectron spectroscope  with monochromatic Al K\u03b1 radiation were employed to examine the film\u2019s chemical bonding states and surface bond nature. The photocatalytic activity for the a-TiOx and annealed TiOx films illuminated by the UV light with a constant intensity of 1 mW/cm2 was evaluated by the decolorization of a 20 ppm concentrated methylene blue (MB) solution using the UV-Vis spectrophotometer from the absorbance of the resulting solution at 665 nm under atmosphere ambient.Film thickness of these a-TiOx film with and without the UV light pre-irradiation for 5 h, and then immersed in the HF etching solution for different etching times. The etching thickness of the a-TiOx film treated by additive UV light pre-irradiation was apparently lower than the a-TiOx film directly etched by the HF solution. The less acidic surface of the a-TiOx film, as a consequence of the Ti(IV)\u2212OH, transformed into a Ti(III)\u2212OH\u2212 group due to the generated electrons under UV light pre-irradiation, was responsible for the alleviation of the sequential etching process [x films with and without the UV light pre-irradiation (approximately 46 nm) as the etching time reached 35 s, while at this time the 100-nm-thick a-TiOx film directly etched by the HF solution was almost completely removed, as shown in x films was obtainable from the fluorination etching with and without UV light pre-irradiation, a selective fluorination etching treatment on the a-TiOx film to modify its surface morphology was carried out by selectively shadowing the surface through a nano-sized mask with UV light pre-irradiation and then processing the fluorination etching. process . A largex film selectively etched by the HF solution for 10, 20, and 35 s, respectively . Both the untreated and SFE-treated base layers, as shown in \u22121 with the functional group of hydroxyl (OH) around high wavenumbers (2800\u20133700 cm\u22121) [\u22121, which was associated with the Ti\u2013F vibration mode [\u22121 due to an increase in the surface acidity. When the a-TiOx film was deposited onto the SFE-treated base layer, the Ti\u2013F bond was hardly observed in the FTIR spectrum (x film deposited onto the untreated base layer (curve c), both spectra only featured Ti\u2013O and O\u2013H bonds with almost the same peak position, except for a higher relative O-H bond intensity obtained from the a-TiOx film deposited onto the SFE-treated base layer. Since the porous structures distributed in the low-temperature deposited oxidation film were the main reason responsible for the formation of the O\u2013H groups [x coated onto the SFE-treated base layer thus implied the increase in the amounts of the pores. In addition, a sharp and intense Ti\u2013O bond with the disappearance of the O\u2013H bond as a consequence of the anatase crystallization was obtained from the FTIR spectrum of the annealed TiOx film.FTIR spectra of the base layer with and without an additive SFE treatment and the a-TiO00 cm\u22121) . The SFEion mode . In addispectrum d. ComparH groups ,16, the s, Ti 2p, and O 1s core levels measured from the surface of the two-step deposited a-TiOx film as well as the base layer are respectively illustrated in x film, indicating the achievement of the fluorine-free surface. In the Ti 2p spectrum ), which was associated with the deficiency in the oxygen atoms on the surface, apparently increased from 0.29 to 0.39 as the a-TiOx film was deposited onto the SFE-treated base layer. Regarding the O 1s spectra ) than those of the film deposited onto the untreated base layer (approximately 0.24).The binding energies related to the F 1spectrum b, both tectively . The com spectra c, both sx film deposited onto the SFE-treated base layer to degrade the MB solution, as depicted in x film with a surface free of the fluorine ions achieved using the two-step deposition even exhibited better efficiency in decomposing the MB solution than the annealed TiOx film with anatase structures. The rate constant, k, which represents the quality of the photocatalytic activity of the film can be evaluated by fitting the curves shown in C and C0 are the concentrations of the MB solution at a UV light irradiation time of t = 0 and t, respectively. The k value evaluated from each curve is denoted in x film deposited onto the SFE-treated base layer corresponded to a rate constant of 0.0267 min\u22121, which was three times higher than the film deposited onto the untreated base layer (0.0076 min\u22121) as well as a little higher than that of the annealed TiOx film (0.0234 min\u22121). According to the investigations into the morphologies and the analysis of the chemical bond configurations of the two-step deposited a-TiOx film, the apparent roughening and nano-textured surface of the upper a-TiOx film without the incorporation of the fluorine ions that grew by conforming along the particle seeds on the base layer surface, which was achieved using an additive SFE treatment, was the mechanism responsible for the great enhancement in the resulting photocatalytic activity.In addition, because the hydroxyl groups are beneficial for trapping hole carriers to suppress the recombination of the photo-generated electron-hole pairs, as demonstrated in previous studies ,24, the x film was realized using an additive SFE treatment for different times. At the initial stage, the size and shape of these particles distributed over the a-TiOx film surface after treatment with the SFE process for 10 s conformed to the patterns of the shadow mask. As the SFE treatment increased to 20 s, the shape of the particles appearing on the a-TiOx film surface evolved from a round-like to a needle-like shape as a consequence of the apparent sidewall etching effect. Eventually, significant channels that corresponded to the film being completely removed from the substrate were observed from the a-TiOx film processed by an additive SFE treatment for 35 s. The fluorinated surface with specific nano-textures of the a-TiOx film acted as seed layer for the subsequently deposited a-TiOx film according to the investigations of the surface and cross-section morphologies of the resulting a-TiOx film. The nuclei of the a-TiOx film self-assembled on the particles distributed over the SFE-treated base layer and then grew up to cause an apparent increase in surface roughness. Such roughening of the surface without the fluorine ion incorporation was achieved using the two-step deposition due to the deposition being selective, which also resulted in the film surface being abundant in the hydroxyl groups that were helpful for suppressing the recombination of the photo-generated electron-hole pairs. Accordingly, the fluorine-free a-TiOx film deposited onto the SFE-treated base layer which had a nanorod-like structure possessing efficient photocatalytic activity, with a rate constant of 0.0267 min\u22121; this was much higher than that of the film deposited on the untreated base layer (~0.0076 min\u22121 ), as evaluated from these films photo-degrading in the MB solution.The depth-dependent morphology of the surface-fluorinated a-TiO"}
+{"text": "Oleaceae known to play a role in the plant\u2013herbivore interaction. However, it is not clear how this molecule is induced to mediate plant responses to microbes and how microbes, in turn, withstand with OLE. To better understand how OLE affects the plant\u2013microbe interaction, the contribution of differential gene expression in the adaptation to OLE was characterized by whole genome transcriptional profiling in Lactobacillus plantarum, a bacterium associated to the olive. OLE downregulated functions associated to rapid growth, remodeled membrane phospholipid biosynthesis pathways and markedly repressed the expression of several ABC transporters from L. plantarum. Genes encoding the plantaricin and lamABDCA quorum-sensing (QS) systems were down-regulated indicating the potential of OLE as a QS-antagonist. Notably, OLE diminished the expression of a set of genes encoding inmunomodulatory components and reoriented metabolic pathways to increase protein acetylation, probably to attenuate plant immunity. Responses were also triggered to repress the transport of acetoin and to buffer reactive oxygen species accumulation, two signals involved in plant development. The results suggest that OLE could act as a signaling molecule in the plant\u2013microbe interaction and facilitate the accommodation of beneficial microbes such as L. plantarum by the plant host, via controlled expression of bacterial molecular players involved in this reciprocal interplay.Oleuropein (OLE) is a secoiridoid unique to  Oleaceae  is the most abundant among a set of secoiridoids that are unique to Oleaceae . This ph tissues , and it  tissues . OLE is g plants . HoweverLactobacillus plantarum (a bacterium associated to the olive), have been also shown to act as signals eliciting immunomodulatory responses . These results are in line with the finding that the Lactobacillales order is one of the bacterial endophytes most abundant that colonize the Mediterranean olive trees . The genome of this strain was the first published among Lactobacillus species and it has a large size of 3.3 M thought to be related to the diversity of environmental niches in which L. plantarum spp. is encountered. Due to the early publishing of its genome sequence this strain has been subject of extensive functional genomic research (L. plantarum WCFS1 was chosen for this study. L. plantarum WCFS1 was grown in Man-Rogosa-Sharpe (MRS) broth  at 30\u00b0C without shaking.The model strain research . Based oresearch , L. planL. plantarum WCFS1 batch cultures (50 mL each) were grown in MRS to an OD600 \u2248 0.8\u20130.9. Then, OLE   was added to a culture of each pair (12 cultures). After 10 min, the cells exposed to OLE and their respective non-induced controls were centrifuged at 4\u00b0C. The pellet was mixed with 2 mL of quenching buffer . Following quenching, the cells were centrifuged at 9,000 \u00d7 g for 10 min at \u221210\u00b0C and suspended in an extraction mixture amino methane, 1 mM EDTA] pH 7.4, 15 mg of polyvinylpolypyrrolidone, and 500 mg of glass beads . The cells were broken under frozen conditions using a cell disruptor (FastPrepTM FP120. Bio101. Savant). Three cycles of 40 s were applied operating at 5 m/s speed with 1 min interval in ice between cycles. The suspension was then centrifuged at 4\u00b0C at 10,000 \u00d7 g for 2 min. After two extractions with 500 mL of chloroform the supernatant containing the RNA was immediately frozen in liquid nitrogen, and stored at \u221280\u00b0C  were applied and the absence of genomic DNA was confirmed by PCR (For RNA isolation twelve paired independent at \u221280\u00b0C . The Nand by PCR .L. plantarum WCFS1 8 \u00d7 15K microarray GE Agilent G2509F Oligo Microarrays (No. 026636) was custom designed and contains 60-mer probes that were based on the gene expression omnibus database (GEO Accession No.GPL5874). The oligo-microarray contained an average of three probes per transcript.Before first-strand cDNA synthesis, RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies). Fluorescently labeled cDNA was obtained by using the SuperScript Indirect cDNA Labeling System (Invitrogen). The Cy3 and HyPer5 fluorescent dyes (Amersham Biosciences) were coupled to the aminoallyl-modified first-strand cDNA, and purification of probes was carried out with the CyScribe GFX Purification Kit. Labeling efficiency was assessed using the NanoDrop ND1000 spectrophotometer. Preparation of probes and hybridization at 65\u00b0C during 17 h was performed as described in the Two-Color Microarray Based Gene Expression Analysis Manual (Quick Amp Labeling) with Tecan HS Pro Hybridization (V. 5.7/Agilent Technologies). Slide \u20131/slope. The expression levels of target genes were normalized. The Bestkeeper analysis  was used as a normalization factor. The expression ratios measured by microarrays and by RT-qPCR assay were plotted, and the linear correlation coefficient was calculated .Real-time RT-qPCR was used to validate the microarray data. Amplification was carried out using a 7500 Fast System (Applied Biosystems). RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems). The specific primers used for the RT-qPCR assays were designed with the Primer Express 3.0 software and listed in the analysis  was applp-values were <0.05 and had a fold change (FC) equal or higher than \u00b11.5. FC was calculated as the average of the FC between significantly regulated probes. Hybridizations and statistical analysis were performed by the Genomics Facility at National Center for Biotechnology, CSIC, Spain.Images were captured with a GenePix 4000B (Axon) and spots quantified using GenPix software (Axon). Background correction and normalization of expression data were performed using the methods normexp and loess in LIMMA, respectively . The expp-values less than 0.1 were considered as significant.Finally, analysis of Gene Ontology (GO) terms and KEGG pathways associated to differentially expressed genes, was performed using DAVID tool . In the GSE90719.The microarray data provided in this study have been deposited in NCBI Gene Expression Omnibus  genomicsL. plantarum WCFS1 after 10 min of OLE stress were determined by global transcriptome profiling using DNA microarrays. The short exposure time to OLE was chosen because we were interested in the very first transcriptional responses which can be masked by secondary effects activated with increasing exposure time. The OLE concentration used [15 mM (0.8%)] is similar to some of the amounts detected in several parts of the olive such as 0.3\u20130.5% found in olive tissues  (t = 0). The DE genes were functionally classified according to their Cluster Orthologous Groups (COGs) categories (The molecular responses of ctively) , 0.7% inctively)  or 0.4% ctively) . The anategories . From thThe enriched GO-DIRECT categories and KEGG pathways resulting from the functional analysis with DAVID  are desclamBDCA operon (The presence of OLE greatly affected the expression of two quorum sensing (QS) systems, one required for the production of plantaricin and other required for the production of LamD558, a cyclic thiolactone autoinducing peptide encoded by the A operon .L. plantarum WCFS1 the enzymatic machinery required for plantaricin production is encoded by the pln locus, which contains five operons  , plnJKLR  operons, which respectively code the EF and JK plantaricins; plnGHSTUVWXY  which codes for the ABC transporter and accessory proteins required to transport and secrete the peptides; and plnMNOP (plnP or lp_0412) coding for proteins of unknown function, were downregulated (In  operons . The moslp_0418) . This opln locus . Accordiegulated .lamC or lp_3581 (up to 3-fold) coding for the sensor histidine protein kinase (HPK) and lamD or lp_3581a (up to 4.6-fold) coding for the precursor auto-inducer peptide (AIP) of this system  QS-systems that are downregulated by OLE have been reported to be involved in immunomodulation  the prophage P2b protein 21 (lp_2460)    and complp_2650) , were allp_2650) .L. plantarum genome, cps1 , cps2 , cps3  and cps4  and cps4 . These genes code for proteins involved in different steps of repeating unit synthesis, export and polymerization of polysaccharides.Genes associated with the GO terms \u201cextracellular polysaccharide biosynthetic process\u201d and \u201ctransferase activity, transferring glycosyl groups\u201d were significantly enriched in the DAVID analysis . These glp_3589), which produces acetyl-P from pyruvate and the pyruvate dehydrogenase complex  which forms acetyl-CoA (the precursor of acetyl-P) from pyruvate. In addition, genes coding for enzymes of the acetyl-CoA complex , which use acetyl-CoA to initiate the fatty acid biosynthesis (fab), and the acetate kinase (lp_0210) which forms acetate from acetyl-P, were downregulated.Genes encompassed within the GO term \u201cpyruvate metabolism\u201d were also significantly enriched in the DAVID analysis . This relp_0171) was upregulated whereas the DHA phosphotransferase encoding genes  were downregulated. In addition, the glycerol-3-phosphate dehydrogenase (glpD or lp_0371) and the glycerol kinase (glpK or lp_0370) were up-regulated , the sugar alcohol mannitol (pts2CB or lp_0230), threalose (pts5ABC or lp_0265), cellobiose  and fructose (pts16ABC or lp_2097) were all induced in presence of OLE. In addition, the maltose proton transporter  was also up-regulated. In contrast, two PTS systems involved in the transport of presently not known \u03b2-glucosides  and PTS systems for the transport of N-acetyl-glucosamine (GlcNAc) (pts18CBA or lp_2531), N-acetyl-galactosamine  (pts19B or lp_2650) and GlcNAc/GalNAc (pts19A or lp_2647) were downregulated.The \u201cphosphoenolpyruvate-dependent sugar phospho transferase system (PTS) transporter\u201d  was one fabZ1 or lp_1670, fabD or lp_1673, accB2 or lp_1676, accC2 or lp_1678, accD2 or lp_1679) and elongation  steps of the KEGG pathway \u201cfatty acid biosynthesis\u201d (FAB) (lp_1672) , were down-regulated  and alkaline (asp1 or lp_0929) or cold shock (lp_0997) proteins, were induced by OLE. Some genes known for its role in countering oxidative stress such as npr2 or lp_2544 (NADH-peroxidase), gshR2 or lp_1253 (glutathione reductase), msrA4 or lp_1979 (methionine sulfoxide reductase) were up-regulated or down-regulated . This response indicates that OLE induces anti-oxidant enzymes/proteins.The DAVID analysis also enriched genes encompassed within several categories related to cell proliferation and stress. For example, strong downregulation was observed for up to 11 clustered genes coding for proteins involved in the initiation (s\u201d (FAB)  as well lp_1672) . Other elp_1672) . Nine ofegulated . In addilp_2739 and lp_2740 genes, which is strongly downregulated by OLE (up to 20-fold), is also highly responsive to resveratrol (p-coumaric acid (lp_2741 to lp_2744) encoding an ABC transporter responsive to gallic acid (oppABCDF or lp_1261 to or lp_1265). Interestingly, genes coding for an acetoin ABC transporter , were down-regulated.The expression of a number of ABC transporters with different functions were differently regulated by OLE . Some ofveratrol , p-coumaric acid  and gallric acid . In the lic acid , were alL. plantarum WCFS1 responded to OLE. The expression of rRNA and fab genes, two major groups of genes associated to rapid growth, was markedly reduced in presence of this compound. L. plantarum strongly downregulated several ABC transporters whose expression was shown to be highly responsive to resveratrol   . Our datmulation  and indulp_0449) . Acetylalp_0449) . The effproteins , but alslamBDCA QS-systems shows the capacity of OLE to act as QS antagonist at the transcriptional level. Interestingly, some of the components of the plantaricin and LamBDCA QS-systems are involved in immunomodulation and regulate the cytokine output in immunomodulatory cells  synthesized by this bacterium. Deletion mutants in the four clusters of L. plantarum WCFS1 have been shown to alter immunomodulation by increasing the exposure of MAMPs to their receptors to induce signaling cascades  increase during the course of many biotic and abiotic stress conditions potentially resulting in damage of macromolecules. Interestingly, ROS have signaling roles in plant adaptation to stress, however, their levels have to be moderated during plant adaptation to act in signaling and not to be channeled to damage . The obsopp genes was not accompanied by induction of endopeptidases (required to digest the oligopeptides for nutrition) or muropeptidases (needed for muropeptide recycling). This profile resembles the transcriptional response of L. plantarum to p-coumaric acid (opp gene cluster is required for functions involved in the establishment of plant\u2013microbe interactions, such as the sensing of plant peptide hormones and/or adhere to plant tissues, as previously reported for other microbial opp genes (L. plantarum in presence of resveratrol (The oligopeptide ABC transporter was upregulated by OLE but induction of the involved ric acid  and may pp genes . The repveratrol . Since averatrol , the repL. plantarum to respond to OLE (opp genes. Notably, OLE may significantly modulate the plant immune responses to microbes such as L. plantarum, via down-regulation of bacterial immunomodulatory components, cps downregulation and increasing bacterial protein acetylation. By diminishing expression of the acetoin transporter and probably buffering ROS accumulation, L. plantarum could not undermine the effectiveness of acetoin and ROS as signaling molecules in plant development whereas the QS-antagonist activity of OLE probably help restrict microbial load in plant endosphere compartments. The controlled expression of all of these molecular players suggests that OLE acts a signaling molecule to accommodate and maintain a close association between beneficial microbes such as L. plantarum and the plant host. More broadly, this study indicates that some plant secondary metabolites, in this case OLE, could be recognized as signaling molecules by plant-associated bacteria and modify the expression of genes to interact with the plant host, thus extending the role of such molecules beyond the control of plant immunity. These understandings could pave the way to use OLE for a better management of the olive phytomicrobiome to obtain improved olive growth and resistance to stress.This study increases the insights into the molecular mechanisms used by d to OLE . AccordiThe datasets generated for this study can be found in NCBI Gene Expression Omnibus, GSE90719.RM, BR, and FF conceived and designed the research plans, and supervised the research. LS and IR performed most of the research. LP-V participated in RT-qPCR, and organized and submitted the data to repository databases. JO performed the functional analysis with DAVID. All authors analyzed the data. FF wrote the manuscript with contributions of all the authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Oryza sativa) variety (IR64) was examined to assess the impact of varying temperatures and relative air humidities during day and night periods on biomass, leaf area, and dry matter partitioning between organs. Three different day and night temperature  and relative air humidity  regimes were established. The effect of relative air humidity on both plant dry matter and leaf area was larger than the effect of temperature, in particular low humidity had a strong negative impact during the night. With high day temperature, the shoot mass fraction increased, whereas the root mass fraction decreased. Specific leaf area increased at high night temperatures and led, along with the high leaf mass fraction at high night humidities, to higher growth rates. The results emphasize the importance of considering relative air humidity when focusing on plant responses to temperature, and strongly suggest that under asymmetric day and night temperature increases in the future, biomass partitioning rather than biomass itself will be affected.Asymmetric changes of day and night temperature have already been observed because of Climate Change. However, knowledge on environmental conditions either during day or night serving as trigger for growth processes is scarce. In this study, one rice ( With climate change, temperature increases are not only expected in the coming decades, but have already been observed . Of partAs shown by the contradictory results of the two studies, the role of night temperature per se is not well understood . ExperimAnother constraint of studies on high night temperature under natural conditions is the physical interaction of temperature and relative air humidity. Since minimum night temperature is usually around the dew point, a smaller daily temperature range is directly correlated with higher relative air humidity during the day. However, changes in relative air humidity have been associated to changes in photosynthetic rates , dry matIn a field study, high night temperature led to biomass reduction in one of the two rice genotypes , whereasAs plants must achieve a balance between carbon assimilation (occurring during the day) and storage and growth (occurring during both day and night) , there aRice plants were grown at three different temperature regimes with either \u201cnatural\u201d , constant , or inverted  day/night temperature in combination with three different relative air humidity (RH) regimes with either \u201cnatural\u201d , constant , or inverted  day/night RH. Two additional treatments with either constantly low  or high  temperature, both at RHcon, were established. After two weeks of different day/night temperature and RH treatment with the same mean temperature and RH, total plant dry matter varied between 6.0 g for Tnat/RHcon and 10.4 g for Tnat/RHnat .Temperature had no statistically significant effect , but RHn2 under Tcon/RHinv to 890 cm2 under Tinv/RHcon. Temperature only had a significant effect under RHcon, with the highest leaf area at Tinv (890 cm2), and the lowest at Tnat (341 cm2). RH had a larger effect on leaf area than temperature, with RHinv leading to a significantly lower leaf area of 356 cm2 on average in comparison to 621 cm2 and 596 cm2 under RHnat and RHcon, respectively. Under constant temperature and relative humidity, both total dry matter and leaf area were significantly lower at 20 \u00b0C  than at 25 \u00b0C  and 30 \u00b0C   .Leaf mass fraction (LMF), the leaf dry weight per plant dry weight, did not respond to temperature, but RH had a statistically significant effect .\u22121, the LMF was significantly lower under RHinv than under RHnat with 0.24 g g\u22121 and RHcon with 0.27 g g\u22121 (\u22121), than at 20 \u00b0C (0.23 g g\u22121), and 25 \u00b0C (0.25 g g\u22121)  than under Tnat and Tcon, which both resulted in a SMF of 0.41 g g\u22121. Also RH had an effect on SMF, which was significantly lower (0.38 g g\u22121) under RHcon than under RHnat (0.40 g g\u22121) and RHinv (0.41 g g\u22121). Constant temperature between 20 and 30 \u00b0C did not have any significant effect on SMF.With an average of 0.20 g g27 g g\u22121 . Under t\u22121 for Tnat/RHnat and Tnat/RHcon and 0.38 g g\u22121 for Tinv/RHnat and Tinv/RHinv and was strongly influenced by temperature. Under Tnat, RMF was on average 0.31 g g\u22121 and significantly lower than under Tcon and Tinv with 0.34 g g\u22121 and 0.37 g g\u22121, respectively. RH as well as different constant temperatures between 20 and 30 \u00b0C had no effect on RMF. The faction of dead leaves (DLMF) varied between 0.00 g g\u22121 for Tinv/RHcon and 0.06 g g\u22121 for Tnat/RHcon and Tnat/RHinv. Nevertheless, there were no significant differences because of large variation within treatments.The root mass fraction (RMF), the root dry weight per plant dry weight, varied between 0.31 g g2 kg\u22121 (Tnat/RHinv) to 32.5 m2 kg\u22121 (Tinv/RHcon) (The SLA varied largely and ranged from 19.7 mv/RHcon) .2 kg\u22121 followed by Tcon with 25.9 m2 kg\u22121 and Tinv with 28.8 m2 kg\u22121. Among the RH treatments, SLA was significantly lower under RHinv with 22.8 m2 kg\u22121 than under RHnat and RHcon with 26.6 and 28.0 m2 kg\u22121, respectively. Further, a significant interaction effect between temperature and RH was found. Under Tnat, RHnat resulted in a significantly higher SLA compared to RHinv, whereas under Tcon and Tinv, RHcon resulted in a significantly higher SLA compared to RHinv. At RHnat, temperature had no significant effect on SLA, while at RHcon, Tinv and Tcon resulted in a significantly higher SLA than Tnat and at RHinv, Tinv resulted in a significantly higher SLA than Tnat. Among the constant temperature treatments, no significant difference was found in regard to SLA.Both temperature and RH had large effects on SLA. On average, Tnat led to the lowest SLA with 22.6 mr = 0.65; p = 0.030)  .r = 0.76; p = 0.006) and the root mass fraction was negatively correlated with day temperature  and positively with night temperature . Leaf and dead leaf mass fractions were not correlated with any of the parameters.The stem mass fraction was positively correlated with day temperature  were germinated in petri dishes on filter paper for one week. Individual seedlings were transferred into pots containing 1 l of half strength nutrient solution in the composition proposed by . After a2 kg\u22121].For each set of plants, destructive samplings were conducted on three plants at the onset of treatments (0 DOT) and at 14 DOT. Plants were individually separated into green leaf blades (hereinafter referred to as leaves), leaf sheaths (hereinafter referred to as stems), roots, and dead leaves. Leaves were considered as dead, when the leaf blade was either completely yellow or by at least 50% dry and dead. Leaf area of all green leaves was measured with a LI-COR leaf area meter (LI-3000C) in combination with a belt conveyer accessory (LI-3050C). All plant material was dried at 70 \u00b0C for at least 48 h and weighed. Specific leaf area (SLA) was calculated as green leaf area per kg of leaf dry matter of the whole plant [mp < 0.05.Data analysis was carried out with STATISTICA 13  using anHigh night temperature resulted in higher leaf area, but only under constant relative air humidity, whereas under low air humidity during the day and high air humidity during the night, high night temperature had no effect on biomass or leaf area. Nevertheless, diurnal temperatures highly affected the partitioning between plant organs. The fraction of stems increased with higher day temperature, whereas the fraction of roots increased with higher night temperature. The leaf mass fraction was only affected by the day and night pattern of relative air humidity, but SLA strongly responded to both, temperature and relative humidity. The higher leaf mass fraction during humid nights, and higher SLA during warm nights were associated with higher leaf growth rates. Therefore, we argue that rice plants might benefit from an asymmetric day/night temperature increase, at least during vegetative growing phases."}
+{"text": "Propionibacterium freudenreichii is a beneficial bacterium, used as a cheese starter, which presents versatile probiotic properties. These properties are strain-dependent. We hypothesized they may also be delivery vehicle-dependent. In this study, we thus explored in healthy piglets how the cheese matrix affects the immunomodulatory properties of P. freudenreichii. During 2 weeks, three groups of weaned piglets consumed, respectively, P. freudenreichii as a liquid culture (PF-culture), P. freudenreichii under the form of a cheese (PF-cheese), or a control sterile cheese matrix (Cheese-matrix). The in vivo metabolic activity of P. freudenreichii was assessed by determining short chain fatty acids (SCFA) concentration and bifidobacteria population in feces. Whatever the delivery vehicle, P. freudenreichii was metabolically active in piglets\u2019 colon and enhanced both bifidobacteria and SCFA in feces. P. freudenreichii consumption decreased the secretion of TNF\u03b1 and of IL-10 by peripheral blood mononuclear cells (PBMC). It did not alter IL-10, IFN\u03b3, IL-17, and TNF\u03b1 secretion in mesenteric lymph node immune cells (MLNC). PF-cheese enhanced significantly Treg phenotype, while PF-culture decreased significantly Th17 phenotype in PBMC and MLNC. Remarkably, only PF-cheese induced an increase of Th2 phenotype in PBMC and MLNC. Ex vivo stimulation of PBMC and MLNC by Lipopolysaccharides and Concanavalin A emphasized the difference in the immunomodulatory responses between PF-culture and PF-cheese group, as well as between PBMC and MLNC. This study shows the importance to consider the delivery vehicle for probiotic administration. It confirms the anti-inflammatory potential of P. freudenreichii. It opens new perspectives for the use propionibacteria-fermented products as preventive agents for inflammatory bowel diseases and intestinal infectious diseases. Propionibacterium freudenreichii is a beneficial bacterium, belonging to the Actinomycetales order. It has been recognized as safe (GRAS status) in the United States of America, and qualified presumption of safety (QPS status) in Europe. P. freudenreichii is a cheese starter used in Swiss-type cheeses manufacture such as Emmental. It moreover revealed versatile, strain-dependent, probiotic functionalities ,which enhances bifidobacteria growth and reduces inflammation in intestinal epithelial cells   in preterm infants  . The devs (Slps) . Underst colitis . The ing infants . These desponses . In addiesponses , 2018. Sin vitro . In thistory one . The impP. freudenreichii CIRM-BIA 129  was provided by the French Dairy Interbranch Organization  and maintained by the International Centre for Microbial Resources . Dairy propionibacteria were routinely cultivated at 30\u00b0C in yeast-extract-lactate medium (YEL). For the PF- culture, P. freudenreichii CIRM-BIA 129 was grown in milk ultrafiltrate supplemented with 100 mM sodium DL-lactate  and 5 g/L casein hydrolysate    at 30\u00b0C,eviously . The bioeviously . The cheeviously . The proThe experimental protocol was performed in accordance with recommendations of the French law (2001-464 29/05/01) and EEC (86/609/CEE) for the care and use of laboratory animals. The protocol was approved by the ethical committee on animal experimentation of Rennes (France), under the certificate of authorization to experiment 2017010922379066-V2. Pigs were sacrificed by electronarcosis followed by exsanguination, and every effort was made to minimize animal suffering.11 CFU of P. freudenreichii) and (3) PF-cheese (1.1011 CFU of P. freudenreichii). PF-cheese and cheese matrix were mixed by a turrax  in four volumes of sterile physiological water. Piglets were gavaged using syringes every morning (between 9.00 and 10.00 am) during 14 days. They were fed with a standard pig diet that was given at 10.00 am. Food was removed from the cage at 4:00 pm to monitor daily food intake. Animals were fasted from 4:00 pm to 9:00 am but had free access to water. Piglets were weighed five times  and fecal samples were collected at day 0, day 7, and day 14. At the end of the 14-day treatment period, pigs were sacrificed 30 min after their last gavage by electronarcosis then exsanguination. Blood was collected in sterile BD vacutainer\u00aeCPTTM tubes (containing sodium heparin as well as FicollTMHypaqueTM density fluid and a polyester gel barrier) at room temperature. Following centrifugation at 1500 g for 20 min without brake, we isolated the peripheral blood mononuclear cells (PBMC) by carefully pipetting the interface above the gel barrier. Additional washing in Hank\u2019s balanced saline solution (HBSS), supplemented with 200 UI/ml penicillin and 200 \u03bcg/ml streptomycin, and centrifugation steps resulted in a suspension of concentrated mononuclear cells. After laparotomy, 4 g of intestinal mesenteric lymph nodes (MLN) were placed in ice-cold HBSS for mononuclear cell isolation, as already described (Twenty one [(Pietrain \u00d7 Landrace) \u00d7 (Large White)] 8-week old piglets (13.3 \u00b1 0.4 kg) from the experimental herd of INRA St-Gilles  were used. Three groups of seven piglets were constituted: (1) Cheese matrix (10 g), (2) PF-culture  and thoroughly mixed. DNA was then extracted in the same way as unknown samples. A standard curve was generated and results are expressed as log [bacteria] per gram of sample. For lactobacilli and bifidobacteria quantification, 10-fold serial dilutions of target genomic DNA extracted from pure cultures of B. longum CIRM-BIA 1336 or L. pentosus CIRM-BIA 660 were performed  and day 14 (end of the treatment). Samples were analyzed in duplicate. For propionibacteria quantification, QIAamp DNA Stool Kit was used to extract DNA, as described previously . For lacShort chain fatty acid concentration was determined in fecal samples at days 7 and 14. Immediately after collection, fecal samples were diluted in ortho-phosphoric acid (50% V/V) to stop fermentation and samples were stored at \u221220\u00b0C until analysis. SCFA were separated on a BP20 (SGE) column and quantified by a flame ionization detector as previously described . Isocapr6 cells/ml for PBMC and 10 \u00d7 106 cells/ml for MLNC in 96-well flat-bottomed plates. Cells were stimulated for 72 h at 37\u00b0C, under an atmosphere containing 5% CO2, in unstimulated condition  or in presence of 200 \u03bcg/ml of P. freudenreichii\u2019 S-layer proteins (Slps). Slps were extracted , or a combination of Slps + LPS and Slps + ConA. Culture supernatants of PBMC and MLNC were harvested and stored at \u221220\u00b0C until assayed for cytokine detection. Remaining cells were re-suspended in FCS 10% DMSO  and stored at \u2212150\u00b0C until mRNA extraction.Immune cells were suspended in complete RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin and 100 mg/ml streptomycin to achieve cell concentration of 5 \u00d7 10xtracted , partialxtracted  and protTM, Kingfisher Biotech, United States). Cytokine concentrations after stimulation were given in pictograms per ml of supernatant.Concentrations of IL-10, IFN\u03b3, and TNF\u03b1 were measured in culture supernatants of PBMC and MLNC, using capture sandwich ELISA porcine ELISA kit  according to the manufacturer\u2019s instructions. IL-17 concentration was measured also using capture sandwich swine ELISA kit , in duplicate analysis for each piglets (n = 7) for the three groups.Quantitative PCR was performed to determine prt gene  . All data were expressed as mean values and standard error of the mean (SEM) (n = 7).We analyzed all data with non-parametric tests after checking the non-Gaussian distribution of data. The effect the consumption of Food intake was similar among the three experimental groups . The impact of P. freudenreichii consumption on piglet\u2019s microbiota was investigated, focusing on two genera, using qPCR. Lactobacilli and bifidobacteria populations between the different treated groups at day 14 were compared to the population level at day 0. P. freudenreichii ingestion significantly enhanced bifidobacteria in feces . Propionibacteria remained also undetectable in the cheese matrix piglets group\u2019s feces Figure . After 2s Figure . By conts Figure .in vivo metabolic activity of P. freudenreichii, SCFA concentration were determined in feces at days 7 and 14. At day 7, SCFA concentrations in feces were equivalent among the different groups    Figure . P. freu) Figure . By cont) Figure . Regardi) Figure . In addi) Figure . The pro) Figure .P. freudenreichii Slps on naive swine immune cells as already demonstrated in other species (p = 0.0763)  Figure . Slps si) Figure . Only Co) Figure .Propionibacterium freudenreichii consumption decreased basal secretion of IL-10 by PBMC, compared to cheese matrix group, whatever the delivery vehicle    Figure . PBMC fr) Figure . By dete) Figure . The Tre) Figure .P. freudenreichii, regardless of the delivery vehicle, significantly increased the Treg/Th17 ratio  to secrete more TNF\u03b1 in response to ConA stimulation  or a single-strain cheese  fermented solely by P. freudenreichii CIRM-BIA 129.The aim of this study was to investigate the impact of the delivery vehicle on the probiotic functionalities of P. freudenreichii was metabolically active in piglet colon. P. freudenreichii metabolically active enhanced SCFA concentration in rats , while inducing branched-chain amino acid degradation (suggesting an increase of BCFA production)  . The modduction) . In a pr piglets . Howeverex vivo the immunomodulatory effect of extracted P. freudenreichii Slps on PBMC and MLNC from naive piglets (cheese matrix group). As already demonstrated with human PBMC  were isolated. At the basal state, cytokines secretion and T lymphocytes phenotypes were analyzed. P. freudenreichii consumption, whatever the delivery vehicle, modulated Treg/Th17 ratio, compared to cheese matrix group. Nevertheless, PF-culture decreased significantly Th17 phenotype, compared to cheese matrix group. Contrastingly, PF-cheese tended to enhance Treg phenotype, compared to the cheese matrix group. In addition, only PF-cheese significantly modulated PBMC Th1/Th2 ratio toward a Th2 phenotype. However, basal cytokine secretions were not consistent with T lymphocytes PBMC phenotypes investigated by qPCR. P. freudenreichii consumption, in both delivery vehicles, decreased basal PBMC secretion of IL-10 (a cytokine produced by Treg and Th2 cells) and of TNF\u03b1 (a cytokine produced by Th1 cells), compared to cheese matrix group. Basal cytokine secretion is a global response of T, B cells and innate immune cells (macrophages and dendritic cells), which may explain these discrepancies. Moreover, we did not evaluate IL-4 basal secretion, a marker of Th2 T cells. Finally in vitro studies showed P. freudenreichii CIRM-BIA 129 as an inducer of IL-10 and TNF\u03b1 in human PBMC (in vivo results obtained in this study. This result suggests that chronic ingestion of P. freudenreichii affects differently immune cells than ex vivo acute stimulation. Noteworthy, P. freudenreichii consumption inhibited IFN\u03b3 secretion by PBMC in response to ConA stimulation, regardless of the delivery vehicle. Further research is needed to understand this delivery vehicle-induced switch in TNF\u03b1/IFN\u03b3 secretion.To explore the immunomodulatory effects of man PBMC , which iP. freudenreichii consumption was also investigated. P. freudenreichii, regardless of the delivery vehicle, did not affect basal secretion of IL-10, IL-17, TNF\u03b1, or IFN\u03b3. Once again, cytokine secretion patterns were not consistent with the phenotype of T lymphocytes in the different piglet groups. The effects of P. freudenreichii on T lymphocytes phenotypes of MLNC were similar to that of PBMC. PF-cheese enhanced Treg phenotype and PF-culture decreased Th17 phenotype, compared to cheese matrix group. Moreover, PF-cheese enhanced Th2 phenotype, compared to cheese matrix group, but without significant difference with the PF-culture group. MLNC responses to LPS and ConA stimulation showed different responses between PF-cheese and PF-culture groups from that of PBMC. Indeed, in response to LPS stimulation, PF-culture consumption enhanced IL-10 and TNF\u03b1 secretion by MLNC, compared to untreated cells, while PF-cheese consumption induced a slight, yet significant increase in IFN\u03b3 secretion. In addition, only MLNC from PF-cheese group showed a high secretion of IL-10 and IFN\u03b3, compared to untreated cells. ConA in naive MLNC did not induce secretion of IFN\u03b3. Moreover, there was no significant difference between the three groups in Th1 phenotypes, an IFN\u03b3-secreting phenotype (P. freudenreichii CIRM-BIA 129 in functional foods to prevent intestinal infections, as already shown with Listeria monocytogenes infection in mice model (in vivo pathogens challenge experiments in piglets.The intestinal immune response to henotype . IFN\u03b3 mahenotype . This rehenotype . In addice model . This shP. freudenreichii showed an anti-inflammatory effect on the systemic and intestinal immune system by enhancing Treg and Th2 phenotypes or decreasing Th17 phenotype, depending on the delivery vehicle. Th2 and Treg responses triggered by B. breve was shown to protect mice from chemically induced colitis (P. freudenreichii in cheese against colitis (P. freudenreichii P.UF1 was shown to increase Th17 population, to sustain Treg population and to decrease Th1 population, via a S-layer protein, in mice (Altogether,  colitis . This ma colitis . Previou in mice , 2018.P. freudenreichii between the two delivery vehicles was observed, we assumed also that the matrix-dependent immunomodulatory effect may be related to surface proteins that would be protected by the cheese matrix (P. freudenreichii CIRM-BIA 129 to interact with host immune system.In this study, since a similar metabolic activity of e matrix . More inP. freudenreichii exerts an anti-inflammatory effect, regardless of the delivery vehicle. The difference between vehicles in term of immunomodulatory modulation was obvious after ex vivo stimulation of immunes cells by LPS and ConA, in particularly at the intestinal level. Our study shows that the delivery vehicle should be carefully considered. It opens the perspective to use P. freudenreichii in cheeses to prevent IBD or intestinal infectious diseases.The present study demonstrated that GB, GJ, and SF-B designed the study. HR and SH prepared all matrices. HR, SH, FG, RJ, SF-B, LLN, GB, and GJ participated in animal experiment and laboratory analyses. HR and GB analyzed data and prepared figures. HR wrote the manuscript with the help of GB, FC, SF-B, and GJ. RJ, GB, and GJ supervised the project. All authors read and approved the final manuscript.FG was employed by company Bioprox. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "In conclusion, the present work provides evidence that glucagon plays an important role in the preventive effect of menthol against HFD-induced weight gain and related complications.Glucagon mediated mechanisms have been shown to play clinically significant role in energy expenditure. The present study was designed to understand whether pharmacological mimicking of cold using menthol (TRPM8 modulator) can induce glucagon-mediated energy expenditure to prevent weight gain and related complications. Acute oral and topical administration of TRPM8 agonists (menthol and icilin) increased serum glucagon concentration which was prevented by pre-treatment with AMTB, a TRPM8 blocker. Chronic administration of menthol (50 and 100 mg/kg/day for 12 weeks) to HFD fed animals prevented weight gain, insulin resistance, adipose tissue hypertrophy and triacylglycerol deposition in liver. These effects were not restricted to oral administration, but also observed upon the topical application of menthol (10% w/v). The metabolic alterations caused by menthol in liver and adipose tissue mirrored the known effects of glucagon, such as increased glycogenolysis and gluconeogenesis in the liver, and enhanced thermogenic activity of white and brown adipose tissue. Correlation analysis suggests a strong correlation between glucagon dependent changes and energy expenditure markers. Interestingly,  Isopropyl alcohol, trizol reagent, fetal bovine serum (FBS), and penicillin/streptomycin (P/S) were purchased from invitrogen . Mouse 3T3-L1 preadipocyte (3T3-L1 PA), Dulbecco's modified Eagle's medium (DMEM), maintenance media (MM) and differentiation medium (DM) were purchased from Zenbio . L-168,049 (4-[3-(5-Bromo-2-propoxyphenyl)-5-(4-chlorophenyl)-1H-pyrrol-2-yl] pyridine; PubChem CID: 5311276) was purchased from Tocris Bioscience .Chemical compounds studied in this article-Menthol; Icilin; AMTB hydrochloride; L-168,049ad libitum. This study was carried out in accordance with the recommendations of \u201cCommittee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)\u201d guidelines on the uses and care of experimental animals. The protocols used in this study were approved by \u201cInstitutional Animal Ethics Committee (IAEC) of NIPER.\u201d After procurement, mice were acclimatized to experimental conditions for one week followed by weight based randomization of animals for each experiment separately.Male Swiss albino mice  were obtained from and housed in the Central Animal Facility, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, Punjab, India. Animals were maintained under standard laboratory conditions  and provided with diets and water All the acute studies were done in 6 h fasted animals. Following experiments were performed during acute studies. Doses and route of administration of agents were determined on the bases of previous literature  Temperature in MiceExperiment 1B: To study the effect of pre-treatment of TRPM8 antagonist, AMTB on orally administered menthol induced increase in serum glucagon levels and core body  temperature in Micep.o.), icilin (20 mg/kg p.o.), and pre-treatment of AMTB (4 mg/kg i.p.) followed by menthol (200 mg/kg) respectively. In groups 1, 2, 3 rectal temperature was measured upto 120 min and serum glucagon levels were measured at 120 min. In pre-treatment group, AMTB was administered 30 min prior to menthol administration and rectal temperature measurement was done for next 120 min and serum glucagon levels were measured at 120 min after the menthol administration.Animals were divided in 4 groups of 6\u20138 animals/group. Group 1, 2, 3, 4 were administered vehicle, menthol , icilin (1 % w/v), and pre-treatment of AMTB (1 % w/v) followed by menthol (10 % w/v) respectively in the selected region of abdominal area. In groups 1, 2, 3 rectal temperatures were measured for 120 min and serum glucagon levels were measured at 120 min. In pre-treatment group, AMTB was administered 30 min prior to menthol administration and rectal temperature measurement was done for next 120 min and serum glucagon levels were measured at 120 min after the menthol application.i.p. dose. 0.2% tween 80 solution was administered/applied as vehicle.Doses of menthol and icilin were prepared in 0.2% Tween-80 using traditional pharmaceutical technique. Briefly, weighed amount of chemical was carefully triturated with required amount of tween 80 and then remaining solvent was mixed step wise with continuous trituration. AMTB was dissolved in 0.9% saline water for Experiment 3: The effect of chronic oral and topical co-administration of menthol against HFD-induced weight gain and associated complicationsp.o.) with HFD, menthol (100 mg/kg p.o.) with HFD, topical menthol (10 % w/v) with HFD, and menthol (100 mg/kg p.o.) with NC, respectively. 0.2% tween 80 was administered as vehicle in NC and HFD fed mice. All the animals were given respective treatments for 12 weeks with weekly measurement of body weight. During 13th week, core body rectal temperature and oral glucose tolerance test was performed . On the day of sacrifice, animals were fasted for 6 h and blood was collected for different biochemical tests and ELISA including glucagon levels (see details in methods section). Animals were sacrificed and tissue , BAT, liver, hypothalamus) were collected, processed, and stored at \u221280\u00b0C for further biochemical, gene expression immunohistochemistry and histological analysis (see details in methods section).Animals were divided in 6 groups of 6\u20138 animals/group. Group 1, 2, 3, 4, 5, 6 were administered NC , HFD (60 % energy by fat), menthol  was prepared in-house by earlier reported method /naso-anal length; , Area under the concentration-time curve (AUC) was calculated using graph pad prism 6 software.During the last week of chronic experiment, oral glucose tolerance test (OGTT) was performed. On the day of experiment mice were fasted for 6 h. Blood glucose concentration was measured after 15, 30, 45, 60, 90, and 120 min of oral administration of 2 g/kg of D-glucose using GlucocardBlood was allowed to coagulate for 20 min on ice followed by centrifugation at 2,500 g for 15 min at 4\u00b0C to obtain serum. Serum triglyceride, glucose , and free fatty acids (FFA) were estimated by colorimetric assay. Glucagon, insulin , leptin, adiponectin , FGF21 and pAMPK  levels in serum and different tissues were determined using ELISA as per the manufacturer's instructions.vWAT and BAT were weighted. Further, these were microwave digested using HNO3 . These digested samples were used for metal analysis by using Inductively Coupled Plasma Mass Spectrometry . Iodine reagent was prepared by dissolving 16.5 ml Lugol's solution (1 g iodine and 2 g of KI in 20 ml water) to 990 ml of 25% KCl solution.Liver glycogen was estimated using classical iodine method  relative to the control group. Sequences of primers are presented in Table Total RNA from vWAT, BAT, and liver was extracted using Trizol based RNA extraction method described elsewhere  and all the images were captured using 20X objective . The mean adipocytes size in adipose tissue sections were estimated in 5 different tissue images using Imagescope  by manual counting.Adipose tissue was UCP1 primary antibody  followed by incubation with FITC leveled goat anti-rabbit secondary antibody  for 2 h at room temperature. DAPI was used to counter stain the nucleus of the cells.For immunohistochemistry, tissue sections were deparaffinised followed by antigen retrival using citrate buffer method. Five percent v/v goat serum was used to prevent nonspecific antibody binding. Slides were incubated overnight with rabbit poly clonal anti-3T3-L1 preadipocytes  were maintained in DMEM supplemented with FBS (10% v/v) and antibiotics (P/S (1 % v/v) until confluence. Differentiation of 3T3-L1 preadipocytes was induced by replacing DMEM with differentiation media for two days. Then, differentiation media was replaced by maintenance media for next 8\u201310 days with media replacement on every second day. On maturation, adipocytes were treated with maintenance media in which FBS was replaced with serum collected from vehicle and menthol treated animals. mRNA expression analysis of energy expenditure genes at different time points of treatment  was carried out. The effective treatment schedule was again repeated in the presence of L-168,049, a non-competitive glucagon receptor antagonist, at reported effective dose (0.3 \u03bcM) . P \u2264 0.05 was considered statistically significant.All the values are expressed as mean \u00b1 SEM. Group comparison was done using One way/Repeated measure ANOVA followed by Tukey's TRPM8 agonists, menthol (200 mg/kg) and icilin (20 mg/kg) elevated serum glucagon levels after 2h of administration Figure . Simultaper se effect)  for 12 weeks significantly prevented HFD induced weight gain Figure  and reve) Figure . HFD fee) Figure . HFD-ind) Figure  was prev) Figure  On the s) Figure , reduced) Figure .PEPCK) and glucose 6 phosphatase (G-6pase) increased significantly after menthol administration in both HFD as well as NC fed mice  expression  increased core body rectal temperature in mouse Figure . Chronice Figure . Signifin Figure . There wn Figure .HFD fed animals display impaired glucose clearance in OGTT, high serum insulin, serum free fatty acids and high liver TG levels Figures . Both orbrowning markers i.e. Uncoupling Protein 1 (UCP 1), Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1\u03b1), PR domain containing 16 (PRDM16), Cell death-inducing DFFA-like effector A (CIDA), Forkhead box protein C2 (FOXC2) and TBX1 in vWAT and BAT  in vWAT  in maintenance media. During the adipogenesis, cells treated with serum of menthol treated animals have significantly higher expression of marker genes responsible for energy expenditure and thermogenesis as compared to untreated cells Figure . Furtherin lieu of their demand of sustained increase in energy supply during these conditions  TRPM8 has significant functional role in environmental temperature detection; and (b) it is non-noxious (between ~15 and 30\u00b0C) in nature (McKemy et al., Acute cold exposure results in increase in oxygen consumption and serum glucagon levels while the levels of serum insulin and blood glucose remains unchanged (Seitz et al., browning\u201d phenotype in 3T3L1 mature adipocytes after 16-h treatment of this serum, hence suggesting that glucagon is potentially playing a role and is sufficient to induced \u201cbrowning\u201d machinery in adipocytes. To confirm this, we repeated the experiment in the presence of specific glucagon receptor antagonist and interestingly the effect was prevented. This substantiate that glucagon secreted during menthol treatment was sufficient and selective in inducing \u201cbrowning\u201d in WAT.In recent years, it has been experimentally proven that non hypoglycaemic release of glucagon is associated with energy expenditure (Billington et al., brite\u201d adipocytes (Wang et al., via their involvement in mitochondrial function is not surprising. Our study also implied that HFD administration significantly decreased the metal concentration and the decrease was significantly prevented in the presence of menthol administration, specifically at higher doses of menthol, in WAT and BAT. We can argue that due to energy expenditure ability and prevention of lipid droplet accumulation, unit weight of adipose tissues must be having more mitochondria, hence more concentration of both metals. Our study has shown similar results in both WAT \u201cbrowning\u201d and BAT activation, hence are in contrast with the previous study. Also, magnesium and zinc concentration is altered during HFD administration and was significantly prevented on co-administration of menthol. Given, the role of magnesium and zinc in glucose utilization, it is not surprising that during \u201cbrowning\u201d or BAT activation there is significant increase in their concentrations due to high energy expenditure state. This is the first report linking these metal concentrations in tissues with HFD and \u201cbrowning.\u201d These have significant implications going forward as these can be common link between obesity/diabetes and micronutrient deficiencies. Although we are able to casually link the possible role of glucagon in menthol induced energy expenditure, but a clear mechanism is still lacking. Earlier studies suggest that glucagon infusion independently increased whole body energy expenditure but unable to increase BAT glucose uptake in human subjects (Salem et al., Recently, there is literature related to the increase in levels of iron and copper during brown adipose tissue activation but in the same study they have mentioned that there is no change in the levels of these metals in \u201cThe present study provides evidence of TRPM8 mediated glucagon dependent mechanism of energy expenditure Figure . InteresMB, KC, and PK generated the hypothesis. MB, KC, PK, SS, and KK designed the experiments and wrote the manuscript. PK, PM, SJ, RB, VK, and DS performed the experiments, diet preparation, and data calculation. MB, KC, SS, KK, RK, SB, and RaKBo helped in manuscript writing and data presentation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "In this work, we aim to investigate whether information based metrics of neural activity are a useful tool for the quantification of consciousness before and shortly after birth. Neural activity is measured using fetal magnetoencephalography (fMEG) in human fetuses and neonates. Based on recent theories on consciousness, information-based metrics are established to measure brain complexity and to assess different levels of consciousness. Different metrics  are, thus, explored in a reference population and their usability is evaluated. For comparative analysis, two fMEG channels were selected: one where brain activity was previously detected and one at least 15 cm away, that represented a control channel. The usability of each metric was evaluated and results from the brain and control channel were compared. Concerning the ease of use with fMEG data, Lempel-Ziv-Complexity (LZC) was evaluated as best, as it is unequivocal and needs low computational effort. The fractality measures have a high number of parameters that need to be adjusted prior to analysis and therefore forfeit comparability, while entropy measures require a higher computational effort and more parameters to adjust compared to LZC. Comparison of a channel with brain activity and a control channel in neonatal recordings showed significant differences in most complexity metrics. This clear difference can be seen as proof of concept for the usability of complexity metrics in fMEG. For fetal data, this comparison produced less clear results which can be related to leftover maternal signals included in the control channel. Further work is necessary to conclusively interpret results from the analysis of fetal recordings. Yet this study shows that complexity metrics can be used for fMEG data on early consciousness and the evaluation gives a guidance for future work. The inconsistency of results from different metrics highlights the challenges of working with complexity metrics as neural correlates of consciousness, as well as the caution one should apply to interpret them. Consciousness is known to be one of the characteristics that make humans unique. But when does this aspect of the human mind arise? Is it possible that consciousness already exists before birth? From the 24th week of gestation, a fetus can process sensory stimuli at a cortical level, as thalamocortical connections are already established  and healthy subjects in different sleep stages. In addition, sedation with different anesthetics can be differentiated , whereby in both cases patients are behaviorally unresponsive but in case of Ketamine they report vivid dreams , placed on one ear. For neonatal recordings, a cradle is attached to the fMEG device. The neonate is attended by one parent inside the measurement room and is measured asleep or quiet awake.fMEG is a non-invasive tool to measure heart and brain activity in fetuses in the last trimester of pregnancy and in neonates shortly after birth  and theta range (4\u20138 Hz) was calculated for further comparisons.To measure the informational complexity of the fMEG signal, multiscale entropy (MSE) and multiscale permutation entropy (MPE) were used, as well as LZC. Additionally, we included the geometrical properties of the signal and measured its amount of fractality. As an approximation for this, the CD, also known as dimensional complexity  and N = 15360 which is a bit lower than the recommended N = 2*104 but was selected in terms of comparability of the different methods and did not show any disadvantage compared to an example analysis with a larger N. We chose a window size between 1 and 20 to calculate the SE for 20 different scales whereas scale one equals the original time series. A time series is considered as more complex than another if a majority of scales show higher entropy values  for multiple time scales. In comparison to other complexity measures, PE is very robust towards noise LZC is a measure closely related to Kolmogorov complexity and Shannon entropy , which in our case is done by a median split as a threshold. In particular, values below the median are indicated as zero, whereas values above the median as one. The median split is relatively robust to outliers compared to other methods , and the description length of the dictionary (lLZC) is defined as the number of included words times the bits needed to encode those words plus the bits needed to define a new symbol in the dictionary , is then normalized by the length of the data string  normalized by the original string length, is the value used to indicate LZC. A higher \u03c10 value indicates higher complexity, thus, less ability to compress. For a more detailed description of the process of compression, the reader is referred to Aboy et al. . 0.5 < H is an indication for long range correlations and H < 0.5 for long-range anti-correlations  is a robust analysis for the estimation of the multifractal spectrum of power-law exponents of a natural time series Ihlen, . Its basH = 1.2 was selected  poses a fast, theoretically efficient and robust analysis method for multifractal properties of real-world data Zilber, . It usesFor the calculation of the WLBMF, the toolbox described in Wendt et al.  was used\u03b1 = 0.006 for the complexity metrics and \u03b1 = 0.025 for the additional power spectral analysis.For statistical analysis, the results of all time windows of a certain subject and condition were averaged. Those mean values were then tested for normality with a Kolmogorov-Smirnoff test. As they showed to be not normally distributed, groups were compared with a Wilcoxon signed rank test. To determine whether there is a trend over gestational ages, a Pearson correlation was calculated. After Bonferroni correction, significance levels were set to After testing several methods for determining complexity of fMEG data, a first step was to evaluate those methods for potential future use. As shown in p < 0.001 in all cases). For fetal data, a significant difference between brain and control was only detected for audio data in the delta range (p = 0.001). Interestingly, fetal audio and spont data differed in their power in the delta range (p = 0.006).p < 0.001) Differences for MSE in spontaneous data were marginally significant (p = 0.007). In all cases, the control channel showed higher values compared to the brain channel. For the scale-free metrics in both cases, H/c1 could not differentiate between brain and control but M/c2 showed a significant difference (p < 0.001). In case of M, the channel with the brain activity appeared more multifractal than the control channel, whereas in case of c2, the opposite was observed. For detailed results, see In the neonatal datasets, the comparison of brain vs. control resulted in a significant difference for both audio and spontaneous data for LZC, MPE and CD . Differences between brain and control within the scale-free metrics did not hold after correction for multiple comparisons expect c1 for audio data (p = 0.006). For detailed results, see For the comparison of brain vs. control of in the fetal datasets, no clear tendencies could be found. The LZC calculation, as well as the MPE and CD metric did not reveal a significant difference, neither for audio data nor for spontaneous data. MSE showed a difference for audio data only . All participants gave written informed consent in accordance with the Declaration of Helsinki and agreed on reuse of data for additional studies.JM conducted the analysis and wrote the manuscript. SB and EK revised the manuscript. JM, FS and HP conceptualized the study. SB, EK, GR, FW and HP advised the analysis. FS provided preceding analysis. All authors read and approved the final version of the manuscript.EK is an employee of Starlab SLU, the company that gave birth to Neuroelectrics in 2011, and GR is a co-founder, shareholder, and employee of Neuroelectrics and Starlab.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Herbal medication use is prevalent and increasing in the general population.\u00a0A comprehensive\u00a0review of complementary and alternative medicine use including herbal medications and\u00a0supplements is often overlooked by physicians. Patients generally believe that all herbal\u00a0products are safe without any side-effects.\u00a0Herbal medications may have complex\u00a0pharmacodynamics and can be associated with various psychiatric symptoms.\u00a0The general\u00a0population, as well as physicians, may be unaware of the risks and side-effects associated with\u00a0herbal supplement use and further research may be needed. The objective is to\u00a0describe a case report of acute onset of symptoms of hypomania associated with the increasing\u00a0use of herbal supplements.\u00a0A 49-year-old man developed symptoms of hypomania after a two-month history of daily use of\u00a0a combination of more than 25 herbal supplements and daily cannabis use.\u00a0Hypomania symptoms were temporally associated with the use of multiple herbal supplements\u00a0that included ginseng.\u00a0We recommend that a thorough history of medication use including\u00a0herbal supplements and other alternative medications and a collateral report from family\u00a0members and other providers including herbalists be obtained on all patients presenting with psychiatric symptoms.\u00a0Further research is\u00a0needed to identify the pharmacodynamics, risks, and adverse effects, and drug and food\u00a0interactions of each herb. Eighty percent\u00a0of the world population relies on traditional medicine for their primary health care needs according to a report by the World Health Organization (WHO) .\u00a0There hComplementary and alternative medication use is common in people with psychiatric illnesses. In a survey of complementary and alternative medicine usage among psychiatric inpatients (n = 82) it was found that 63% used at least one complementary and alternative medication modality within the previous 12 months, including 44% who used herbal medications\u00a0[Even though there is a widespread use of herbal medications, individuals do not inform their physicians of this history of medication use, nor do many physicians routinely obtain this history from their patients\u00a0-6.\u00a0PeoplIn the United States, herbal supplements are regulated by the U.S. Food and Drug Administration (FDA) as food products. They are not regulated as strictly as prescription drugs or over-the-counter (OTC) drugs. Supplements fall under the category of dietary supplements and therefore are not subject to the phases of clinical testing. Scientific evidence on the efficacy and safety of herbal supplements is sparse as there are many challenges and a lack of clear guidelines to conduct research on herbal products. Diagnostic And Statistical Manual Of Mental Disorders, Fifth Edition (DSM-5) introduced the diagnostic category of \u201csubstance/medication-induced bipolar and related disorder\"\u00a0.\u00a0In ordeA 49-year-old man with a history of depression for nine years, treated with escitalopram, presented to the emergency department with complaints of anger outbursts, insomnia, difficulty with short-term memory, decreased attention span, dizziness, and a herpes outbreak on his lips.\u00a0Collateral information was obtained from the patient\u2019s wife and mother. The patient's personality and behavior change started about two\u00a0months ago.\u00a0He was noted to have anger outbursts, mood swings, excessive crying, talkativeness, flight of ideas, grandiosity, distractibility, indiscretion, hyperactivity, and decreased sleep with subsequent worsening of these symptoms.\u00a0The patient\u2019s erratic and disorganized behavior had worsened about 10 days before he came to the Emergency Department. The patient was noted by the family to be vigorously cleaning the house, boarding up the attic windows, having post-it notes all over the house, and driving impulsively to various locations.\u00a0He was also emotionally and verbally abusive towards his wife.\u00a0Loss of 20 pounds of weight over the past 10 months and increase in social anxiety was also noted. He had been working from home for the last two years.\u00a0As per the patient and the patient\u2019s family, these symptoms had never occurred before.\u00a0There was no family history of mental health conditions. The patient denied any suicidal ideation, intent, or plan and denied any auditory or visual hallucinations. He reported a three-year history of myalgias and cannabis use to help with the pain. There was a significant increase in the frequency of cannabis usage for the past three months. He had also recently changed his supplier from a licensed dispensary in MA to a local street dealer. On further evaluation of the patient\u2019s medication history, the\u00a0patient reported that he had also been taking herbal medications from a licensed acupuncturist for the past two months for his myalgias. He was consuming unknown quantities of these supplements. The patient had a history of alcohol use in the past and had been abstinent since 2003. The patient's father had a history of Alzheimer's dementia and alcohol use disorder. There was no other family history of psychiatric disorders.\u00a0The patient\u2019s consumption of multiple herbal medications along with daily use of possibly tainted cannabis coincided with the onset of hypomanic symptoms. A complete list of the herbal supplements taken by the patient over the past two months  is shown in Table\u00a0In the emergency department, the\u00a0patient was noted to have hypotonic hyponatremia with an initial sodium level of 125 and low serum osmolality of 257 with hypochloremia with chloride level of 94 and mildly elevated creatinine kinase level of 913.\u00a0The toxicology screen was negative except for cannabinoids.\u00a0Head CT was negative.\u00a0The patient received 1 liter of the saline bolus in the emergency room and his sodium level subsequently improved to 132 and creatinine kinase level to 508.Given that the patient\u2019s behavior had become increasingly disorganized with symptoms of hypomania and concern for the patient\u2019s ability to adequately care for himself at home, he was admitted to the inpatient psychiatric unit.During the hospitalization, the patient was started on\u00a0olanzapine and the dose of escitalopram was reduced from 20 mg to 15 mg.\u00a0Throughout the patient\u2019s hospitalization, he was pleasant and cooperative and stayed in good behavioral control. He did not require any emergent medications, restraints, or seclusions.\u00a0Over the course of hospitalization, the\u00a0patient reported improvement in the presenting symptoms including insomnia, flight of ideas, irritability, memory loss, poor concentration, and distractibility. There was an improvement in sodium level to 140 and chloride level to 103.\u00a0B1, B6, and B12 levels were normal and Lyme and syphilis serologies and heavy metal panel were negative.\u00a0MRI brain was negative as well.\u00a0The patient was then discharged home with olanzapine and escitalopram.\u00a0Symptoms of hypomania can be associated with the use of herbal supplements, cannabis use, hyponatremia, and treatment-emergent hypomania associated with the use of escitalopram.The above-mentioned patient developed symptoms of hypomania only after the start of herbal medications with no prior episodes.\u00a0In our review of the literature on herbal medication use associated with psychiatric symptoms, it was notable that the symptoms that our patient presented resembled the symptoms and associations with herbal medicine use as mentioned in the literature.ConsumersLab.com is a website that can be used by clinicians to find information on herbal supplements. It can provide information about the side effects, uses, and the several brands of a specific supplement.Various case reports have been reported with the use of herbal supplements and symptoms of mania and hypomania.\u00a0Bostock et al did a systematic review and quality assessment of case reports on mania associated with herbal medications\u00a0. FourteePotential harms of the use of cannabis in patients with psychotic and mood disorders have been documented in the literature\u00a0.\u00a0AdolescTreatment-emergent mania/hypomania is an infrequent event, occurring in 2.3% of patients treated for major depression\u00a0. Given tWith regards to the association of hyponatremia with hypomania, very few case reports have been reported.\u00a0One was a case report of recurrent mania in a patient with prior history of bipolar disorder type 1 with recurrent hospital admissions about four times within three weeks presenting with hyponatremia and with manic symptoms during each medical admission\u00a0.\u00a0The othThe above-mentioned patient\u2019s hypomania symptoms occurred in the context of starting a concoction of herbal medications including the use of Ginseng.The main conclusion from the present case report is the importance of understanding which herbal supplements a patient is taking and what potential adverse reactions these supplements could cause. In our case, the previously high-functioning man became acutely manic that caused not only severe functional impairment , but a dangerous physiological and emotional impairment - i.e., weight loss, hyponatremia, elevated CK, paranoia, agitation, and sleeplessness. We recommend that a thorough history of medication use including herbal supplements and other alternative medications, and a collateral report from family members and other providers including herbalists be obtained on all patients presenting with psychiatric symptoms.\u00a0Further research is needed to identify the site, mode, and mechanism of action of commonly used herbal supplements in combination and as monotherapy. More information is required on the desired effects, adverse effects, and interactions of herbs with each other, with prescription drugs and OTC medications, and with food.\u00a0The scientific community should come together to make policies on conducting clinical trials and research on herbal supplements as their use and popularity are increasing among our patients. The limitation of the current case report is the unknown contribution of the street-purchased cannabis that this patient was consuming along with the herbal supplements."}
+{"text": "Gallstone disease is a major health problem addressed by general surgeons, with approximate incidence of 10-15% in the Western world. With increasing focus in the healthcare literature on cost containment, controlling excess lengths of hospital stay (LOS) in this population is paramount. The aim of this study was to determine the factors that influence LOS in cholecystectomy patients to examine whether results would indicate a possible improvement in perioperative patient care and decrease costs at our community hospital in a suburban setting.This is a retrospective review during a two-year period from 1/1/2013-12/31/2014 of patients admitted from the emergency department and undergoing cholecystectomy during the same admission. The study team analyst conducted univariate analysis for significant predictors of length of stay.The authors identified a total analytic sample of 312 subjects who met inclusion criteria. Sample patients admitted to the surgical service had a statistically significant shorter LOS than those patients who were not . There was also a moderate positive correlation between decreased time to surgery and LOS . Patients admitted to non-surgical services were more likely to have comorbidities like COPD, DM, arrhythmia, CAD, anticoagulation, CHF and previous abdominal surgeries. However, when placing each comorbidity into an analysis of covariance, patients admitted to surgical services still had a significantly shorter LOS .Admission to a non-surgical service and increased length of time to surgical intervention were associated with prolonged LOS and potentially increased cost in cholecystectomy patients in this study sample. Though patients admitted to non-surgical services are \u201csicker,\u201d they still had prolonged LOS when controlling for comorbidities. Based on these findings, the establishment of an acute care surgery service may help to address this disparity in care. Gallstone disease  is a major health problem frequently addressed by general surgeons. This condition affects approximately 10-15% of adults in the Western world population during their lives.Gallstone disease can present in a wide variety of clinical scenarios. Patients may experience symptomatic cholelithiasis  with intermittent obstruction of the cystic duct resulting in the classic symptoms already described. Complete obstruction results in bacterial infection of the gallbladder . Obstruction of the common bile duct  results in obstructive jaundice, and may lead to reflux of the pancreatic duct . Choledocholithiasis may also result in infection of the biliary system , which has been associated with severe sepsis and high mortality.In patients with asymptomatic cholelithiasis, 1-4% will have their conditions progress annually to complications.One older article estimated that more than 700,000 cholecystectomies were annually performed in the US.One major factor used to control costs in surgical patients is decreasing the time period to surgical intervention when needed. Previous studies have demonstrated that decreased time to surgical intervention results in lower hospital costs and is not associated with worse outcomes.In 2014, a research group analyzed the factors associated with increased LOS for acute cholecystitis patients , and not the entire range of biliary disease. As a result, there is still limited information regarding the factors impacting LOS in all cholecystectomy patients. In this study, we were therefore interested in examining the factors that influenced LOS for all cholecystectomy patients at a community hospital with the goal of improving perioperative patient care and decreasing hospital costs. Given that the vast majority of cholecystectomies performed at our institution were laparoscopic , the authors made no distinction made between open and laparoscopic cholecystectomy cases.Saint Joseph Mercy Oakland Hospital is 443-bed facility located in Pontiac, MI. The community has a population of 59,889 people, with an average age of 33.5 years, and an estimated per capita income of $16,087. Racial make-up includes 50.9% black, 25.7% white, 15.8% Hispanic, 4.9% 2 or more races, and 2.9% Asian.Before data collection, the hospital institutional review board had approved the study. This retrospective review examined data from a two-year time period from January 2013 through December 2014 that included patients admitted from the emergency department and undergoing cholecystectomy during their same hospital stay. We identified adult sample patients using a program called Discern Analytics\u00ae, searching for the term \u201ccholecystectomy.\u201d Exclusion criteria included patients less than 18 years of age, undergoing a scheduled cholecystectomy or one due to prior work-up, patients not admitted through the emergency department, cholecystectomy performed as a secondary procedure, and pregnant women.We performed an additional review of 33 subjects with prolonged LOS, defined in this study as a LOS \u226510 days. Thirty-two of these patients were admitted to a nonsurgical service and one subject was admitted to a surgical service. Based on consensus of independent review by the authors, subjects admitted with non-gallbladder disease or had prolonged LOS for reasons unrelated to gallbladder disease or postoperative complications were excluded. Data were collected concerning patient demographics such as patient age, gender, self-reported race, and comorbidities. Further collected data included type of admitting service, time to surgery, pre and postoperative diagnosis, LOS, postoperative morbidity and mortality, and 30-day readmission rates.All statistical analyses were conducted using SPSS version 22 software. Univariate analysis examined potential predictive factors suggested in the literature to have an effect on LOS.After we determined that sample patients\u2019 admitting service was a significant predictor of LOS, we conducted further analyses. We used a series of Chi-square tests to compare the LOS of patients admitted to surgical or non-surgical services controlling for presence of comorbidities. We entered these comorbidity analytic terms in an analysis of covariance (ANCOVA) as equally weighted covariates with the dependent LOS variable and additional independent variable type of admitting service. Finally, we completed additional t-tests to examine the significance of differences in time to surgery between surgical vs.\u00a0non-surgical admitting service patients.There were 854 cholecystectomies performed at Saint Joseph Mercy Oakland Hospital during the two-year analytic period. Of these, 542 (63.5%) subjects were excluded based on our selected exclusion criteria. The majority of excluded patients were outpatient and/or elective cholecystectomies. Data from 312 (36.5%) sample patients who met inclusion criteria were analyzed.The demographic characteristics of sample patients can be seen in Table 1. Upon chart review, the average age of the subjects was 54.5 years of age . Two hundred and eleven (68%) subjects were female, and one hundred and one (32%) were male. The racial profile of the sample was skewed toward Caucasians since although ~51% of the given community population self-reported being African-American, the profile of this sample was: 72.6% white (n = 230), 13.8% African American (n = 43), 6.7% Hispanic (n = 21), 1.0% Asian (n = 3), and 5.4% unknown or other (n = 17). Patients were about evenly split between admission to a surgery service  or non-surgical service .This was a relatively healthy sample of patients. The incidence of individual comorbidities documented were as follows:Chronic obstructive pulmonary disease (COPD) ,Other lung disease ,Some form of diabetes (DM) ,Cirrhosis ,Other liver disease ,Heart valve disease ,Cardiac arrhythmias ,Coronary artery disease (CAD) ,Taking an anticoagulate ,Congestive heart failure (CHF) , andPrevious abdominal surgery .As previously mentioned, intraoperative conversion to an open cholecystectomy was n = 14 (4.5%) in this sample. Postoperative complications were also rare in study patients. The incidence of each study complication was as follows: postoperative bile leak , surgical site infection , pneumonia , myocardial infarction , intra-abdominal infection , death . There were zero instances of biliary duct injury observed in this cohort. We concluded that the size of this sample subgroup with complications was too small to conduct further analyses.Post-hospital patient disposition frequencies were as follows: home n = 306 (98.1%), subacute rehabilitation n = three (1.0%) and acute rehabilitation n = two (0.6%). Since most sample patients were discharged home, there was no further statistical evaluation. Similarly, the 30-day readmission rate for sample patients was relatively low, at n = 50 (16.0%). Of these readmissions, 25 (50.0%) were for reasons related to gallbladder disease .We conducted a univariate analysis to assess factors that affected LOS. Factors found to be significantly associated with increased LOS included admission to a non-surgical service  and increased time to surgery . Non-significant factors included comorbidities and preoperative diagnosis. Due to these findings, the authors performed further analyses concerning evaluating admitting service. (Table 2)As seen in Table 2, patients admitted to non-surgical services were also significantly \u201csicker,\u201d meaning they were more likely to have one of each of the following comorbidities: COPD, DM, CAD, on an anticoagulation medication, CHF and previous abdominal surgeries (p < 0.0005). However, none of these individual comorbidities were statistically significant when placed into an ANCOVA model. The only p value that remained significant was the type of admitting service, with patients admitted to the non-surgical services having longer LOS (p < 0.005) when controlling for comorbidities. Patients admitted to a non-surgical service also had increased time to surgical intervention  compared to surgical service admission patients .In our study, admission to a non-surgical service and increased length of time before surgical intervention were significantly associated with longer LOS in cholecystectomy patients. Based on prior studies, the estimated total savings for the patients admitted to the surgical service in this study setting may have totaled $4,156 in surgery and $6,390 in extra hospital LOS costs.The overall findings of this study are similar to other surgical conditions in the literature. For example, two studies have demonstrated that patients admitted to a medical hospitalist service with small bowel obstruction had increased length of stay and increased charges as compared to patients admitted to the surgical service.In this community-based setting, patients admitted to non-surgical services were more likely to have documented comorbidities with longer LOS and increased time to surgery when controlling for those comorbidities. Since this was a retrospective review, we could not make a clear distinction between admission time and time of diagnosis, or stratify by the type of varied symptoms that patients may have had at time of presentation. For example, providers may have admitted some patients to the hospital with sepsis of unknown origin with the diagnosis of cholecystitis later made. Such patients may have been admitted to a non-surgical service with increased time to surgical evaluation, increased time to surgery and prolonged LOS. Although we did conduct ANCOVA analyses to attempt to control for certain comorbidities, it would have been ideal if we could have actually matched patients in the surgical and non-surgical admitting services to control for these comorbidities. Further studies of this type are warranted.One possible means of decreasing LOS and hospital costs in this population is implementation of an acute care surgery (ACS) service.Several suggested benefits of ACS services include improved scheduling and operating suite predictability for surgeons, improved patient access to surgical intervention and improved post-discharge follow-up.There are several limitations to this study. The study was set at a single institution using a smaller retrospective convenience sample. The generalizability of our results to other organizations may be limited, and we may have had an inadequate level of statistical power to detect meaningful relationships possibly detectable in a larger sample. Additionally, the results of this study may have been subject to unmeasured confounding influences.In conclusion, these results indicate that providers may expect longer LOS for many cholecystectomy patients admitted to a non-surgical service with increased length of time to surgical intervention. These results and the findings of earlier studies suggest that certain factors can be targeted to decrease LOS in this gallbladder disease population. One may argue with this conclusion since our sample patients admitted to non-surgical services were \u201csicker,\u201d although we still found then to have longer LOS and greater time to surgical intervention when controlling for comorbidities. Ideally, ACS can have helped providers achieve a more rapid evaluation of potential \u201cgallbladder patients\u201d in the emergency department by surgical team providers. Further studies are required to evaluate the actual cost savings derived from these types of hospital admission services to optimize the outcomes in this growing population of patients.The authors declare no conflict of interest."}
+{"text": "Background: We examined the longitudinal relationships between hemoglobin concentrations or the severity of anemia and depression and whether baseline cognitive function modifies these longitudinal relationships over 4 years of follow-up. Methods: A total of 1608 community-dwelling older adults from the International Mobility in Aging Study (IMIAS) aged 65 to 74 years were recruited in Natal (Brazil), Manizales (Colombia), Kingston , and Saint-Hyacinthe . The study outcome was depression, defined by a score of 16 or over in the Center for Epidemiologic Studies Depression Scale (CES-D). Longitudinal associations over four years follow-up were examined using generalized estimating equations. Models reported were either unadjusted and adjusted for research sites, alcohol drinking status, body mass index, chronic conditions, activities of daily life disabilities, and polypharmacy. Results: Longuitinal relationships suggested an evidence of multiplicative interaction by baseline global cognition in which 1g/dL increase in hemoglobin concentrations there was a significant reduction in the risk of depression with a stronger effect among participants with good cognitive function (Odds Ratio (OR)=0.85, 95% CI: 0.78-0.92) compared to those with poor cognition . Anemia and poor cognition at baseline were associated with an increased risk of depression over 4 years of follow-up . Global cognition was an effect modifier of the longitudinal association between the severity of anemia and depression. Conclusion: In international samples of older adults, hemoglobin concentrations, as well as the severity of anemia, were independent risk factors for depression and these associations differed by global cognitive function."}
+{"text": "Scientific Reports 10.1038/s41598-020-65922-0, published online 03 June 2020Correction to: This Article contains an error in Reference 41 which was incorrectly given as:Plants8, 305 (2019).\u201d\u201cVictoria, F. B., Jairo, A. P., Chen, Y. L. & Kadambot, H. M. Early season drought largely re-duces grain yield in wheat cultivars with smaller root systems. The correct Reference is listed below.Plants8, 305 (2019).Figueroa-Bustos, V., Palta, J.A., Chen, Y. L. & Siddique, K.H.M. Early season drought largely reduces grain yield in wheat cultivars with smaller root systems."}
+{"text": "Mean inhibition decreased by 16.4%, and mean change in COPM performance and satisfaction scores were 2.1 and 2.4, respectively. The photovoice narrative focus group supports findings evidenced with improved daily routines. Conclusions: The Octopus watch is an innovative early intervention that can promote purposeful ADLs, fostering family resilience by enhancing occupational engagement. Further research is required.Background: Children with spina bifida and/or hydrocephalus (SB&/H) often experience difficulties with activities of daily living (ADLs) due to impaired executive functioning, increasing sedentary behaviours. The HeyJoy Octopus watch, a child-friendly icon-based smartwatch could be used as an enabler to promote purposeful ADLs . Objective: to investigate the effectiveness of the Octopus watch in promoting purposeful ADLs for children living with SB&/H (<8 years). Methods: Mixed-methods engaging parents and children in four phases: (1) Administered demographic questionnaire, semi-structured interview, childhood executive functioning inventory (CHEXI) and the Canadian occupational performance measure (COPM); focus group one introducing the study, information pack using smartwatch and photovoice data collection methods. (2) Measured baseline movement for four days with smartwatch without using functions. (3) Measured activity for 16-days while using the smartwatch. (4) Re-administered assessments and conducted a second focus group based on photovoice narratives. Results: movement data recorded for four participants, three of four showed mean activity increase (36%). N-of-1 analyses found one participant showed clear improvement ( Spina bifida (SB) is a congenital deformity of the neural tube and is the most common neural tube defect (NTDs) in children . In IrelStr\u00f6mfors et al. identified three significant barriers to independence in ADLs, including motivation, preparedness and planning . These pIn enhancing health behaviours for individuals living with SB&/H, physical activity (PA) is an important aspect to consider . PA inteResearchers are increasingly recognising the significance of self-management interventions in supporting living with chronic illness (including SB&/H) to enhance independence with ADLs and ultimately improve health outcomes . SystemaIn recent years, wearable assistive technology such as PA trackers has grown tremendously in popularity ,31. HealAccordingly, an intervention that promotes a play element into ADLs is both a meaningful and purposeful activity for a child . Thus, aTherefore, the primary objective of this pilot study is to address whether the Octopus watch is a feasible intervention to promote purposeful ADLs in a group of children (<8 years) living with SB&/H. Thus, the study aims were: (1) to investigate the potential of the watch to increase PA; (2) to examine whether the watch compensates for impaired EF; (3) to explore the potential effects of the intervention on purposeful ADLs, and (4) to explore user experience and technology acceptance of the Octopus watch.Quantitative pretest-posttest and repeated measure single-case n-of-1 designs were used, combined with photovoice methodology to supplement the findings . PhotovoParticipants were enrolled if they met the inclusion criteria: (1) a child under eight years old who has SB&/H and is supported by a parent or guardian, (2) has English as their first language, (3) meets the minimum requirement of functional ability to self-propel a wheelchair see . ParticiFive families (parents/guardians of children living with SB&/H) were recruited through the gatekeeper (SBHI) via convenience sampling, with four families (80%) completing the full study see . WrittenThe Octopus watch developed by HeyJoy is designed to empower children by teaching the concept of time and routine, while also encouraging them to stay active with a built-in fitness tracker . The watThe study commenced over four phases: pre-test, baseline, intervention and post-test.Phase one: the pre-test, participants completed a demographic survey , the chiPhase two: Comprised of collecting baseline activity while carrying out normal day-to-day routines without using Octopus-watch icons/schedule functions. Trost et al. recommend that four days or more is considered adequate for gathering accurate reliability of accelerometer data, providing the rationale for a four-day baseline measurement .Phase three: Consisted of two parts (1) the intervention and measuring activity for 16-days while using the icon functions on the Octopus watch during the daily routine  and internal consistency (Cronbach\u2019s \u03b1 > 0.85) using parent scoring approximately three weeks apart [The CHEXI is a 24-item inventory of EF for children . The twoks apart . Parentsks apart .The COPM is a client-centred semi-structured interview that measures the self-perceived evaluation of occupational performance (OP) and occupational satisfaction (OS) domains for three activity categories: productivity, self-care and leisure . For thiThe proprietor describes the smartwatch activity monitoring as using 3-axis digital accelerometry and a digital gyroscope. Proprietary technologies are used to compute a measure of activity, known as \u2018octo-points\u2019 which we used as a proxy for PA. 6000 \u2018octo-points\u2019 equate to about 60 min of activity . Thus, tQuantitative CHEXI and COPM data were analysed using descriptive statistics, including means and standard deviations, completed using Excel formulas on a Microsoft Excel spreadsheet. Paired samples t-tests were performed to investigate changes. All statistical analysis was conducted on IBM SPSS.26 using a 5% level of significance.r2 \u2265 0.25, r2 \u2265 0.09, r2 \u2265 0.01 representing large, medium and small effect sizes, respectively [The PA time series data were plotted and visually examined to explore changes in activity during baseline and intervention phases for each participant. Although visual inspection of this data is recommended , recent ectively .n = 9), due to the accelerometer data not recording any PA. A further data point was deemed missing as a parent violated the experimental protocol by wearing the watch. Missing data points were imputed using a simple averaging approach either side of the missing data [Across all PA outputs, 10 random data points of 80 were missing (12.5%), with the majority recording zero octo-points . Three of the four participants used assistive devices for mobility  with one child independently ambulant and one child independently ambulant for short distances. All participants attended the initial focus group with two of the four participants attending the second focus group, with individual feedback provided by participants unable to attend.Based on the primary objective to assess the feasibility of the Octopus watch as an early-intervention, findings are presented with quantitative results first, followed by supporting photovoice narrative themes.Research aim 1. To investigate the watch\u2019s potential in promoting PA, visual plots were used to display the mean change in PA outputs (octo-points) from baseline to intervention see . Overallp = 0.021) with a large effect size (r2 = 0.28). Changes recorded for Rachel, Alice and Ethan were not statistically significant.A corresponding n-of-1 analysis for each participant on the pre-whitened data ,46 is prResearch aim 2. To examine if the watch compensates for impaired EF see . There wResearch aim 3. To explore the potential effects of the intervention on purposeful ADLs, every participant created five user-centred goals based on the COPM assessment see . OverallResearch aim 4. To explore user experience and technology acceptance of the watch. The semi-structured interviews, qualitative COPM components  morning routine, and (b) night-time routine.Three participants reported that the Octopus watch impacted positively on their morning routine, the outlier being Alice. David displayed strong improvement in this area, with his mother Ellen explaining that after three weeks he no longer needed to use the timer function they had been using for breakfast.\u201cBefore, David was up and down and had no concentration at breakfast-time\u2026now David sits and eats. He doesn\u2019t need to use the timer anymore\u201d.After learning to recognise the icons on the watch, Rachel also made progress with developing a morning routine of her own. While previously she relied on instruction from her family, she now uses the Octopus watch to prompt her for tasks such as retrieving her breakfast bowl from the cupboard and making sure she has her bag ready for school. Claire explained that this is a great help to her in the mornings. Cathy also explained that while Ethan still requires physical assistance, the Octopus watch has helped with his anxiety in the mornings and he \u201chas been going on his bus much easier since he has the watch and getting the bus icon\u201d.Both Rachel and David incorporated reading a story-book into their night-time routine. Both showed improvement in this task. By the end of the third week, Ellen explained, each night David changed into his pyjamas and independently retrieved a book from the shelf see . SimilarA big success of the project was that three of the four participants reported an increase in their child\u2019s independence while wearing the Octopus watch. David\u2019s improvement is evident in each of the photographs. Ellen explained that by the end of the third week, David no longer needed the prompts from his watch in the morning as he had already completed his tasks.\u201cThe timer would go off and David would have task completed. He asks why it is going off if he had done task already\u201d.She also explained that David felt safer when wearing his watch as Ellen had enabled the in case of emergency (ICE) function to include both hers and her husband\u2019s phone numbers.\u201cDaniel got lost one day about 2 years ago, he was crying, he was very upset. Now he tells me that he can go up to somebody and tell them to ring mammy or daddy and show them the watch\u201d.\u201cAt the start, there was a lot of coaxing to go out and I would have to go with her or she wouldn\u2019t stay out for long but by the end of the third week\u2026she would look for me to open the door and she would go out herself\u2014not for long but we are building it every day. I\u2019m delighted to see her achieve this\u201d.Claire expressed in the initial focus group that she was eager for Rachel to \u201cbe more independent\u201d and her progress is evident through the photographs she provided. Claire expressed her joy at Rachel\u2019s progress with spending time outside after dinner, an activity that Rachel previously did not like to partake in see . She expTwo of the participants reported that their child was eager to show their watch to their friends. During the discussion at the second focus group, Anna explained that Alice had previously distanced herself at a birthday party and usually preferred to be alone rather than join a group. Since wearing the watch, Anna has noticed that Alice has become more sociable at play-school and likes showing her watch off to her friends, depicted in \u201cThis one was her going into play school in the morning and wanting to show the boys her watch\u201d  living with SB&/H. The great heterogeneity in the needs of children living with SB&/H makes it challenging to form an effective intervention that fosters healthy behaviour. However, the novel Octopus watch, tailored to meet individual children\u2019s needs provides an appropriate platform to encourage purposeful activity. Our mixed-methods findings show promising preliminary evidence for the feasibility of the Octopus watch. Children exhibited improved inhibitory control, increased independence, and routine development in purposeful ADLs. In addition, the process of introducing the Octopus watch provides a method of technology transfer to promote effective use and avoidance of abandonment. Encouraging outcomes were also reflected in user acceptance, with the families responding positively to the intervention. Some evidence was found in terms of increased PA for participants. This is a critical first step, considering the dearth of early intervention evidence for promoting healthy lifestyles in children living with SB&/H.Firstly, from the CHEXI assessment, supporting interviews and photovoice narratives, the participants appeared to compensate for executive dysfunction. Inhibitory control improved across all participants, although working memory did not change. From previous research, it could have been anticipated that working memory would not improve because no specific memory training was conducted . A notewSecondly, the satisfaction domain on the COPM showed some evidence of an increase, in contrast to the focus group and interviews where parents described positive ADL performance effects. This discrepancy between quantitative and qualitative findings is possibly due to the small sample size, restricting our ability to judge significance . HoweverFrom the COPM assessment, supporting content analysis and photovoice narratives, the key findings were that the Octopus watch increased independence and supported routine. The results suggest that, possibly due to its user-centred nature with icon prompts promoting ADL goals, the Octopus watch enables the child to lead the intervention ,77. SimiThirdly, accelerometer data show that David had a significant increase in PA from baseline to intervention, supported with a large effect size. Hence, the Octopus watch benefited this participant\u2019s PA considerably. Alice displayed a mean decrease in PA output from baseline to intervention. One explanation was that the vibration notification function on the watch did not work for this participant, thus hindering intervention benefits. In contrast, both Rachel and Ethan increased mean PA from baseline to intervention. However, these changes were not statistically significant . Power iBesides personal barriers, it is feasible that our 16-day intervention was too short to elicit significant changes in the complex task of increasing PA for each participant. Supporting this explanation, in the O\u2019Brien et al. review the shortest randomised controlled trial (RCT) PA intervention for children who mobilise with wheelchairs was three months . SimilarFourthly, consistent with Puri et al. , technolFinally, the Octopus watch is one helpful component for strengthening routines and independence which may contribute to family resilience. Family resilience is generally understood as the capacity of the family to remain positive when faced with a disruptive situation affecting their stability and integrity. Resilience enables the family to come out stronger . HolmbecAlthough this case series used the participants as their own controls , the smaA reliability study should be conducted on the Octopus watch with a subsequent RCT to support more conclusive findings. To enable generalisability, a large sample size is recommended, thus, facilitating a power analysis before data collection is necessary . Other cIn conclusion, the Octopus watch appears to be a feasible early intervention for children living with SB&/H and is a promising user-friendly smartwatch that places the user\u2019s ADL goals at the centre, thus enabling the child to increase independence and autonomy through icon prompts. The consistent goal-based icon repetition of the smartwatch acted as a compensatory strategy for EF and thus ameliorated impaired inhibition which appeared to yield increased purposeful ADL participation. Further, all participants adopted the smartwatch and showed potential to increase PA. Aspects which contributed to technology acceptance were aesthetics, real-time feedback rewards and play-based activities. Overall, the increase in purposeful ADLs and evident technology acceptance strengthened family resilience by fostering occupational engagement. This is a substantial finding as sustainability assistive technology use for children living with SB&/H was questionable due to associated difficulties with goal-directed skills. This study is a small but noteworthy step toward finding a translational early intervention that is beneficial to promoting better routines and habits for children living with SB&/H. Hence, the findings provide direction for further large-scale studies."}
+{"text": "Autism spectrum disorder (ASD) is a chronic neurodevelopmental condition with a prevalence rate above 1%, characterized by deficits in social communication and interaction; restrictive, repetitive patterns of behavior, interests, or activities; and a preference for sameness and routines. The majority of adult ASD patients suffer from comorbid conditions such as depression and anxiety. Therapy options for adult ASD patients are lacking, with presently no available evidence-based interventions in Germany. Recently, two interventions to improve social responsiveness have been published. FASTER (\u201cFreiburger Asperger-Spezifische Therapie f\u00fcr ERwachsene\u201d = Freiburg Asperger-specific therapy for adults) is a manualized group psychotherapy program including three modules on psychoeducation, stress regulation management, and non-verbal and verbal social communication training with videotaped tasks. SCOTT&EVA  is a computer-based training program to enhance social cognition including video and audio material of emotional expressions and complex real-life social situations. Initial studies for both programs have shown good feasibility and efficacy.Three hundred sixty adult participants with an autism spectrum disorder (ASD) will take part in a randomized controlled three-armed multi-center trial to prove the efficacy of manualized group psychotherapy and a manualized computer-based training program. Both interventions will be compared with a treatment as usual (TAU) group, aiming to establish evidence-based psychotherapy approaches for adult individuals with ASD. The primary outcome is evaluated by parents, spouses, or others who have sufficient insight into the respective participant\u2019s social communication and interaction, and will be measured with the Social Responsiveness Scale. First, each of both interventions will be compared to TAU. If at least one of the differences is significant, both interventions will be compared against each other. The primary outcome will be measured at baseline (T0) and 4\u00a0months after baseline (T1).The trial is the first to validate psychiatric therapeutic and training interventions for adult ASD patients in Germany. A trial is needed because the prevalence of ASD in adulthood without intellectual disability is high, and no evidence-based intervention can be offered in Germany.DRKS00017817. Registered on 20 April 2020.German Clinical Trial Register  Autism spectrum disorders (ASD) are neurodevelopmental conditions behaviorally characterized by impairments in social communication and interaction as well as the presence of repetitive and restricted patterns of behaviors, interests, and activities , 2. WhilIn past clinical practice, ASD in adulthood has was regarded as a neurodevelopmental disorder, which generally goes along with overt and severe language deficits, learning problems and low IQ. Only few adult high-functioning patients received the diagnosis ASD, but instead were often been diagnosed only by their secondary psychiatric conditions . In thesAdults with ASD show high rates of psychiatric comorbidity such as depression (53%), anxiety (50%), attention deficit hyperactivity disorder 43%), obsessive-compulsive disorder (24%), tic disorders (20%), and psychotic disorders (12%) 3%, obses\u20138. This Given the deficits in social cognition and social interaction, individuals with ASD often have significant interpersonal problems. Difficulties in understanding pragmatic aspects of language frequently lead to social withdrawal, depression, and anxiety. This may result in intensified social withdrawal, isolation, and unemployment . UnemploIn summary, these results show that there is a need (i) to implement valid diagnostic investigation of adults with symptoms of ASD in the diagnostic routines of APP and (ii) to develop specific psychotherapy programs addressing the core features of ASD. Presently, such therapy options for adult patients are rare , 5, 9. TTo advance the process of empirical validation of these intervention methods, we designed a combined controlled, randomized trial for both treatment approaches, which has received funding from the German Research Foundation.The main objective of this trial is to prove the effectiveness and efficacy of the therapy programs FASTER and SCOTT&EVA in a combined phase-III trial.The primary hypothesis is if there is superiority of at least one of the treatments FASTER and SCOTT&EVA over treatment as usual (TAU) regarding social responsiveness.If this is confirmed, the difference between FASTER and SCOTT&EVA regarding the primary endpoint will be tested confirmatorily. Otherwise, the comparison between FASTER and SCOTT&EVA will be done descriptively.This trial is a controlled, three-armed, cluster-randomized, observer-blinded, multi-center study to compare FASTER and SCOTT&EVA with TAU as parallel groups see Fig.\u00a0. The allhttps://www.drks.de/drks_web/navigate.do?navigationId=trial. HTML&TRIAL_ID=DRKS00017817).The comparison between the FASTER and SCOTT&EVA interventions and TAU will be implemented as a national interventional multi-center trial at six centers in Germany. Six centers in Germany take part in the recruitment and implementation of the study. The list of recruiting study sites can be obtained from the German Clinical Trial Register with the register number DRKS00017817 , high-functioning autism , or atypical autism, if the DSM-IV criterion A and deficits in social communication and social interaction are fulfilled . All three diagnoses are current standard diagnoses in Germany, encoded with the F-keys from the International Statistical Classification of Diseases and Related Health Problems, Book V . EquivaFurther inclusion criteria for our study are an age between 18 and 65\u2009years, an IQ higher or equal 80, relevant psychosocial impairment lower or equal to a score of 60 as measured with the Global Assessment of Functioning .Psychiatric comorbidities for exclusion are as follows: florid schizophrenia , florid psychosis , acute manic episode within a bipolar disorder , acute severe depression , or acute suicidality. Further, substance abuse or substance dependencies within the last 12\u2009months , present or past gambling disorder, not corrected severe vision or hearing impairment, a history of severe group disturbing behavior (documented therapy exclusions from previous group therapies), any neurological disorder or medical condition interfering with group therapy, group-based social skills training or other structured psychotherapy for the core symptoms of ASD , inpatient treatment at baseline (T0) is also an exclusion criterion; however, inclusion is possible after the completion of inpatient treatment.Patients will receive information on the content and process of the study by mail and will be asked to give preliminary consent for data storage if they are interested in the study. In the next step, potential participants will be contacted by phone or invited into the study center for a first screening interview.This first interview only comprises some short questions about eligibility. It will be performed by trained study personnel. If no exclusion criterion is applicable, the participants will be invited to come to a face-to-face interview to the study center. During the second interview, trained study staff (physicians or psychologists) provide verbal information about the trial, answer questions, and obtain written informed consent from patients.On the consent form, participants will be informed that their data will be anonymized in case they decide to withdraw from the trial and request of their personal information to be deleted. Participants will also be asked for permission that the research team can share relevant data with people from the universities taking part in the research or from regulatory authorities where relevant. This trial does not involve collecting biological specimens for storage.Given the high therapeutic need and the high prevalence of ASD and the recognized urgent need for evidence-based therapy approaches, we chose to evaluate both FASTER and SCOTT&EVA against TAU within one combined pivotal study.The two interventions FASTER and SCOTT&EVA will be compared to a TAU group. The TAU group can continue to use the usual outpatient treatments, but not a therapy that targets the core symptomatology of ASD. In Germany, \u201ctreatment as usual\u201d is in fact rarely an ASD-specific intervention.The SCOTT&EVA concept focuses on emotions and social interactions whereas FASTER additionally aims at areas such as stress management and a deeper understanding of social communication and interaction.Thus, the study with two intervention groups evaluates two approaches including different aspects and range of important areas of interventions for people with ASD, both with the aim to ultimately improve social responsiveness and social competence.After the screening phase, eligible study participants will be included into the study and assessed at baseline (T0). Following baseline measurement of six participants, all six will be assigned to one of the three groups  via cluster randomization.The Freiburg Asperger-Specific Therapy for adults (FASTER) intervention , 5, 13 hEach single group psychotherapy session comprises three different parts. A short review of last week\u2019s outstanding events or experiences of each participant will be shared with the group for the first 20\u2009min of the session. If participants require clarification of urgent or important group-appropriate issues, they can receive feedback from other group members. This will be followed by a short mindfulness exercise that is especially adapted to individuals with ASD. Subsequently, the actual topic of the session will be introduced.After completion of the 4-month trial, participants will receive four monthly fresh-up group meetings in the follow-up period to keep up a therapeutic structure and motivation, to foster transfer of new knowledge and strategies into everyday life, and to allow for feedback and exchange of information. The topics of the refresher sessions comprise participants\u2019 daily stress management, and communication issues in everyday social situations. All sessions are substituted by specific ongoing homework including self-monitoring, introspection, and analysis of social interactions that will be discussed in the next group meeting.The Social COgnition Training Tool (SCOTT) is a computer-based manualized training program that was developed in 2008 to target social cognitive impairments in high-functioning adults with ASD. It aims to foster the recognition of 40 different emotions , 22. TheFace Task: short video sequences of faces expressing emotions are displayed in three different variants of this task: video sequences of faces are either (a) simply displayed or (b) slowly revealed and the user is asked to match the faces by emotional state. A third variant of this task is that (c) the video sequences displaying faces are cut into upper and lower face parts (same identity faces) and the user is asked to match the pieces accordingly.Voice Task: To improve the understanding of prosody, users are asked to match auditory vocal sequences with emotional states depicted as emotional terms.Social Interaction Task: short videos (<\u20092\u2009min) portraying the interaction of 2\u20134 actors in demanding real-life contexts are divided into sub-sequences, which are displayed simultaneously on the computer screen. The user is asked to reconstruct the correct chronology of the short movies by socio-emotional content using drag and drop functions. In a next step, the user is asked to answer questions referring to the actors\u2019 mental states.The SCOTT&EVA training comprises three visually and technically appealing training modules based on diverse video and audio material:Furthermore, SCOTT&EVA contains a large didactic platform, detailing semantic embedding and physiological and expressional features of all emotions targeted. In this so-called Emotion Treasury module, all 40 emotions are systematically ordered and described in detail . Furthermore, all emotions are depicted by face and voice stimuli.Additionally, users receive feedback about their performance for each task and in total (Performance Scores) and time they have spent with the training (Experience Score).The SCOTT&EVA training will be conducted by each participant individually from home via Internet. The treatment is designed to be approximately equivalent to the FASTER treatment concerning training time . The time every participant actually spends with SCOTT&EVA will be assessed by online tracking in SCOTT&EVA\u2019s central database. In order to ensure correct software use and to motivate independent training at home, the participants randomized to the SCOTT&EVA group will be introduced and supervised in dealing with the computer training at the beginning of week one and ongoing once per month in the study center (with an approximate duration per supervised session of 60\u2009min). During the follow-up phase, patients also receive one supervised fresh-up session per month (minimum duration of 30\u2009min).Subjects are allowed to withdraw their consent to participate in the study interventions at any time without personal disadvantages and without having to give a reason. Subjects will take part in the further assessments , if they do not deliberately resign from the follow-up assessments.If a subject withdraws from the trial, no additional data will be collected, but the existing data will be used for statistical analysis. If, in addition, the subject requests the collected data to be destroyed, their personal information will be erased from the database and cannot be used for further analysis.The time of treatment or trial discontinuation and, if known, the reason for withdrawal will be documented on the CRF and in the patient file.The investigator or Data Safety and Monitoring Board (DSMB) can also discontinue the intervention after considering the risk-to-benefit ratio, if they no longer consider further treatment of the patient according to study protocol justifiable. The date of and the primary reason for the termination and the observations available at the time of withdrawal are to be documented on the CRF. If possible, the measurements of those patients will be used. They will still be considered for the final analysis.Drop-out patients will not be replaced.Rater for the screening, baseline, post-treatment, and follow-up is trained in the used tests, interviews, and computer programs: Global Assessment of Functioning (GAF), Structured Clinical Interview for DSM-5 for clinical diseases (SCID-5-CV), Autism Diagnostic Observation Scale, Version 2 (ADOS-2), Intelligence test (MWT-B and CFT 20-R), and Multifaceted Empathy Test (MET).Therapists are trained in the application of Freiburg Asperger-Specific Therapy (FASTER) and in the administration and supervision of the Social Cognition Training Tool (SCOTT&EVA).Video recordings from the trainings together with all manualized materials will be provided on a login-protected internet platform for raters and therapists. Thus, each study center can refresh their trainings or train new study members.Once a month, there will be a telephone supervision for potentially emerging rating and therapy questions. Additionally, for all FASTER group therapy sessions, therapists are videotaped. These recordings will be used for supervision and adherence checks. Video cameras are placed in such a way that the study participants are not visible, only their voice is recorded. For that reason, written consent for the recording of study participants audio will be obtained.In case of acute problems or comorbid disorders that have to be treated, all participants irrespective of their group are allowed to make use of outpatient visits, such as family doctor visits, outpatient psychiatric visits for comorbid mental disorders, doses adaptation of medication, and psychotherapy for comorbid disorders outside the study center. However, patients are not allowed to participate in specific therapy targeting the core problems of ASD during the whole trial phase and are not allowed to have received specific ASD therapy  for the core problems of ASD up until 6\u00a0months before inclusion into the study. It is also not permitted to change the active substance of psychotropic drugs during the study period. Participants may receive an overall maximum of up to 120\u2009min of individual, non-ASD-specific consultation with study personnel within their period of participation.If serious changes occur during the study period, e.g., in relation to comorbid disorders requiring treatment, these are also followed up after the study period. Appropriate measures are taken for this purpose, such as outpatient or inpatient care or referral to other specialists.Since there are hardly any specific measures for people with ASD, participants who were randomized initially to the treatment as usual group (TAU) are offered SCOTT&EVA or FASTER after the end of the study.All outcomes will be assessed at baseline (T0), post-treatment , and follow-up .Social Responsiveness Scale  to 4\u2009months after baseline (T1) in social responsiveness as measured with the e/other, , 19).The secondary outcome measures aim to differentially assess aspects of self-reported social responsiveness, social cognition, social skills, empathy, common comorbid psychopathology, self-esteem, life satisfaction, and quality of life. Almost all questionnaires and tests  at the follow-up measurement time point (T2) is also a secondary outcome.SRS-A [For social responsiveness, the SRS-A , 24 is uSRS-A . Due to SRS-A . Good reThe SRS-A cut-off score for adults is 67 with a sensitivity of .85, and a specificity of .83 for autism spectrum disorder in comparison to patients with other mental disorders and typical developed individuals. There is no normed German version of SRS-A available so far, but a validation has already been published .Additional SRS-A measurements will be done for our secondary objectives.Social Responsiveness Scale for adults, second edition , is also available in the form of a self-report. Compared to the SRS-1, the SRS-A has the additional variant for self-assessment and is eligible from 18\u2009years onwards. The 65 items of the SRS-A are almost identical to the SRS-1 in terms of content, but in the SRS-A self-assessment, the statements are given in an ego perspective. The questionnaire comprises five subscales: social consciousness, social cognition, social communication, social motivation, and autistic mannerism. The Cronbach\u2019s alpha for the total score is .89 for individuals within the autism spectrum.The As secondary outcome, we will assess changes in social responsiveness via the SRS-A self-report \u201326 for tFurthermore, we apply the Global Assessment of Functioning (GAF), which is part of the Structured Clinical Interview for DSM-IV  and freqMultifaceted Empathy Test , which The MET is an objective photo-based test that has specifically been designed and validated in adults with ASD . It alloBeck Depression Inventory, second edition . It is  . It has . It hasLife Satisfaction Questionnaire . It conQuality of Life Questionnaire from the WHO . It conTwo other questionnaires have been constructed in Freiburg especially for adults with autism and are not validated until now. We use specifically formulated questions for potentially occurring life events and a partnership questionnaire where either partners or only one may be autistic. To date, no comparable questionnaires are available in Germany.Freiburg Event List (FEL) will be used to assess non-treatment life events and is included to evaluate possible moderation effects at post-treatment and follow-up.The Autism Spectrum Quotient  and theent . The auToronto-Alexithymia-Scale-26 . It. ItToronWender-Utah Rating Scale  for sym-report, ) for curCulture Fair Intelligence Test, second edition , measurMultiple choice vocabulary intelligence test  is a veNEO-Five-Factors-Inventory  will beNEO-FFI, ) may leaadditional information about the participants is collected: age, gender, native speaker, marital status, age and sex of siblings and own children, current life situation, school leaving certificate, education, profession, family members with ASD diagnosis, possible addiction-related Internet use in various areas, current psychiatric and psychotherapeutic treatments, time point and diagnostic center at ASD diagnosis, and years of therapeutic ASD treatment.The following The measurement points are displayed in Table\u00a0The sample size calculation is based on the primary outcome measure \u201cmean difference of the SRS-A score between baseline and 4 months after baseline\u201d. The aim is to show that at least one of the two treatment protocols (SCOTT&EVA or FASTER) is superior to TAU which results in a union-intersection test. The sample size calculation is based on an ANCOVA model adjusted for the baseline SRS-A score. Since both models are well comparable, the power of the finally applied mixed model repeated measures (MMRM) is expected to be close to the intended value used for sample size calculation based on an ANCOVA. To obtain an estimate for the correlation between baseline and 4-months value, we analyzed internal data of 17 patients with a time difference of 9\u2009months between measurements of SRS-A score. This yielded a correlation coefficient of .27 . Hence, we use a conservative correlation of .2 for sample size calculation. The standardized treatment effects for SCOTT&EVA versus TAU and FASTER versus TAU are both assumed to be equal to 0.4. These prior assumptions are based on previous research results .The global two-sided significance level is 0.05. The two test statistics referring to the comparison of TAU versus SCOTT&EVA and TAU versus FASTER are correlated due to the common comparator TAU. This correlation is determined by the ratio of variances of the group effect estimators. We assume equal variances in TAU and SCOTT&EVA and a small intra-group correlation of 0.05 in FASTER. Since SCOTT&EVA and TAU are no group therapies, we expect, if at all, a very small intra-group correlation caused by cluster randomization. Conservatively, we assume an intra-group correlation of 0.01 in these treatments. This results in a design effect of 1.25 inflating the variance in FASTER and a design effect of 1.05 inflating the variances in SCOTT&EVA and TAU. Hence, the correlation of test statistics is given by 0.026632 . ApplyinThe required sample size to find a significant effect in at least one of the two comparisons with a power of 0.8 under the above-described assumptions and at correlation-adjusted local levels of 0.026632 is given by 87 patients per group. Based on experiences in our previous FASTER and SCOTT&EVA trials, drop-out rates of about 20\u201325% are expected , 21. ThuParticipant recruitment is expected to begin in March 2021 and will continue for 28\u2009months.Each of the participating centers has consented to include a certain number of patients according to their usual quantities of diagnostics and treatment of autistic patients. The study centers keep waitlists for diagnostics and treatment and will also use existing patient files for screening. Flyers with cover letters for medical practices and autism centers as well as press information have been prepared with general information about the study. These materials can be adapted with each study center\u2019s contact information and distributed within their local networks. Some of the study centers have well-established web pages in place to advertise clinical trials especially for their autistic patients. In addition, Germany\u2019s largest society for autistic people and their caregivers, Autismus Deutschland e.V., has been requested to include information about the study on their webpage for advertising trials looking for autistic participants in the German speaking area.http://www.randomizer.at) will be used and the allocation sequence is created by computer-generated random numbers.Subjects are randomized by cluster randomization in an allocation ratio of 1:1:1. The cluster size is set to 6 patients per cluster. This means that groups of 6 participants are built within each center, and these groups are randomly assigned to one of the three study arms. The randomization will not be stratified by center or other factors. An Internet-based randomization system . Details of the randomization scheme will be specified in an external document accessible to the Institute of Medical Biometry and Informatics  only to minimize selection bias.http://www.randomizer.at). Study staff which do not have to be blind will insert always six in the study included study participants\u2019 identification codes into the randomizer online. All already randomized groups can be viewed by the study staff of the corresponding center and from the IMBI. Subjects withdrawn from the trial will retain their identification codes . New subjects must always be allotted a new identification code.The allocation sequence is implemented and concealed by the randomization tool \u201crandomizer\u201d . All study participants who give written consent and meet the eligibility criteria will be randomized in clusters. Raters from each center decide on the inclusion of potential study participants based on clearly defined criteria. Randomization will be requested from the rater of each study center via the online randomization tool \u201crandomizer.at\u201d from the University of Graz, Austria. Only the data manager of the Institute of Medical Biometry and Informatics  has complete access to the stratification and randomization process. All centers add six new participant identification numbers into the randomizer for a complete cluster and will then receive the random allocation to one of the three groups .The study-specific randomization tool of Randomization takes place after baseline. Study staff from each study center who measure post-treatment (T1) and follow-up (T2) are blinded regarding group assignment. Rater and therapy rooms are far apart, to support the blinding of the raters throughout the whole course of the study. Raters log at the measurement time points T1 and T2 for each study participant whether they are blinded or not. Due to study design and significantly different interventions in the study arms, therapists, study participants, and additional persons who are responsible for the external assessment for social responsiveness are not blinded. The biometrician and his representative will remain blinded until data base closure.No procedure for unblinding of therapists is necessary. Therapists always have information about the allocation of each study participant. If unblinding of raters takes place by accident, the unblinding is recorded and another rater will perform the future measurements (T1 or T2).A detailed description of the study instruments and their validity can be found in the section \u201cOutcomes\u201d.Raters are trained for 2\u00a0days in the Structured Clinical Interview for DSM-5 for clinical diseases (SCID-5-CV), the Autism Diagnostic Observation Scale (ADOS-2), the Global Assessment of Functioning (GAF), the Multifaceted Empathy Test  and IQ testing for the Culture Fair Intelligence Test (CFT 20-R) and the complete process of data acquisition.Additionally, all trained procedures are video recorded and are available via Internet access for all raters. Also available are frequently asked questions about the procedures via Internet access. Monthly supervisions via video conference will be held for best knowledge approximation between centers.If new raters have to be trained, they will use the video-recorded information about training and take also part in the monthly supervisions via video-conference.Therapists for FASTER and SCOTT&EVA are trained for 2\u00a0days regarding all sessions and procedures. Additionally, all trained procedures are video-recorded and are available for all therapists via Internet access. Frequently asked questions about the procedures are also available via Internet access. Monthly supervisions via video conference are offered for best knowledge approximation between centers.If new therapists have to be trained, they use the video-recorded information about training and take also part in the monthly supervisions via video conference.The training measures for raters and therapists of the study are documented in training logs. Raters and therapists can only be accepted into the study team after successful completion of the training. The recording must be authorized by the center\u2019s principal auditor. The training logs are checked via the monitoring visits.Data collection forms for each center can be found in the investigator site file (ISF) as well as in an internet-based platform that is accessible to all raters and therapists. In addition, frequently asked questions (FAQ) about study contents are available for all participating members of the study centers.Between T1 and T2, both FASTER and SCOTT&EVA participants are offered monthly refresher sessions in the respective study center. The refresher sessions are implemented to support the transfer of skills and knowledge gained during the intervention phase into daily life.TAU patients are offered a compensatory FASTER or SCOTT&EVA program after completion of their trial participation. SCOTT&EVA participants will also be offered an additional FASTER group therapy after completion of their trial participation if requested. Currently, there are few treatment options for the study participant group, so participation in this study is most likely motivating. In case of protocol violations or withdrawals, the subjects will still take part in the further assessments if they do not deliberately resign also from the follow-up assessments.Case report forms (CRFs) are paper-based. Source data will be collected in each center from the rating and therapy staff and will be transferred into the CRFs. Source data is stored in the respective center. Source data and transmission to the CRFs will be checked via monitoring by on-site visits in each study center. A copy of each CRF will be made and the original will be sent to the Institute of Medical Biometry and Informatics .The Institute of Medical Biometry and Informatics  is responsible for the data management within the trial. In order to ensure that the database reproduces the case report forms (CRF) correctly, the IMBI accomplishes a double entry of data. In order to guarantee high quality of data, the completeness, validity, and plausibility of data as defined in a data validation plan will be checked using validating programs, which will generate queries. A tracking system for CRF data and queries will be established to guarantee that data is managed in a timely manner. The investigator or the designated representatives are obliged to clarify or explain the queries. If no further corrections are to be made in the database it will be closed and used for statistical analysis. All data management procedures will be carried out on validated systems and according to the current Standard Operating Procedures (SOP) of the IMBI that guarantee an efficient conduct which is in compliance with GCP.At the end of the study, the data will be transformed into different data formats  to ensure that it will be possible to reuse it. The principle investigator will retain the originals of all CRFs and the trial data for long-term preservation. After completion of the study, we plan to make the publication data and the primary data publicly available for re- and meta-analyses.The data obtained in the course of the trial will be treated pursuant to the Federal Data Protection Law .During the clinical trial, subjects will be identified solely by means of their individual identification code . Trial findings stored on a computer will be stored in accordance with local data protection law and will be handled in strictest confidence. Distribution of these data to unauthorized persons is strictly prevented. The appropriate regulations of local data legislation will be fulfilled in its entirety.The subject consents in writing to release the investigator from his/her professional discretion in so far as to allow inspection of original data for monitoring purposes by authorized persons . Authorized persons  may inspect the subject-related data collected during the trial ensuring the applicable data protection law.The investigator will maintain a subject identification list (subject numbers with the corresponding subject names) to enable records to be identified. Subjects who did not consent to circulating their pseudonymized data will not be included into the trial.This protocol, the CRFs, and other trial-related documents and material will be handled with strictly confidentiality and will not be disclosed to third parties except with the express prior consent of the Lead Investigator. Staff of the investigators involved in this study are also bound by this agreement.There will be no biological specimens collected .In the recently released ICH E9 (R1) addendum on estimands and sensitivity analysis in clinical trials, the estimands framework is recommended as clear and transparent definition of \u201ca structured framework to strengthen the dialogue between disciplines involved in the formulation of clinical trial objectives, design, conduct, analysis and interpretation, as well as between sponsor and regulator regarding the treatment effect(s) of interest that a clinical trial should address\u201d . Such anThe population is defined through appropriate inclusion/exclusion criteria to reflect the targeted patient population.The variable is the SRS-A score (parent/spouse/other).Possible intercurrent events and the strategies to handle them are as follows. Serious adverse events and adverse events will be ignored for the primary analysis (treatment policy strategy). The same strategy will be applied for all other intercurrent events as, e.g., treatment withdrawal or unforeseen in-patient stay.The summary measure is the difference in the SRS-A (parent/spouse/other) between 4\u2009months after baseline (T1) and the baseline value (T0). The applied test is described in the next subsection.Let \u03bc denote the unknown true mean difference of the SRS-A score between baseline and 4\u2009months after baseline. The following two primary local test problems are assessed:andThe aim is to show that at least one of the two local alternative hypotheses holds true. For each pairwise comparison (TAU versus FASTER and TAU versus SCOTT&EVA) a Mixed Model Repeated Measures (MMRM) will be applied. It includes the difference of the SRS-A score between baseline and 4\u2009months after baseline as the dependent variable, the baseline SRS-A score, and the group allocation as fixed effects, and the center and the cluster as random effects. The MMRM uses all available changes from baseline SRS-A score for all patients for model estimation. In the \u201cSample size {14}\u201d section, the local significance level of .026632 has been deduced. In case that at least one of the null hypotheses specified above can be rejected at the local significance levels of .026632, we hierarchically assess additionally the following test problem:at the full two-sided level of 0.05 in a confirmatory manner. In case neither H0, SCOTT&EVA nor H0, FASTER can be rejected, the comparison between the FASTER and the SCOTT&EVA arm is done descriptively. The confirmatory analysis of the primary efficacy endpoint corresponds to the primary estimand and will be conducted in the full analysis set.As a sensitivity analysis to the primary efficacy analysis, corresponding MMRMs with additional covariates/factors given by IQ, age, gender, ASD symptom severity, MET test result, and center will be conducted. Furthermore, the separate items of the SRS-A score are analyzed individually to investigate which items differ between the treatment groups. Details of these analyses will be specified in the statistical analysis plan (SAP).Exploratory analyses will investigate moderation of treatment effects by conducting the primary MMRM considering the following additional variables: IQ, age, gender, center, years of therapy experience with autism, depressiveness (BDI), self-confidence (MSWS), social competence (SASKO), and life events (FEL). To optimize treatment selection for individual patients, these covariates will be used to estimate individualized treatment effects from parameter estimates to construct optimal individualized treatment rules and to clarify for whom a treatment works best .p-values will be reported. Graphical methods will be applied to visualize the findings. The safety analysis includes calculation of frequencies and rates of adverse and serious adverse events. Additionally, as supplementary analysis the primary endpoint will be evaluated in the per-protocol population. Furthermore, statistical methods are used to assess the quality of data. All analyses will be done using Statistical Analysis Software (SAS) version 9.4 or higher.Descriptive methods will be used for the analysis of the secondary outcomes, including the calculation of appropriate summary measures of the empirical distribution as mean, median, standard deviation, 1st and 3rd quantile for continuous outcomes, and absolute and relative frequencies for count data. Furthermore, 95% confidence intervals and descriptive two-sided Analyses will be defined in detail in the SAP which has to be authorized before data base closure by the two involved biometricians and the lead investigator.No interim analysis is planned.Subgroup analyses will be performed regarding the indication .The trial populations including the per protocol analysis set are defined in the section \u201cStatistical methods\u201d.The MMRM applied for the analysis of the primary endpoint takes into account missing data. It uses all available changes from baseline SRS-A score for all patients for model estimation. If a patient has a missing change from baseline SRS-A score, the model assumes that the patient\u2019s missing SRS-A score is comparable to the observed SRS-A score of another patient having similar baseline characteristics and a comparable course of change from baseline.After completion of the trial, the data obtained by the study will be summarized and analyzed according to the protocol and hereafter published in a peer-reviewed journal. This will include an appendix with the full study protocol. Requests for data sharing will be reviewed on an individual basis by the Steering Committee. The data sharing process will comply with the good practice principles for sharing individual participant data from publicly funded clinical trials, and data sharing will be undertaken in accordance with the required regulatory requirements. Especially, the privacy of the patient data  will be followed throughout the entire study.Lead InvestigatorProf. Dr. Ludger Tebartz van ElstMedical Center \u2013 University of FreiburgDepartment of Psychiatry and PsychotherapyHauptstrasse 5, 79104 Freiburg, GermanyBiometrician and Data ManagementInstitute of Medical Biometry and Informatics, University Hospital HeidelbergProf. Dr. Meinhard KieserMarsilius-Arkaden, Turm West,Im Neuenheimer Feld 130.369120 Heidelberg, GermanyProject ManagerMedical Center \u2013 University of FreiburgDepartment of Psychiatry and PsychotherapyDr. Thomas FangmeierHauptstrasse 579104 Freiburg, GermanyMonitoringCoordination Center for Clinical Trials (KKS), University Hospital HeidelbergDr. Steffen P. LuntzMarsilius-Arkaden - Turm WestIm Neuenheimer Feld 130.369120 Heidelberg, GermanyLead Site InvestigatorsBerlin:Prof. Dr. Isabel DziobekBerlin School of Mind and Brain, Department of PsychologyFaculty of Life Sciences, Humboldt-UniversitaetUnter den Linden 6, 10099 Berlin, GermanyDresden:Prof. Dr. Veit RoessnerFaculty of Medicine, TU DresdenDepartment of Child and Adolescent Psychiatry and PsychotherapyFetscherstra\u00dfe 74, 01307 Dresden, GermanyEssen:Prof. Dr. med. Katja KoelkebeckLVR-Hospital Essen, Department of Psychiatry and Psychotherapy, Medical Faculty, University of Duisburg-Essen,Virchowstr. 174, 45147 Essen, GermanyMannheim:Dr. Oliver HennigCentral Institute for Mental HealthJ 5, 68159 Mannheim, GermanyT\u00fcbingen:Prof. Dr. Dr. Dirk WildgruberDepartment of Psychiatry and PsychotherapyUniversity of TuebingenCalwerstr. 14, 72076 Tuebingen, GermanySteering CommitteeProf. Dr. L. Tebartz van Elst, Prof. Dr. I. Dziobek, Dr. T. Fangmeier, Prof. Dr. M. KieserThe independent Data Safety and Monitoring Committee (DSMC) will consist of two medical scientists and one statistician with longstanding experience in clinical trials: Prof. Dr. Sven B\u00f6lte , Prof. Dr. Konrad , and Prof. Dr. Martin Hellmich . The function of the DSMC is to monitor the course of the study and, if necessary, to give a recommendation to the coordinating investigator regarding discontinuation, modification or continuation of the study. The underlying principles for the DSMC are ethical and safety aspects for the patients. The DSMC is responsible to examine whether the conduct of the study is still ethically justifiable, whether security of the patients is ensured and whether the process of the study is acceptable. The DSMC will be informed about adherence to the protocol, patient recruitment, and observed serious adverse events. The DSMC will receive the corresponding reports at regular intervals (every 6\u2009months). Recommendations on the further continuation or modification of the study will be given to the coordinating investigator. The composition and responsibilities of the DSMC, the structure and procedures of its meetings, and its relationship to other study team members will be documented in a separate DSMC charter.Following ICH-GCP, the definition of an adverse event (AE) is adapted as follows: an AE is any untoward occurrence in a subject participating in a study, which does not necessarily have a causal relationship with the study treatment. An AE can therefore be any unfavorable and unintended sign, symptom, or disease temporally occurring alongside with the treatment (FASTER/SCOTT&EVA/TAU), whether or not related to the treatment.Increase of comorbid symptoms, e.g., substantial depression symptoms from none or mild to moderate symptoms \u226519 (BDI-II) or a significant worsening from moderate to severe symptoms (\u2265 30); severe obsessive-compulsive or anxiety disorder; constant and excessive interactional problems (dispute with group members) or sensory overflow that prevent participation in group therapy.Significant recurrence of comorbid disorders.Appearance of new comorbid symptoms/disorders as described in 1 or new diagnosis.Occurrence of new passive suicidal thoughts .Occurrence of active suicidal thoughts or plans .Changes in medication class (not dose adjustment) of antidepressants, benzodiazepines, typical or atypical neuroleptics, stimulants, and mood stabilizers.Occurrence of constant and excessive problems in the relationship between patient and therapist.Private burdens like divorce, death of a family member, and relocation.Occupational burdens like job change, loss of employment, or significant changes in frame conditions of existing employment.Vocational reintegration burdens .The following will be defined as adverse events for the treatment study FASTER/SCOTT&EVA:A pre-existing disorder or symptom will not be considered an adverse event unless there will be an untoward change in its intensity, frequency or quality. This change will be documented by an investigator.Surgical procedures themselves are not AEs; they are therapeutic measures for conditions that require surgery. The condition for which the surgery is required may be an AE. Planned surgical measures permitted by the clinical trial protocol and the condition(s) leading to these measures are not AEs if the condition leading to the measure was present prior to inclusion into the trial.AEs are classified as \u201cnon-serious\u201d or \u201cserious.\u201dMonitoring will be done by on-site visits and frequent communication  by a clinical monitor according to SOPs of the KKS. The monitor will ensure that the trial is conducted according to the protocol and regulatory requirements by review of source documents, entries into the CRFs and essential documents. Therefore, the investigator will allow the monitor to verify these documents and will provide support to the monitor at all times. The monitor will document the visits in a report for the sponsor. The site will be provided with a follow-up letter of the findings and the necessary actions to be taken.In total, on average one to two monitoring visits are carried out per center and year, depending on the number of study participants included. The monitoring is carried out by the Coordination Center for Clinical Trials in Heidelberg (KKS).As the monitoring strategy will consider current aspects of risk-based quality management, frequency of monitoring activities per site will vary depending on recruitment, experience, and general performance, e.g., quality of documentation of the individual trial centers. Details of monitoring will be defined in the monitoring plan.If there are major findings during monitoring or an audit, the investigational site might be closed by the Lead Investigator.Any modifications to the protocol which may have an impact on the study, potential benefit for the patient or may affect patient safety, including changes of study objectives, study design, patient population, sample sizes, study procedures, or significant administrative aspects, will require a formal amendment to the protocol. This will be decided jointly with the Steering Committee, the study centers, and will undergo approval by the Ethics Committee.Administrative changes of the protocol are minor corrections and/or clarifications that have no effect on the way the study is conducted. These administrative changes will be approved within the Steering Committee and the study centers.The primary outcome publications of the study will present outcome data regarding social responsiveness between baseline and post-treatment, as well as secondary outcomes for social responsiveness, social cognition, psychological well-being, quality of life, self-worth, and life satisfaction for all three measurement points.In addition, outcomes will be presented on congresses, symposia, workshops, etc., if applicable.http://www.drks.de) ensure both the validity of the methodical procedure as well as the transparency of the whole trial for the scientific community and is a prerequisite for future publications in peer-reviewed journals. A comprehensive independent monitoring strategy including on-site visits and frequent communication according to the standard operating procedure of the Coordination Center for Clinical Trials, Heidelberg will assure data quality and security of patients. In consideration of reducing experimental biases, data collectors of each center (rating staff) are blinded in this study and separated from therapists or other intervention related work. Thus, they will have no knowledge about individual group assignment. Both intervention programs will be implemented by trained therapists with clinical background. Using intention-to-treat-analyses and per-protocol-analyses, the study represents a conservative and clinically useful strategy of data analysis.The purpose of this study is to validate existing data for the two different treatment concepts FASTER and SCOTT&EVA in a multi-center, randomized phase-III trial. FASTER and SCOTT&EVA concepts are among the most elaborated German treatment concepts for adults with ASD in line with modern therapy standards and have demonstrated feasibility and yielded good phase-II data. The study protocol meets the essential standards of modern research in psychotherapy. Since the required sample size to find a significant effect with a power of 0.8 is given by 87 patients per group, a number of 120 participants per group and a total sample size of 360 participants will be sufficient to find an effect (assuming there is one). In order to minimize the differences among groups, the participants will be randomized to treatment and control conditions . The use of standardized diagnostic instruments for the assessment of eligibility as well as primary and secondary efficacy analysis facilitates ecologically valid outcomes. A defined primary endpoint before trial initiation and a registration at Current Controlled Trials on the German Clinical Trials Register . Due to the delay caused by the SARS-CoV-2 pandemic, recruitment will start in March 2021. Last patient out has been planned 28\u2009months after first patient inclusion.LTvE and TF conceived the study. LTvE, TF, and UMS initiated the study design. TF, MK, ID, and CK helped with implementation. MK provided statistical expertise in clinical trial design. All authors contributed to refinement of the study protocol and approved the final manuscript."}
+{"text": "The discovery of atrial, brain, and C-type natriuretic peptides  and their cognate receptors has greatly increased our knowledge of the control of hypertension and cardiovascular homeostasis. ANP and BNP are potent endogenous hypotensive hormones that elicit natriuretic, diuretic, vasorelaxant, antihypertrophic, antiproliferative, and antiinflammatory effects, largely directed toward the reduction of blood pressure (BP) and cardiovascular diseases (CVDs). The principal receptor involved in the regulatory actions of ANP and BNP is guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which produces the intracellular second messenger cGMP. Cellular, biochemical, molecular, genetic, and clinical studies have facilitated understanding of the functional roles of natriuretic peptides (NPs), as well as the functions of their receptors, and signaling mechanisms in CVDs. Transgenic and gene-targeting (gene-knockout and gene-duplication) strategies have produced genetically altered novel mouse models and have advanced our knowledge of the importance of NPs and their receptors at physiological and pathophysiological levels in both normal and disease states. The current review describes the past and recent research on the cellular, molecular, genetic mechanisms and functional roles of the ANP-BNP/NPRA system in the physiology and pathophysiology of cardiovascular homeostasis as well as clinical and diagnostic markers of cardiac disorders and heart failure. However, the therapeutic potentials of NPs and their receptors for the diagnosis and treatment of cardiovascular diseases, including hypertension, heart failure, and stroke have just begun to be expanded. More in-depth investigations are needed in this field to extend the therapeutic use of NPs and their receptors to treat and prevent CVDs. The natriuretic peptides (NPs) family contains a group of hormones that are pivotal in the control of cardiovascular, endocrine, renal, and vascular homeostasis . Atrial Importantly, NPs not only regulate BP and CVDs, but also maintain natural antagonistic actions to the renin-angiotensin-aldosterone system (RAAS), exert antimitogenic effect, and inhibit myocardial hypertrophy and fibrosis. Moreover, NPs play roles in endothelial cell function, cartilage growth, immunity, and mitochondrial biogenesis . The demDifferent NPs receptors have been classified namely; NP receptor-A (NPRA), NP receptor-B (NPRB), and NP receptor-C (NPRC). NPRA and NPRB exhibit an intrinsic intracellular guanylyl cyclase (GC) catalytic domain and are designated as GC receptor-A (GC-A/NPRA) and GC receptor-B (GC-B/NPRB) . Both ANin vitro and transgenic and gene-targeted (gene-knockout and gene-duplication) mouse models in vivo have greatly advanced our understanding of NPs and their receptors by delineating the normal and abnormal control of physiological and pathophysiological functions in CVDs  now designated as ANP in cardiac myocytes . ANP, thDendroaspis angusticeps) as a 38-amino acid biologically active peptide molecule  and their three distinct receptor subtypes  have widespread cell and tissue distributions, indicating the pleotropic actions at the systemic and local levels . ANP andmolecule . ANP shomolecule . In humamolecule . The clemolecule . CNP hasmolecule .The design of chimeric NPs has led to synthesis of the biologically active novel NPs of clinical importance, which represent single-chemical molecule with combined structural and functional properties . ChimeriNppa and Nppb promoters in a competitive mode instead in a cooperative manner  than ANP (0.5\u22124), BNP exhibits extended action and leads to enhanced natriuretic and diuretic actions as compared with ANP . The carBecause of the longer half-life and stability, the measurement of NT-proBNP as a diagnostic marker is preferred over measurements of BNP and ANP. Moreover, the NT-proBNP molecule is considered to be a predictive marker after cardiac transplantation . In contin vivo probably exerts a chloride-dependent feedback-control on receptor function  and FQQI (Phe790-Gln791-Gln792-Ile793) have been identified, which serve as consensus internalization signal motifs for endocytosis and intracellular trafficking of this receptor protein  Hypertension and Cardiovascular Diseases(b) Renovascular Dysfunction(c) Cardiac remodeling and dysfunction(d) Metabolic Syndrome and Diabetic Conditions(e) Immunogenic Responses and Cardiovascular Disorders(a) Hypertension and Cardiovascular DiseasesNPPA), BNP (NPPB), and NPRA (NPR1) polymorphisms with hypertension and CVDs in humans Nppa mice fed standard or intermediate-salt diets, BP was elevated by 8\u221210 mmHg. Haplotype (/)Nppa mice on a standard-salt diet contained a normal amount of plasma ANP and had normal BP, while on a high-salt diet these animals were hypertensive, and their BP was elevated by 25\u221227 mmHg. Global deletion of the Nppa allele can lead to salt-sensitive hypertension even when plasma ANP level are not significantly decreased. Global ablation of Npr1 increases BP by 36\u221240 mmHg in null mutant  mice as compared with wild-type  mice  mice significantly reduced BP and enhanced kidney and heart function  mice . In contfunction .Npr1 gene copies, determining the excretion of urine and sodium, renal blood flow (RBF), glomerular filtration rate (GFR), and BP after blood volume expansion in Npr1 0-copy, 2-copy, and 4-copy mice in a gene-dose-dependent manner (Npr1 gene-knockout (0-copy), wild-type (2-copy), and gene-duplicated (4-copy) mice. In addition, Npr1 null mutant (0-copy) mice retained significantly higher levels of Na+ and water; however, gene-duplicated (4-copy) mice as compared to wild-type (2-copy) mice had greatly reduced levels of Na+ and water. Our findings demonstrated that the ANP/NPRA axis is predominantly responsible for regulating the renal hemodynamics and Na+ excretory responses to intravascular blood volume expansion.Earlier, we examined the mechanisms that may mediate the function of increasing numbers of t manner . The vol(b) Renovascular DysfunctionXenopus 2F3 cells , Npr1+/+ (2-copy), and Npr1++/++ (4-copy) mice in a gene-dose-dependent manner (1R and angiotensin-converting enzyme 1 (ACE 1) in the kidneys of Npr1\u2013/\u2013 null mutant mice  Cardiac Remodeling and DysfunctionNPPA, NPPB, and NPR1 genes are overexpressed in hypertrophied failing heart, suggesting that the autocrine and/or paracrine effects of NPs predominate and acts endogenously to protect against maladaptive pathology of CVDs  in the cardiac tissues  is progressively decreased and the level of cytosolic Ca2+ is increased in the hypertrophied heart of Npr1\u2013/\u2013 mice  Metabolic Syndrome and Diabetic ConditionsThe growing evidence suggest that ANP-BNP/NPRA signaling regulates whole body metabolism and controls diabetic conditions . The preFurthermore, ANP/NPRA signaling seems to regulate obesity, type 2 diabetes, and resistance to insulin . On the (e) Immunogenic Responses and Cardiovascular DisordersProinflammatory cytokines play a central role in the development of hypertension, cardiac hypertrophy, and CHF in experimental animal models and in humans . ElevateNpr1 gene-knockout and gene-duplicated mouse models in efforts to determine whether genetically determined differences in Npr1 expression changes the levels of cardiac proinflammatory cytokines , which seem to be associated with cardiac hypertrophy, fibrosis, and extracellular matrix remodeling - and IFN- gamma- induced expression of proinflammatory cytokines and nuclear factor-kappa B (NF-\u03baB) in macrophages . Ablatiomodeling , 2014. Nnditions . A signiant mice . The inc of CVDs .Npr1 gene copies  in mice by genetically altering protein product levels. It has been found that common genetic variants of NPPA, NPPB, and NPR1 are associated with circulating ANP and BNP levels and BP, contributing individual variations in the regulation of BP and CVD risk factors  facilitated the recovery of increased blood flow in ischemic conditions and exerted antihypertrophic and antifibrotic effects . Recombi and BNP . Genetic factors .1R) blocker, which is also referred to as Ang II receptor-neprilysin inhibitor (ARNi). Enhancing endogenous ANP-BNP/NPRA signaling has proven to be critical in the first line of therapeutic targets for hypertension, cardiac dysfunction, and CHF in decades  , which lower BP and pulse pressure  and BNP (nesiritide) for the treatment of hypertension, renal diseases and CVDs. Unfortunately, synthetic NPs as a drug have not yet been recommended for the therapeutic treatments in the United States . Howeverpressure . InteresNpr1 function in both normal and disease states. Both ongoing and future clinical studies will be needed to identify more functionally significant markers of NPPA, NPPB, and NPR1 variants in a larger human population. The chimeric designer peptides of ANP, BNP, and DNP have opened new avenues for studies of CHF, cardiac disorders, and remodeling therapy. The future progress in this area of research should significantly strengthen and advance our understanding of the genetic and molecular approaches used to evaluate diverse pathophysiological conditions in CVDs. We need to expand the potential clinical implications of ongoing investigations on the next generation personalized medicine and pharmacogenomics of NPs and their receptors that are currently in progress. The resulting knowledge will yield novel therapeutic targets and new treatment strategies for CVDs.Future studies are expected to lead a better understanding of the genetic and molecular basis of the ANP-BNP/NPRA system in regulating CVDs, including high BP, stroke, CHF, and neurological disorders. The results of future investigations of NPs and their receptors should certainly help resolve the complexities of CVDs. These future studies need to be designed to elucidate the genetic and molecular basis of KP wrote the manuscript and complied information and references.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Neurofeedback training (NFT) enables users to learn self-control of EEG activity of interest and then to create many benefits on cognitive function. A considerable number of nonresponders who fail to achieve successful NFT have often been reported in the within-session prediction. This study aimed to investigate successful EEG NFT of upregulation alpha activity in terms of trainability, independence, and between-session predictability validation. Forty-six participants completed 12 training sessions. Spectrotemporal analysis revealed the upregulation success on brain activity of 8\u201312\u00a0Hz exclusively to demonstrate trainability and independence of alpha NFT. Three learning indices of between-session changes exhibited significant correlations with eyes-closed resting state (ECRS) alpha amplitude before the training exclusively. Through a stepwise linear discriminant analysis, the prediction model of ECRS\u2019s alpha frequency band amplitude exhibited the best accuracy (89.1%) validation regarding the learning index of increased alpha amplitude on average. This study performed a systematic analysis on NFT success, the performance of the 3 between-session learning indices, and the validation of ECRS alpha activity for responder prediction. The findings would assist researchers in obtaining insight into the training efficacy of individuals and then attempting to adapt an efficient strategy in NFT success. NFT is a safe, inexpensive, and accessible technology that is a valuable intervention. Several lines of evidence have demonstrated NFT as a promising and nonpharmacological supportive treatment for neurological and psychiatric disorders, such as attention deficit hyperactivity disorder (ADHD)4, depression5, anxiety6, or insomnia7. NFT has also been applied in healthy participants to enhance several aspects of cognitive functions11. These studies have indicated that a well-trained performance is very important in either cognitive enhancement or symptom amelioration.Neurofeedback training (NFT) enables users to learn self-regulation of their cortical oscillations by receiving moment-to-moment feedback from their electroencephalogram (EEG)12. Of these studies, participants were classified into the category of responders or nonresponders. The rate of responders varies in a range of 50\u201380%13. Responders express a significant improvement in performance after training in a variety of NFT protocols17. In contrast, nonresponders often show less improvement in behavioral outcomes than responders19 or even no improvement after NFT20. Thus, responder identification may play an important role in the efficacy of NFT.Ideally, participants can learn to control their brain activities through NFT assistance. Although many participants can gain successful EEG learning by a variety of NFT protocols, some participants fail to achieve the required control of brain activity in a desired direction22. A previous study proposed successful training for responders in terms of trainability (amplitude change throughout the NFT) and independence 9. In addition to these two characteristics, an important contribution to training success is prediction from psychological or neurophysiological variables (for review24). The prediction of successful NFT would have great advantages in reducing potential frustration, saving cost on nonresponders, modifying the training protocol, and further understanding the clue of poor learning ability on NFT.Regarding responder identification, numerous studies focus on the learning ability of an NFT. Previous NFT studies have shown controversial results in training performance and outcomes25. Additionally, learning of the sensorimotor rhythm or alpha rhythm can be predicted by the amplitude/power of initial sensorimotor rhythm26 or alpha activity27 combined with other frequency bands, respectively. EEG NFT learning is evaluated by the training parameter change within sessions29, across sessions27, within sessions compared to baselines30, or across sessions compared to baselines31. The within-session performance indicates changes of a testing day but not the overall progression for an NFT. Responders of an NFT are typically defined by significant changes across sessions, e.g., between the first session and the last session32 or between the first session and other sessions2. This is a cross-day evaluation. In contrast to those prediction studies using within-session assessment, there is largely a lack of clarity regarding the performance of between-session alteration by the predictor from initial amplitude of brain activity2.The neurophysiological features, particularly initial brain activity before training, have been investigated to predict NFT success. For instance, learning beta/theta control can be predicted by resting beta activity prior to training24. One of the most popular learning indices is the alteration of EEG amplitude or power between the first session and the last session32. A previous study demonstrated the power of sensorimotor rhythm in the middle process of training, which is calculated as the average difference between the power of the first session and the other sessions, as a great feature2. Another between-session learning index is the regression level from changes in alpha amplitude throughout all sessions, which exhibits a remarkable linear relation with initial alpha amplitude27. Although these learning indices exhibit strong correlations with the initial amplitude of trained rhythm individually27, there is no systematic comparison of these learning indices for prediction with the initial EEG amplitude of interest.Several aspects of between-session learning indices have been investigated to evaluate trained performance regarding initial amplitude29. A previous study of beta/theta ratio NFT used a 2-parameter model  to validate the responder of a learning index25. The upregulation of sensorimotor NFT used a topographic model of sensorimotor amplitudes from 40 channels to validate responders regarding the within-session learning index26. The downregulation alpha NFT used a 4-parameter model from amplitudes of theta, lower alpha, sigma, and beta1 to validate responders from a within-session learning index29. The features and learning indices used in the validation model are very divergent among these studies. In addition, NFT success can be predicted by the relative alpha amplitude prior to an NFT of upregulation alpha activity through regression analysis of the within-session and between-session learning indices27. However, the relation between the learning indices and amplitudes of other frequency bands is unknown in the upregulation alpha NFT. Moreover, the predictability of the upregulation of alpha NFT by learning indices has not yet been validated.Stepwise linear discriminant analysis (LDA) combined with leave-one-out cross-validation (LOOCV) is commonly used for responder validation regarding the learning index and initial EEG amplitude. An inconsistent phenomenon between the trained brain activity and validated model of brain activity has been exhibited in previous studies33. We anticipated trainability and independence of alpha activity in the NFT process. The present study calculated 3 between-session learning indices and assessed their correlations with eyes-closed resting state (ECRS) EEGs before training. Furthermore, we validated the prediction of ECRS EEGs on the 3 between-session learning indices through stepwise LDA with LOOCV. We hypothesized a successful alpha NFT with good trainability and independence and predictability of NFT success from prior ECRS brain activity.The present study carried out NFT to upregulate alpha activity because previous studies have demonstrated advantages of alpha NFT in memory enhancement11,495\u2009=\u20091.53, P\u2009=\u20090.12) and no significant difference between the first session and other sessions. Figure\u00a011,495\u2009=\u20099.49, P\u2009<\u20090.05). The MRAAs from the 5th to 12th sessions were significantly higher than those of the first session. Moreover, the amplitude spectra of the first and 12th sessions were remarkably different in the alpha frequency band (AFB) exclusively . In particular, amplitudes of the 12th session exhibited significant increments in the range of 8\u201312\u00a0Hz compared with those of the first session. Thus, our results exclusively indicated successful regulation over EEGs of 8\u201312\u00a0Hz.Statistical analysis of baseline alpha amplitude revealed no significant main effect of session (F2 was 0.410 for L1 (P\u2009<\u20090.001), 0.492 for L2 (P\u2009<\u20090.001), and 0.30 for L3 (P\u2009<\u20090.001). Therefore, the ECRS alpha amplitude was identified as a significant predictor that accounted for 41.0% of the variance in L1, 49.2% of the variance in L2, and 30% of the variance in L3. Accordingly, the ECRS alpha amplitude provided the best prediction in L2.All learning indices were derived from the progression of alpha activity during NFT in this study. The learning index L1 ranged from\u2009\u2212\u20090.45 to 1.56 (mean\u2009\u00b1\u2009SD: 0.39\u2009\u00b1\u20090.43), L2 ranged from\u2009\u2212\u20090.16 to 1.12 (0.29\u2009\u00b1\u20090.30), and L3 ranged from\u2009\u2212\u20090.12 to 0.56 (0.16\u2009\u00b1\u20090.16). We evaluated the correlation between the learning indices and ECRS EEGs prior to the training and found that the ECRS alpha amplitude exhibited significant positive correlations with the learning indices of L1 , L2 , and L3 (r\u2009=\u20090.55 P\u2009<\u20090.001) , 35 (76.1%), and 41 (87.0%), respectively. Furthermore, stepwise LDA was applied to extract valuable features and build a prediction model according to ECRS EEGs for responder classification. First, this study examined the amplitudes of 4 typical frequency bands  of ECRS EEGs. Alpha amplitude was the only significant parameter for responder classification on all learning indices. Then, the amplitude of AFB in ECRS EEGs was extracted to build the prediction model.Moreover, we further validated the prediction ability of the AFB model on responder identification regarding the three learning indices through LOOCV. A participant with a negative discriminant score of the AFB model was considered a responder and vice versa. We found a large portion of participants who were responders through cross-validation between the learning indices and discriminant score of the prediction model Fig.\u00a0. Table 2Considering the importance of alpha specification on outcome measures and predictions, we aimed to investigate whether ECRS alpha activity prior to NFT exclusively predicts learning ability in successful NFT progression. This study achieved a successful NFT, presenting spectral changes of interest frequency band (independence) and temporal progression (trainability) throughout 12 training sessions. Only initial ECRS alpha activity was positively correlated with 3 learning indices that were derived from temporal alteration of alpha activity throughout the training course. Stepwise LDA exhibited alpha activity of ECRS EEG being a significant feature exclusively to discriminate responder from participants for all learning indices. Moreover, the prediction accuracy of the AFB model was high (>\u200982% in accuracy) in the leave-one-out cross-validation, particularly for the responder classification using the L2 index. The results suggest that a success of upregulation alpha NFT needs to exhibit an independent alpha change and that prior ECRS alpha activity predicts training success.27. The current study also found similar regression results. Moreover, this study provided additional evidence of the cross-validation on predictability for the 3 between-session learning indices. The best correlation with the ECRS alpha amplitude is the L3 learning index in a previous study27. However, the L3 learning index was worse than the other two indices in terms of the correlation coefficient and regression power in the present study. Possible reasons for the discrepancy between these two studies of the upregulation alpha NFT are different training sessions (12 vs. 20), trial amount of a session (6 vs. 12), and trial duration (6\u00a0min vs. 20\u00a0s). A meta-analysis study indicated that a long trial duration is good to increase alpha amplitude and duration during NFT33. The MRAA of the previous study ranged from 0.9 to 1.05 throughout 20 sessions27. MRAA throughout the training increases slowly with a linear trend. However, the MRAA of the present study ranged from 1 to 1.35 throughout 12 sessions. The increasing trend looks like a sigmoid curve rather than a linear trend, which may reduce the predictability of the ECRS alpha amplitude with the L3 index.NFT success can be predicted by relative alpha amplitude prior to an NFT of upregulation alpha activity through regression analysis of the within-session and between-session learning indices13. These previous studies use the performance of outcomes between the first and last sessions or the last 3 sessions as a statistical comparison to assess responders, which is similar to the learning index L1 here. The current study exhibited a high responder ratio (82.6%) for the L1 learning index, which reflects a large increase in MRAA during NFT. The high responder rate may contribute to high prediction and cross-validation accuracy by ECRS alpha activity.Responders to NFT have been reported to account for 50\u201380% of NFTs in previous studies according to different evaluations9, e.g., changes in amplitude and/or duration of trained brain activity. This study provided evidence of temporal amplitude changes in alpha activity throughout 12 sessions and significant alterations in the alpha frequency range exclusively to support trainability and independence for NFT. Recently, a previous study proposed 3 learning indices to investigate predictions of between-session, within-session, and across-all-sessions learning in an NFT27. Additionally, the same group has used a within-session learning index to validate the predictability of NFT responders using a 4-frequency-band model29. We also validated the predicted accuracy as 82.6\u201389.1% using the AFB model with 3 learning indices. The prediction of successful NFT would have great advantages in reducing potential frustration, saving costs on nonresponders, etc. In addition to trainability and independence, predictability for NFT participants can be considered performance parameters to motivate the learning driving force from participants for trainers in an NFT.A successful NFT has been proposed to use trainability and independence of training outcomes34. Reducing training sessions and attaining effective advantages are always concerns and appreciations for NFT. The prediction protocol using the participant\u2019s ECRS activity can help the researcher understand the potential characteristics of each participant and strengthen valuable instructions to increase the training progress of an NFT. For instance, ECRS alpha activity may be related to activation levels of the default mode network (DMN), the central execution network and the salience network35. As we showed, responders of alpha NFT had the ability to control their brain\u2019s self-regulation ability, which is related to remarkable alteration of the DMN36. A greater level of ECRS alpha amplitude reflects the inhibition of nonessential activity, which in turn may facilitate performance on the task37. In other words, a higher ECRS alpha amplitude may strongly inhibit irrelevant processes during NFT. These findings support the prediction and validation of higher learning ability during an upregulation alpha NFT regarding resting alpha activity.From a practical viewpoint, this study provided direct and economic advantages for trainers in the prediction of participant training through convenient and time-saving ECRS EEG recordings before training. In general, NFT usually needs tens or hundreds of sessions for patients with disorders, such as attention-deficit-hyperactivity-deficit symptoms27. Furthermore, this study extended the observation that the correlation was exclusively associated with ECRS\u2019s alpha amplitude for 3 between-session learning indices  vs. individual peak alpha frequency range (7.5\u201312.5\u00a0Hz), between-session learning index vs. within-session learning index, etc.). Second, there was a different sample size (46 of this study vs. 29 of the previous study). Third, different recording sites  were used. Centroparietal alpha activity plays a more important role in several cognitive functions, including attention and memory33.A previous study showed a significant correlation between 3 learning indices and ECRS alpha activity under an NFT of increasing alpha activityes Table . Another24. The prediction results of these potential psychological factors are obscure and very subjective. For a quantitative assessment, neurophysiological measures, such as ECRS alpha activity here, may be a good choice to be a determining factor for the prediction of NFT success.We reported high accuracy >\u200982%) in discriminating responders with 3 different learning indices . Additionally, the accuracy of the current study (89.1%) was higher than the accuracy of 86.2% on NFT of downregulation alpha activityIn summary, we provided trainability, independence and predictability for alpha upregulation NFT. Three between-session learning indices exhibited a significant correlation with ECRS alpha activity exclusively. Moreover, the AFB model of the ECRS EEG presented better cross-validation parameters with the 3 learning indices for responder identification. This study provides a systematic analysis of NFT performance, learning indices, and cross-validation for the prediction of responders from an ECRS EEG of 2\u00a0min. Our results suggest a simple way to predict alpha training. It would be very helpful for participants and researchers to save time and may set a better route for adapting the training protocol as follows.The study recruited 46 healthy and NFT-na\u00efve participants . The average age was 22.6 . All subjects signed written informed consent prior to participation and received monetary compensation for their participation after the experiment. The study was approved by the Institutional Review Board of National Cheng Kung University Hospital, and it was conducted in accordance with the relevant guidelines and regulations.Participants stayed in a quiet room and sat comfortably with our apparatus. An ECRS EEG of 2\u00a0min was recorded at the beginning. Subsequently, there were 12 NFT sessions. Each session was composed of a baseline recording of 2\u00a0min followed by six training trials of 6\u00a0min with an interval of 1 min between trials. Each participant completed 12 sessions of NFT within 4\u00a0weeks (3 days per week and a session per day).11. Scalp voltage was recorded using a cap  embedded with six Ag/AgCl electrodes. The six electrodes formed three pairs of bipolar recordings in an anteroposterior direction , which is beneficial for characterizing frontoparietal activity, as shown in our previous topographic mapping analysis11. The ground electrode was at the right mastoid. Bipolar recording was used to reduce possible artifacts of head motion or eye blink31. All electrode impedances were\u2009\u2264\u20095 k\u03a9. The acquired signal was amplified  through a multichannel amplifier with batteries39, which diminished 60-Hz electromagnetic interference. These bipolar EEGs were digitized at 500\u00a0Hz . The entire program, including acquisition and online feedback processing, was performed in the LabVIEW environment .EEG recording and processing of NFT in this study has been published previouslyInitially, ECRS EEG recording was performed for 2\u00a0min. ECRS EEG data were transformed into the frequency domain every second using a fast Fourier transform (FFT) algorithm with a Hamming window. Amplitudes within specific frequency bands, i.e., delta (1\u20133\u00a0Hz), theta (4\u20138\u00a0Hz), alpha (8\u201312\u00a0Hz), and beta (13\u201330\u00a0Hz), were calculated each second. Then, the amplitudes of these 4 frequency bands for 2\u00a0min were averaged to obtain the ECRS amplitude for each band.11. Participants were asked to avoid entering a training condition of alpha NFT during baseline recording to maintain a stable EEG quality for further analysis of MRAA.In the present study, a 2-min baseline activity was recorded before each session of alpha NFT. During the baseline recording, participants freely explored the environment in front of them and were informed that eye closure was inappropriate. The baseline activity was examined to confirm less electromagnetic interference from the power line and little artifact of head or eye movement31. The instantaneous alpha amplitude is indicated as a horizontal bar with a cartoon Bonny-like rabbit symbol. The length of the horizontal bar reflected the alpha amplitude of the 1-s EEG and fluctuated every second. When the Bonny index moved to the right direction, the averaged alpha amplitude was high and vice versa. Participants were instructed to move the bar to the rightmost position of the screen and to hold it there as long as possible. There was a resting period of 1\u00a0min between two consecutive trials. During each resting period, a researcher entered the room and identified timestamps of high amplitude from cumulative alpha events. Additionally, the researcher tried to understand each participant\u2019s strategy to obtain a high alpha amplitude. Subsequently, the researcher encouraged participants to continue using the valuable strategy or provided constructive strategies according to our previous experience31. In addition, participants were informed that eye closure was not a valid strategy during the training phase. A digital camera was set in the recording room to rule out the influence of the wrong behaviors, for example, falling asleep/drowsiness, less attention during the training, or inadequate strategy involving body movement.During an NFT, EEG data were transformed into the frequency domain in a second-by-second manner using an FFT algorithm with a Hamming window. Amplitudes of 8\u201312\u00a0Hz from three bipolar EEGs for each 1-s epoch were averaged as feedback information, which contained an instantaneous amplitude of alpha activity on the top panel and cumulative information of available alpha amplitudes within a trial on the bottom panel11. Next, the processed EEGs were visualized and manually marked to remove other contaminants, such as eye blink.Off-line EEG signals were analyzed to ascertain the training effect of an NFT. Spectral analysis of 1-s EEG was performed using an FFT algorithm and a Hamming window for the ECRS, baseline, and training trials. The epoch duration was 1000\u00a0ms, and the crossover percent was 0%. Then, the mean and standard deviation (SD) of 1\u201330\u00a0Hz amplitudes of 360 epochs in each trial were calculated. Possible artifacts were automatically marked if the EEG amplitude of a selected 1-s epoch was 2.5-fold over the amplitude SD of the trial, which worked fine to reject head or body motion artifactsThe present study used the MRAA of a session as the learning performance of an NFT throughout 12 sessions. We first calculated the relative alpha amplitude through the averaged alpha amplitude of a trial divided by the averaged baseline alpha amplitude. Furthermore, the mean relative alpha amplitude (MRAA) was calculated from averaged relative alpha amplitudes across 6 trials. The data processing and analysis were performed in MATLAB .27. For alpha upregulation NFT, participants with a positive learning index were defined as responders, and participants with a negative learning index were defined as nonresponders. The first learning index (L1) was defined as the difference in the MRAAs between the first and 12th sessions. It can be written asIn the present study, we assessed learning ability from 3 different aspects of between-session factors, i.e., training parameter changes between the 1st/12th sessions, across 11 sessions on average, and across the whole training with regressionThe second learning index (L2) was calculated as the MRAA changes of the second to 12th sessions from the first session and then averaged. It can be written as11, the third learning index (L3) was the slope of the regression line calculated by a logarithmic regression model in which the session number was taken as the independent variable and the MRAA in each session was the dependent variable.Moreover, we further focused on the learning speed over sessions. Considering the possible nonlinear trend of the MRAA over sessionshttp://www.rstudio.com/).The statistical analysis was conducted by SigmaPlot . A two-tailed significance level was set at P\u2009<\u20090.05. Data distribution was assessed by the Kolmogorov\u2013Smirnov test. All data were found normally distributed. We examined successful training of an NFT in terms of trainability and independence. Trainability was considered a significant change in alpha amplitude across training sessions. Thus, we used one-way repeated measures analysis of variance (ANOVA) to assess the baseline alpha amplitude and MRAA across 12 sessions of an NFT. Independence was defined as a significant increase exclusively in the alpha frequency range in the present study. EEG spectra of the first and 12th sessions were compared using two-way repeated-measures ANOVA (session\u2009\u00d7\u2009frequency). If appropriate, post hoc comparisons were performed using Bonferroni correction. Pearson correlation coefficients between ECRS amplitudes of 4 frequency bands  and each learning index  were compared using false discovery rate (FDR) correction by RStudio  of the ECRS EEG. Second, the coefficients of selected feature variables were determined to achieve maximum separation of two groups from the discriminant function40. Likewise, a discriminant function  of the two groups. Consequently, we built a prediction model using the discriminant function to predict responder classification with respect to L1, L2, and L3. We assessed the performance of the prediction model using a LOOCV. The LOOCV has a smaller bias compared with other validation methods  in estimating the true prediction error because each observation has an equal chance of being in a training set and a test set41. For the prediction model, we reported its accuracy, sensitivity, and specificity, which are defined below.To predict responders and nonresponders, we selected a stepwise LDAn as Eq.\u00a0) is formTrue positive (TP): correctly classifying the person as responder; True negative (TN): correctly classifying the person as a nonresponder; False positive (FP): incorrectly classifying the person as responder; False negative (FN): incorrectly classifying the person as nonresponder."}
+{"text": "The Palliative Prognostic Index (PPI) was developed to improve survival prediction for advanced cancer patients. However, there is limited data about the PPI application in a real-world scenario. This study aimed to assess the accuracy of PPI > 6 in predicting survival of cancer inpatients.A prospective observational cohort in an inpatient palliative care service at a tertiary hospital in S\u00e3o Paulo-SP, Brazil, between May 2011 and December 2018.p < 0.001). PPI \u2265 6 predicted survival of <3 weeks with a positive predictive value (PPV) of 72% and an negative predictive value (NPV) of 68% . PPI > 4 predicted survival of <6 weeks with a PPV of 88% and an NPV of 36% . When PPI was <4, the mortality rate over 3 weeks was 39% with a relative risk (RR) of 0.15 , and the 6-week mortality rate was 63% with a RR of 0.18  compared to PPI \u2265 4.We included 1,376 critically ill cancer inpatients. Patients were divided into three PPI subgroups: PPI \u2264 4, PPI 4\u20136, and PPI \u2265 6. Their respective medium overall survival values were 44 days , 20 days (95% CI 15.40\u201324.59), and 8 days (95% CI 7.02\u20138.98), (PPI was a good discriminator of survival among critically ill cancer inpatients and could assist in hospital discharge decision. PPI may help healthcare policymakers and professionals in offering high-quality palliative care to patients. Physicians often overestimate the survival time of seriously ill patients. These inaccurate prognoses lead to substantial hospice underuse and later hospice enrollment in end-of-life settings , 2. HencIn recent years, the significant development and validated use of prognostic tools have improved the clinical prediction of survival . These pThe Palliative Prognostic Index (PPI) is a well-known scoring system for predicting the survival of terminally ill cancer patients, and it was initially developed in a hospice inpatient unit in Japan . PPI is Therefore, we aimed to evaluate PPI accuracy for predicting the survival of inpatients at a palliative care service in a Brazilian tertiary hospital. This study assessed a substantially extensive database of patients in a real-world scenario.We conducted this prospective observational study with a cohort of terminally ill cancer in patients who were referred for a palliative care service at a tertiary hospital and assessed the accuracy of PPI in predicting the survival and discussed its potential implications in clinical practice.The data was collected prospectively under the study.et al  scales. According to the original publication by Morita et al  PPI was The oral intake was subjectively assessed as normal, moderately reduced or severely reduced by the medical evaluator. In case the patient had received total or partial parenteral nutrition, he or she has been included in the normal oral intake group. Dyspnea at rest was evaluated due patient\u2019s symptom referral or need of supplementary oxygen. Edema was documented as any swelling of limbs with indentation after gently thumb pressure during physical exam.Delirium was diagnosed with the criteria from the American Psychiatric Association . If the Survival curves and 95% confidence intervals (CIs) of the median survival of the patients were calculated using the Kaplan\u2013Meier method, and survival analyses were performed using the log-rank test. We arranged patients into three prognostic groups according to defined intervals: \u22644, 4\u20136, and \u22656 [We estimated the optimal PPI values for predicting 3- and 6-week survival based on receiver operating characteristics (ROC) curves and statistics. In this case, we included patients who either died or had a censored survival during the study.The mortality rates at 3 and 6 weeks were evaluated according to different PPI cut-offs: PPI \u2265 6 versus PPI < 6 and PPI \u2265 4 versus PPI < 4. The statistical analysis of the odds ratio for mortality was performed using the chi-square test.Among the patients who died, positive predictive values (PPVs) and negative predictive values (NPVs) were calculated for surviving <6 weeks with PPI > 4, and surviving <6 weeks with PPI > 6. Censored patients were not included in this analysis.A significance level of 5% was considered for all statistical analyses. When information regarding PPI was not available, the patient was excluded from the statistical analysis for clinical outcomes. Statistical analyses were performed using IBM\u00ae SPSS Statistics version 24.Overall, 1,381 cancer patients were included in this study; their baseline characteristics are summarized in At the time of analysis, an actual survival date was available for 1,037 patients (75.4%) who died, whereas censored survival was registered for the remaining 338 patients (24.6%). Because six patients underwent only an initial assessment, they were excluded from the survival analysis.p < 0.001).The study sample was divided into the three subgroups as follows: Group 1 included patients having PPI \u2264 4, Group 2 included those having PPI of 4\u20136, and Group 3 included those having PPI \u2265 6. The Kaplan\u2013Meier survival curves for the three groups are shown in p < 0.001), and the 6-week mortality rate was 93% with a RR (compared with PPI < 6) of 5.44   of 6.11  .p < 0.001), and the 6-week mortality rate was 91% with a RR (compared with PPI < 4) of 5.61 . Alternatively, when PPI was <4, the mortality rate over 3 weeks was 39% with a RR (compared with PPI \u2265 4) of 0.15  and that over 6 weeks was 63% with a RR (compared with PPI \u2265 4) of 0.18   of 6.68  .Compared to the PPI original publication , in our Using ROC curves of PPI, PPI <5.5 best predicted 6-week survival with a sensitivity of 79%, a specificity of 55%, and area under the curve (AUC) of 0.714 . PPI \u22655.PPI is a useful scoring system for predicting the survival of terminally ill cancer patients in several settings, such as palliative care units and home care settings \u20139. SigniA PPI single measure has good accuracy for clinical practice with terminally ill patients; however, this might be improved with serial assessments . If the et al [et al [et al [In our study, the prediction of survival of <6 weeks with PPI > 4 had high sensitivity and NPV, which were comparable to those reported in the original publication by Morita Considering 3 and 6 weeks mortality rate, PPI equal or greater than 6 and equal or greater than 4 had almost identical results . Conversely, the 3 weeks mortality among patients with PPI < 4 was only 39% and we consider that PPI < 4 can be considered a good prognostic factor.Patients with PPI < 4 could likely benefit from a program and have an early hospital discharge. Moreover, clinicians should consider other factors, such as the primary site and treatment perspective, for decision-making regarding the care plan of patients with low PPI values. On the other hand, PPI \u2265 4 could be used as an indicator of a high likelihood of death and could help clinicians provide the best supportive care for patients.We also performed a ROC curve analysis in order to find another PPI cut-off accurate and that include a larger number of patients in an early hospital discharge program validated in a future study. We found that a PPI cut-off of 5.5 would be the most accurate value for survival prediction within 3 and 6 weeks.Our study\u2019s strengths were the large sample size, prospective design, and diversity of primary cancer sites. In addition, the study database included a great number of inpatients evaluated by the palliative service in our institution and over a long-term period. This extended study duration has led the care team to a better understanding and proper use of PPI.In contrast, our study\u2019s limitations were the single-center study design and restriction of the study population to include inpatients under evaluation by a palliative care service from a tertiary hospital. The reality is that convenience sampling highly likely led to a biased sample, which had a predominance of seriously ill subjects and refractory symptoms. Hence, these results would be less applicable to an outpatient or hospice scenario.However, the study context reflects the current healthcare, which is hospital-centered, expensive, and ineffective, whereas palliative care and hospice remain new concepts .This study prospectively assessed PPI\u2019s effectiveness in a large cohort of cancer inpatients from a real-world scenario; patients enrolled in a palliative care team in a Brazilian tertiary hospital were included in this study. The routine use of PPI has been previously demonstrated to improve the clinical survival prediction of terminally ill cancer inpatients .In this study, we demonstrated that PPI was a reliable and useful tool for predicting survival of critically ill cancer inpatients. Therefore, PPI will help physicians in making decisions regarding clinical practice and end-of-life care for cancer patients.PPI is a useful scoring system for predicting the survival of terminally ill cancer patients in several settings. PPI\u2019s significant advantages include relying only on clinical variables, without the need for laboratory measures, simple utilization, and reproducible results. This study results demonstrated that PPI is a good discriminator of survival among critically ill cancer inpatients, and it could assist in the decision about hospital discharge. PPI may help healthcare policymakers and professionals in providing high-quality optimized palliative care for patients.All authors have no conflicts of interest to declare and the study was funded by the authors."}
+{"text": "The SlowMo study demonstrated the effects of SlowMo, an eight\u2010session digitally supported reasoning intervention, on paranoia in a large\u2010scale randomized\u2010controlled trial with 362 participants with schizophrenia\u2010spectrum psychosis.The current evaluation aimed to investigate the impact of Patient and Public Involvement (PPI) in the SlowMo study.PPI members were six women and three men from Sussex, Oxford and London with experience of using mental health services for psychosis. They received training and met at least 3\u2010monthly throughout the project. The impact of PPI was captured quantitatively and qualitatively through (i) a PPI log of recommendations and implementation; (ii) written subjective experiences of PPI members; (iii) meeting minutes; and (iv) outputs produced.The PPI log revealed 107 recommendations arising from PPI meetings, of which 87 (81%) were implemented. Implementation was greater for recruitment\u2010, data collection\u2010 and organization\u2010related actions than for dissemination and emergent innovations. Qualitative feedback revealed impacts on study recruitment, data collection, PPI participants' confidence, knowledge, career aspirations and society more widely. Outputs produced included a film about psychosis that aired on BBC primetime television, novel webpages and journal articles. Barriers to PPI impact included geography, travel, funding, co\u2010ordination and well\u2010being.A future challenge for PPI impact will be the extent to which peer innovation (innovative PPI\u2010led ideas) can be supported within research study delivery.Planned Patient and Public Contribution in SlowMo comprised consultation and collaboration in (i) design, (ii) recruitment, (iii) qualitative interviews and analysis of service users' experiences of SlowMo therapy and (iv) dissemination. However, it is notoriously challenging to demonstrate the added value or the impact of PPI in research, and few studies report this impact. The current evaluation reports the impact of PPI in the SlowMo study.1.1The SlowMo study investigated the effects of SlowMo, an eight\u2010session digitally supported cognitive\u2010behavioural reasoning intervention, on paranoia and the mechanisms of change over 24 weeks in a large\u2010scale randomized\u2010controlled trial with 362 participants with schizophrenia\u2010spectrum psychosis and distressing, persistent paranoia across three UK sites .1.2The NIHR INVOLVE guidance (2020)1.3The theoretical rationale behind PPI in the SlowMo study was the expectation of epistemic improvements in the rigour, relevance and reach (three Rs) of the research.Consistent with the epistemic framework for PPI, the study incorporated a consideration of these three Rs in the impact of PPI, and the PPI outcomes are reported in this paper, with reference to the GRIPP\u20102 reporting checklist for PPI in research.1.4Ives et al.1.5Before the current project, an extensive research programme incorporating both feasibility and an interactive human\u2010centred design approach was undertaken as outlined in Hardy et al.PPI input for the current project commenced with the grant application. The PPI consultants influenced the choice of the primary outcome measure, which assessed distress and paranoia. They also advised that the intervention should address well\u2010being, functioning and distress, such that these were incorporated into the outcome measures, alongside a secondary outcome measure of self\u2010esteem. All the PPI consultants felt strongly that there was a need to improve treatments and access to treatments for distressing paranoia.1.6One challenge in the identification of suitable PPI members lay in the well\u2010documented tension between the recruitment of lay service users, versus professionalized \u2018expert\u2019 PPI members,1.7The aims of PPI in the SlowMo study were that the PPI team would be involved in three specific aspects of work: (1) Assisting study recruitment, by presenting the research to teams and participants and giving their perspective on the study, and helping with the development of materials such as leaflets; (2) Designing and conducting qualitative interviews on participants' experiences of receiving SlowMo therapy; and (3) Assisting in the future dissemination of findings.Funding was secured to provide for 8\u2009hours of consultation per month on average throughout the duration of the project. To assist in meeting these aims, the PPI team received regular training and supervision, met as a group regionally and also project\u2010wide, and were invited to study management meetings.The aim of the current evaluation was to investigate the impact of PPI in the SlowMo study.22.1PPI members for the SlowMo PPI teams were identified through a combination of (i) recruitment from pre\u2010existing PPI research and consultation groups, (ii) identification of people who had themselves taken part in the previous or current SlowMo research and (iii) direct expressions of interest, in response to publicity. The PPI teams comprised nine people: two women and one man in Sussex, two women in Oxford and two women and two men in London. They were aged between 30 and 56 years; one woman and two men  were from a BAME background, whilst all others were White British. All members had previous experiences of using mental health services for a psychosis spectrum condition. The principles of ethics were maintained including anonymity and confidentiality, and first names have been used with permission.2.2Involvement commenced with a whole PPI team introduction and training session, co\u2010facilitated by the study PPI lead (K. G.) and local site leads. This was followed by a second training 6 months later. Thereafter, regional teams met approximately every 1\u20133 months, with group discussion and activity facilitated by the respective site lead, and also by a designated Expert by Experience PPI lead at the Sussex site (S.R.). The PPI team together, made a plan to meet as a whole study group, once or twice per year. Finally, PPI members were invited to key study meetings including the study launch, study steering meetings and the results meeting , based on previous training programmes that were co\u2010produced with service user involvement leads. The training focused on (i) an introduction to PPI and the \u2018critical friend\u2019 model, (ii) discussing, disclosing and using experiences, (iii) an introduction to research methods, PPI and peer research in the SlowMo study and (iv) supervision and safe\u2010guarding. Subsequent whole\u2010group training was more consultative and PPI\u2010led, and included (i) site updates, (ii) specific project work, (iii) role play practice and feedback in preparation for qualitative interview data collection and (iv) the development of personalized role boundaries, disclosure, keeping well and supervision plans. As recommended by Friesen et al.,2.4The core tasks for the PPI team, outlined at the start of the study, were to (i) support recruitment activity, (ii) conduct qualitative interviews with service users regarding their experiences of SlowMo therapy and (iii) support dissemination activity. The Sussex PPI lead (S. R.) contributed to the design of the evaluation, advising on the creation of the PPI log, sharing the GRIPP\u20102 reporting tool and supporting the decision to report the PPI evaluation. Early PPI activity comprised consultation regarding recruitment materials and activities, and content of the qualitative interview topic guide. Subsequent input used a more formal collaborative PPI model. It involved PPI members acting as peer researchers to collect interview data, analyse sections of transcribed data, and co\u2010produce resulting themes from the qualitative substudy with the research team, as well as co\u2010producing the Plain English results summary, and providing written project summaries for use in lay journals and future publications.2.5The impact of PPI on the project was captured in a number of ways. First, a PPI log in the form of an excel spreadsheet was created in consultation with the PPI team. This log enabled the PPI team to create a written record of (i) recommendations that arose from site and whole\u2010team meetings, (ii) the study team response to these recommendations, (iii) whether recommendations were implemented and (iv) the PPI team's perspective on the outcome. Recommendations were proposed by PPI members during PPI meetings, and recorded in the log by the PPI lead or research assistant. This log provided the opportunity for a quantitative record of the recommendations made and the percentage of these that were adopted. Second, at various stages throughout the project, both early in relation to consultation and later during the qualitative substudy, the PPI team provided written feedback on their qualitative subjective experiences of involvement. Third, PPI members attended study meetings and their impact was documented in meeting minutes. Finally, there were tangible impacts in the form of observable outputs produced by and as a result of the PPI group. Factors that enabled or hindered PPI, comprised reflections over the course of the evaluation from the PPI leads.33.1The PPI team made substantial contributions to the SlowMo study across all phases of the study, as captured through the measurement of PPI impact. First, the PPI log . Based on the Humans of New York website, it aimed to tell individual stories of how fast thinking can trip you up, and how slowing down for a moment can be helpful. Drawing on both service user and researcher stories, the aim was to normalize the fast thinking style whilst also presenting real\u2010life personal experiences of the impact of slowing down.The PPI team developed the concept of the \u2018SlowMo People\u2019 webpages (see http://slowmotherapy.co.uk/news-2/). The BBC One show has an audience of 5 million people. Feedback via twitter suggested that the film had a major community value in providing a normalizing portrayal and hopeful outcome for psychosis and voice hearing, both for the general public, and for people across the country who are suffering with these experiences. The short film was also subsequently shown to people at the start of the therapy.Finally, the Sussex PPI lead, team and therapist worked with one PPI member, the NHS communications team and a local newspaper to produce an article about the experience of paranoia and voice hearing and the positive impact of receiving the SlowMo intervention. This was picked up nationally, resulting in a short film that was aired on prime\u2010time television on the BBC One show in April 2018 , and other sites had more limited capacity to co\u2010ordinate local PPI groups, given different staffing and other challenges. In addition, several PPI members struggled with travel. As a result, PPI meetings at one site were less frequent, and preparation and conduct of qualitative interviews were more challenging, with fewer interviews conducted. The study management meetings were in the central site (London), which involved significant time and travel, and again created challenges for some PPI members in attending meetings. There were also variations in PPI members' confidence and capacity to use technology to join meetings remotely. As a result, PPI team members' input to study management meetings was limited, although it was represented as a standing agenda item at each meeting with written reports from the PPI team or verbal reports provided by the lead author.3.3.2The study team welcomed PPI involvement in the study and responded creatively and flexibly to ideas and challenges as they arose. The PPI team remained relatively stable, with five PPI members contributing for the entire project. Flexible individual support plans were put in place to enable meaningful contributions from all members despite fluctuations in well\u2010being. An agreement was reached to fund costed service user consultants' time beyond the end of funding for specific dissemination activities.However, funding was comparatively limited for PPI co\u2010ordination and input at this multisite level. This may have affected the robustness of data collection for the PPI log, although redistribution of funds across sites based on activity level ameliorated other impacts. There was a potential challenge with respect to the aim for meaningful PPI input to dissemination, as these activities occurred beyond the funding window. These included the drafting of the Plain English summary, other dissemination materials, the qualitative project publication, website updates and presentation at the stakeholder event. There was variation in attendance at the whole\u2010site PPI training and consultation sessions, related to factors such as mental well\u2010being. Finally, there were understandable fluctuations in the life circumstances, health and well\u2010being of PPI members in all sites, which impacted participation in meetings and other PPI activities. Three PPI members stopped attending PPI meetings before the end of the study: one after a period of illness; one due to workload on other projects; and a third was not contactable by their original phone number towards the end of the study. A fourth PPI member sadly passed away. Naturally, this had a significant impact on the team and was discussed both individually within the local site and as a wider team in the subsequent all\u2010site meeting.44.1The main impacts of PPI within and beyond the SlowMo trial were within the qualitative substudy4.2Overall, the PPI contribution to the SlowMo study was well supported, with clear impacts on the research and wider society and positive experiences for individual PPI members, who felt valued, supported, empowered, rewarded and understood and that their contributions mattered. PPI members also described personal growth in knowledge, skills and confidence. The PPI in the SlowMo study met five of the six UK standards for PPI (2020)4.3There are many advocates of the need to evaluate the impact of PPI in research.In the SlowMo study, we planned to evaluate the impact both quantitatively in terms of the proportion of PPI recommendations that were adopted of those that were recorded in the log and qualitatively in terms of subjective feedback and study group document review. The log was relatively well maintained, but due to resourcing issues and challenges of updating across multiple sites, it is possible that some more minor entries were omitted. It is also acknowledged that the proportion of recommendations that were adopted is a blunt measure of impact, being dependent on the number and nature of recommendations made and the ease with which they could be achieved. Some recommendations had greater potential impact and value than others, and a future log might also consider the nature and relative weight of the recommendations adopted and the reasons for them not being enacted. Subjective qualitative experiences were limited to PPI members who were more engaged, thus being open to the criticism levied by Petit\u2010Zeman and LocockPerhaps, the clearest and most tangible impacts were not those that emerged from the narrow epistemic focus on enhanced research quality, but those that arose as unique outcomes with added value from the PPI, such as the BBC One Show film and the SlowMo people webpage. There is often limited scope for these emergent community\u2010based impacts within a funded research study, and several other such innovations such as the use of thunderclaps, twibbons and a public facebook page that were also proposed by the PPI team were not taken forward. Whilst a variety of factors affected these decisions, funded research studies may necessarily be forced to limit unanticipated innovation.4.4Traditional PPI roles utilized in SlowMo included the critical friend model of consultation, and the peer researcher model of collaboration. These roles impacted on the design, ethics and delivery of the research as well as on participants, researchers, PPI members, organizations and the wider community.We propose that an important and novel role for PPI in research is that of emergent \u2018Peer Innovator\u2019. Experience within the SlowMo study and other studies4.5Future projects would benefit from a requirement for a comprehensive PPI plan, alongside the detailed project plan, at the grant application stage, costed with reference to INVOLVE guidance.Prof. Freeman reported receiving personal fees from Oxford VR outside of the submitted work. No other disclosures were reported.PPI plan: Prof. Kathryn Greenwood, Prof.\u00a0Philippa Garety, Drs. Thomas Ward, Amy Hardy and Sam Robertson. Conduct of the PPI plan, acquisition, analysis or interpretation of data: Prof. Kathryn Greenwood, Prof. Philippa Garety, Drs. Thomas Ward, Amy Hardy, Sam Robertson, Alison McGourty, Cat Sacadura, Mar Rus\u2010Calafell, Nicola Collett as well as Evelin Vogel, Claire Vella and the SlowMo PPI team. Drafting of the manuscript: Prof. Kathryn Greenwood and SlowMo PPI team members. Critical revision of the manuscript for important intellectual content: All authors including SlowMo PPI team members.Prof. Kathryn Greenwood had full access to all the data in the study and takes responsibility for the integrity of the data and the analysis."}
+{"text": "The mol\u00adecular and crystal structure of a ferrocenyl derivative with an undecyl-1,11-diol chain on one cyclo\u00adpenta\u00addienyl ring is reported: O\u2014H\u22efO, C\u2014H\u22efO and C\u2014H\u22ef\u03c0(ring) contacts occur in the extended structure. 5H5)(C16H27O2)], comprises an \u03b1,\u03c9-diol-substituted undecyl chain with a ferrocenyl substituent at at one terminus. The alkane chain is inclined to the substituted ring of the ferrocene grouping by 84.22\u2005(13)\u00b0. The ferrocene rings are almost eclipsed and parallel. The crystal structure features O\u2014H\u22efO and C\u2014H\u22efO hydrogen bonds and C\u2014H\u22ef\u03c0 contacts that stack the mol\u00adecules along the c-axis direction. A Hirshfeld surface analysis reveals that H\u22efH inter\u00adactions (83.2%) dominate the surface contacts.The racemic title compound, [Fe(C It was synthesized to provide a ferrocenyl-substituted diol for the preparation of polyesters with regular pendant electroactive groups. Similar ferrocenyl neo-pentyl diol-derived terephthalate polymers have been shown to display inter\u00adesting electrochemical properties  using LiAlH4. Enanti\u00adomeric selection of the individual chiral forms should be possible using more complex synthetic methodology (C16H27O2)], comprises a ferrocene unit that carries a well-ordered undecane chain (atoms C11\u2013C21) with hydroxyl substituents at the 1 and 11 positions along the chain \u00b0 between them. The C11 undecyl chain in 1 is conformationally extended with the typical anti\u00adperiplanar \u2005\u00c5. The C1\u2013C5 and C6\u2013C10 cyclo\u00adpenta\u00addienyl rings of the ferrocenyl group are approximately 3\u00b0 from being eclipsed and are almost coplanar with a dihedral angle of 1.7\u2005(2)\u00b0 between them; the separation of the ring centroids is is 3.298\u2005(2)\u2005\u00c5.The title compound, , 1.7\u20131.3 . 13C NMR (CDCl3): 94.7 (Fc ipso), 69.7 (\u2013CHOH\u2013), 68.3 (Cp), 67.9, 67.7, 67.3, 65.2 (Fc\u2014C\u03b1 & \u03b2), 63.2 (\u2013CH2OH), 38.3, 32.9, 29.6, 29.6, 29.5, 29.5, 26.1, 25.8 (\u2013CH2\u2013). UV\u2013vis (CH2Cl2): 325 (90), 440 (110) nm (\u025b).The title compound iso(H) = 1.5 Ueq(O). All H-atoms bound to C were refined using a riding model with C\u2014H = 0.95\u20131.00\u2005\u00c5 and Uiso(H) = 1.2Ueq(C). Despite repeated attempts to grow crystals of better quality, the crystals obtained were weakly diffracting and the extent of diffraction observed is poor with sin\u2005(\u03b8max)/\u03bb = 0.544 (2\u03b8max = 44.5\u00b0). Despite this, the structure solved and refined adequately.Crystal data, data collection and structure refinement details are summarized in Table\u00a0310.1107/S205698902101358X/hb8007sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698902101358X/hb8007Isup2.hklStructure factors: contains datablock(s) I. DOI: 2130725CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:"}
+{"text": "The long-term outcome of psychosis in association with systemic lupus erythematosus (SLE) has been insufficiently characterised. We used a specialist centre cohort of patients with SLE and psychosis to investigate their clinical outcome and phenotypic and laboratory characteristics.Retrospective cohort study of 709 SLE patients seen at a specialist centre between January 1978 and November 2018. Clinical, biochemical and immunological characteristics (Bonferroni corrected), and serum neuronal surface antibody profile using novel cell-based assays, were compared between patients with and without psychosis.vs 26.5%) and less likely to have anti-cardiolipin antibodies (5.6% vs 30.0%), but this was not significant in our small sample. Neuronal surface autoantibody tests found GABABR autoantibodies in 3/10 (30.0%) lupus psychosis patients compared with only 3/27 (11.1%) in age- and sex-matched SLE controls using fixed cell-based assays (P\u00a0=0.114). However, GABABR antibodies were not replicated using a live cell-based assay. NMDAR-antibodies were not detected with fixed or live cell assays in any samples.Eighteen  patients developed lupus psychosis over a mean \u00b1 SD of 17.5\u2009\u00b1\u200911.0\u2009years follow-up. Psychosis fully remitted in 66.7% (12/18) with a combination of antipsychotic (in 38.9%) and immunosuppressive therapy . Patients who developed lupus psychosis may be more likely to have anti-RNP antibodies (50.0% Lupus psychosis is rare but treatable. In this rare sample of eighteen patients from a 40-year cohort, no significant biomarker was found, but some preliminary associations warrant further exploration in a larger multicentre analysis. Lupus psychosis is rare and highly responsive to treatment with a good prognosis.In this preliminary study, no significant serological or neuronal surface autoantibody biomarker was found.However, a possible autoantibody mediated mechanism in at least some patients requires further exploration.Neuropsychiatric manifestations of SLE (NPSLE) include diffuse, focal, psychiatric, central, peripheral and autonomic nervous system disorders due to primary SLE disease . NPSLE hWithin NPSLE, psychiatric syndromes are present in at least half of SLE patients, most commonly mood disorders  . DespiteBR, DPPX, AMPAR1/2, NMDAR, LGI1, CASPR2) [The present study aimed to identify SLE psychosis from a large case series of SLE patients followed up at a single specialist centre for over 40\u2009years and to assess clinical and immunological characteristics. We tested serum from SLE psychosis patients for an array of antibodies directed against neuronal cell surface antigens that have been implicated as targets in psychosis (GABA CASPR2)  in order CASPR2) .University College London Hospital (UCLH) NHS Foundation Trust has followed up 709 patients with SLE during a 40-year censorship period (January 1978 to November 2018). All patients met at least four of the revised American College of Rheumatology (ACR) classification criteria for SLE . ClinicaPatient data were retrospectively collected from medical and psychiatric clinical records at UCLH. Detailed clinical information was collected at every outpatient appointment and every inpatient admission and stored on electronic and paper databases. This forms part of the British Isles Lupus Assessment Group (BILAG) index; a reliable and valid scoring system for assessing clinical disease activity in SLE . Both BIFor patients with lupus psychosis, the investigation variables  and treatment variables  were collected. Treatment with prednisolone was divided into low 0\u20137.5\u2009mg/day), medium 7.5\u201319\u2009mg/day) and high (\u226520\u2009mg/day) dose. For patients with lupus psychosis, the short  and long-term (one year and beyond) outcome of psychosis was established, as guided by previous literature [.5\u2009mg/day\u2009mg/day a\u00ae AG .Serum samples of lupus psychosis patients were tested at the Neuroimmunology and CSF laboratory, National Hospital for Neurology and Neurosurgery, Queen Square  by E.A. and M.C. using a multiplex system provided by EuroimmunSerum samples are collected routinely at UCLH during follow-up and stored. While we endeavoured to test sera for all patients who developed lupus psychosis, this was not always possible. For example, the patient may have had blood tests done at another hospital other than UCLH, they may not have been under UCLH follow-up at the time of psychosis, they may have refused at the time.n\u2009=\u200927). They were aliquoted and frozen at \u201380\u00b0C according to standardised procedures. As described elsewhere [\u00ae was used for the detection of serum IgG antibodies binding to the following neuronal antigens: (i) NMDAR NR1/NR2b subunits; the VGKC-complex-associated proteins (ii) LGI1 and (iii) CASPR2; (iv) GABAB Receptors B1 and B2; (v) AMPAR receptors type 1 and 2 and, finally; and (vi) DPPX. Five positive controls and one negative control were provided. At the time of the study, this kit did not include a non-transfected cell chip or AMPAR receptor antibody positive control. According to instructions provided, each mosaic was incubated for 30\u2009min with a human serum at an initial 1:10 dilution, followed by a 0.2% PBS-tween wash for 5\u2009min, and finally, incubation with the secondary antibody (flourescin-labelled goat anti-human IgG). Sample IgG binding to the surface of the transfected cells was revealed by green fluorescence and scored qualitatively . For interpretation, four independent assessors  scored each sample separately and blinded. Positive results were repeated to verify positivity, and to obtain a semi-quantitative measure of the antibody titre .We used all the available sera; 10 of the 18 lupus psychosis patients in all. Sera from each available lupus psychosis patient were tested at two time points: the time of psychosis, and a paired sample one to five years later (depending on availability). Samples were individually matched for age, sex, ethnicity and time/date of the sample to two or three non-psychosis SLE controls . Live cells were incubated with the patient serum  in DMEM supplemented with HEPES and 1% Bovine Serum Albumin (BSA) for 1\u202fh at room temperature. Coverslips were then washed and fixed in 4% PFA. After further washes, they were incubated with secondary antibodies  washed and mounted onto glass microscope slides with DAPI. Antibody binding to the expressed antigen was observed using a fluorescence microscope.All the samples were further tested in live cell-based assays for NMDAR and GABAescribed , 22. BriPermission to complete the clinical analysis was given by the Divisional Clinical Director for Medical Specialities at UCLH NHS Foundation Trust. Neuronal surface antibody tests were approved by the UCLH NHS Foundation Trust local Health Research Authority (HRA) Research Ethics Committee (REC), National Health Service (NHS), reference 16/SC/0494.t tests to compare continuous variables and Fisher exact tests to compare categorical variables. Taking into consideration the small sample size of patients with this rare but important complication, P-values are to be interpreted with extreme caution. To control false discovery, a Bonferroni correction was utilised and a level of significance of P\u00a0<0.002 denoted significance . In this preliminary study, P-values are intended to be used conservatively and in an explorative manner. Data were analysed using STATA (version 15.1).Clinical data were analysed descriptively. Continuously distributed data were expressed as mean\u00b1SD. We used Of 709 patients with SLE, 18 (2.5%) were diagnosed with lupus psychosis . There were no significant differences in mean age at diagnosis of SLE, duration of follow-up, gender or ethnicity between those who developed psychosis and those who did not. The mean time delay from the diagnosis of SLE to the diagnosis of psychosis was short (0.6\u2009\u00b1\u20092.9\u2009years). Ten of the 18 patients developed lupus psychosis within 12\u2009months of the diagnosis of SLE. Of the remaining patients, seven developed psychosis one to four years after the diagnosis of SLE, and the final patient unusually had their first psychotic episode nine years prior to the diagnosis of SLE.P\u00a0=0.3). All lupus psychosis patients had at least one other neuropsychiatric manifestation in addition to psychosis. Although we were able to obtain only limited data on neuropsychiatric features in the SLE cohort without psychosis, the proportion of seizures between the two groups was not significantly different (P=0.167).There were no different clinical SLE features (as defined by ACR) between those who developed psychosis and those who did not . Class Ivs 26.5%) and fewer anti-cardiolipin (5.6% vs 30.0%) antibodies, but these findings were not significant in our small sample with Bonferroni correction. Lupus psychosis patients had fewer instances of low lymphocyte count, but again, the sample is very small .In terms of serology, the majority of lupus psychosis patients tested positive for ANA , followed by anti-RNP antibodies  and anti-double-stranded DNA . Lupus psychosis patients may have more anti-RNP (50.0% The distribution of the classification of reported psychotic symptoms is shown in BR antibodies were positive in 3/10 (30%) psychosis cases  and 3/27 controls (11.1%) . BR autoantibody in the serum of \u2018patient 1\u2019. BR antibodies at either time point, one was positive at the time of psychosis only and one was positive on the later sample only, but notably had two further psychotic episodes during follow-up. The final patient was antibody positive on both the sample taken at the time of psychosis and persistently positive three years later. All GABABR autoantibody positive samples were also reproducibly positive on repeated fixed cell-based assay testing using the same methodology, and became demonstrably weaker on diluting the serum providing titres ranging from 1:10 to >1:80. Due to the different fixation method used for the NMDAR autoantibody, these cells were permeable to ANA and all SLE psychosis samples and ANA-positive controls showed intense antibody binding to the nuclei, but not to the cell membrane where the NMDAR was expressed . Similar to other NPSLE phenomena, the prevalence of lupus psychosis varies wildly in existing studies depending on the use of the ACR case definition . HowAs previously established, we confirmed that lupus psychosis is usually an early complication with just over half 10/18, 55.6%) developing psychosis concurrently with diagnosis of SLE , 55.6% d, 24. It P\u00a0=0.033) and lower proportion of anti-cardiolipin antibodies (P\u00a0=0.032) is in contrast to recent meta-analysis of NPSLE, but may be explained by the suggestion that \u2018focal\u2019 NPSLE such as stroke is more likely to be related to thrombogenic antibodies than \u2018non-focal\u2019 NPSLE such as psychosis [In terms of psychosis symptoms, the majority of presentations included paranoid and grandiose delusions, as well as auditory and visual hallucinations. This is in keeping with previous literature on organic psychosis , 29, butsychosis , 31. ThiBR antibodies may be more frequently found in lupus psychosis using the commercially available fixed cell-based assay. However, this needs to be interpreted with caution as positivity was not statistically significant in our small sample and, moreover, it was not replicated on the live cell-based assay. The levels of the antibodies titre were also low . It is also possible that this was a chance finding due to higher levels of immunosuppression in the matched controls, as perhaps suggested by the higher prevalence of proliferative nephritis. This may also explain the lower lymphocyte count found in SLE patients who did not have psychosis. However, it is not possible to draw definitive conclusions from this sample and this result remains exploratory.Our results suggest that GABABR have been previously identified in patients with NPSLE, including two patients with lupus psychosis, using ELISA [BR antibodies were first identified by a cell-based assay, similar to that used here, in limbic encephalitis [BR system dysfunction has been implicated in post-mortem studies in schizophrenia [BR in SLE controls suggests that other mechanisms, which may be SLE specific, are important in the development of lupus psychosis. The lack of NMDAR surface antibodies in lupus psychosis on both live or fixed cell-based assays is surprising, as NR2 has been reported to be a target of antibodies in NPSLE [Antibodies binding to short peptides of the GABAng ELISA . GABABR phalitis  and GABAophrenia . If implin NPSLE , 35 and in NPSLE , 37. It Our study has a number of limitations. Firstly, despite the fact that the cohort had undergone a lengthy and detailed follow-up at a single specialist centre, lupus psychosis is a rare complication and our sample size is small. While our findings may point towards important targets for further research, they are exploratory and must be interpreted tentatively. In addition, while efforts were made to retrieve historic information, this was not always possible. Psychiatric information was relatively scarce and validated rating scales for psychotic symptoms  rarely utilised. Additionally, as psychosis is a very early complication of SLE\u2014sometimes occurring prior to formal diagnosis\u2014we do not have information on the nature of any prodromal or initial symptoms that pre-dated the formal diagnosis of SLE in this cohort. In terms of biomarker testing, we used a commercially available fixed cell-based assay kit for the detection of antibodies, which has methodological superiority to peptide ELISA, but permeability of the fixed NMDAR transfected cell to ANA antibodies can make interpretation challenging to inexperienced groups. Samples required verification on live cell-based assays, which measure only those antibodies binding to the cell surface antigen but use a higher dilution with lower sensitivity. The study would benefit from other centres attempting to replicate the findings on their own archived samples.To conclude, our study of a large cohort of patients with SLE followed up for a mean of 14.1\u2009years demonstrates that lupus psychosis is a rare and early complication of SLE, with a good prognosis. There is an urgent need for more comprehensive psychiatric evaluation of patients with lupus psychosis. This preliminary study demonstrates that more work is needed to identify potentially pathogenic biomarkers in SLE and psychosis, which may be immunotherapy responsive."}
+{"text": "Big Data and Artificial Intelligence (AI) are rapidly transforming modern healthcare. The combination of these technologies allows for the collection, analysis, and utilization of large amounts of healthcare data in ways that were previously not possible. While there are several challenges to overcome, the potential benefits of using these technologies are significant and include improved patient outcomes, efficient and effective healthcare delivery, and potential of improving access and affordability of healthcare interventions. Ophthalmology is a field that generates a large amount of healthcare data, including images of the retina, cornea, and other eye structures . The widThis Research Topic focuses on the utility and the potential of big data and AI in ophthalmology. Authors from a broad spectrum of vision science and ophthalmology associated specialties from several countries, have contributed to this Research Topic. They have highlighted novel uses of large datasets, introduced new perspectives, and have reported AI algorithms with immense translational potential. In this editorial we provide a thematic overview of the exciting and diverse content covered under this Research Topic.Yang et al. have performed a bibliometric analysis of the publication trends in AI in retina from 2012 to 2022 and report interesting findings. Countries like US and China have been leading the research output with maximum number of publications , citations , and H-index . However, several issues such as lack of real-world testing of algorithms, meaningful economic impact assessment, use of multimodal imaging data for algorithm development and ethical, regulatory, and legal complexities associated with dataset curation need to be addressed by the researchers. Additionally, adequate representation of different populations in training datasets and algorithm generalizability are other areas of concern that need attention.Kwak et al. used the KNHIS-Senior database  to demonstrate that the rate or risk of surgical complications of cataract surgery did not change with tamsulosin use in the Korean elderly population. These findings contradict conventional understanding that intraoperative floppy iris syndrome (IFIS) is frequent in patients taking tamsulosin and can cause significant perioperative or postoperative complications during cataract surgery  demonstrated the value of big data by showing that surgically induced astigmatism (SIA) was higher in the femtosecond assisted cataract surgery + arcuate keratotomy group than the conventional phacoemulsification group (0.886 vs. 0.631 p < 0.001). The overcorrection ratios were also higher in the in the femtosecond group (58.9%) vs. the conventional group (48.8%). Though the femtosecond laser was effective when target induced astigmatism (TIA) values are greater than 0.75 D, overcorrection in patients with a lower degree of astigmatism and the angle of error in patients with higher astigmatism may lead to higher postoperative corneal astigmatism. Further research is thus needed to understand the factors that affect astigmatism in femtosecond laser assisted cataract surgery.In this Research Topic we observe the utility of big data to answer perplexing clinical problems. n = 558,17 to demoAhn et al. (b) also demonstrated that multi-source ASOCT images can be used for estimating preoperative best corrected visual acuity (BCVA). This AI biomarker can be used as a surrogate for cataract grade as well. The authors also reported that in the subgroup which had an absolute error (AE) \u2265 0.1, subjects had significant vision impairing disease like macular disease/glaucoma or another optic neuropathy. Thus, a more intuitive approach would be to use both anterior and posterior segment imaging to formulate an algorithm that provides an estimate of both pre and postoperative BCVA. Surgeons and patients would then be able to effectively manage expectations and deliver satisfying outcomes.Qian et al. showed that anterior chamber depth (ACD) can be predicted using smartphone captured images of the anterior segment. The MAE reported by the authors was 0.16 \u00b1 0.13 mm, and R2 between the predicted and measured ACD was 0.40. The central corneal region was highlighted in the saliency maps indicating that the predicted ACD was correlated with the clinically used site for ACD measurement. Such algorithms that utilize easily available consumer technology for image capture and subsequently can predict important ocular biomarkers have potential for rapid deployment in the real world.Ahn et al. (c) also demonstrated the possibility of automating hospital workflows by predicting pupil dilation based on medical interview and basic eye examinations data. Using a large well-curated dataset of 56,811 patients over a period of 3 years, the authors demonstrated a sensitivity of 94.2\u201375.7% and specificity of 96.2\u201396% for predicting the need of a pupil dilation test only based on basic clinical information. The authors identified that asymptomatic lesions however led to reduced performance of the model, though it is still interesting to see how big data and innovative AI algorithms may improve and automate hospital and clinic workflows.Lin et al. showed that OCT images could be used to infer VA information using a deep learning algorithm in patients with diabetic macular edema (DME). Traditionally VA is documented using chart based methods that are prone to subjectivity and depend on chart quality and illumination. AI based methods for VA estimation can be thus used for subjects with poor cooperation and provide surrogate functional vision endpoints for monitoring.Lu et al. demonstrated prediction of axial length classes using choroidal thickness measures from 2D OCT images. The model however requires 6 point choroidal thickness measurement and variables like age, gender, height, and weight for axial length classification. Development of future AI models that can predict axial length without additional demographic information can be explored using larger and multimodal datasets.Wang et al. demonstrate a deep learning model for DME classification with AUC range of 98.1\u201395.2%, sensitivity of 96.4\u201387.4%, and specificity of 90.2\u201390.1% in three large datasets. The model is novel as it can localize hard exudates along with anatomical landmarks. Diabetic retinopathy (DR) and its associated complications offer several opportunities for AI based algorithm development and potential use due to ease of retinal image capture using portable fundus cameras, well-defined disease labels and high disease prevalence.Kim et al. showed that in elderly patients with retinal diseases, the vitrectomy group showed the lower mortality from pulmonary causes when compared to those without vitrectomy. The associations were different based on underlying vitreoretinal disease: higher risk of all-cause mortality and vascular causes in patient subgroup with retinal vascular diseases and lower risk of all-cause mortality, vascular causes, and pulmonary causes in those with macular diseases. Such serendipitous results are difficult to explain as in this study a greater proportion of the patients with macular diseases who underwent vitrectomy were current or ex-smokers who regularly consumed alcohol. However, such results inspire discussion and future research into the impact of surgical interventions for ocular disease on patient mortality.In another study using big data from the Korean NHIS-Senior database, Gunasekeran et al. conducted the Acceptance and Perception of Artificial Intelligence Usability in Eye Care (APPRAISE) survey to evaluate the global perspective of ophthalmologists (n = 1176) regarding AI, focusing on four major eye conditions, namely DR, age related macular degeneration, glaucoma, and cataract. This survey highlighted that most respondents (80.9%) believed that the pandemic had played an important role in the willingness to adopt AI tools due to global focus on teleophthalmology and digital solutions for patient care while lockdowns were being implemented. While the goal of AI developers is to provide comprehensive AI solutions for patient care, ophthalmologists are more willing to use AI as clinical assistive tools (88.1%), when compared to clinical decision support tools (78.8%) or diagnostic tools (64.5%). The survey also highlighted the perceived advantages of AI based tools in patient screening (94.5%), improved access (84.7%), affordability (61.9%), quality (69.4%), targeted referrals (87.1%), and reduction of monotonous work (82.7%). Some potential disadvantages of AI like concerns over medical liability for errors (72.5%) and data security/privacy concerns (64.9%) were also mentioned. While AI is often mentioned as a threat to jobs in mainstream media, most survey responders were confident their roles will not be replaced (68.2%). The survey thus provides a comprehensive insight into the current perspective of ophthalmologists regarding AI based tools. The results can be utilized by all stakeholders to facilitate effective communication and formulate targeted interventions to address barriers that hamper development, adoption, and use of AI tools in ophthalmology.Zhang X. et al. report alarming projections of myopia affecting 8.57 million children (7\u201312 years) and 15.77 million adolescents (13\u201318 years) by the year 2050 in eastern China. Simple low cost interventions like outdoor activities, frame glasses and eye exercises have high utilization prevalence and can significantly reduce the burden of myopia. AI can also be used as a tool for myopia detection, monitoring and management. In their review Zhang C. et al., highlighted the potential uses of AI in addressing myopia holistically. With the advent of virtual reality (VR) and augmented reality (AR) into consumer realm, there are also opportunities to also develop intelligent digital tools that can aid in behavioral interventions for myopia control. However early detection of myopia and its complications followed by timely therapeutic interventions remain critical for myopia management.This Research Topic has a diverse array of publications that cover a spectrum of topics dealing with the use of big data and applications of AI in ophthalmology. The ideas, algorithms and perspectives discussed and published by the researchers in this topic have potential for considerable impact on shaping the future landscape of ophthalmology. Collaborative efforts built on these foundations can help in translating these innovations across the frontiers of ophthalmology and medicine for effective patient care and welfare.ST contributed to conception and design of the study and wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version."}
+{"text": "On the other hand, the CPs also inhibited the expression of recognized receptor CD14 and TLR4, down-regulated P-JNK, P-ERK, and P-p-38, and thus inhibited inflammatory cytokine levels in Kupffer cells (KCs). Furthermore, four kinds of dipeptides with a leucine residue at the C-terminus that might exhibit down-regulated inflammatory cytokines in the NF-\u03baB/AMPK signaling pathway functions were detected using HPLC-MS/MS. These results indicated that CPs have a potential application value in acute alcoholic liver disease.Alcohol can cause injury and lead to an inflammatory response in the liver. The NF-\u03baB/AMPK signaling pathway plays a vital role in regulating intracellular inflammatory cytokine levels. In this study, corn oligopeptides (CPs), as the research objects, were obtained from corn gluten meal, and their regulation of the activation of the Kupffer cell NF-\u03baB/AMPK signal pathway induced by LPS was investigated. Results showed that ALT, AST, and inflammatory cytokines in mice serum after the administration of CPs at 0.2, 0.4, and 0.8 g/kg of body weight displayed a distinct ( Alcoholic liver disease (ALD) is the hepatic manifestation of alcohol overconsumption. ALD could be generally aroused by excessive and acute ingestion of alcohol ,2. AccorALD has also given rise to a high mortality rate because of ineffective treatments. The Veterans Administration Cooperative Studies have shown that the mortality rate of patients with alcoholic hepatitis and cirrhosis exceeds 60% after 4 years ,9. UntilFood-derived oligopeptides are functional components derived from food protein and exist in dietary protein with a specific amino acid sequence. After the protein is degraded, these functional peptides are released and exhibit superior biological activities in the process, including antioxidant, anti-inflammatory, immune regulating, antibacterial, etc. Recently, several bioactive peptides extracted from natural products were studied, such as ganodermalucidum peptides  and cassw/w) protein [Corn gluten meal (CGM) is a byproduct in the starch processing industry, and it includes roughly 60% . The suspension was then hydrolyzed at pH 11.0 and 90 \u00b0C for 1 h. The suspension was neutralized and centrifuged to recover the insoluble protein precipitate. The insoluble protein precipitate was resuspended and subjected to the procedures above. The wet corn protein isolate (CPI) was thus obtained.CPs were prepared from CGM . The CPsw/w) and subjected to a two-step enzymatic hydrolysis. In the first step, the enzymatic hydrolysis was performed with crude alkaline proteinases at pH 8.5 and 55 \u00b0C for 3 h. The second step was performed with crude neutral proteinases at pH 7.0 and 45 \u00b0C for 2 h . The obtained hydrolysates were centrifuged to remove the insoluble impurities. The supernatant was filtered successively through 10 and 1 kDa MWCO ceramic membranes. The step of nanofiltration was carried out to remove the mineral salt. The salt-removed solution was concentrated by cry concentration under vacuum at 70 \u2103, with an evaporation rate of 500 kg/h. When the solution concentration was about 30 Baum\u00e9 degrees, it was decolored with 12% active carbon at 75 \u00b0C for 1 h. The carbon was then removed by normal filtration after de-coloration. Most of the water was removed by spray drying with a pressure of 20 MPa. The CP powder was obtained, and it was applied in the following experiments.The wet CPI was resuspended with a concentration of 6% . The molecular weight distribution of the corn oligopeptide was established using HPLC  according to the previously reported method. A total amino acid analysis was conducted with an amino acid analyzer  ,22. The Male Kunming mice were used for this study . The mice were aged 6 to 12 weeks and weighed 20 \u00b1 2 g. All the mice were maintained in an environmentally controlled room at 22 \u00b1 1 \u00b0C, with a 12 h light/dark cycle (light from 7:00 to 19:00). The treatment and maintenance of animals were conducted according to the Principle of Laboratory Animal Care  and the Peking University Animal Research Committee guidelines.n = 5), an alcohol control group , and 3 CPs intervention groups with different doses .All mice were fed a normal AIN-93M rodent diet , and the main protein source was casein. The animals were randomly assigned to 5 groups: a normal control group  ). The mice in the control group were administrated with ethanol, without any treatment. All the mice fasted for 12 h after ethanol treatment. Then, all the mice were anesthetized with pentobarbital. Blood was taken from the mouse\u2019s heart, and serum was obtained from the blood by centrifugation at 3000\u00d7 The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice serum were analyzed by the Mouse Aspartate Aminotransferase ELISA Kit  and Mouse Alanine Aminotransferase ELISA Kit  following the manufacturer\u2019s instructions.Mouse serum samples were analyzed for TNF-\u03b1, IL-1, and IL-6 levels with ELISA following the manufacturer\u2019s instructions. The involved ELISA kit was obtained from Jiancheng Bioengineering Institute .Total RNA was extracted from the liver with the SV Total RNA Isolation System. cDNA was synthesized from 1 \u03bcg of RNA by the First Strand cDNA Synthesis Kit for RT-PCR (AMV). Real-time PCR was performed for TNF-a, IL-1, IL-6, and the housekeeping gene, encoding glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). It was carried out with ABI PRISM 7000 Sequence Detection systems . The reaction mixture was composed of Absolute TM QPCR SYBR Green Mixes (12.5 \u03bcL), forward and reverse primers (5 \u03bcM and 1 \u03bcL each), nuclease-free water (8 \u03bcL), and a cDNA sample (2.5 \u03bcL).All primers were synthesized by Invitrogen . The GAPDH gene was used as an internal control. The real-time PCR primer sequences for these genes are shown in g at 4 \u00b0C for 5 min, and the pellet was washed three times with 40 mL cold HBSS containing 10 mmol/L HEPES and 10 mg/mL DNase (HBSS + HEPES/DNase). The final pellet was resuspended in 40 mL HBSS + HEPES/DNase and centrifuged at 100\u00d7 g at 4 \u00b0C for 1 min. The resulting supernatant  was layered on a sterile Percoll gradient (15 mL 25% Percoll over 15 mL 50% Percoll), which was then centrifuged at 900\u00d7 g for 20 min. The lower zone, including the interface zone, was collected and resuspended in 40 mL cold HBSS + HEPES/DNase and centrifuged at 900\u00d7 g for 5 min. The pellet was resuspended, washed twice with HBSS + HEPES/DNase, and then resuspended in RPMI 1640 medium with 10 mmol/L HEPES. The final cell pellets were resuspended in the appropriate volume of RPMI 1640 medium supplemented with 10% endotoxin-free FCS, 100 U/mL penicillin, 100 U/mL streptomycins, 15 mM L-glutamine, and 10 mM HEPES to achieve a final concentration of 1 \u00d7 106 viable cells/mL. The cell suspensions were seeded in 90 mm culture dishes and incubated at 37 \u00b0C in 5% CO2 air for 3 h to allow the adhesion of KCs. Non-adherent cells were removed by vigorous washing with Hank\u2019s solution. Over 90% of the adherent cells were identified to be KCs by positive peroxide staining. The adherent cells were then trypsinized for cell detachment. They were sub-cultured at 2 \u00d7 106 cells/mL in 35 mm dishes for 24 h to recover from isolation and adherence for in vitro stimulation. The cells were exposed to E. coli LPS (60 ng/mL); at the same time, the CPs with a concentration of 0.1, 0.5, and 1 mg/mL were added respectively and incubated at 37 \u00b0C for 1 h or 12 h. Kupffer cell viability with CPs was assessed using the MTT, as described previously [KCs were isolated according to the method described previously . Briefly6) were washed with ice-cold phosphate-buffered saline (PBS) and were lysed by adding ice-cold SDS sample buffer containing 62.5 mM Tris-HCL (pH 6.8), 2% SDS, 10% glycerol, 50 mM DTT, and 0.1% bromphenol blue. The cell extract was collected into a microfuge tube and was sonicated for 10 to 15 s to shear DNA and reduce sample viscosity. The sample was followed by heating to 95 \u00b0C to 100 \u00b0C for 5 min, and it was spun for 20 min at 4 \u00b0C at 15,000 g in a microfuge. The supernatant was decanted, and a small aliquot was removed for protein assessment. The rest of the sample was aliquoted and frozen at \u221270 \u00b0C until the application for western blotting.After 1 h incubation, the cells (2 \u00d7 10w/v) at 4 \u00b0C overnight. After that, the membranes were incubated with the primary antibodies:inhibitor of \u03baB kinase-\u03b1 , TLR4, CD14 , p-p38, p-JNK, p-ERK, total p38, total JNK, total ERK , and \u03b2-actin ; the samples were incubated overnight at 4 \u00b0C. Then, membranes were treated with HRP-conjugated anti-rabbit IgG (H + L) as the second antibody . Chemiluminescent HRP Substrate examined the immunoblotting  according to the manufacturer\u2019s instructions. Membranes were exposed by a FUJIFILM Luminescent Image Analyzer LAS-1000 . The intensities of the resulting bands were quantified by Quantity One software on an AGS-800 densitometer .Protein samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes . These nitrocellulose membranes were blocked with 5% skimmed milk powder/Tris-buffered saline with 0.1% Tween20  and toll-like receptor 4 (TLR4). The downstream signaling pathways are activated and end in activating transcription factors, including nuclear factor (NF)-\u03baB ,29.The cell viability of Kupffer cells was determined after treatment with different concentrations of CPs . The results are shown in The western blot analysis was used to compare the expression of CD14 and TLR4 in three groups: standard group without any treatment, LPS treated group, and both LPS and CPs treated group 1 mg/mL, A. In theIn the LPS treated group, the NF-\u03baB was activated, and JNK, ERK, and p-38 protein phosphorylation was enhanced. This led to an inflammatory response and the upregulated expression of inflammatory cytokines, such as TNF-\u03b1 and IL-1. It also led to additional liver cell damage. In the western blot analysis, after adding both CPs and LPS, the expression level of IkB-\u03b1 was increased, and its inhibition effect on NF-\u03baB was enhanced D. As a rThe pathogenesis of ALD involves many factors, such as genetics and nutrition, in addition to many injurious factors such as oxidative stress, bacterial lipopolysaccharides (LPS) and cytokines ,31. The Maize is a popular food for people all over the world. Studies have proven the safety of CPs. They also have various functions, such as antioxidant, anti-inflammatory, anti-hypertensive, immune boosting, and anti-fatigue ,17. SomeIn recent years, an increasing number of studies have focused on the inflammatory mechanisms (innate immune mechanisms) of ALD. It is believed that activation of KCs is a major trigger of hepatotoxicity and liver injury . KCs areIn previous studies, ethanol ingestion led to damage to the gut barrier. Gut barrier dysfunction results in an elevation of circling bacterial endotoxins, which plays a key role in triggering LPS as recognized by the ethanol-induced expression of the CD14/TLR4 receptor. This leads to the production of pro-inflammatory cytokines. In our study, we found that liver tissue inflammatory factors were abundantly expressed. We speculated that this was caused by elevated LPS . TherefoIn this study, the role of natural CP in protecting the liver from alcohol damage by inhibiting LPS-induced activation of KCs cells was successfully demonstrated. It proves to be potential food therapy for patients with ALD. The primary culture was involved in evaluating the effect mechanism from CPs on KCs. The KCs-related signal pathway could also be applied to evaluate other treatments for ALD. In addition, the KCs are closely related to liver transplants. Further studies should be performed to develop treatments based on the functions of CPs and other natural ingredients."}
+{"text": "A novel highly pathogenic avian influenza A(H5N6) clade 2.3.4.4b virus was isolated from a poultry market in China that a person with a confirmed case had visited. Most genes of the avian and human H5N6 isolates were closely related. The virus also exhibited distinct antigenicity to the Re-11 vaccine strain. H5 viruses have spread to Eurasia since 2003, Africa since 2005, and North America since 2014\u20132015. These viruses cause huge economic losses to the poultry industry and pose substantial threats to human health. By March 2022, a total of 75 confirmed cases of human infection with influenza A(H5N6) virus had been reported, including 48 cases in China since 2021 .On July 9, 2021, a human case of H5N6 infection was reported in Chongqing, China. One day later, we conducted an epidemiologic survey in the poultry market the patient had visited and collected swab samples from poultry. We identified the samples as H5N6 subtype by using H5- and N6-specific primers and probes. We propagated the virus in 10-day-old specific pathogen\u2013free chicken embryos and designated the isolate as A/chicken/Chonqqing/H1/2021(H5N6) (CK/CQ/H1). We sequenced the viral genome by using the Sanger method and deposited the sequences in GISAID  genes showed that CK/CQ/H1 and A/Chongqing/02/2021 were closely related genetically and belonged to subclade 2.3.4.4b, along with H5N6 human isolates from Sichuan (2021) and Hunan (2021) Provinces, indicating that their HA genes likely derived from wild bird strains that arrived in China in 2020 . PhylogeSequence analysis suggested that the polymerase basic protein 1, polymerase acidic protein, and nucleoprotein genes of CK/CQ/H1 were closely related to those of H5N6 viruses in China, such as A/Environment/Guangdong/C18277136/2018(H5N6) and A/Muscovy duck/China/FJFZ21/2020(H5N6). The matrix protein gene was most closely related to those of H5N8 viruses in Korea and China such as A/wild bird/Korea/H496\u20133/2020(H5N8) and A/Cygnus columbianus/Hubei/49/2020(H5N8), the polymerase basic protein 2 gene to those of A/Environment/Guangxi/28753/2014(H3N2), and the nonstructural protein gene to those of A/Environment/Jiangxi/47054/2016(H4N2)  Table 1.Analysis based on the HA amino acid sequence revealed the presence of a cleavage site (PLREKRRKR/GLF), suggesting that the isolate was highly pathogenic in chickens. The presence of receptor binding sites Q226 and G228 (H3 numbering) indicate that the isolate would preferentially bind to avian-like receptors  of the HA gene; D30, M43, and A215 of the matrix protein 1 gene; S42, E55E, E66, M106, and F138 of the nonstructural protein 1 gene; the nonstructural protein 1 C-terminal ESEV motif of the PDZ domain at position aa 227\u2013230; V89, D309, K339, G477, V495, E627, and T676 of the polymerase basic protein 2 gene; V3 and G622 of the polymerase basic protein 1 gene; and D383 of the polymerase acidic protein gene  Figure.2 lower than that against the homologous Re-11 antigen, indicating that CK/CQ/H1 exhibited greater antigenic drift relative to the Re-11 vaccine strain. The variations of antigenicity-associated amino acid sites on HA might indicate the potential antigenic drift of CK/CQ/H1  has been used in China to control clade 2.3.4.4 viruses. We analyzed differences in antigenicity between CK/CQ/H1 and Re-11. The hemagglutination inhibition titer of Re-11 antiserum against CK/CQ/H1 was 5 logCK/CQ/H1  Table 2.6 50% egg infectious dose of CK/CQ/H1. All vaccinated birds displayed no clinical signs and survived, but 2 of them shed virus  viruses in poultry and humans, China, 2021."}
+{"text": "Background: Polypharmacy has become a major and growing public health issue, with significant implications for health outcomes and expenditure on healthcare resources. In this study, a risk prediction model of polypharmacy represented by a nomogram for community-dwelling elderly patients based on the Chinese population was constructed.Methods: A cross-sectional study was conducted in Shanghai, China. The variables data affecting polypharmacy were fetched from the information system database of health government departments in Shanghai. The Least Absolute Shrinkage Selection Operator (LASSO) regression analysis was used to select the predictor variables, and multivariate logistic regression was used to establish the prediction model. A visual tool of the nomogram was established for predicting the risk of polypharmacy in the elderly population. In addition, the receiver operating characteristic (ROC) curve, calibration curve, and decision curve analysis (DCA) were used to estimate the performance of the model.Results: A total of 80,012 elderly patients were included in this study. Eight variables, containing age, residential area, preferred medical institutions, number of visits to tertiary hospitals, number of visits to secondary hospitals, number of visits to community health centers, number of diagnoses, and main types of disease, were included in the risk prediction model of nomogram. The area under the curve (AUC) of the nomogram was 0.782 in both sets, demonstrating that the model has a good discriminant ability. The calibration chart shows that the prediction model fits well with the validation set. DCA results displayed that the threshold probabilities of the two sets in the prediction model reached up to 90%, implying that the model had a preferable application value.Conclusion: This study explored the risk factors for polypharmacy among the elderly in Shanghai, China, and applied the nomogram to establish a predictive model via eight variables, which provided an effective tool for early screening and timely prevention of polypharmacy. Family physicians or pharmacists could scientifically use the tool to closely observe community-dwelling elderly patients, decreasing the adverse health effects caused by medication for the elderly. The concurrent use of multiple medications, also referred to as polypharmacy, was most commonly applied to situations where patients took 5 or more medications, which is normal among elderly patients with multimorbidity . PolyphaGiven the high degree of aging in China, polypharmacy may pose greater challenges to health outcomes and economic burden, due to the complexity of patients\u2019 healthcare needs and frequent interactions with medical services that are fragmented, ineffective, and incomplete . BecauseSignificant progress has been made on risk factors for polypharmacy. For example, age, living areas, number of diagnoses, number of visits, preference for medical institutions, etc., may increase the risk of polypharmacy . Based oThe nomogram is a novel risk prediction model based on multivariate logistic analysis with multiple indicators. Several studies have shown that it is widely used for risk prediction of various diseases, carrying significance for screening and clinical practice . In thisA cross-sectional study was conducted in Shanghai by the Department of Health Policy and Management, School of Public Health, Fudan University. Based on simple random sampling in cross-sectional studies, the sample size to estimate the overall incidence rate was calculated as follows:As the total population aged 65 and above in Shanghai was known, which was 3,616,600 as of 31 December 2019 , the corThe following formula could be obtained through integration:a is 0.05. According to the previous survey  A confidentiality agreement was signed and does not allow the database to be shared in any form; 2) The database received by the research group is anonymous, therefore it excludes name, ID card, medical insurance card, home address, contact information, etc.; 3) All personnel in the research group who have access to the database are prohibited from online processing of the database, and need to use the office computer for data analysis without an external network. This study was approved by the Ethics Committee of School of Public Health, Fudan University .The outcome variable, polypharmacy, was defined as the simultaneous use of five or more medications  for at least the last week.For the independent variables, based on previous research results , we usedThere are 3 types of living districts: countryside, suburb, and central-city. As for medical institutions, community healthcare centers, secondary hospitals, and tertiary hospitals were included, and the preferred institution was defined as the type of institution in which the patient had the greatest number of visits. Number of visits were grouped as follows: less than or equal to 5, ranging from 6 to 10, and greater than 10. The main type of disease was defined as the disease with the highest number of recurrences or, in case of a tie, the disease with the highest duration. According to the ICD-10 , including the circulatory system (I00-I99), the digestive system (K00-K93), the respiratory system (J00-J99), the musculoskeletal system and connective tissue (M00-M99), endocrine, nutritional and metabolic diseases (E00-E90), etc.R software (version 4.2.0) was used for statistical analysis.Initially, 80,012 participants were randomly divided into a training set  and a validation set  at a ratio of 3:1 using the R \u201ccaret\u201d package. Specifically, a series of test/training partitions are created using \u201ccreateDataPartition\u201d while \u201ccreateResample\u201d creates one or more bootstrap samples. The \u201ccreateFolds\u201d splits the data into k groups while \u201ccreateTimeSlices\u201d creates a cross-validation split for series data. The \u201cgroupKFold\u201d splits the data based on a grouping factor. The mean \u00b1 standard deviation was used to describe data with a normal distribution, and number and percentages were used for categorical values.p-value <0.05 was considered significant. The statistically significant predictors in the two groups were selected to establish the risk prediction model for polypharmacy, representing a nomogram.Least Absolute Shrinkage Selection Operator (LASSO) regression is a contraction and variable selection method for regression models that shrinks the regression coefficient of certain variables to zero by imposing constraints on model parameters. Thus, the LASSO method was used to analyze data from the training set to select the best predictors of polypharmacy. The above included the eight variables used for the preliminary screening of risk factor variables. By introducing the selected features in the LASSO regression model, we constructed the prediction model by multivariate logistic regression analysis, and a In addition, the accuracy of the risk prediction model was estimated based on several validation methods by using the data of the training set and validation set. The receiver operating characteristic curve (ROC) was used to identify the quality of the nomogram to distinguish true positives from false positives based on the area under the curve (AUC). The calibration curve was drawn and calculated to evaluate the calibration of the polypharmacy risk nomogram. According to the net benefits of different threshold probabilities, the decision curve analysis (DCA) was used to determine the clinical utility of nomograms in this population.p < 0.0001). After randomization, there was no statistical difference between the training set and the validation set. The homogeneity between the two sets supports the credibility of the model construction and related verification. A total of 80,012 patients with an average age of 75.29 \u00b1 7.73\u00a0years were included in this study, of whom 50.06% were males. The patients\u2019 mean number of diagnoses was 3.50 \u00b1 1.99, receiving a median number of 6.10\u00b14.02 drugs. The prevalence of polypharmacy (\u22655 medications) was 60.13%, and the prevalence of extreme polypharmacy (\u226510 medications) was 14.49%. Based on the ICD-10, the most frequent types of disease were in the circulatory system (35.27%), digestive system (10.04%), and respiratory system (7.22%). There were significant differences in age, sex, living district, preference for medical institutions, number of visits, number of diagnoses, and main types of disease between patients with polypharmacy and non-polypharmacy   . A p-valDCA results display that the threshold probabilities of the two sets in the prediction model reach up to 90%, implying that the model had a good application value .Recognizing the scale of avoidable harm associated with unsafe medication practices, WHO launched the third Global Patient Safety Challenge: Medication Without Harm in March 2017 . Given tAs an economically developed area with a high degree of aging in China, the polypharmacy prevalence of Shanghai elderly patients included in this research was 60.13%. This study used institutional data from a representative sample of the elderly population in Shanghai, representing the polypharmacy level of the elderly in more developed regions of China. Other studies related to the prevalence of polypharmacy have shown different results. In a survey of 2,707 elderly European patients with an average age of 82.2\u00a0years, 51% used more than 6 drugs ; accordiElderly patients have a variety of characteristics that make them more prone to polypharmacy. These indicators can help develop models for early identification of the patients at risk of polypharmacy and help to prevent polypharmacy along with its related problems. In this study, we built and validated a risk prediction model for polypharmacy with eight variables to identify elderly patients potentially at risk.Greater age is an essential indicator . The noThe current study shows that elderly patients living in central areas are more likely to suffer from polypharmacy, which is related to China\u2019s relatively loose referral system. With more medical institutions having better medical conditions and doctors in the central-city area, the elderly living in the central city could go to any specialized or general hospital for treatment in a closer geographical location, which increases the risk of polypharmacy, while for elderly patients living in the countryside more visits mean further transportation and higher costs.Patients who prefer to see a doctor in the community have a lower risk of polypharmacy, which implies that medication review through primary pharmaceutical care based on the Family Physician Service optimizes the use of medicines for individual patients .In addition, for any level of the medical institution, a lower number of patient visits is a protective factor against polypharmacy. In this study, the number of visits is grouped in the nomogram, which may weaken the accuracy of the prediction model, but it will broaden the practicality and applicability.Undoubtedly, the number of diagnoses was a strong predictor of polypharmacy . ResearcGiven the convenience of application and high diagnostic performance, this study applied the nomogram to polypharmacy risk prediction. Based on the results of the above eight risk factors, we visualized the multivariate prediction model as a nomogram and verified it in three aspects of discrimination, calibration, and Clinical usefulness.It should be considered that regular and comprehensive medication reviews, coordinated by the physician or pharmacist in primary care, are necessary interventions to keep polypharmacy under control . The nomSeveral limitations of this study should be acknowledged. First, the data on the medication used in the study was institutional. Since the medical system is not completely interconnected, the drugs of private medical institutions are not involved, which means that the risk of polypharmacy is underestimated. Second, for the institutional data from sampling the elderly population, this study only collects the most basic demographic and medical history characteristics, lacking the sociological and economic characteristics of elderly patients. Nevertheless, the applied indicators are reliable and easy to obtain, so the model has high representativeness and strong applicability. Third, the model needs certain application conditions, such as economically developed areas, mature family physician services, pharmacist equipment, etc.via eight variables, which provided an effective tool for early screening and timely prevention of polypharmacy. Early identification of these patients, even before polypharmacy arises, is the first step in avoiding negative effects on their health. Family physicians or pharmacists could scientifically use the tool to closely observe community-dwelling elderly patients, decreasing the adverse health effects caused by improper medication use for the elderly in the future.In conclusion, this study explored the risk factors for polypharmacy among the elderly in Shanghai, China, and applied the nomogram to establish a predictive model"}
+{"text": "Longitudinal studies increasingly collect rich \u2018omics\u2019 data sampled frequently over time and across large cohorts to capture dynamic health fluctuations and disease transitions. However, the generation of longitudinal omics data has preceded the development of analysis tools that can efficiently extract insights from such data. In particular, there is a need for statistical frameworks that can identify not only which omics features are differentially regulated between groups but also over what time intervals. Additionally, longitudinal omics data may have inconsistencies, including non-uniform sampling intervals, missing data points, subject dropout and differing numbers of samples per subject.OmicsLonDA, a statistical method that provides robust identification of time intervals of temporal omics biomarkers. OmicsLonDA is based on a semi-parametric approach, in which we use smoothing splines to model longitudinal data and infer significant time intervals of omics features based on an empirical distribution constructed through a permutation procedure. We benchmarked OmicsLonDA on five simulated datasets with diverse temporal patterns, and the method showed specificity greater than 0.99 and sensitivity greater than 0.87. Applying OmicsLonDA to the iPOP cohort revealed temporal patterns of genes, proteins, metabolites and microbes that are differentially regulated in male versus female subjects following a respiratory infection. In addition, we applied OmicsLonDA to a longitudinal multi-omics dataset of pregnant women with and without preeclampsia, and OmicsLonDA identified potential lipid markers that are temporally significantly different between the two groups.In this work, we developed https://bioconductor.org/packages/OmicsLonDA), to enable widespread use.We provide an open-source R package (Bioinformatics online. Human health is highly dynamic, and there is great interest in better understanding how wellness and disease states fluctuate over time in relation to different variables such as lifestyle or treatment perturbations. While genetics provides a blueprint for life, health states are also reflected by many other \u2018omics\u2019 such as transcriptomics, proteomics, metabolomics, lipidomics, and microbiomics. With rapid advances and decreasing costs in sequencing and mass spectrometry, many studies are beginning to measure comprehensive omics profiles at frequent timepoints across many individuals. Longitudinal omics studies generate enormous datasets; however, there is currently a major bottleneck in analyzing these data to extract and interpret meaningful findings. In particular, there is a need for robust statistical methods for longitudinal omics.Longitudinal omics data have their own properties that differentiate them from cross-sectional experiments, including high dimensional feature space, temporal and intrapersonal variation, and samples characterized by heterogeneity of various natures. These heterogeneities include a different number of samples per subject, uncaptured data points, variable time of sample collection \u2018sampled non-uniformly\u2019 and omics features often represent a biological process that usually exhibits temporal variation. Another aspect is the variability in temporal dependence structure, \u2018variance\u2013covariance structure\u2019, between repeated measurements. All of these characteristics of longitudinal omics data make the analysis a challenging task. Methods developed for longitudinal omics data analysis can be categorized into the following groups: (i) methods that extract omics biomarkers for a specific phenotype .bootLong, was developed for extracting microbial biomarkers from longitudinal microbiome data based on a moving block bootstrap approach , for widespread availability.In this article, we introduce a robust method to perform longitudinal differential analysis on omics features in order to identify time intervals of differences between study groups. The method is based on a semi-parametric approach, where we use smoothing splines to model longitudinal data and infer significant time intervals of changes in omics features based on an empirical distribution constructed through a permutation procedure. The proposed method can handle all types of inconsistencies in sample collections and adjust for subjects' specific baseline. Identifying biomarkers and their significant time interval differences can inform intervention strategies , and most importantly, may indicate the best time for interventions to be administered to patients. The method achieved a correctly calibrated type-I error rate and is robust to data collection inconsistencies that commonly occur in longitudinal human studies. Application of the proposed method to iPOP cohort revealed a multitude of sex differences in dynamic respiratory infection response. To our knowledge, this is the first study to investigate sexual dimorphism in infection response with frequent temporal sampling and delineation of the dynamic infection response for each sex. We also applied OmicsLonDA, aims to find the feature's significant time intervals (FSTI) of differences between each pair of the tested groups . The method works on unpaired experiment design, where subjects' longitudinal samples are related to only one of the tested groups. We model the longitudinal data in a time-series model using a spline kernel. Although, in theory, longitudinal data should be correlated, the first-order auto-correlation is not high [e.g. 0.19 in the longitudinal microbiome data from the Human Microbiome Project  represents the quantity from sample j that is annotated to feature i. The proposed method is based on four main steps as shown in The proposed method,  Project , metabolf under consideration, we first adjust for the difference in the personal baseline. Our strategy is to calculate the log-ratio between omic feature's level of each timepoint t to the level of the same omic feature at the subject's chosen baseline bt  as shown in  is an indicator function. The pvalue is adjusted for multiple testing using Benjamini\u2013Hochberg (BH) to control for the false discovery rate. For each feature f, significant time intervals are those with We perform a permutation procedure by permuting the sample group labels ed using  when testive and  when it SimStudy , which follows . OmicsLonDA based on the model they use to fit the longitudinal data for each group; (i) OmicsLonDA with smoothing spline ANOVA (OmicsLonDA_SSANOVA) and (ii) OmicsLonDA with Gaussian additive mixed models (OmicsLonDA_GAMM). GAMM allows fitting smoothing terms to model time-series data, and it uses subject ID as a random effect. No covariates were added in this simulation study. We also benchmarked OmicsLonDA against MetaLonDA and splinectomeR. We choose to benchmark against MetaLonDA and splinectomeR since, to our knowledge, they are the two methods that were developed mainly to identify time interval of significance between the two tested groups. There are three key differences between OmicsLonDA and MetaLonDA: (i) MetaLonDA does not correct for personal baseline, (ii) MetaLonDA uses negative binomial smoothing spline when used with microbiome data and LOESS regression otherwise and (iii) MetaLonDA uses a different formula for testStatistic that only include the area between the curves of the two groups without adjusting of the standard error in their estimation. In our benchmarking experiments, we ran MetaLonda with LOESS regression and 1000 permutations, and all other parameters were left as default. For splinectomeR, we ran the benchmarking evaluation with cut_low\u2009=\u20090 inorder not to filter out any subjects based on small number of time points, and the rest of parameters were left as default.We evaluated the performance of the OmicsLonDA (>0.99) among all five tested patterns, with OmicsLonDA_GAMM has slightly more specificity over the first four patterns, and OmicsLonDA_SSANOVA has slightly more specificity in Pattern 5. On the other hand, MetaLonDA\u2019s specificity is \u223c0.80 among all first four patterns, and 0.97 in Pattern 5. splinectomeR followed similar trends to MetaLonDA, high sensitivity and low specificity. \u00a0splinectomeR has the highest sensitivity (\u223c1) among the compared methods. This high sensitivity can also be seen as a trade-off with the low specificity of splinectomeR. In general, there is a decrease in sensitivity for all methods from Pattern 1 to Pattern 4. This decrease in sensitivity is expected due to the fact that as the time intervals that are significantly different between the two groups shift to the right , there are more participants dropping out of the study, and hence there is lower power of each method to detect the significantly differential time intervals. OmicsLonDA_SSANOVA maintains reasonably high sensitivity across all patterns . OmicsLonDA_GAMM has a similar sensitivity pattern to OmicsLonDA_SSANOVA, but surprisingly, the sensitivity drops significantly at Pattern 4 (0.72). These results demonstrate that OmicsLonDA_SSANOVA is a better choice than OmicsLonDA_GAMM when few samples cover the tested time interval. Additionally, OmicsLonDA performance. OmicsLonDA when running on the five simulated patterns after each of the baseline adjusting methods. While log-ratio and min\u2013max baseline adjusting methods have yielded a similar effect on OmicsLonDA specificity, min\u2013max has yielded higher sensitivity.Additionally, we evaluated the two implemented personal baseline adjusting methods  on OmicsLonDA depends primarily on the number of permutations used to construct the empirical distribution for each feature. Additionally, OmicsLonDA can be run in parallel on multi-core platforms. However, we used one thread in our time evaluation. In our analysis of the simulated data with 1000 permutations, for each feature, OmicsLonDA analysis took, on average, 43\u2009min and 47\u2009s. The evaluation was conducted on a MAC machine with a 2.5\u2009GHz Intel Core i7 processor and 16 GB 1600\u2009MHz RAM.The running time of 2, 55 females and 50 males) were profiled during healthy periods and extensively during periods of respiratory viral infection (RVI), immunization and other situations that perturb human host\u2013microbial physiology.As an application for our proposed method, we used the integrative Personal Omics Profiling (iPOP) cohort, a longitudinal cohort that aims to characterize the complex host\u2013microbial interactions in type 2 diabetes mellitus (T2DM)  participants who were followed before and after RVI  exhibit temporal differences between males and females following RVI. OmicsLonDA on a longitudinal lipidomics study on preeclampsia . In our analysis, we used 1000 times permutations to construct the empirical distribution for each lipid. All the results were adjusted for multiple testing using BH method with a significance level Following n method  and normWe identified 19 lipid species, accounting for eight lipid classes, with significant temporal differences between preeclampsia and control groups during pregnancy. OmicslonDA has achieved a correctly calibrated type I error rate and is robust to data collection inconsistencies that commonly occur in longitudinal human studies. Moreover, the sensitivity is high in Pattern 1 and then declines slightly through Pattern 4. This decrease in sensitivity can be explained due to the decreasing number of samples collected towards the end of the study (i.e. patient dropout).In this work, we have developed a statistical method that provides robust identification of time intervals where omics features are significantly different between groups in longitudinal multi-omics. The method is able to simultaneously identify time intervals and differential signatures by analyzing each feature separately, but across all patients. The proposed method is based on a semi-parametric approach, where using smoothing splines to model longitudinal data and infer significant time intervals of omics features based on an empirical distribution constructed through the permutation procedure. A critical need in longitudinal omics is for robust frameworks that incorporate the time dimension in statistical significance analysis. Our method was evaluated through extensive simulations (five patterns). The performance evaluation demonstrated that OmicsLonDA on two real-world datasets: (i) the iPOP longitudinal omics study for investigating sexual dimorphism on molecular response following RVI and (ii) preeclampsia cohort to identify time intervals that lipids are significantly different between pregnancy with and without preeclampsia. Recently, OmicsLonDA has been utilized to identify the seasonal time intervals of differentially abundant/expressed omics features between insulin-resistant and insulin-sensitive individuals  such as CE (20:4), PC (17:0/20:4), PC (18:0/20:4) and PC (18:1/20:4), which may serve as clinically meaningful biomarkers for preeclampsia early diagnosis. Intriguingly, all of these four lipid biomarkers share the same fatty acid chain arachidonic acid (fatty acid 20:4). Arachidonic acid is a polyunsaturated fatty acid-containing 20 carbons and four double bonds with a final double bond in the \u03c9\u2009\u2212\u20096 position. It is well-documented that arachidonic acid and its products eicosanoids play important roles in inflammatory processes  baseline adjustment method and (ii) number of permutations. Firstly, if subjects have samples right before perturbation, baseline adjustment based on log-ratio .btac403_Supplementary_DataClick here for additional data file."}
+{"text": "In tennis, previous studies have shown differences in plantar pressure depending on tennis-specific movements , playing surface , and serve type . However, the influence of stroke direction on plantar pressure in tennis players with diverging skill level is unknown. Thus, the purpose of this study was to determine the effect of stroke direction on plantar pressure in each foot during the forehand and backhand stroke among players of different performance levels.Thirty-nine female and male healthy adult tennis players (mean\u2009\u00b1\u2009SD age: 23.5\u2009\u00b1\u20096.4\u00a0years) representing athletes from three performance levels  participated in this study. The players performed longline/cross forehand and backhand groundstrokes (topspin) on a clay court while plantar pressure distribution was measured in each foot using flexible instrumented insoles.The three-way ANOVA (performance level\u2009\u00d7\u2009stroke direction\u2009\u00d7\u2009foot dominance) showed (a) no significant differences in plantar pressure data between cross and longline strokes in almost all cases, (b) in part, significantly larger pressure values in advanced compared to intermediate and recreational players, and (c) significantly larger pressure data for the dominant compared to the non-dominant foot in nearly all comparisons.Regarding an appropriate plantar pressure distribution, our results suggest that during training of especially recreational and intermediate players attention should be paid to the feet rather than to stroke direction. N\u2009=\u200910, mean\u2009\u00b1\u2009SD age: 23.8\u2009\u00b1\u20096.0\u00a0years) [N\u2009=\u200915, age range: 10\u201316\u00a0years, tournament level) showed higher plantar pressure values for the flat compared to the topspin and the slice serve [In tennis, stroke direction  in combination with stroke technique  and stroke type  is a significant factor for variable groundstrokes during a match . Accordice serve .Although the previously reported studies have increased the knowledge on the influence of different characteristics of tennis serve and foot placement on plantar pressure distribution, studies on the influence of stroke direction  are lacking. In addition, all previous studies only examined players representing a single performance level and based on studies on physical , 6 and bTherefore, the aim of the present study was to investigate the effect of stroke direction  on plantar pressure distribution in each foot  during the forehand and backhand groundstroke among healthy adult tennis players of different performance levels . We hypothesised that plantar pressure data will differ between cross and longline stroke direction as well as among dominant and non-dominant foot, and this will further be affected by players\u2019 performance level. To the best of our knowledge this is the first study that investigated the effect of stroke direction  on plantar pressure per foot during the forehand and backhand stroke in tennis players of different performance levels. From a practical point of view, the present study has the potential to influence the design of training programs for performance enhancement. Precisely, with a better understanding of plantar pressure differences in terms of stroke direction and foot dominance depending on performance level, specifically tailored exercises can be designed.Thirty-nine female and male healthy adult tennis players from different local tennis clubs agreed to participate in this study Table . PlayersDuring a single visit to the clay court, tennis players performed a standardized 15-min tennis-specific warm-up including ground strokes with submaximal speed. Afterwards, the players were familiarized with the instrumented insoles followed by the execution of different groundstrokes (topspin) using a standardized sequence: a) forehand cross, b) backhand cross, c) forehand longline, and d) backhand longline. The players were free to decide their stance condition . A trial included the feed at submaximal speed by player 1, followed by the return stroke at submaximal speed by the investigated player 2 into a predetermined 4.12\u2009\u00d7\u20096.50\u00a0m landing zone  with a sampling frequency of 200\u00a0Hz Fig.\u00a0. Preciset-tests) were performed. Further, effect size (\u03b7p2) was calculated and reported as small (0.02\u2009\u2264\u2009\u03b7p2\u2009\u2264\u20090.12), medium (0.13\u2009\u2264\u2009\u03b7p2\u2009\u2264\u20090.25), and large (\u03b7p2\u2009\u2265\u20090.26). The significance level was set at p\u2009<\u20090.05.All statistical analyses were performed with SPSS version 27.0 . Descriptive data are reported as group mean values and standard deviations. For all analyses, assumptions of normality (Shapiro\u2013Wilk Test) and homogeneity of variance/sphericity (Mauchly Test) were checked and met prior to the application of analysis of variance (ANOVA). A 3 \u2009\u00d7\u20092 \u2009\u00d7\u20092 , non-dominant) repeated measures ANOVA was conducted for the forehand and the backhand, separately. If a significant performance level by stroke direction interaction occurred, Bonferroni-adjusted post-hoc tests , performance level , and foot dominance  during the forehand and backhand groundstroke are illustrated in Table p\u2009=\u20090.030\u20130.042, \u03b7p2\u2009=\u20090.16\u20130.18) and foot dominance  but not of Stroke direction  and foot dominance  but not of stroke direction , performance level , and foot dominance   but not of stroke direction and performance level  among healthy adult tennis players of different performance levels. The main results can be summarized as follows: (a) in all cases (except for the rearfoot during backhand stroke), there were no significant differences in plantar pressure data between cross and longline strokes; (b) in part, significantly larger pressure values were found in advanced compared to intermediate and recreational players as well as in intermediate compared to recreational players; (c) nearly all comparisons showed significantly larger pressure data for the dominant compared to the non-dominant foot.Contrary to our hypothesis, there were almost no differences in plantar pressure data between the cross and longline stroke , regardless of the foot dominance and performance level considered. Accordingly, in contrast to other factors  stroke direction does not seem to have a significant influence on plantar pressure distribution. Alternatively, it could be argued that the assessment of plantar pressure data is not sensitive enough to detect existing differences. However this seems unlikely, as several studies have shown pressure differences using insoles with respect to types of tennis serve , 14, serN\u2009=\u200913, mean\u2009\u00b1\u2009SD age: 20.1\u2009\u00b1\u20090.9\u00a0years) versus intermediate  players. Besides others, they observed significantly larger pressure data  at backward-end and forward-end in superior compared to intermediate players. Further, Zhao and Li [N\u2009=\u200910, mean\u2009\u00b1\u2009SD age: 23.7\u2009\u00b1\u20092.4\u00a0years) and amateur  badminton players. Among others, significantly larger pressure values  were found in professional compared to amateur players. Referring to the present study, the presence of a greater force level could be a possible reason for the larger plantar pressure values in advanced compared to intermediate and recreational players. For example, in a recent systematic review with meta-analysis, Lambrich and Muehlbauer [In accordance with the hypothesis, partial differences in plantar pressure data were shown regardless of the stroke direction and foot dominance considered. Specifically, the largest pressure values  were observed in the advanced players followed by the intermediate players and then the recreational players. The available literature does not indicate research related to plantar pressure data in tennis players of different performance levels. Therefore, we compared the results of our research to other types of sport . Qian et al.  examinedo and Li  investigConsistent with the hypothesis and other studies , 4, diffZ). Therefore, future investigations should be carried out using a force plate in order to be able to make statements on the other force components . Third, only pressure but no kinematic data of the lower extremities were collected. Therefore, no statements can be derived regarding changes in position data .There are some limitations in this study that need to be addressed. First, the feed at submaximal speed was not standardized  but performed by a player. This results in some variability in the fed ball speed, spin, and depth, which may have influenced the plantar pressure data. Second and due to the use of instrumented pressure-detecting insoles, the present results are limited to the vertical force component (FWe investigated the effect of stroke direction on plantar pressures in each foot during the forehand and backhand groundstroke (topspin) among healthy adult tennis players of different performance levels. Our findings suggest that out of the investigated parameters (a) stroke direction has almost no, (b) performance level has partial and (c) foot dominance has most influence on plantar pressure. Therefore, during the training of especially recreational and intermediate players, less attention should be paid to differences in plantar pressure between both stroke directions, but rather between the feet."}
+{"text": "The Impact-Echo (IE) test is an effective method for determining the presence, depth, and area of cracks in concrete as well as the dimensions of the sound concrete without defects. In addition, shallow delamination can be measured by confirming a flexural mode in the low-frequency region. Owing to the advancement of non-contact sensors and automated measurement equipment, the IE test can be measured at multiple points in a short period. To analyze and distinguish a large volume of data, applying supervised learning (SL) associated with various contemporary algorithms is necessary. However, SL has limitations due to the difficulty in accurate labeling for increased volumes of test data, and reflection of new specimen characteristics, and it is necessary to apply semi-supervised learning (SSL) to overcome them. This study analyzes the accuracy and evaluates the applicability of a model trained with SSL rather than SL using the data from the air-coupled IE test based on dynamic preconditions. For the detection of delamination defects, the dynamic behavior-based flexural mode was identified, and 21 features were extracted in the time and frequency domains. Three principal components (PCs) such as the real moment, real RMS, and imaginary moment were derived through principal component analysis (PCA). PCs were identical in slab, pavement, and deck. In the case of SSL considering a dynamic behavior, the accuracy increased by 7\u20138% compared with SL, and it could categorize good, fair, and poor status to a higher level for actual structures. The applicability of SSL to the IE test was confirmed, and because the crack progress varies under field conditions, other parameters must be considered in the future to reflect this. Over time, concrete buildings and civil infrastructure are subjected to continuous stochastic damage processes such as continuous loads, vibrations, and various degradation factors over a long period . Their dFrom this viewpoint, nondestructive testing and its fusion in the safety diagnosis of concrete structures have gained much popularity over the past 20 years ,10. HoweThe Impact-Echo (IE) test, developed by Carino et al. , is one To overcome these drawbacks, methods such as machine and deep learning have emerged, and through these methods, various features can be derived in addition to differences in dynamic components based on the dynamic or wave theory. Most of these methods are approaches based on supervised learning (SL). In addition, its application has a high potential to complement various weaknesses, such as nonlinearity and damage degree of concrete, and to check the current state . AlthougSince the introduction of the IE method in the 1980s, it has been widely known as a representative nondestructive method for finding the integrity and defects of concrete members and structures . ExampleAmong these advancements have been advanced analysis goals, such as improved filtering of IE signals, development of signal processing techniques, and machine and deep learning. More specifically, it was primarily signal processing in the past, but recently, machine and deep learning methods have been employed. In signal processing, to examine the non-static characteristics of IE signals, time-frequency analysis such as short-time Fourier transform (STFT) , empiricRecent studies ,41 proviHowever, proper labeling for the entire temporal domain is very expensive and time-consuming , and theh, the P-wave velocity Cp, and the peak frequency in the frequency spectrum are related to each other in the following Equation (1) [\u03b2 is a shape factor of approximately 0.96 in an infinite plate structure.The IE method was established as a reliable method to check dimensions or lamellar cracks in concrete slabs and pavements ,56. The tion (1) :(1)h=\u03b2CPa/h value is large), it is difficult to distinguish the thickness mode because the bending mode is dominant [The non-contact IE test system replaces the contact sensor with a non-contact sensor. The basic principle of the non-contact IE test method is to measure a leaky wave generated by a surface wave with a microphone. Various studies have shown that the non-contact IE data provide the same results as a conventional contact sensor . The IE dominant .a) and depth (h) under a semi-clamped boundary condition over the horizontal crack. According to the plate theory, plates can be classified as thick plates, a thin plate, and a membrane, which can be classified as shown in a/h) [a/h decreases, , the effect of shear deformation and rotational stiffness cannot be ignored, so the bending behavior of the plate is difficult to express numerically [a/h values, and the relative dominance of the bending mode and Impact-Echo mode (thickness mode) changes according to the change in a/h. Generally, when the a/h value is large, the bending mode dominates, and the amplitude value of the thickness mode is quite small, making it difficult to measure. However, as the a/h value decreases, the size of the thickness mode starts to increase, and as the value approaches the thickness of the plate, the thickness mode becomes clear and can be easily distinguished from the values of other modes. It is important to study the dynamic behavior of the plate in the bending and the thickness modes, which are sensitive to changes in a/h values.In the case of delamination cracks inside the structure in the IE method, plate theory can be applied by assuming the plate with the width ( in a/h) . Generalerically . Since dw = w is the transverse deflection, which corresponds to the thickness , the dominant frequency for delamination cracks is up to 6 kHz in the typical case of area dimensions (100\u20132000 mm) and depth (10\u2013100 mm) ranges assuming a thin plate with the clamped boundary condition according to Equation (2) . Additio\u03bc) of each row of the X matrix is calculated to construct the average matrix, and in the third step, the deviation matrix is constructed by subtracting the mean from the element values of the basic matrix X. Thereafter, in step 4, the covariance matrix may be derived by multiplying the deviation matrix by the transposed matrix (DT) of the deviation matrix. Additionally, eigenvector and a eigenvalue (\u03bb) are calculated using the obtained covariance matrix, and the eigenvectors are arranged in ascending order according to the size of the corresponding eigenvalue to derive the principal component.Principal component analysis is a method for reducing high-dimensional data with various variables to low-dimensional data. It was proposed by Pearson and later improved by Hotelling and Jolliffe to establish a modern theory ,64. PCA It is difficult to quantify the extent to which the theoretically occurring main frequency in the IE spectrum is shifted according to the degree of delamination defect. In this study, various features of the flexural mode were derived in a spectral region of a certain range by considering the dynamic characteristics in the IE spectrum, and PCA was employed to analyze them.Semi-supervised learning (SSL) is a method that enables the effective use of unlabeled data in machine and deep learning. Chapelle et al.  summarizIn this study, instead of SL with high prediction probability, as presented in The algorithm first trains on the labeled data through a designated classifier and then performs a label prediction on the unlabeled data. It checks the score for the prediction and, if it exceeds the predetermined ScoreThreshold, treats the prediction as the actual label for the next training cycle. This process is repeated until the label prediction converges. Each iteration of the algorithm performs label predictions on unlabeled observations and computes a score for these predictions. Unlabeled observations with a predicted score greater than or equal to the score threshold are treated as labeled observations in the next iteration.To identify shallow delamination of concrete, air-coupled IE tests were conducted on sections of concrete slabs, pavements, and bridge decks with simulated artificial defects. The three specimens had various artificial defects and voids. 2 test points was defined on the surface, and an air-coupled IE test of 266 points at the grid location was conducted. In Slab B, the unreinforced pavement section consisted of a thin layer of Portland cement concrete 50 mm deep on top of a thick (>150 mm) asphalt concrete base layer. The pavement contains six artificial delamination defects (double-layered polymer sheets) of various areas and depths, as presented in 2 in the longitudinal and transverse directions. Air-coupled IE data of 324 points were collected over a large area along a defined test grid. In Slab C of 3, respectively, to simulate a real-scale RC bridge. The bridge deck slab includes two uncoated steel reinforcement mats, 60 and 150 mm deep. Defects were simulated using embedded foam pieces of various sizes and depths. The shallow delamination (DL-C1 to C6) and deep delamination (DL-C7) had a depth of 60 and 150 mm, respectively. A 15 \u00d7 15 cm grid of test points was defined on the surface, and an air-coupled IE test of 455 points at the grid location was conducted. IE data of 1045 points were collected for the three slabs, which were used for training the SL and SSL models. For the primary performance verification of the additionally developed model, DL-A2, DL-A5, and DL-A6 of Slab A were remeasured with a detailed grid, as presented in The slab was reinforced with two-dimensional and double rebars with depths of 60 and 200 mm. Slab A had artificial delamination and voids of various sizes and depths, as presented in A mobile microphone was installed near 10 mm of the concrete surface, as presented in a/h was calculated to confirm the occurrence of the flexural mode by type of detachment defect, and a/h, \u201co\u201d means dominant flexural mode, and \u201cx\u201d is non-dominant. When a/h is less than eight, such as DL-A1\u2013A3, the thickness and flexural modes for defects may occur simultaneously among IE modes according to the thick plate theory. Additionally, when a/h is greater than eight, as in DL-A4 according to the thin plate theory, the flexural mode is expected to dominate. The thickness mode is expected to dominate, except for fabrication errors in the sound region and interfaces between the sound and delamination regions.Previously, studies on the occurrence of IE mode and flexural mode were conducted for plate-shaped (delamination) defects. If the flexural mode is not considered, detection of delamination defects based on the IE mode may be difficult due to the limitations of high-frequency generation and experimental errors. This study described various artificial delamination defects in the thick and thin plate theories for training SL and SSL models, and the flexural mode was intensively analyzed. In addition, according to the technological development trend, field applicability was increased using a non-contact microphone rather than an in-lab test using an accelerometer. First, a/h of delamination in this study ranges from 3 to 40 depending on the structural shape, and according to the theory of natural frequency, only a strong flexural mode occurs in the range of several hundred Hz to 5 kHz, or weak flexural and thickness modes coexist. Furthermore, the flexural mode does not appear in the sound region, and the thickness mode is dominant. It has been verified that strong frequencies within the range occur through IE test results. tf), whereas Slabs B and C are 9.6 kHz. Except for the area of delamination artificially depicted in the specimen, most showed similar results to the theoretical ft. However, there were cases in which other features appeared in some sound regions, which are analyzed to occur at the interface between delamination and sound due to insufficient fabrication. In the case of the thickness mode for delamination defects, it is analyzed that it occurs at 35 kHz or higher for the defect types in this study according to Equation (1). However, as there is a limit to having a frequency of 30 kHz or higher, this study used an 18 mm steel ball hammer (~15 kHz) to detect and analyze the defect through the generation of a flexural mode for the delamination defect.The a/h of four; however, the flexural mode may not occur easily with 150 mm, the deepest defect tested. In the case of Slabs A and C, when looking at the amplitude of the flexural mode, the artificial defect seems to be described very well, and in the case of Slab B, the amplitude of the flexural mode is weak, so it is expected that the artificial defect production is somewhat insufficient. Although the characteristics of the flexural mode varied depending on the slab type , the occurrence of the main flexural mode was confirmed below 5 kHz. Therefore, it is expected that using various features derivation and PCA in the time signal and FFT domains will allow for sufficient differentiation.In The existing IE spectrum analysis detects the shift or energy attenuation of the thickness mode or detects a new peak occurring at a high frequency according to the depth of the delamination defect according to Equation (1). However, if the physical properties of concrete change or some factors interfere with the propagation of elastic waves, it may be difficult to discriminate them using the theoretical formula. In the case of shallow defects, high-frequency generation due to general excitation is limited, and the shape of the flexural mode varies depending on the impact location; thus, an accurate analysis may be difficult. Rather than analyzing an inaccurate peak frequency of a spectrum, it is necessary to consider various features that can distinguish normality within a detectable frequency range. Moreover, analysis of uncertain results may require a professional ability to distinguish between defects and non-defects; however, through feature extraction and PCA, the distribution range of defects and sound regions can be distinguished, and quick and easy judgment is possible. In the dynamic response, according to the thickness of the three types of slabs considered in this study, the theoretically possible thickness mode is 6 kHz or higher, and the flexural mode, according to Equation (2), appears below 6 kHz. Therefore, a feature that maximizes the distinction between sound and defective is extracted by filtering the frequency in advance through a low-pass filter of 6 kHz. Twenty-one features are extracted through the procedure in Principal components (PCs) were derived through PCA for 21 features, PC1 , PC2 , and PC3 (imaginary moment) were chosen as the main components with the largest variance in data, and comparable results were shown in the three slabs. a/h of delamination. The actual model was trained using PC1\u20133 derived earlier, but predicting the three-dimensional area uses excessive memory; thus, Supervised learning was trained and verified using 1045 data from Slabs A, B, and C, as presented in The SSL method is employed for mixing labeled and unlabeled data. First, an SL model is developed using the labeled data, and the label of unlabeled data is fitted using the created model. In this process, the optimal label is assigned through 1000 iterations. Therefore, it is important to use accurate label data. In this study, as presented in As a result of Cases 1 to 3, it was analyzed that Case 2 could fit unlabeled data with high accuracy due to the high data density for each label. The SSL model shows lower accuracy than the SL model; however, as this accuracy may be due to an initial set incorrectly labeled, further verification comparing the performance of the developed SL and SSL models is required. Therefore, in this study, two validation sets were composed by testing for three defects  in Slab A and the actual Virginia bridge in Three defects in Additional analysis was conducted to confirm the applicability of the developed model to the actual bridge. The conventional SSL method has been applied to reduce the labeling effort in the field of image deep learning, which requires a large dataset. However, specifying labeling through a core test requires more effort and cost in the nondestructive test for concrete structures. Furthermore, a model developed with little labeling data may have less applicability to actual structures. Therefore, in this study, by applying the SSL method to minimize this problem, an SSL model was developed using labeled data with high accuracy, and the applicability by predicting the condition of the actual structure was verified. As the SSL model can be continuously improved using new unlabeled data, the proposed flowchart in Various research progressed on the utilization of the SL method using the existing IE test. However, these studies can be applied to concrete slabs under specific conditions, and their use is limited due to differences in the characteristics of artificial specimens and actual structures. In addition, it is difficult to reflect the new characteristics of the SL model developed in the artificial specimen after being developed. However, this study clearly presented the thickness mode or the flexural mode on delamination by the reflection of the body wave of classical IE, unlike previous similar studies. Afterward, it is meaningful that various types of concrete  with different thicknesses and conditions were extracted simultaneously through PCA. The application of SSL to the extracted features has been verified through the results of The limitations of previous studies that could not reflect various physical characteristics from valuable cases without synthetic data were resolved using verified reliable data.PCA efficiently and clearly extracted frequency domain features from typical IE data that reflect dynamic behavior based on repetitive body waves.Non-experts can readily use a conventional SSL algorithm, which can also be applied to actual bridge data.Moreover, an SSL model was developed in advance in this study using the specimens with simulated defects as unlabeled data, the unlabeled field data were verified, and the following summaries were derived.In comparison with the previous model, which used the entire frequency domain, an algorithm that can quickly and accurately determine the presence or absence of defects through dynamic preconditions was proposed.Compared with SL, the proposed SSL model can accurately determine the presence or absence of defects by about 7\u20138% or more through domain correction of inaccurate label data and updating of new specimen characteristics.In the future, the field data that is difficult to label can be applied by reflecting the characteristics of the new specimen.Cost-effective and optimal maintenance and repair methods require information on material defects and deterioration conditions. Although the IE method effectively identifies internal defects of concrete, it requires expertise to analyze the signal. In this regard, this study analyzed the main features derived from the PCA for the pattern such as peak, energy area, and moment in the frequency domain of IE signals and proposed a semi-supervised detection model that can classify the presence or absence of defects in platelike structures, such as concrete slabs, pavements, and bridge decks, into three categories: good, fair, and poor. The SSL method has the advantage of updating the properties of new specimens and judging signals that are not appropriately labeled or that are difficult to label. As such, the novelty of this study can be summarized as follows:Additionally, while the main purpose of this study has been achieved, further study is required to overcome various limitations. The detection performance of the current method requires additional measures to consider the field conditions, such as concrete strength and modulus, crack distribution, and defect severity. A more detailed study into whether other features in the time or frequency domain can perform early detection of the defect propagation and the development of various methodologies to improve the accuracy of unsupervised learning is required. If the limitations of these studies are minimized, the SSL method can be an effective method for nondestructive methods based on mechanical waves, such as IE, SASW, and MASW."}
+{"text": "Drosophila mushroom body (MB), the fly\u2019s center for learning and memory. Our model combines the electron microscopy-based architecture of one MB output neuron (MBON-\u03b13), the synaptic connectivity of its 948 presynaptic Kenyon cells (KCs), and its membrane properties obtained from patch-clamp recordings. We show that this neuron is electrotonically compact and that synaptic input corresponding to simulated odor input robustly drives its spiking behavior. Therefore, sparse innervation by KCs can efficiently control and modulate MBON activity in response to learning with minimal requirements on the specificity of synaptic localization. This architecture allows efficient storage of large numbers of memories using the flexible stochastic connectivity of the circuit.The ability to associate neutral stimuli with valence information and to store these associations as memories forms the basis for decision making. To determine the underlying computational principles, we build a realistic computational model of a central decision module within the  Operating successfully within a complex environment requires organisms to discriminate between a vast number of rewarding and stressful interactions. Learning to avoid potentially harmful interactions will promote survival of individual animals and requires long-term memory mechanisms. In recent years, significant progress has been made toward our understanding of the cellular and circuit mechanisms underlying learning and memory. Despite these advances, at the computational level it remains largely unknown how the cellular and circuit architectures contribute to the efficient formation and storage of multiple memories.Drosophila melanogaster mushroom body (MB) as a simple model system to investigate the computational principles underlying learning and memory in the context of decision making. A key advantage of the MB, the learning and memory center of the fly, is its relative simplicity in terms of the number and types of neurons compared to the mammalian brain. It has been demonstrated that the MB is required for the acquisition and recall of associative olfactory memories  to the Kenyon cells (KCs) of the MB. PNs are connected in a largely stochastic manner to the approximately 2000 KCs per brain hemisphere, resulting in a unique KC odor representation in individual flies . The excElectrophysiological and calcium imaging studies demonstrated that any given odor activates approximately 3\u20139% of KCs . KCs theDANs of the PPL1 cluster preferentially provide input to the vertical lobe of the MB and are activated during negative reinforcement, while DANs of the PAM cluster are activated during positive reinforcement and modulate activity in the horizontal lobe of the MB . The majIn vivo calcium imaging and electrophysiological experiments demonstrated that the activity of individual MBONs can be altered by conditioning paradigms . This moDrosophila projection neurons to gain first insights into the computational processes of central neurons. Using linear cable theory, they demonstrated that voltage differences are much smaller within the dendritic trees than between the dendrites and the soma. Using a similar approach, Scheffer and coworkers simulated voltage distributions in the dendritic tuft of another Drosophila neuron (EPG) but used its measured morphology, rather than random geometries  data of an individual MBON. This approach allows us to determine the impact of populations of KCs on MBON activity and to resolve the potential computational mechanisms underlying memory encoding. We focus on MBON-\u03b13  data  data  or partied) data . We go bed) data  with funed) data , it currTogether, we obtain a realistic in silico model of a central computational module of memory-modulated animal behavior, that is a mushroom body output neuron.Drosophila, this indicates that in contrast to large vertebrate neurons, local active amplification or other compensatory mechanisms .Flies were reared at 25\u00b0C and 65% humidity on standard fly food. The following stocks were used in this study: 10XUAS-IVS-mCD8::GFP  BDSC 32 . Flies were then washed for at least 3x30min at RT in PBST before dissection. Dissected brains were then labelled with primary antibodies rabbit anti-GFP  and mouse anti-Brp  for two nights at 4\u00b0C. After incubation, brains were washed at least 6x30 min in PBST. Secondary antibodies (Alexa488 (goat\u03b1rabbit) and Alexa568 (goat\u03b1mouse) coupled antibodies, Life technologies, Carlsbad, USA, 1:1000) were applied for two nights at 4\u00b0C. After a repeated washing period, brains were submerged in Vectashield , mounted onto microscope slides, and stored at 4\u00b0C.Images were acquired using a LSM 710 confocal scanning microscope with a 25 x Plan-NEOFLUAR, NA 0.8 Korr DIC, oil objective  and a Leica STELLARIS 8 confocal microscope with a 20 x HC PL APO NA 0.75 multi-immersion objective . Raw images were projected with Fiji  and cropStreptomyces griseus, CAS# 90036-06-0, Sigma-Aldrich, St. Louis, USA) containing extracellular saline \u201310 P, Science Products, Hofheim, Germany) and a horizontal puller  to obtain pipettes with a resistance of solution    in Igor In current-clamp mode, a step-protocol was performed and recorded at In current-clamp mode, no current was injected throughout a In current-clamp mode, a short current pulse . These data describe the structure of its dendritic arborization . The axon and synaptic terminal of this neuron were approximated as five separate sections, distinguished by the major branch points identified in the electron microscopic reconstruction R , obtainetruction . Each setruction . The dimtruction . Synaptitruction .Drosophila central neurons  and Matlab . Before a statistical comparison was performed, individual groups were tested in Prism for normality and lognormality with an Anderson-Darling test. Statistical significance was tested with an unpaired parametric t-test or in case of multiple comparisons, with an ordinary corrected parametric one-way ANOVA test. Further information is provided in figure legends. Drosophila melanogaster contribute with equal weight to the depolarization of the neuron, independently of their location on the arbor, a phenomenon known as synaptic democracy. These important findings establish the validity of computational models based on passive dendritic propagation for simulating fly brain circuits and highlight the differences between the much larger mammalian neurons that present active propagation strategies as part of their approach to synaptic democracy.In light of the ongoing emergence of volume electron microscopy connectomics, detailed morphologies at the nanometre scale for many neurons are now available, ready for functional and computational analysis. Building on these foundational resources, this work delivers compelling evidence that synaptic inputs onto the dendritic arborisations of readout neurons (MBONs) of the learning and memory system of the adult  public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:Drosophila supports stochastic input integration\" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and K VijayRaghavan as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Albert Cardona (Reviewer #1).Thank you for submitting your article \"The cellular architecture of memory modules in The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:Please follow carefully the many detailed comments by the reviewers. To emphasize comments with regard to the caveats of using partially EM-reconstructed neuronal morphologies, and in the statements regarding electrotonic compactness.Reviewer #1 (Recommendations for the authors):Hafez and collaborators describe the construction and analysis of a computational model of a mushroom body neuron. The anatomy derives from a combination of electron microscopy reconstructions of MBON-\u03b13 and also from light microscopy. The physiological parameters derive from publications that measured them, in addition to the author's own electrophysiological recordings with patch-clamp.There are two main findings. First, the dendritic arbor of MBON-\u03b13 is electrotonically compact, meaning, individual connections from Kenyon cells will similarly elicit action potentials independently as to where, spatially, the synapses lay on the arbor. Second, in simulation, exploration of changes in the strength of Kenyon cell inputs illustrate two possible ways to alter the strength of the KC-MBON physiological connection, showing that either could account for the observed synaptic depression in the establishment of associative memories. The properties of each approach differ.Overall, the manuscript clearly describes the journey from connectomics and electrophysiology to computational modeling and exploration of the physiological properties of a circuit in simulation.The discussion ought to be expanded to include the implications of two possible approaches to physiologically altering the KC-MBON synapse and the consequence of their combination in expanding the space of alterations induced by associative memory paradigms.In general, the results are clear, but some details remain underdetermined and I have listed them below in the detailed comments. The introduction and discussion present some inaccuracies that can be swiftly addressed by the authorsDetailed comments:Line 45: Language: \"potential rewarding\": potentially.Line 49: Language: \"cellular and circuit architecture contributes\": architectures contribute.Line 72: instead of 5%, the number of KCs active at any one time seems to be 6% as per Turner et al. 2008 and Campbell et al. 2013. What is the robustness of the analysis to this small change? Did you explore a range of possible single-digit percent KC activations?Line 99: Aso & Rubin 2016 belongs with citations in line 95.Drosophila\" needs italics, throughout the manuscript.Line 122: \"The authors devise a computational model of an MBON using a neuronal arbor reconstructed from volume electron microscopy by Takemura et al. 2017. That paper details that only 93% of all synapses were connected to an arbor, and only 86% of the synapses had known pre- and postsynaptic arbors. For the MBON that was used for modeling, what was the fraction of terminal ends labeled as uncertain, and where these clustered or scattered across the arbor? Furthermore, the volume imaged with FIBSEM did not fully enclose the vertical lobe of the MB. Any estimate of what fraction of the chosen MBON's arbor is contained within the imaged volume? In other words, what analysis has been done here to ensure that the modeled arbor is representative of an MBON arbor in vivo, and what mitigating measures were taken to account for the potentially missing 14% or more of the arbor synapses and terminal dendrites?The authors report using the 10XUAS-IVS-mCD8::GFP to label the MBON, so that they can then record electrophysiologically with patch-clamp. What is the effect of inserting so many mCD8 proteins (a large transmembrane protein) into the neuron's membrane on the voltage potential and action potential formation and transport? The 10XUAS is particularly strong. How does the morphology of the imaged neuron differ from that of the EM-reconstructed neuron, regarding calibers and amount of cable? For this purpose, a cytosolic GFP targeting the soma or nucleus and poorly diffusing into the arbor would have been far preferable, as the effect of inserting transmembrane proteins in neurons' membranes on resting potentials is well reported.Line 155: average resting potential for the MBON is reported at -56.7 mV +/- 2.0 mV. In Hige et al. 2015a, cells were held at -70 or -60 mV. Nowhere does Hige et al. 2015a report on the resting potential.Line 174: amplitude of action potentials was rather small, but in Hige et al. 2015a action potentials typically exceeded 200 pA. Is this what the authors mean by small? Just how small were the recorded action potentials?Line 176: by small amplitudes and the explanation on the long neurite connecting the dendritic arbor with the soma, you mean that the signal is attenuated over long distances?Line 179: how was the membrane capacitance calculated?Table 1: 5 neurons were used. How do you know they are all MBON-\u03b13? Has it been confirmed that the GAL4 line doesn't have stochastic expression among similar yet different sibling neurons of the same lineage? How many other MBONs innervate the tip of the \u03b1 lobe and do any of them share neuroblast lineage with MBON-\u03b13? The large differences in measured values listed in Table 1 could be explained by having measured similar yet different neurons. Did you run a battery of tests before and after the measurements to ensure the recorded neurons remained in good health throughout the measurement session? Such tests often consist in a ramp of current injections and the recording of the neuron's responses, which are then compared between before and after the experimental measurements of membrane properties .Drosophila, recording from e.g. 10 cells, all homologous cells across 10 individuals, gives e.g., 8 responding with excitation to a sensory stimuli with some variation and 2 responding with inhibition. There is a lot of variability in the responses. Recording from only 3 cells seems risky, statistically speaking. What justifies this low number?Line 184: why only 3 cells? In Line 186: \"To to further\".Drosophila, what do you mean exactly? How similar, how far off, by what parameters?Line 199: when you say that the measurements are in \"good agreement with prior recordings\" of other neurons in eLife where the whole mushroom body is reconstructed, including the olfactory projection neurons, so such correlations if any may be evident in that data set.Line 203: might as well mention that there were 948 reconstructed KCs synapting onto MBON-\u03b13, so 5% is 50. Spare the reader remembering where the 50 was picked from. . And you seem to not keep in mind that the KCs responding to a specific odor may be correlated in their synaptic connectivity strength onto MBON-\u03b13. Data to this end may be included in Li et al. 2020 Line 210: a complete reconstruction of MBON-\u03b13 now exists, from either the FAFB volume or the Hemibrain volume. In the methods you mention you used the Hemibrain data set for the axon.Line 209: the 12,770 synaptic connections aren't \"all\", these are the ones reported from the anatomical reconstruction from volume electron microscopy. According to the source papers  about 10% of all synapses are missing. An analysis of how these missing synapses impacts the structure of the arbor is absent from the paper.eLife \"Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation\" and the follow up paper Tamada et al. 2020 eLife \"Ultrastructural comparison of dendritic spine morphology preserved with cryo and chemical fixation\". What measures were taken to correct or mitigate these artifactual differences with in vivo neurons?In addition, sample preparation for electron microscopy with chemical fixation alters the fine anatomical details, including the length of terminal dendrites and the calibers of neurites throughout. See e.g., Korogod et al. 2015 Later, Figure 2F strongly supports the appropriateness of the model, yet, the above points merit discussion and even exploration: how much of the dendritic arbor can you miss and still get the same result? What does the response to current injection depend on, cable, number of synapses, synapse spatial location, cable calibers, tapering of cable? What cable truncations are tolerable? This is very important information towards future computational studies based on neuronal morphologies reconstructed from volume electron microscopy.Figure 2 legend: what is the evidence that the \"proximal neurite\" in green in Figure 2B is the site of axon potential generation? Gouwens and Wilson 2009 pointed at a region anywhere between the root of the dendritic tree and half-way through the axon of the uniglomerular olfactory projection neuron they modeled.Does the site of axon potential generation emerge from your model, or did you specify it in the model?Why is the two-tailed non-parametric Spearman correlation the correct statistic to compare the modeled and the experimentally measured membrane potential in Figure 2F?Figure 2 legend reads \"see appendix\" but there isn't any appendix to the manuscript?Line 249: \"the average number of synaptic contacts from a KC to this MBON is 13.47\". This statement ought to be qualified: for the single MBON-\u03b13 measured in Takemura et al. 2017, and with the caveat of ~10% of synapses potentially missing. You could just as easily apply a correction factor and say the average number is about 14.7 + 1.47 = 16.2. Would this change the outcome of your model?eLife doesn't restrict the length of your test. Second, PN is an established acronym, universally across all neuroscience literature, for projection neuron. Plus, the \"proximal neurite\" (as per figure 2B) might as well be called the putative AIS  where the integration of inputs across the entire dendritic tree take place and the axon potential is initiated.Please don't use \"PN\" as an acronym for proximal neurite. First, eLife and Tamada et al. 2020 eLife.Figure 3H: in the measurement of \"local dendritic section volume\", did you correct for volume artifacts induced by using (in purpose!) an incorrect osmolarity of the buffers when fixing the tissue in the sample preparation protocol for electron microscopy? See Korogod et al. 2015 Line 291: \"this value is in good agreement with in vivo data for MBONs\". Please could you specify what this agreement is, how close, some details.Line 293: in line with analyzing all KCs with exactly 13 synaptic inputs onto MBON-\u03b13, what's the result of analyzing the voltage excursion from drawing random subsets of 13 synapses? . Are the natural groups of 13 synapses different in their effect on the neuron's voltage than artificial groupings?Line 299: inaccurate statement: \"Given that \u2248 5% of the 984 KCs innervating MBON-\u03b13 are typically activated by an odor\". Instead, what is known from the literature is that, given the presence of the GABAergic neuron APL in the mushroom body which acts as the inhibitory unit of a winner-take-all configuration, only 6% (not 5%) of KCs simultaneously respond to any one odor. Plus when the APL neuron is inhibited, a huge double-digit percent of KCs are active in response to an odor.eLife, and also, for larvae, Eichler et al. 2017 Nature.Line 311: there is now far better evidence of stochastic odor encoding by KCs than Caron et al. 2013. See Zheng e t al. 2020 bioRxiv \"Structured sampling of olfactory input by the fly mushroom body\", and Li et al. 2020 Drosophila mushroom body calyx accompanies memory consolidation. Cell reports. 2021 Mar 16;34(11):108871.Line 319: see also Baltruschat L, Prisco L, Ranft P, Lauritzen JS, Fiala A, Bock DD, Tavosanis G. Circuit reorganization in the Line 337: the differences in the somatic amplitudes may be significant statistically, but are they meaningful? In other words, the effect size looks like near zero. The real, and important difference, is in Line 345 where it is stated that \"we observed some differences in the slope of the responses between the different tuning modalities .\"Line 350: Scheffer et al. 2020 is not an appropriate citation for the statement \"Te ability of an animal to adapt its behavior to a large spectrum of sensory information requires specialized neuronal circuit motifs\". Rather, a textbook such as Kandel et al. Principles of Neural Science, or no reference at all, would be appropriate. You could also delete the sentence without loss.Drosophila must go in italics, it's a species name. Multiple occurrences throughout.Line 353: eLife.Line 356: through both short-term and long-term memory. A good example is Aso & Rubin 2016 Line 359: note the olfactory system of the fly has a sort of \"fovea\", Zheng et al. 2020 bioRxiv.Line 362: this sentence needs work, I am not sure what it means: \"Individual flies display idiosyncratic, apparently random connectivity patterns that transmit information of specific odors to the output circuit of the MB.\"Line 366: MBONs can only each be classified as approach or avoidance within specific behavioral paradigms. In different contexts, including different physiological states , the classification changes.eLife, where EM-reconstructed dendrites of olfactory projection neurons were modeled to understand the impact of dendritic arbor size on neuronal function.Line 381: prior work includes Tobin et al. 2017 Line 386: again, these numbers aren't precise. There's about 10% of missing synapses to consider, and potentially additional Kenyon cells. And some of the KCs, particularly those with low number of synaptic connections to MBON-\u03b13, may have been connected in error.Line 397: Strongest finding of this work: \"The location of an individual synaptic input within the dendritic tree has therefore only a minor effect on the amplitude of the neuron's output, despite large variations of local dendritic potential.\" Would be best to surface it more.eLife \"When complex neuronal structures may not matter\", where the authors \"quantify animal-to-animal variability in cable lengths (CV = 0.4) and branching patterns in the Gastric Mill (GM) neuron\". And also Otopalik et al. 2019 eLife \"Neuronal morphologies built for reliable physiology in a rhythmic motor circuit\".Line 402: a comparison would be appropriate with neurons from the crayfish stomatogastric ganglion (STG) as described by Eve Marder lab's, with published findings such as neurons being electrotonically compact despite their large size in mature adult animals. For example, Otopalik et al. 2017 Line 406: you forgot to cite Jackie Schiller's work on cortical pyramidal cells and their tuft dendrites, some of which predates all the cited work in this statement.Line 412: your model, as per Figure 2F, rather very closely matches the observed electrophysiological responses of the neuron under study. In what further ways could you model more closely match experimental observations? This would be very instructive to the reader.Line 415: by ensembles of both KCS and MBONs, not just KCs, if you are including the memory part and not only the input representation part.Line 416: for most of the paper, you quoted these papers to justify the 5% of KCs being simultaneously active in response to an odor. Now the range is shown as 3 to 9%. This discrepancy ought to be reconciled.Line 422: again, nowhere in the manuscript so far did you detail in what way your simulation and experimental findings match those of prior experimental reports regarding neuron response and physiological properties.Line 425: this statement is inaccurate. An individual KC does not encode for a single odor. That would almost never be the case even if a KC was single-claw, as in, exclusively integrated inputs from a single projection neuron: individual projection neurons rarely encode an odor; it's the population of projection neurons that does. Similarly, ensembles of co-active KCs together represent an odor, and do so more narrowly and accurately than the population of projection neurons that excited them.Line 452: left unresolved remains the question of why would both mechanisms exist, as in, is the combination of altering the KC-MBON synapse and altering the PN-KC synapse better in some dimension than altering either alone? Does this relate perhaps to a possible dynamic nature of the olfactory \"fovea\" proposed by Zheng et al. 2020 bioRxiv as presumably static?Line 466: what is standard fly food? Please define it. Choice of food affects very much behavioral assays, for example.Line 476: no antigen saturation steps in the immunohistochemistry protocol? Please revise.Line 505: what were the settings of the puller? These would be necessary to reproduce your glass capillaries. Did you grind the tips, and if so, how?Line 534: how were the R_series, R_input and R_m determined with Igor Pro?Line 561: why was a threshold of 600 M\u03a9 used to exclude cells? How many cells were recorded in total, and how many were excluded?Line 584: once again the data is the best human effort in proofreading a semiautomatic segmentation. It is not the absolute truth. Also, each individual fly is somewhat different, so this is merely one fairly complete yet partial reconstruction, and not assured to be error free, particularly of errors of omission, of a single MBON from a single individual. Would be appropriate to remark this clearly.Did you provide the NeuroML or similar files necessary to run the model in the NEURON simulation software? These should be appended to the manuscript as supplemental data.Line 596: if \"parameters were tuned until the computed voltage excursion at the soma matched our electrophysiological recordings\", what is the rationale for comparing the voltage potential of the simulation with those of the experimental observations?Line 607: where do these ranges of physiologically plausible values come from? Citation, or measurements done in the lab and therefore a figure is needed to show them? Are these ranges from the literature listed in Table 2? Likely yes, would be appropriate to cite them here too or at least explicitly point to Table 2.Line 616: innervation of MBONs is cholinergic because KCs are cholinergic, but that's not from Takemura et al. 2017, instead from Barnsted et al. 2016 Neuron.Line 622: would be appropriate to include the python scripts used to configure and run the NEURON simulation as supplemental material. Likewise for the matlab scripts used to load the analyzed data and plot it. The raw data ought to be included as CSV files or similar.Reviewer #2 (Recommendations for the authors):Drosophila supports stochastic input integration\" is a classical biophysical compartmental modelling study. It takes advantage of some simple current injection protocols in a massively complex mushroom body neuron called MBON\u2013a3 and compartmental models that simulate the electrophysiological behaviour given a detailed description of the anatomical extent of its neurites.\"The cellular architecture of memory modules in This work is interesting in a number of ways:\u2013 The input structure information comes from EM data (Kenyon cells) although this is not discussed much in the paper.\u2013 The paper predicts a potentially novel normalization of the throughput of KC inputs at the level of the proximal dendrite and soma.\u2013 It claims a new computational principle in dendrites, this didn't become very clear to me.Problems I see:\u2013 The current injections did not last long enough to reach steady state , and the model current injection traces have two time constants but the data only one . This does not make me very confident in the results and conclusions.\u2013 The time constant in Table 1 is much shorter than in Figure 1FG?\u2013 Related to this, the capacitance values are very low maybe this can be explained by the model's wrong assumption of tau?\u2013 That latter in turn could be because of either space clamp issues in this hugely complex cell or bad model predictions due to incomplete reconstructions, bad match between morphology and electrophysiology (both are from different datasets?), or unknown ion channels that produce non\u2013linear behaviour during the current injections.\u2013 The PRAXIS method in NEURON seems too ad hoc. Passive properties of a neuron should probably rather be explored in parameter scans.Questions I have:\u2013 Computational aspects were previously addressed by e.g. Larry Abbott and Gilles Laurent (sparse coding), how do the findings here distinguish themselves from this work.\u2013 What is valence information?\u2013 It seems that Martin Nawrot's work would be relevant to this work.eLife but also passive normalization. The equal efficiency in line 427 reminds me of dendritic/synaptic democracy and dendritic constancy.\u2013 Compactification and democratization could be related to other work like Otopalik et al. 2017 \u2013 The morphology does not obviously seem compact, how unusual would it be that such a complex dendrite is so compact?\u2013 What were the advantages of using the EM circuit?\u2013 Isn't Figure 4E rather trivial if the cell is compact?Overall, I am worried that the passive modelling study of the MBON\u2013a3 does not provide enough evidence to explain the electrophysiological behaviour of the cell and to make accurate predictions of the cell's responses to a variety of stochastic KC inputs.Reviewer #3 (Recommendations for the authors):This manuscript presents an analysis of the cellular integration properties of a specific mushroom body output neuron, MBON-\u03b13, using a combination of patch clamp recordings and data from electron microscopy. The study demonstrates that the neuron is electrotonically compact permitting linear integration of synaptic input from Kenyon cells that represent odor identity.Strengths of the manuscript:1) The study integrates morphological data about MBON-\u03b13 along with parameters derived from electrophysiological measurements to build a detailed model.2) The modeling provides support for existing models of how olfactory memory is related to integration at the MBON.Weaknesses of the manuscript:1) The study does not provide experimental validation of the results of the computational model.2) The conclusion of the modeling analysis is that the neuron integrates synaptic inputs almost completely linearly. All the subsequent analyses are straightforward consequences of this result.3) The manuscript does not provide much explanation or intuition as to why this linear conclusion holds.In general, there is a clear takeaway here, which is that the dendritic tree of MBON-\u03b13 in the lobes is highly electrotonically compact. The authors did not provide much explanation as to why this is, and the paper would benefit from a clearer conclusion. Furthermore, I found the results of Figures 4 and 5 rather straightforward given this previous observation. I am sceptical about whether the tiny variations in, e.g. Figures 3I and 5F-H, are meaningful biologically.1) My biggest question is about the claim of extreme electrotonic compactness of this neuron. Figure 3D,E suggests that the voltage change at the proximal neurite and at the soma varies by only about 1% depending on stimulation location. Since this is supported only by simulation, it is worth asking how robust this conclusion is.a) Given that the variability in 3I is so small in magnitude, any dependence would be swamped by other sources of heterogeneity, so the statement that this correlates with distance (line 278) is likely irrelevant.b) Can the authors provide a confidence interval for the fit of biophysical parameters to their recordings?c) On lines 271-274, the authors state, \"This architecture with the smallest dendritic sections at the most distant sites may contribute to the compactness of the dendritic tree, ensuring that even the most distant synaptic inputs result in somatic voltage deflections comparable to the most proximal ones.\" Is a dependence of dendritic size on distance required for the results? It seems like the result is simply that there is no attenuation within the dendritic tree at all. In general, the authors don't actually provide an explanation for the compactness. In the discussion, it is stated that, \"The compactification of the neuron is likely related to the architectural structure of its dendritic tree,\" which again is rather vague. The authors should strive to provide a clear explanation for this, since it is their key result.d) Can the authors report what the electrotonic length for such a dendrite would be? How long before we expect to see a significant spread in 3E?2) My other concern is that, once we assume this perfect integration, the subsequent analyses are all a straightforward consequence. In particular, Figures 4 and 5 just repeatedly convey that it doesn't matter which synapse is being activated, the effect on the somatic voltage is the same. I was particularly confused about the conclusions of 5F,G,H. The authors claim \"small but significant differences\" here, but practically speaking, I can't imagine any of the differences in these plots being meaningful.3) Reference to the data used to constrain the model is confusing.a) At various places in the manuscript references are made to in vivo recordings, but it appears that all of the recordings were done ex vivo.b) On lines 389-392, the authors state: \"Near-perfect agreement between experimentally observed and simulated voltage distributions in the dendritic tree shows that linear cable theory is an excellent model for information integration in this system.\" What recordings of the voltage distribution in the dendritic tree were performed?4) It seems like the conclusions are different than those of Gouwens and Wilson (2009), who described their reconstructed PNs as electrotonically extensive. The authors should comment on what about MBONs and PNs is different. Reviewer #1 (Recommendations for the authors):Hafez and collaborators describe the construction and analysis of a computational model of a mushroom body neuron. The anatomy derives from a combination of electron microscopy reconstructions of MBON-\u03b13 and also from light microscopy. The physiological parameters derive from publications that measured them, in addition to the author's own electrophysiological recordings with patch-clamp.There are two main findings. First, the dendritic arbor of MBON-\u03b13 is electrotonically compact, meaning, individual connections from Kenyon cells will similarly elicit action potentials independently as to where, spatially, the synapses lay on the arbor. Second, in simulation, exploration of changes in the strength of Kenyon cell inputs illustrate two possible ways to alter the strength of the KC-MBON physiological connection, showing that either could account for the observed synaptic depression in the establishment of associative memories. The properties of each approach differ.Overall, the manuscript clearly describes the journey from connectomics and electrophysiology to computational modeling and exploration of the physiological properties of a circuit in simulation.The discussion ought to be expanded to include the implications of two possible approaches to physiologically altering the KC-MBON synapse and the consequence of their combination in expanding the space of alterations induced by associative memory paradigms.We now extended this discussion. However, as we do not provide any experimental evidence supporting either one of these hypothesis, we think that this point would be better addressed in a review format as we can only speculate at this point of time.In general, the results are clear, but some details remain underdetermined and I have listed them below in the detailed comments. The introduction and discussion present some inaccuracies that can be swiftly addressed by the authorsDetailed comments:Line 45: Language: \"potential rewarding\": potentially.We have changed the text accordingly.Line 49: Language: \"cellular and circuit architecture contributes\": architectures contribute.We have changed the text accordingly.Line 72: instead of 5%, the number of KCs active at any one time seems to be 6% as per Turner et al. 2008 and Campbell et al. 2013. What is the robustness of the analysis to this small change? Did you explore a range of possible single-digit percent KC activations?We now state the number as 3-9% of KCs and highlight that different studies observed reliable calcium signals in 5 or 6% of KC upon odor stimulation. Our own results  indicate that approximately 5% of KCs are activated by each odor and we used this number as an approximation for our simulations. It is correct, however, that the single-cell study by Turner et al. (2008) reported an average 6% of activated KCs for each odor. A later optogenetical study by the same group  reported 5%; importantly for the present discussion, they however considered this result \"extremely similar\" to their earlier results . We take from their formulation that they consider the difference between 5% and 6% as very small It would then make sense to assume that small differences obtained in different experimental preparations, on the order of a percentage points, as a kind of noise, and to evaluate the robustness of results under such perturbations. However, in absolute terms this difference by one percentage point, between 5% and 6%, represents a fifth difference  in the synaptic input to the MBON. And, of course, in our results this difference can not be due to different experimental conditions since they are obtained in the same \"experimental preparations,\" namely the same simulations. To answer directly the Reviewer\u2019s question, we have considered a range of single-digit percent KC activations, however our \"range\" only had 3 entries. We have not simulated a 20% change of the number of activated synapse but we did simulate a slightly larger increase, by 25% . Our results show that the 25% increase leads to a substantial change in the somatic MBON voltage . Given the nearly linear dependence of the voltage increase with the number of synapses , we are confident that a 20% increase can be obtained with high precision from a linear interpolation based on the 25% increase . An increase from 5% to 6% of activated synapses therefore increases the voltage by about 80% of the value shown in Figure 5B, D which is a substantial difference. We would consider such a difference not a question of robustness but as a change that is likely physiologically relevant.We have added a short discussion of this topic in the revised manuscript Line 99: Aso & Rubin 2016 belongs with citations in line 95.Many thanks for pointing this out. We moved the citation to the correct place.Line 122: \"Drosophila\" needs italics, throughout the manuscript.Drosophila\" are now set in italics.All instances of \"The authors devise a computational model of an MBON using a neuronal arbor reconstructed from volume electron microscopy by Takemura et al. 2017. That paper details that only 93% of all synapses were connected to an arbor, and only 86% of the synapses had known pre- and postsynaptic arbors. For the MBON that was used for modeling, what was the fraction of terminal ends labeled as uncertain, and where these clustered or scattered across the arbor?The reviewer rightfully highlights the limitations of the EM-dataset that we used for our reconstruction. Indeed, Takemura and colleagues report that not all postsynaptic sites within the \u03b1-lobe could be assigned to identified cells. Importantly, however, the authors state that these synaptic sites very likely belong to identified cells within the dataset. Thus, for our dataset this would mean that the MBON itself may have up to 7 percent more input synapses but these synapses would belong to the identified 948 KCs. An individual KC may therefore at most have one or two additional input synapses, e.g. KCs currently annotated with 13 input synapses may form 14 input synapses. These differences would not significantly change any of the conclusions of our manuscript. We now addressed this point experimentally and analyzed the activation of KCs with 12, 13 and 14 input synapses and compared this to the activation of 13 random synapses . We observed significant differences in MBON responses when changing the number of input synapses but not between KCs with 13 synapses and the activation of 13 random synapses. Thus, the number of input clearly changes postsynaptic responses, however, as we assume that these non-annotated synapses are likely uniformly distributed, these changes would not affect our physiological simulations of different sets of 50 KCs. Unfortunately, Takemura and colleagues did not specify the location of these non-annotated sites and we simply cannot re-trace the entire dendritic tree of the MBON. As they state however that \"the small size and discontinuous nature of these neurites indicates that they are fragments of identified cells rather than collectively constituting an additional cell type\", we assume that these sites were not clustered.A similar rate of \"tracing shortcomings\" has been observed in the hemibrain dataset and in the current FlyWire approach. We decided that the most conservative approach would be to use the data as published as any modification, e.g. addition of 10% of synapses, would make later reproductions of our data more difficult. We now state the potential problems clearly in our paper .Furthermore, the volume imaged with FIBSEM did not fully enclose the vertical lobe of the MB. Any estimate of what fraction of the chosen MBON's arbor is contained within the imaged volume? In other words, what analysis has been done here to ensure that the modeled arbor is representative of an MBON arbor in vivo, and what mitigating measures were taken to account for the potentially missing 14% or more of the arbor synapses and terminal dendrites?Indeed the dataset of Takemura et al. did not include the entire vertical lobe of the MB. However, it included the entire \u03b1-lobe of the MB \u2013 only the \u03b1 prime lobe is missing. As beautifully shown by Takemura et al., the entire dendritic tree of MBON-3alpha that only innervates the \u03b1-lobe is included and presented in Figure 2H and videos 4, 6 and 7 with the position of synaptic input sites. The striking visualization of Takemura and colleagues of this dendritic tree was one of the main reasons why we chose this MBON as an example for our combined in vivo and in silico analysis. While Takemura states that they could only precisely identify both pre- and postsynaptic partners for 86% of all synapses, this does not necessarily mean that 14% of KC to MBON synapses are missing in the dataset as e.g. only 7% of the postsynaptic sites remained unassigned (and these are the relevant ones for our MBON). We again carefully compared the annotations of KC to MBON-alpha3 synapses in Takemura et al. and Scheffer et al. (Hemibrain dataset) to evaluate the effects of a potential under-representation of synapses within the dendritic compartment of MBON-alpha3. Interestingly, the numbers for both MBON-alpha3 cells in the hemibrain dataset are below the numbers of Takemura et al. Specifically the numbers are for Takemura: MBON-alpha3A 12,770 synaptic connections from the 948 KCs (average 13.47 synapses), MBON-alpha3B 13,129 from 948 KCs (average 13.85 synapses) vs Scheffler: MBON-alpha3A 11,950  synapses from 894 KCs (average 12.44 synapses), MBON-alpha3B 11,121  synapses from from 897 KCs (average 13.32 synapses).These numbers are highly similar between the two data sets and also between the two different MBON-alpha3 neurons. Importantly however, the numbers are lower in the Hemibrain data set. Thus, the variation between individual flies (or data sets) seems to be in the range of 10%. As such, the Takemura dataset likely represents a very good approximation despite some shortcomings due to either the EM fixation (see below) or due to annotation problems. As we did not want to introduce any arbitrary mistakes in our analysis we chose to adhere to the numbers provided in the Takemura data set. We now clearly state the potential shortcomings of using a single EM data set as the basis for a simulation (line 206ff). We still think that this is currently the best possible approach.The authors report using the 10XUAS-IVS-mCD8::GFP to label the MBON, so that they can then record electrophysiologically with patch-clamp. What is the effect of inserting so many mCD8 proteins (a large transmembrane protein) into the neuron's membrane on the voltage potential and action potential formation and transport? The 10XUAS is particularly strong. How does the morphology of the imaged neuron differ from that of the EM-reconstructed neuron, regarding calibers and amount of cable? For this purpose, a cytosolic GFP targeting the soma or nucleus and poorly diffusing into the arbor would have been far preferable, as the effect of inserting transmembrane proteins in neurons' membranes on resting potentials is well reported.We would like to thank the reviewer for this suggestion. We now performed additional recordings with a cytosolic GFP construct. The data from 4 additional recordings is now presented in Figure 1 Supplement 2. In this new Figure we now provide a direct comparison between the mCD8-GFP based recordings and the cytosolic GFP based recordings and present all raw data traces. This analysis demonstrated that the averaged traces of the new recordings fall within the data range of our initial dataset . Thus, the source of GFP used to label these cells did not have any significant consequences for the recordings. In addition, with this new data set we could clearly demonstrate that our electrophysiological data are reproducible and that the original data set provides accurate values for our computer simulations.Line 155: average resting potential for the MBON is reported at -56.7 mV +/- 2.0 mV. In Hige et al. 2015a, cells were held at -70 or -60 mV. Nowhere does Hige et al. 2015a report on the resting potential.Drosophila . We now state more appropriately (line 154ff): This value for the restring membrane potential is in line with prior measurements of MBONs in vivo and of other central neurons in Drosophila The average resting membrane potential of the recorded MBONs was at -56.7 mV +/- 2.0 mV. To our knowledge, we are the first to report precise electrophysiological values for this particular cell type. It is correct that Hige et al., 2015a do not report values however, it is possible to estimate these values from the raw traces provided in their publication. Importantly, our value of approximately -60 mV is in line with data from a number of central neurons in Line 174: amplitude of action potentials was rather small, but in Hige et al. 2015a action potentials typically exceeded 200 pA. Is this what the authors mean by small? Just how small were the recorded action potentials?\u00b1 0.029 mV) represent relatively low values compared to previously published data from different cell types in the central brain of the adult fruit fly (see references above). Even between different MBONs a high variability in action potential amplitudes has been observed . While precise values were not reported in this publication, based on the traces the values range between very small (gamma2), 10 mV  and 25 mV (gamma1). The small amplitudes observed for MBON alpha3 are likely a consequence of the unipolar morphology of the neurons with a very long neurite connecting the dendritic input region to the cell soma. This morphology is especially pronounced for MBON alpha3 .The amplitude of the action potentials . We now state this in the text (line 179).Table 1: 5 neurons were used. How do you know they are all MBON-\u03b13? Has it been confirmed that the GAL4 line doesn't have stochastic expression among similar yet different sibling neurons of the same lineage? How many other MBONs innervate the tip of the \u03b1 lobe and do any of them share neuroblast lineage with MBON-\u03b13? The large differences in measured values listed in Table 1 could be explained by having measured similar yet different neurons. Did you run a battery of tests before and after the measurements to ensure the recorded neurons remained in good health throughout the measurement session? Such tests often consist in a ramp of current injections and the recording of the neuron's responses, which are then compared between before and after the experimental measurements of membrane properties .The MB082C split-Gal4 line we used for our recordings is a highly specific driver line which is only expressed in the two MBON-alpha3 neurons per hemisphere across all observed preparations. These neurons do not share any overlap (position of dendrites plus position of soma) with any other MBON. Thus we are certain that all our recordings were performed on MBON-alpha3 neurons. However, the two MBON-alpha3 neurons innervating the same position in the \u03b1 lobe are indistinguishable from each other. As there is no possibility to differentiate between the two and as their morphological features are almost identical (see numbers in our answer to the EM dataset above) we treated them simply as MBON-alpha3. We now state this clearly in our manuscript. As we are certain about the identity of the MBON, any differences likely result from inter-individual biological differences.Line 184: why only 3 cells? In Drosophila, recording from e.g. 10 cells, all homologous cells across 10 individuals, gives e.g., 8 responding with excitation to a sensory stimuli with some variation and 2 responding with inhibition. There is a lot of variability in the responses. Recording from only 3 cells seems risky, statistically speaking. What justifies this low number?We agree. We now added 4 additional recordings using a cytoplasmic GFP as a labelling source to the manuscript . These values of these recordings fall in between the values of our prior recordings and thus validate our prior results. We now have 7 detailed recordings for these cells that likely show the range of biological variation. It is technically extremely challenging to perform these recordings.Line 186: \"To to further\".We corrected this mistake.Line 199: when you say that the measurements are in \"good agreement with prior recordings\" of other neurons in Drosophila, what do you mean exactly? How similar, how far off, by what parameters?We revised this section. We now simply report the results of our recordings and state that MBONalpha3 is a spike-frequency adapting neuron like other MBONs. We removed the prior sentence with comparisons to other neurons.Line 203: might as well mention that there were 948 reconstructed KCs synapting onto MBON-\u03b13, so 5% is 50. Spare the reader remembering where the 50 was picked from. . And you seem to not keep in mind that the KCs responding to a specific odor may be correlated in their synaptic connectivity strength onto MBON-\u03b13. Data to this end may be included in Li et al. 2020 eLife where the whole mushroom body is reconstructed, including the olfactory projection neurons, so such correlations if any may be evident in that data set.We now state directly that the number 50 is deduced from the 5% of KCs at the appropriate position in the text.The reviewer is certainly correct that there is now clear evidence that the PN>KC input is not entirely random. Importantly, Li et al. demonstrated that inputs from different sensory modalities are especially segregated \u2013 however, MBON-alpha3/14 receives almost exclusively olfactory input . In their models Li et al., still find some evidence for non-random sampling at the PN>KC synapse but this was modest at best and they observed only minor persistance to the KC>MBON level. In addition Zheng et al., 2022 demonstrate that some specific odors may be overrepresented compared to others to enable preferential encoding of naturally meaningful stimuli. In our first analysis presented here we decided to focus on the robustness of encoding principles by assuming random connectivity. We certainly aim to pursue a targeted analysis in a future study focussing on multiple MBONs.Line 210: a complete reconstruction of MBON-\u03b13 now exists, from either the FAFB volume or the Hemibrain volume. In the methods you mention you used the Hemibrain data set for the axon.In the FAFB volume our MBON is not fully reconstructed. The reconstructions from Takemura and the Hemibrain are very similar and consistent regarding the number of KC>MBON synapses (see above). As we started our first analysis before the publication of the Hemibrain focusing only on the dendritic tree  we build our current study on this analysis and complemented the morphology reconstruction with data from the Hemibrain.Line 209: the 12,770 synaptic connections aren't \"all\", these are the ones reported from the anatomical reconstruction from volume electron microscopy. According to the source papers  about 10% of all synapses are missing. An analysis of how these missing synapses impacts the structure of the arbor is absent from the paper.Please see our responses above to this topic. We now highlight these issues in the results when first describing our approach (line 206ff).In addition, sample preparation for electron microscopy with chemical fixation alters the fine anatomical details, including the length of terminal dendrites and the calibers of neurites throughout. See e.g., Korogod et al. 2015 eLife \"Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation\" and the follow up paper Tamada et al. 2020 eLife \"Ultrastructural comparison of dendritic spine morphology preserved with cryo and chemical fixation\". What measures were taken to correct or mitigate these artifactual differences with in vivo neurons?Korogod et al. showed that the main effect of fixating somatosensory mouse cortex using the standard aldehyde method was a substantial decrease of the extracellular space and an increase of astrocytic volume, compared to a \u2019fresh\u2019 (not fixated) preparation. This was not the case, however, for neurites. They found that \"the volume fraction occupied by axons and dendrites was similar between the fixation conditions\" (their p 3).\u2248 43%), a difference they explained by the fact that there are no synapses in structures like cell bodies and blood vessels. It seems therefore likely that the increase of the synapse density is primarily due to a decrease in the denominator (the volume) due to the loss of recorded extracellular space, rather than in the numerator (number of synapses).There was also no indication of systematic loss or erroneous addition of synapses (the shape of synaptic vesicles was modified but we do not make use of this feature in our study). Synaptic density in the chemically fixed neuropil was 38% higher than cryo-fixated neuropil, i.e. they measured 38% more synapses per volume. This is slightly lower than the total tissue shrinkage they observed  came to a similar conclusion.Given that the main effect of chemical fixation is a sharp reduction of extra-cellular space, with no significant changes of neurite dimensions, we feel that the EM data we use are at least a good first approximation of the physiological state. A previous study of Tamada et al. focused on fixation-induced morphology changes of dendritic spines, again in mouse cerebral cortex. They found that the length of the spines as well as the spine head volume was not significantly changed by traditional  fixation preceding EM. They did find that chemical fixation resulted in significantly (by 42%) thicker spine necks, however. Assuming identical cytoplasmatic specific resistivity, using the measured  spine neck diameter would result in significantly lower resistance between spine head and dendrite than the actual physiological value.Drosophila nervous system : 221-234). However, the relevance of these results for our model is unknown. In the published data no spine-like structures or locations on the dendritic tree of MBON-\u03b13 have been characterized; so we are not able to evaluate details of the impact that different diameters of the spine necks would have. In the absence of such information, we can not address  differences in the resistance between individual synaptic contacts and the dendrites. Instead, we fit a common value for the cytoplasmatic resistivity for the whole neuron (our Table 2). We agree that it would, indeed, be very interesting to study the effect of synapses on spines (for those neurons where they exist) compared to that of synapses located directly on the dendritic surface, but this requires the availability of data characterizing location and, ideally, geometry of dendritic spines on neurons of interest. Due to these reasons we did not include any correction factor in our analysis.Spine-like structures have been observed in the Later, Figure 2F strongly supports the appropriateness of the model, yet, the above points merit discussion and even exploration: how much of the dendritic arbor can you miss and still get the same result? What does the response to current injection depend on, cable, number of synapses, synapse spatial location, cable calibers, tapering of cable? What cable truncations are tolerable? This is very important information towards future computational studies based on neuronal morphologies reconstructed from volume electron microscopy.We agree that these are important points. It is important to note that we provide results for our simulations at two different points. First, at the proximate neurite, the \"exit\" point of the dendritic tree and potential site of action potential initiation, and second at the soma region. Importantly, the region of the proximate neurite is still within the EM-dataset of Takemura et al. \u2013 thus, no relevant dendritc arbor sections are missing and no assumptions regarding the diameter of any neuronal structures have been made. We provide a direct comparison to the soma part that is based on a different dataset and approximations as it is currently not included in any EM dataset (the upcoming FlyWire dataset will hopefully resolve this issue). Our comparison demonstrates very few differences between these two points of analysis indicating that our dataset is very robust \u2013 but of course the main computation occurs within the dendritic compartment that was not modified in our analysis . We therefore cannot draw any significant conclusions regarding physical alterations of the dendritic compartment and would like to stick to our \"conservative\" approach for this initial study. We will certainly test more challenging models in future studies.Figure 2 legend: what is the evidence that the \"proximal neurite\" in green in Figure 2B is the site of axon potential generation? Gouwens and Wilson 2009 pointed at a region anywhere between the root of the dendritic tree and half-way through the axon of the uniglomerular olfactory projection neuron they modeled.Drosophila para channel in other neurons the position is likely in close proximity to the dendritic arbor. Here, we used this position for our comparative analysis with the soma as this region was still confined within the Takemura EM reconstruction \u2013 we state this now more clearly in the revised manuscript in the legend to Figure 2. As we observed consistent results between the two regions it would not make a significant difference if this region would be moved. In future work we hope to precisely determine the site of action potential generation using genetic methods.We currently do not have any evidence that this is the position of action potential initiation. Based on immunohistochemical analyses of the Does the site of axon potential generation emerge from your model, or did you specify it in the model?Please see above, we specified the site in the model.Why is the two-tailed non-parametric Spearman correlation the correct statistic to compare the modeled and the experimentally measured membrane potential in Figure 2F?Many thanks for highlighting this. This comparison is not necessary as the fitting was done in NEURON using the mean squared error. We now state this appropriately in the Methods section.Figure 2 legend reads \"see appendix\" but there isn't any appendix to the manuscript?We changed this and now only refer to the figure supplement.Line 249: \"the average number of synaptic contacts from a KC to this MBON is 13.47\". This statement ought to be qualified: for the single MBON-\u03b13 measured in Takemura et al. 2017, and with the caveat of ~10% of synapses potentially missing. You could just as easily apply a correction factor and say the average number is about 14.7 + 1.47 = 16.2. Would this change the outcome of your model?Please see our discussion of this issue above. We now clearly state that 10% of synapses potentially missing at the beginning of the section and provide our reasoning. We also now tested the responses to 12, 13 or 14 synapses in Figure 4F.Please don't use \"PN\" as an acronym for proximal neurite. First, eLife doesn't restrict the length of your test. Second, PN is an established acronym, universally across all neuroscience literature, for projection neuron. Plus, the \"proximal neurite\" (as per figure 2B) might as well be called the putative AIS  where the integration of inputs across the entire dendritic tree take place and the axon potential is initiated.Many thanks for pointing this out. We now use PN as an acronym for projection neuron and do not abbreviate proximal neurite. We now also highlight that this is the likely site of action potential initiation. However, as this is not based on experimental evidence we would like to remain conservative and refer to it only as the proximal neurite.Figure 3H: in the measurement of \"local dendritic section volume\", did you correct for volume artifacts induced by using (in purpose!) an incorrect osmolarity of the buffers when fixing the tissue in the sample preparation protocol for electron microscopy? See Korogod et al. 2015 eLife and Tamada et al. 2020 eLife.Please see our response to potential fixation artifacts above.Line 291: \"this value is in good agreement with in vivo data for MBONs\". Please could you specify what this agreement is, how close, some details.Many thanks for pointing this out. We now removed this sentence as we only deduced this value from odor evoked activations in vivo that activate approximately 5% of KCs and result in action potential generation. Thus a direct comparison is currently not possible.Line 293: in line with analyzing all KCs with exactly 13 synaptic inputs onto MBON-\u03b13, what's the result of analyzing the voltage excursion from drawing random subsets of 13 synapses? . Are the natural groups of 13 synapses different in their effect on the neuron's voltage than artificial groupings?Many thanks for suggesting this very interesting experiment. We now performed the experiment and provide a comparison between KCs with exactly 12, 13 and 14 synapses to a random set of 13 synapses . This experiment demonstrated no significant difference between the activation of KCs with 13 synapses and 13 random synapses. Interestingly though, slightly more variation could be observed in the dataset of KCs with 13 synapses. This may indicate some biological relevance however, the differences compared to activating one additional synapse are negligible in line with our model.Line 299: inaccurate statement: \"Given that \u2248 5% of the 984 KCs innervating MBON-\u03b13 are typically activated by an odor\". Instead, what is known from the literature is that, given the presence of the GABAergic neuron APL in the mushroom body which acts as the inhibitory unit of a winner-take-all configuration, only 6% (not 5%) of KCs simultaneously respond to any one odor. Plus when the APL neuron is inhibited, a huge double-digit percent of KCs are active in response to an odor.We now corrected this statement and highlight that only 5-6% (range 3-9%) of neurons reliably respond to individual odors. This is the value that is likely relevant for memory association. As we do not address the inhibitory role of the APL neuron in our current study we would like keep our study focused and potentially explore these issues in a future study.Line 311: there is now far better evidence of stochastic odor encoding by KCs than Caron et al. 2013. See Zheng e t al. 2020 bioRxiv \"Structured sampling of olfactory input by the fly mushroom body\", and Li et al. 2020 eLife, and also, for larvae, Eichler et al. 2017 Nature.We are sorry for our oversight and now include these citations.Drosophila mushroom body calyx accompanies memory consolidation. Cell reports. 2021 Mar 16;34(11):108871.Line 319: see also Baltruschat L, Prisco L, Ranft P, Lauritzen JS, Fiala A, Bock DD, Tavosanis G. Circuit reorganization in the We added this citation.Line 337: the differences in the somatic amplitudes may be significant statistically, but are they meaningful? In other words, the effect size looks like near zero. The real, and important difference, is in Line 345 where it is stated that \"we observed some differences in the slope of the responses between the different tuning modalities .\"We agree that the observed differences may not be biologically relevant but we can not formally exclude this possibility at the moment. We now highlight the differences in the slopes of the responses further in the discussion.Line 350: Scheffer et al. 2020 is not an appropriate citation for the statement \"Te ability of an animal to adapt its behavior to a large spectrum of sensory information requires specialized neuronal circuit motifs\". Rather, a textbook such as Kandel et al. Principles of Neural Science, or no reference at all, would be appropriate. You could also delete the sentence without loss.We agree and deleted the sentence.Drosophila must go in italics, it's a species name. Multiple occurrences throughout.Line 353: Drosophila\" is now set in italics throughout.As mentioned above, \"Line 356: through both short-term and long-term memory. A good example is Aso & Rubin 2016 eLife.Many thanks for this useful suggestion \u2013 we adjusted the text accordingly.Line 359: note the olfactory system of the fly has a sort of \"fovea\", Zheng et al. 2020 bioRxiv.As the study is now peer-reviewed and published we included it in the reference list and altered the statement to reflect the fact that processing of odor information does not exclusively depend on stochastic connectivity (line 65ff) \u2013 in addition we refer to this paper in our discussion of the two possible plasticity modes (line 504ff).Line 362: this sentence needs work, I am not sure what it means: \"Individual flies display idiosyncratic, apparently random connectivity patterns that transmit information of specific odors to the output circuit of the MB.\"We agree and deleted the sentence.Line 366: MBONs can only each be classified as approach or avoidance within specific behavioral paradigms. In different contexts, including different physiological states , the classification changes.Yes, we now state this more appropriately in the revised version of the manuscript (line 409ff).Line 381: prior work includes Tobin et al. 2017 eLife, where EM-reconstructed dendrites of olfactory projection neurons were modeled to understand the impact of dendritic arbor size on neuronal function.Many thanks for pointing this out. We now cite this work.Line 386: again, these numbers aren't precise. There's about 10% of missing synapses to consider, and potentially additional Kenyon cells. And some of the KCs, particularly those with low number of synaptic connections to MBON-\u03b13, may have been connected in error.in silico reconstruction.\"We now highlight this point in the discussion and state (line 432ff): \"While this EM reconstruction may contain some mistakes in synaptic connectivity as e.g. up to 10% of synaptic sites remained unassigned within the dataset , it currently represents the best possible template for an Line 397: Strongest finding of this work: \"The location of an individual synaptic input within the dendritic tree has therefore only a minor effect on the amplitude of the neuron's output, despite large variations of local dendritic potential.\" Would be best to surface it more.We agree. We think that the importance of this finding is appropriately highlighted in the current form.Line 402: a comparison would be appropriate with neurons from the crayfish stomatogastric ganglion (STG) as described by Eve Marder lab's, with published findings such as neurons being electrotonically compact despite their large size in mature adult animals. For example, Otopalik et al. 2017 eLife \"When complex neuronal structures may not matter\", where the authors \"quantify animal-to-animal variability in cable lengths (CV = 0.4) and branching patterns in the Gastric Mill (GM) neuron\". And also Otopalik et al. 2019 eLife \"Neuronal morphologies built for reliable physiology in a rhythmic motor circuit\".Many thanks for pointing this out. We now include this work in our discussion and cite these papers.Line 406: you forgot to cite Jackie Schiller's work on cortical pyramidal cells and their tuft dendrites, some of which predates all the cited work in this statement.We regret this oversight and now appropriately cite the work.Line 412: your model, as per Figure 2F, rather very closely matches the observed electrophysiological responses of the neuron under study. In what further ways could you model more closely match experimental observations? This would be very instructive to the reader.Drosophila in the future. We now make this more clear by starting the sentence with \"In case\u2026\"This may be a misunderstanding. We simply highlight the possibility to incorporate active currents within the dendritic compartment if these should be discovered in Line 415: by ensembles of both KCS and MBONs, not just KCs, if you are including the memory part and not only the input representation part.Yes, we changed the sentence accordingly.Line 416: for most of the paper, you quoted these papers to justify the 5% of KCs being simultaneously active in response to an odor. Now the range is shown as 3 to 9%. This discrepancy ought to be reconciled.As stated above we now make this point clear throughout the paper and discuss it more carefully (line 340ff).Line 422: again, nowhere in the manuscript so far did you detail in what way your simulation and experimental findings match those of prior experimental reports regarding neuron response and physiological properties.We agree that the sentence was misleading. We now compare this activation to the observed odor evoked changes in action potential frequency in MBONs in the studies by Hige and colleagues (line 469ff).Line 425: this statement is inaccurate. An individual KC does not encode for a single odor. That would almost never be the case even if a KC was single-claw, as in, exclusively integrated inputs from a single projection neuron: individual projection neurons rarely encode an odor; it's the population of projection neurons that does. Similarly, ensembles of co-active KCs together represent an odor, and do so more narrowly and accurately than the population of projection neurons that excited them.This seems to be a misunderstanding. In line 425 of the original version we did not talk about individual KCs but about sets of 50 KCs. This is in line with the statement of the reviewer.Line 452: left unresolved remains the question of why would both mechanisms exist, as in, is the combination of altering the KC-MBON synapse and altering the PN-KC synapse better in some dimension than altering either alone? Does this relate perhaps to a possible dynamic nature of the olfactory \"fovea\" proposed by Zheng et al. 2020 bioRxiv as presumably static?We now extended this part of the discussion and speculate about the relevance of these different plasticity modes (line 504ff).Line 466: what is standard fly food? Please define it. Choice of food affects very much behavioral assays, for example.As we did not perform any behavior assay, we did not provide a detailed recipe. We basically follow BDSC with small modifications.Line 476: no antigen saturation steps in the immunohistochemistry protocol? Please revise.Yes, we did not perform any antigen saturation steps as this is not necessary for our anti-GFP stainings.Line 505: what were the settings of the puller? These would be necessary to reproduce your glass capillaries. Did you grind the tips, and if so, how?We now provide the detailed settings of the puller.Line 534: how were the R_series, R_input and R_m determined with Igor Pro?We now specify this in the Methods section.Line 561: why was a threshold of 600 M\u03a9 used to exclude cells? How many cells were recorded in total, and how many were excluded?We now specified the inclusion criteria more carefully. Cells not matching those criteria were not included in any analysis.Line 584: once again the data is the best human effort in proofreading a semiautomatic segmentation. It is not the absolute truth. Also, each individual fly is somewhat different, so this is merely one fairly complete yet partial reconstruction, and not assured to be error free, particularly of errors of omission, of a single MBON from a single individual. Would be appropriate to remark this clearly.We now state this clearly throughout the manuscript and again explicitly in the methods section.Did you provide the NeuroML or similar files necessary to run the model in the NEURON simulation software? These should be appended to the manuscript as supplemental data.Yes, all code necessary to run the model in the NEURON simulation software is included and documented in the Dataverse deposit, doi.org/10.7281/T1/HRK27V, as listed under .Line 596: if \"parameters were tuned until the computed voltage excursion at the soma matched our electrophysiological recordings\", what is the rationale for comparing the voltage potential of the simulation with those of the experimental observations?The described part in the methods refers to the establishment of the basic electrophysiological properties of the simulated neuron. Our analysis then address the activation of this neuron.Line 607: where do these ranges of physiologically plausible values come from? Citation, or measurements done in the lab and therefore a figure is needed to show them? Are these ranges from the literature listed in Table 2? Likely yes, would be appropriate to cite them here too or at least explicitly point to Table 2.We now point to Table 2 in this section.Line 616: innervation of MBONs is cholinergic because KCs are cholinergic, but that's not from Takemura et al. 2017, instead from Barnsted et al. 2016 Neuron.We adjusted it accordingly.Line 622: would be appropriate to include the python scripts used to configure and run the NEURON simulation as supplemental material. Likewise for the matlab scripts used to load the analyzed data and plot it. The raw data ought to be included as CSV files or similar.All code and raw data necessary to run the model in the NEURON simulation software and to recapitulate our work is included and documented in the Dataverse deposit, doi.org/10.7281/T1/HRK27V, as listed under .Reviewer #2 (Recommendations for the authors):Drosophila supports stochastic input integration\" is a classical biophysical compartmental modelling study. It takes advantage of some simple current injection protocols in a massively complex mushroom body neuron called MBON-a3 and compartmental models that simulate the electrophysiological behaviour given a detailed description of the anatomical extent of its neurites.\"The cellular architecture of memory modules in This work is interesting in a number of ways:\u2013 The input structure information comes from EM data (Kenyon cells) although this is not discussed much in the paper\u2013 The paper predicts a potentially novel normalization of the throughput of KC inputs at the level of the proximal dendrite and soma\u2013 It claims a new computational principle in dendrites, this didn't become very clear to meProblems I see:\u2013 The current injections did not last long enough to reach steady state , and the model current injection traces have two time constants but the data only one . This does not make me very confident in the results and conclusions.These are two important but separate questions that we would like to address in turn.As for the first, in our new recordings using cytoplasmic GFP to identify MBON-alpha3, we performed both a 200 ms current injection and performed prolonged recordings of 400 ms to reach steady state . For comparison with the original dataset we mainly present the raw traces for 200 ms recordings in Figure 1 Supplement 2. In addition, we now provide a direct comparison of these recordings (200 ms versus 400 ms) and did not observe significant differences in tau between these data . This comparison illustrates that the 200 ms current injection reaches a maximum voltage deflection that is close to the steady state level of the prolonged protocol. Importantly, the critical parameter (tau) did not change between these datasets.\u03c4\u2248 16ms, from Table 1; see Figure 1 Supplement 2 for details), the voltage decays and rises much faster immediately following the onset and offset of the current injections. We believe that this is due to the morphology of this neuron. Current injection, and voltage recordings, are at the soma which is connected to the remainder of the neuron by a long and thin neurite. This \u2019remainder\u2019 is, of course, in linear size, volume and surface (membrane) area much larger than the soma, see Figure 2A. As a result, a current injection will first quickly charge up the membrane of the soma, resulting in the initial fast voltage changes seen in Figure 2D,F, before the membrane in the remainder of the cell is charged, with the cell\u2019s time constant \u03c4.Regarding the second question, the two different time constants, we thank the reviewer for pointing this out. Indeed, while the simulated voltage follows an approximately exponential decay which is, by design, essentially identical to the measured value ; see Table 1 of the manuscript. We chose the capacitance of the soma as 1.5pF, making the time constant of the soma (1.5ms) an order of magnitude shorter than that of the cell.. The circuit is shown in \u2248 15ms is visible at both onset and offset of the current injection. While we did not try to quantitatively assess the deviation from a single-exponential shape of the voltage in Figure 2E, a more rapid increase at the onset and offset of the current injection is clearly visible in We have added a short discussion of this at the end of Section 2.3 to briefly point out this observation and its explanation. We there also refer to the simplified circuit simulation and comparison with raw voltage traces which is now shown in the new Figure 2 Supplement 2.\u2013 The time constant in Table 1 is much shorter than in Figure 1FG?No, these values are in agreement. To facilitate the comparison we now include a graphical measurement of tau from our traces in Figure 1 Supplement 2 J.\u2013 Related to this, the capacitance values are very low maybe this can be explained by the model's wrong assumption of tau?Indeed, the measured time constants are somewhat lower than what might be expected. We believe that this is because after a step change of the injected current, an initial rapid voltage change occurs in the soma, where the recordings are taken. The measured time constant is a combination of the \u2019actual\u2019 time constant of the cell and the \u2019somatic\u2019 (very short) time constant of the soma. Please see our explanations above.Importantly, the value for tau from Table 1 is not used explicitly in the model as the parameters used in our simulation are determined by optimal fits of the simulated voltage curves to experimentally obtained data.\u2013 That latter in turn could be because of either space clamp issues in this hugely complex cell or bad model predictions due to incomplete reconstructions, bad match between morphology and electrophysiology (both are from different datasets?), or unknown ion channels that produce non-linear behaviour during the current injections.Please see our detailed discussion above. Furthermore, we now provide additional recordings using cytoplasmic GFP as a marker for the identification of MBON-alpha3 and confirm our findings. We agree that space-clamp issues could interfere with our recordings in such a complex cell. However, our approach using electrophysiological data should still be superior to any other approach . As we injected negative currents for our analysis at least voltage-gated ion channels should not influence our recordings.- The PRAXIS method in NEURON seems too ad hoc. Passive properties of a neuron should probably rather be explored in parameter scans.We are a bit at a loss of what is meant by the PRAXIS method being \"too ad hoc.\" The PRAXIS method is essentially a conjugate gradient optimization algorithm . This seems to us a systematic way of doing a parameter scan, and the procedure has been used in other related models, e.g. the cited Gouwens & Wilson (2009) study.Questions I have:\u2013 Computational aspects were previously addressed by e.g. Larry Abbott and Gilles Laurent (sparse coding), how do the findings here distinguish themselves from this work.>MBON) play within the circuitry. As we use functional and morphological relevant data this study builds upon the prior work but significantly extends the general models to a specific case. We think this is essential for the further exploration of the topic.In contrast to the work by Abbott and Laurent that addressed the principal relevance and suitability of sparse and random coding for the encoding of sensory information in decision making, here we address the cellular and computational mechanisms that an individual node . Valence information is provided by the dopaminergic system. Dopaminergic neurons are in direct contact with the KC>MBON circuitry and modify its synaptic connectivity when olfactory information is paired with a positive or negative stimulus.\u2013 It seems that Martin Nawrot\u2019s work would be relevant to this work.We are aware of the work by the Nawrot group that provided important insights into the processing of information within the olfactory mushroom body circuitry. We now highlight some of his work. His recent work will certainly be relevant for our future studies when we try to extend our work from an individual cell to networks.\u2013 Compactification and democratization could be related to other work like Otopalik et al. 2017 eLife but also passive normalization. The equal efficiency in line 427 reminds me of dendritic/synaptic democracy and dendritic constancy.Many thanks for pointing this out. This is in line with the comments from reviewer 1 and we now highlight these papers in the relevant paragraph in the discussion (line 442ff).\u2013 The morphology does not obviously seem compact, how unusual would it be that such a complex dendrite is so compact?\u03bb). The degree of a dendritic structure being electrotonically compact is determined by the interaction of morphology, size and conductances . We don\u2019t believe that one of these factors alone (e.g. morphology) is sufficient to characterize the electrical properties of a dendritic tree. We have now clarified this in the relevant section.We should have been more careful in our terminology, making clear that when we write \u2019compact\u2019 we always mean \u2019electrotonically compact,\" in the sense that the physical dimensions of the neuron are small compared to its characteristic electrotonic length  model. Our approach tries to make the least assumptions about the electrophysiological properties of the cell. We think that based on the current knowledge our approach is the best possible approach as thus far no active components within the dendritic or axonal compartments of Reviewer #3 (Recommendations for the authors):This manuscript presents an analysis of the cellular integration properties of a specific mushroom body output neuron, MBON-\u03b13, using a combination of patch clamp recordings and data from electron microscopy. The study demonstrates that the neuron is electrotonically compact permitting linear integration of synaptic input from Kenyon cells that represent odor identity.Strengths of the manuscript:1) The study integrates morphological data about MBON-\u03b13 along with parameters derived from electrophysiological measurements to build a detailed model.2) The modeling provides support for existing models of how olfactory memory is related to integration at the MBON.Weaknesses of the manuscript:1) The study does not provide experimental validation of the results of the computational model.The goal of our study is to use computational approaches to provide insights into the computation of the MBON as part of the olfactory memory circuitry. Our data is in agreement with the current model of the circuitry. Our study therefore forms the basis for future experimental studies; those would however go beyond the scope of the current work.2) The conclusion of the modeling analysis is that the neuron integrates synaptic inputs almost completely linearly. All the subsequent analyses are straightforward consequences of this result.We do, indeed, find that synaptic integration in this neuron is almost completely linear. We demonstrate that this result holds in a variety of different ways. All analyses in the study serve this purpose. These results are in line with the findings by Hige and Turner (2013) who demonstrated that also synaptic integration at PN>KC synapses is highly linear. As such our data points to a feature conservation to the next node of this circuit.3) The manuscript does not provide much explanation or intuition as to why this linear conclusion holds.We respectfully disagree. We demonstrate that this linear integration is a combination of the size of the cell and the combination of its biophysical parameters, mainly the conductances across and along the neurites. As to why it holds, our main argument is that results based on the linear model agree with all known (to us) empirical results, and this is the simplest model.In general, there is a clear takeaway here, which is that the dendritic tree of MBON-\u03b13 in the lobes is highly electrotonically compact. The authors did not provide much explanation as to why this is, and the paper would benefit from a clearer conclusion. Furthermore, I found the results of Figures 4 and 5 rather straightforward given this previous observation. I am sceptical about whether the tiny variations in, e.g. Figures 3I and 5F-H, are meaningful biologically.Please see the comment above as to the \u2019why\u2019 we believe the neuron is electrotonically compact: a model with this assumption agrees well with empirically found results.We agree that the small variations in Figure 5F-H are likely not biologically meaningful. We state this now more clearly in the figure legends and in the text. This result is important to show, however. It is precisely because these variations are small, compared to the differences between voltage differences between different numbers of activated KCs  or different levels of activated synapses  that we can conclude that a 25% change in either synaptic strength or number can represent clearly distinguishable internal states, and that both changes have the same effect. It is important to show these data, to allow the reader to compare the differences that DO matter  and those that DON\u2019T .The same applies to Figure 3I. The reviewer is entirely correct: the differences in the somatic voltage shown in Figure 3I are minuscule, less than a micro-Volt, and it is very unlikely that these difference have any biological meaning. The point of this figure is exactly to show this!. It is to demonstrate quantitatively the transformation of the large differences between voltages in the dendritic tree and the nearly complete uniform voltage at the soma. We feel that this shows very clearly the extreme \"democratization\" of the synaptic input! Please see also the reply to your second comment below.1) My biggest question is about the claim of extreme electrotonic compactness of this neuron. Figure 3D,E suggests that the voltage change at the proximal neurite and at the soma varies by only about 1% depending on stimulation location. Since this is supported only by simulation, it is worth asking how robust this conclusion is.The results of the simulation are very robust. The linearity of the system makes the solution of the underlying equations very stable; small perturbations in the input are guaranteed (by the nature of linear systems) to only result in small changes in the output.As for how well the results reflect biological reality, this is a question that eventually needs to be answered by empirical investigations. The results of any theory, like the one in this study, are subject to verification/falsification. We consider our theoretical results as predictions that are easily falsifiable given suitable experimental techniques. Future empirical studies will determine the validity of these predictions.a) Given that the variability in 3I is so small in magnitude, any dependence would be swamped by other sources of heterogeneity, so the statement that this correlates with distance (line 278) is likely irrelevant.The reviewer is precisely right, the differences in the somatic voltage shown in Figure 3I are minuscule, less than a micro-Volt. But that is exactly the point of this Figure!What we show in Figure 3H is that there are substantial differences (by at least an order of magnitude) between \"local\" voltages, i.e. at the synaptic locations in the dendritic tree. This is expected because of the much smaller diameter of distal parts of the tree than more proximal ones. What is unexpected (at least it was to us) was that these differences are EXACTLY (within a fraction of a micro Volt) compensated by the electrotonic decay from the synapses to the soma. This is the case for ALL 3121 synaptic segments in the dendritic tree. It thus appears as if the morphology of the neuron is fine-tuned to result in extreme \u2019democratization\u2019 of all synapses.\u03bb) which is also present in this neuron . While large \u03bb compresses the range of voltage distributions at distant locations, it is a linear phenomenon and as such it preserves the relative voltage values. The interplay of segment size and, more generally, morphology of the dendritic tree allows for nonlinear phenomena, such as the compression of the voltage range in the dendritic tree which vary over a factor of \u2248 30  towards a much smaller range (variation by about 1%) in Figure 3I.Note that this goes beyond the equalizing effect resulting from a large space constant .b) Can the authors provide a confidence interval for the fit of biophysical parameters to their recordings?The fitting was performed as an iterative approach in the NEURON environment until we got the best fit of simulated results to neurophysiological data was achieved. Please see details in comments to reviewer 2 above. As such we cannot provide a confidence interval for the fit.c) On lines 271-274, the authors state, \"This architecture with the smallest dendritic sections at the most distant sites may contribute to the compactness of the dendritic tree, ensuring that even the most distant synaptic inputs result in somatic voltage deflections comparable to the most proximal ones.\" Is a dependence of dendritic size on distance required for the results? It seems like the result is simply that there is no attenuation within the dendritic tree at all. In general, the authors don't actually provide an explanation for the compactness. In the discussion, it is stated that, \"The compactification of the neuron is likely related to the architectural structure of its dendritic tree,\" which again is rather vague. The authors should strive to provide a clear explanation for this, since it is their key result.We agree that we should have been more explicit in describing the origin of the electrotonic compactness of the neuron. We also agree that the main reason is the low attenuation of electrical signals in the dendritic tree (large space constant \u03bb). We now add a paragraph (second paragraph of Section 2.3) where we compute an estimate for the characteristic length of the cell and show that it substantially exceeds the cell size, resulting in low decay of distal input.However, we believe that there is a second effect that contributes to the compactness of the cell. Analysis of the dendritic morphology reveals that the volume of dendritic segments is inversely correlated with the distance from the soma . For a given current input (or synaptic conductance change), this results in much higher voltage excursions in the distal parts of the dendritic tree, with small neurite diameters, than in more proximal parts, with larger diameters, Figure 3H. As a consequence, the additional voltage decreases due to longer path length from distal synapses vs. proximal synapses is nearly exactly compensated by the increase of local dendritic voltage in the distal locations. This is shown in the nearly identical voltages experienced in the soma by distal and proximal synapses, Figure 2. Therefore, it is not only the large space constant which is important but also the interplay between small distal dendritic volumes and long distance between them and the soma.d) Can the authors report what the electrotonic length for such a dendrite would be? How long before we expect to see a significant spread in 3E?\u03bb= 1,340\u00b5m for the dendritic tree only, and \u03bb= 1,510\u00b5m for the whole neuron, when we base it on the mean of the diameters of all neuronal segments.We now report the electronic length in the rewritten Section 2.3, see also the previous point. Our simple analytical calculation gives 2) My other concern is that, once we assume this perfect integration, the subsequent analyses are all a straightforward consequence. In particular, Figures 4 and 5 just repeatedly convey that it doesn't matter which synapse is being activated, the effect on the somatic voltage is the same. I was particularly confused about the conclusions of 5F,G,H. The authors claim \"small but significant differences\" here, but practically speaking, I can't imagine any of the differences in these plots being meaningful.It is correct that the neuron\u2019s compactness determines its behavior. It is indeed one of our main conclusions that the influence of all synapses on the somatic voltage is very similar which, as we discuss, is important for the odor representation by KCs and its interpretation by the MBON. We document that with different analyses, e.g. those reported in the figure panels referred to by the reviewer. They could be considered redundant but we feel that they elucidate different aspects of the main message of our study. If the reviewer feels strongly that these panels are not needed, we are willing to do without them but we feel that they convey the message very clearly.3) Reference to the data used to constrain the model is confusing.a) At various places in the manuscript references are made to in vivo recordings, but it appears that all of the recordings were done ex vivo.We apologize for this mistake. Yes, all our recordings were performed ex vivo. We now state this explicitly throughout the manuscript. However, we also refer to some data by other groups that were collected in vivo.b) On lines 389-392, the authors state: \"Near-perfect agreement between experimentally observed and simulated voltage distributions in the dendritic tree shows that linear cable theory is an excellent model for information integration in this system.\" What recordings of the voltage distribution in the dendritic tree were performed?Of course the reviewer is correct: Neither we nor, as far as we know, any other groups have recorded detailed voltage distributions in these neurons. We meant to refer to the voltage traces (not distributions) shown in Figure 2D-F that show excellent agreement between recorded and simulated data. The text has been corrected.4) It seems like the conclusions are different than those of Gouwens and Wilson (2009), who described their reconstructed PNs as electrotonically extensive. The authors should comment on what about MBONs and PNs is different.This is an excellent question, and it can be answered at several levels.First, the most obvious one: why do the simulations result in neurons with different (compact vs extended) model neurons? In both cases, the model parameters used in the simulation are determined by finding the optimal fit to electrophysiological (patch clamp) data. Even the numerical methods are identical: both studies use the PRAXIS method in NEURON. In both cases, the fits obtained are in excellent agreement with the measured data. One difference is that we construct the dendritic tree from the morphological (EM) data while Gouwen and Wilson constructed synthetic dendrites since the exact morphology was not available at the time when they conducted their study. We do, however, not believe that this difference is of importance for the question at hand.\u03bb of a neurite depends on three variables, its diameter a, membrane resistance rm and inverse cytoplasmatic resistance ra: \u03bb2 = ar2rma. For the diameter, we used the EM data in the dendritic tree and, as available, for other neurites, and the light-microscopic data where EM data was not available. Gouwen and Wilson used synthetic segments in the dendritic tree and light-microscopical data elsewhere. We could not find the exact values they used in their paper, therefore we can not say whether there are substantial differences between their values and ours. We consider it unlikely that this is the case.The electrotonic length \u2126m) is about three times smaller than Gouwen and Wilson\u2019s . There is also a factor of about five between the fits for the membrane resistance in the numerator: our value is about five times larger (\u2248 100K\u2126cm2) than theirs (\u2248 20K\u2126cm2). Since these numbers multiply, we find a factor of about fifteen. Assuming similar diameters, after taking the square root we find that their \u03bb is about 4 times smaller than ours. Would we take their fitted values for rm and ra, we would likewise obtain a neuron whose characteristic length is substantially smaller than its physical length, i.e.an electrotonically extended neuron. The computations are therefore consistent.There is a factor of nearly three between our best fit for cytoplasm resistance in the denominator; our value  but functionally segregated between compartments (their page 46ff). This is consistent with the electrotonically extended projection neuron studied by Gouwen and Wilson, which receives its input in one of the glomeruli and project its output in the mushroom body, clearly two different compartments. In contrast, MBON\u03b1-3 receives input from KCs that represent odor input in a highly distributed sparse code. We have hypothesized in this study that this code is read by the MBONs by having all synapses count equally. This is not compatible with an extended structure since the impact of a synapse would depend on its location on the cell. In contrast, the PN studied by Gouwen and Wilson receives input in its glomerulus from the olfactory receptor neurons, from between 10 and 50 synapses (release sites). Indeed, their simulations showed that a volley of this size (they assumed 25 simultaneously active synapses) is required to generate a physiologically meaningful effect at the soma, with input from small numbers of synapses having little effect. An electrotonically compact neuron would faithfully transmit the effect of each individual synapse which due to stochastic variability will likely result in a high level of noise. Therefore, the electrotonically extended neuron enforces that only robust input from a large number of near-simultaneous inputs is registered at the soma.Finally, on a third level, does the difference between these neurons make sense functionally? We believe that it does. MBONWe therefore conclude, that these two neurons have very different decoding roles which require exactly the difference in biophysical properties that are found in these two complementary studies by Wilson and Gouwen and our own. It will of course be interesting to directly evaluate this topic again in a comparative study in which both simulations depend on the EM-based reconstructions and are performed side-by-side. Only then, we can provide definitive answers \u2013 or alternatively we aim to highlight it in a review format. Therefore we kept this discussion for now to this place."}
+{"text": "The workload of a vascular surgeon is evolving, with a reduction in the prevalence of aortic and symptomatic carotid disease and an escalating burden of chronic limb-threatening ischaemia (CLTI) and lower limb wounds, associated with diabetes and an ageing population. It is estimated that nearly 15 per cent of US Medicare beneficiaries have a wound,Non-healing wounds, stalled in a chronic inflammatory phase, increase the risk of infection, hospitalization and amputation. Failure to identify the wound aetiology appropriately, institute appropriate specific therapies including compression or off-loading, and refer promptly to specialist services have been identified as a primary cause of delayed wound healingThere is significant variability in delivery of wound care assessment and management, with multiple pathways involving disparate groups of physicians, even within the same healthcare system. The push to improve outcomes for people with diabetes with expedited multidisciplinary care provision has led to an inequitable service for those without diabetes. There is a need to reform services to address such healthcare inequalities, and meet the demands of hard-to-reach groups.,So, what is the role of the vascular surgeon in improving outcomes in lower limb wounds? Whilst advanced training in wound care has not been a priority in the vascular surgery curriculum of most nations, the delivery of timely, appropriate vascular intervention remains key to the management of the leading causes of lower limb wounds. There have been welcome recent advances in guidance, not least the change in terminology from \u2018critical limb ischaemia\u2019 to \u2018chronic limb-threatening ischaemia\u2019. Finally, common sense has prevailed, recognizing that a one-size-fits-all definition of \u2018adequate\u2019 wound perfusion is clinically incorrect. The global vascular guidelines also provide a structured framework for revascularization decision making, emphasizing the need for objective assessments of perfusionThe second factor influencing vascular surgery is the identification of urgency in delivery of CLTI interventions. The Vascular Society of Great Britain and Ireland CLTI Quality Improvement Programme has identified targets from initial consultation to revascularization of 5 days and 14 days for inpatients and outpatients respectivelyThe third challenge is ensuring prompt referral of patients into the service, provision of holistic care and optimal wound management. The NHS National Wound Care Strategy Programme has identified this as a key factor in improving wound outcomes, recommending that all wounds without immediate red flag symptoms are referred to \u2018a clinician with advanced wound care capabilities/competencies working within a multidisciplinary team (MDT) system for assessment, diagnosis and treatment planning\u2019There is a strong clinical rationale for inter-disciplinary management of the patient with a chronic wound, but what role should the vascular surgeon play within the team? A skilled vascular clinician with detailed knowledge of both wound care and vascular assessment/interventions is pivotal. This may be a vascular surgeon, in combination with a podiatrist, tissue viability nurse or vascular nurse specialist to provide wound care expertise. The \u2018toe and flow\u2019 model of joint working between vascular surgery and podiatry has been promoted internationally for those with chronic foot wounds but will not suit those patients with wounds above the ankle. Models with a vascular nurse specialist providing both wound care and vascular expertise have also been reported to deliver excellent outcomes and may be more cost effective. In Belgium and Germany external peer review of services takes the form of formal credentialling of diabetic foot clinicsVascular disease is changing and the key challenge facing patients, healthcare professionals and healthcare systems are wounds of the lower limb, diabetes and CLTI. Vascular surgeons are well placed to coordinate and deliver care to this population. However, the demands that these patients place on the healthcare system are unique and require the development of innovative and evidence-based interventions and pathways of care.The author declares no conflict of interest."}
+{"text": "Legal documents serve as valuable repositories of information pertaining to crimes, encompassing not only legal aspects but also relevant details about criminal behaviors. To date and the best of our knowledge, no studies in the field examine legal documents for crime understanding using an Artificial Intelligence (AI) approach. The present study aims to fill this research gap by identifying relevant information available in legal documents for crime prediction using Artificial Intelligence (AI). This innovative approach will be applied to the specific crime of Intimate Partner Femicide (IPF). A total of 491 legal documents related to lethal and non-lethal violence by male-to-female intimate partners were extracted from the Vlex legal database. The information included in these documents was analyzed using AI algorithms belonging to Bayesian, functions-based, instance-based, tree-based, and rule-based classifiers. The findings demonstrate that specific information from legal documents, such as past criminal behaviors, imposed sanctions, characteristics of violence severity and frequency, as well as the environment and situation in which this crime occurs, enable the correct detection of more than three-quarters of both lethal and non-lethal violence within male-to-female intimate partner relationships. The obtained knowledge is crucial for professionals who have access to legal documents, as it can help identify high-risk IPF cases and shape strategies for preventing crime. While this study focuses on IPF, this innovative approach has the potential to be extended to other types of crimes, making it applicable and beneficial in a broader context. AI, as a computer science discipline, encompasses the design, development, and implementation of intelligent systems capable of efficiently processing large volumes of data, identifying patterns, solving complex problems, and making informed decisions based on available information4. In the legal field, the two main branches of AI, Natural Language Processing (NLP) and Machine Learning (ML), have been extensively applied.In recent decades, the exponential growth of electronic legal information produced in courts has prompted the use of artificial intelligence (AI) to process and manage this vast amount of valuable data. AI aids legal professionals in various tasks such as retrieving, classifying, translating, summarizing, and reviewing legal documents, as well as predicting case outcomes6. Legal professionals need to read previous similar cases for reference in their own defenses, and NLP assists in this search of legal documents of interest as well as producing summaries to avoid spending long time reading long documents and picking out the useful information8. The studies of Hachey and Grover9 and Jain et al.10 are instances of using NLP for automatically extracting relevant features of legal text judgments for creating summaries of legal documents without human intervention. In the field of legal debates, researchers such as Atkinson, Eliot, and Greenwood, among others15 have delved into the analysis of optimal argumentation strategies with the use of NLP. This line of research focuses on developing methods capable of exploring the argumentation employed by individuals in criminal proceedings and unraveling the underlying patterns of argumentation in terms of the structure and content of their arguments. This analysis of previous debates and arguments allows us to learn how to make better and more convincing argumentative syntheses.NLP, in particular, involves algorithms to analyze extensive volumes of text-based data, including extracting relevant information as well as interpreting and generating human language19. Most legal studies that apply ML focus on predicting the final outcome of charges and decision in a legal case. Several studies22, for instance, detect in light of the alleged crime, available evidence, and applicable laws from large sentences the relevant factors related to specific charges .ML is also a sub-field of AI that focuses on developing algorithms that allow machines to automatically process large data sets and detect patterns or relationships in data to make future predictions or make decisions based on data23 analyzed textual patterns and features related to legal arguments and facts influencing judicial decisions of the European Court of Human Rights. The obtained models identified relevant patterns leading to specific judicial decisions, and they allowed predicting courts\u2019 decisions with an accuracy of approximately 79%. Similar studies predicting courts\u2019 decisions of Philippine Supreme Court24, Brazilian Court25, Moroccan Court26 among others, have been carried out to improve and streamline the work of legal professionals when raising a defense, filing appeals, and filing lawsuits.Another connected area of law research applying ML focuses on the analysis of legal text for forecasting judicial outcomes. For example, the study of Aletras et al.27 analyzed court documents to identify common characteristics of homicide perpetrators such as mental health, educational level, criminal history, and substance abuse, and, based on them, developed profiles of criminals homicides. Another example is the study of Vatnar et al.28 which determined by court judgments a significant association between substance use by offenders and crime perpetration.The application of AI in the analysis of legal documents has showcased significant potential in enhancing various aspects of law enforcement and holds promise in the field of criminology as well. Legal documents contain valuable information about criminal offenses, and their analysis can contribute to a better understanding of the dynamics and patterns of specific criminal behaviors. As a matter of fact, some studies have demonstrated the effectiveness of extracting data from legal documents to delve into the characteristics of offenders and their profiles, as well as the risk factors associated with criminal behaviors. For example, the study of Khoshnood et al.To date and to the best of our knowledge, no studies in the field examine legal documents for crime understanding using an Artificial Intelligence (AI) approach. The incorporation of AI in this field of research could aid in automatically identifying relevant characteristics associated with criminal activities, such as criminal records, modus operandi, and locations, thereby assisting in crime prediction. Identifying the factors associated with criminal incidents expands our understanding and serves to anticipate potential crimes in similar conditions. One of the key advantages of AI is its capacity for rigorous and efficient handling of large-scale data. It excels in identifying complex and non-linear patterns within data, making it exceptionally well-suited for thoroughly analyzing extensive legal documents. Hence, the current study aims to analyze pertinent information available in court judgments using NLP and ML methods to predict crime. The specific focus will be on the crime of intimate partner femicide (IPF) due to its prevalence and severe consequences. The findings from this study will be useful for judges, lawyers, police, and victim support services who can utilize the analyzed information to identify high-risk IPF cases and shape effective prevention strategies.The sample of this study consists of cases of lethal violence against women by their current or former male intimate partners (referred as IPF and LV) and cases of non-lethal violence against women by their current or former male intimate partners (labelled as N-LV) determined by court judgements. The interest of the study focuses on the detection of factors associated with LV present in court judgements, and it requires the inclusion of N-LV cases for comparison and validation purposes. The study will also determine which factors are exclusive to LV cases. This comparative analysis will contribute to a better understanding of the differentiated factors of both groups that assist in the detection of IPF.The cases were extracted from the Vlex legal database by conducting an extensive search of legal documents within this particular database using NLP. The search of cases was conducted on February 13th 2022 and updated on January 3rd 2023. Search criteria includes crime, judicial resolution, jurisdiction, jurisdictional organ, time, and location. By applying NLP algorithms, we were able to perform a sophisticated and context-aware search that filtered through a vast amount of legal documents, pinpointing cases that were directly relevant to our research objectives.According to the \u201ccrime\u201d search criteria, the keyword \u201cgender violence\u201d was introduced in the the database. The inclusion of this term was made by the fact that, within the Vlex database, this term encompasses both LV and N-LV. To be more precise, the specific offenses of the Spanish penal code with their respective penal articles applicable to the crime of LV and N-LV were also incorporated: homicide and murder , injuries , illegal detention , threats (article 169), constrains (article 172), habitual violence (article 173), sexual aggression and abuse , prostitution and sexual exploitation (article 187), unauthorized access to privacy (article 197), and insults (articles 208 and 209), which are applicable to LV and N-LV. Consequently, we exclude all crimes not related to LV or N-LV.With regards to \u201cjudicial resolution\u201d search criteria, only final court judgments were selected, whereas provisional resolutions were excluded. The \u201cjurisdiction\u201d search criteria was limited to penal field, while civil, fiscal, social, constitutional, commercial and business, public and administrative, and procedural law fields were excluded.Concerning \u201cjurisdictional organ\u201d search criteria, only provincial and supreme courts were considered given their competence in various types and severity of LV and N-LV cases. In Spain, instruction courts and specialized courts of violence against women are competent for diligence labor and prosecution of minor offences. Penal courts prosecute crimes with prison punishment of up to 5 years, and thus are excluded due to the medium to severe crimes of N-LV, and LV (that exceed the 5 year limit). Supreme courts can prosecute any LV and N-LV case appealed from lower courts, providing an updated sentence. They mostly have the final decision in these cases. The inclusion of this court increases the diversity of types and severity of LV and N-LV crimes. Moreover, the provincial courts were also included for the same reason: being competent for prosecuting crimes with prison punishment above five years and other N-LV cases that have been appealed, some of them not going to the high courts, such as the supreme court.Regarding \u201clocation\u201d as a search criterion, all the included cases were restricted to Spain as its legal resolutions are differentiated from those in other countries, for both LV and N-LV. Finally, the \u201ctime\u201d search criterion concerns with cases from 2019 to 2022 in order to obtain pre and post COVID pandemic data.After conducting the search strategy, 8481 cases of LV and N-LV were obtained. We read the proven facts and judgement from each sentence. Exclusion criteria were: (i) acquittal sentences , (ii) condemnatory sentences involving adult aggressors and minor victims, (iii) condemnatory sentences for cases of reciprocal violence among men and woman, (iv) sentences that nullify previous judicial decisions of recourse and ordering the continuation of proceedings by the competent judicial organ, (v) sentences that do not provide proven facts to refer the previous sentence of judicial recourse and (vi) sentences written in co-official languages of Spain.Consequently, a total of 491 cases met the inclusion criteria of LV and N-LV established by penal court judgements and declared by the Spanish provincial and supreme courts. Out of the 491 cases, 330 are N-LV and 161 are LV. Accordingly, the 330 cases of N-LV involve crimes against personal freedom, physical and psychological integrity, sexual integrity, and privacy. The remaining 161 cases are related to homicides and murders. All of them perpetrated against women by men who are their husbands, ex-husbands, partners or an ex-partners.Regarding the sampling strategy, for those mentioned above, a stratified random sampling approach was adopted to ensure the dataset\u2019s representativeness. The process randomly selects sentences from the LV and N-LV categories while preserving the original class proportions found in the population. This approach allowed us to maintain a balanced dataset and prevent biases arising from an imbalanced class distribution.The variables and associated data have been gathered from the 491 penal court judgments declared by the Spanish provincial and supreme courts, as detailed in the preceding \u201cThe dependent variable is IPF which is categorized into two values: 0 and 1. A value of 0 means absence  of LV (or presence of N-LV), whereas a value 1 signifies presence of LV. A total of 33 independent variables were included in this study, which have been organized into 3 groups. The first group includes variables related to past criminal behaviours and sanctions imposed for their commission. The second group involves variables referred to environment and situation where the LV and N-LV crimes occur. The third group consists of variables related to characteristics of the violence against women perpetrated by their intimate partners.30. Therefore, examining past criminal behaviors in the study of LV and N-LV is essential. Previous studies have delved into this issue and indicate that criminal records are associated with LV33. However, to our knowledge, it has not been analyzed which specific types of past criminal behaviors are related to LV. Furthermore, no evidence of the impact of previously imposed sanctions on the commission of LV and N-LV has been analyzed. For this reason, the current study analyses the variables of past criminal behaviors and sanctions imposed for their commission. General criminal history .Specific criminal history .Criminal history of injuries  .Criminal history of threats  .Criminal history of constraints  .Criminal history of habitual violence  ).Criminal history of insults   that seriously injures a person\u2019s dignity).Criminal history of illegal detention  .Criminal history of sexual aggression  .Criminal history of sexual abuse  .Criminal history of invasion of privacy  .Prison sentence .Deprivation of the right to possess and carry weapons sentence .Prohibition to approximate the victim sentence .Prohibition to communicate with the victim sentence .Community service sentence .Fine sentence .Time of prison (in years).Time of deprivation of the right to possess and carry weapons (in years).Time of prohibition to approximate the victim sentence (in years).Time of prohibition to communicate with the victim sentence (in years).Time of community service sentence (in days).Time of fine sentence (in days).The second group consists of 5 independent variables that capture the environment and situational circumstances of LV and N-LV crimes (number 24\u201328). Although past research underscores the significance of considering the environment and its contextual elements in the study of criminal behavior35, a limited number of studies have explored this association with LV. The existing ones have exclusively focused on geographic context and characteristics of the neighborhood where LV occurs40. The current study strives to broaden the scope by including additional environmental and situational variables, thus contributing to a better understanding of the environment and situation with regard to LV. 24.Place of crime indicates whether the commission of LV or N-LV crime occurred in a rural  or urban area .25.Presence of people concerns whether the LV or N-LV crime occurs in an area where commonly there is nobody or few people around  , or on the other hand, if there are more people present  .26.Social control refers to the presence of people who are aware of the occurrence of a LV or N-LV crime while it is being committed, and have the influence to discourage criminal behavior. It involves formal control including professionals  and informal control referring to non-professionals  .27.Time of crime refers to whether the LV or N-LV crime was committed in dawn , morning , afternoon , night , or at various moments .28.Dispute refers to conflicts between offender and victim during the commission of the LV or N-LV crime or just before the crime began .The third group involves 5 independent variables that capture the characteristics of the violence against women perpetrated by their intimate partners (number 29 to 33). Prior research has delved into various variables encompassing the frequency and severity of violence, the intensification of violent incidents, and the types of violence associated with LV. These studies consistently highlight the significant association between these factors and LV, underscoring their relevance to the current study43. By contrast, concerning coping strategies, previous studies have explored their impact on N-LV. Their findings suggest that disengagement coping strategies are related to elevated risk and prevalence of N-LV45. However, to the best of our knowledge, there is a lack of research analyzing the influence of coping strategies on LV. This critical gap in the literature has driven our decision to incorporate this variable into our present study. 29.Frequency of violence involves the number of violent incidents occurs. It have been categorized into three levels: low (1 violent incident) , medium (2\u20133 violent incidents) , and high (4 times and above) .30.Escalation of violence refers to the gradual rise in frequency and severity of violence as time goes on. The temporal sequence of the criminal behaviors related to intimate partner violence was analyzed, and in the presence of a crime considered by the Spanish code as a minor crime at time point 1 and a severe crime at time point 2, the variable takes value 1. Otherwise, the variable takes a value of 0.31.Physical violence refers to assaults that inflict harm or pose a threat to victims\u2019 physical integrity such as punching, strangling, and shooting .32.Psychological violence involves diverse actions that impact on victims\u2019 mental health such as acts and expressions that inflict humiliation, fear, and undermine the value and dignity .33.Victim\u2019s coping strategies in the face of violence refers to the actions taken by the victim to deal with the violence: disengagement coping strategies  and engagement coping strategies . Disengagement coping strategies are assigned when the victim takes little or no action to deal with the violence, such as submission attitudes and conflict avoidance. On the other hand, engagement coping strategies refers to when the victim deals with violence by using active behaviors such as seeking for information and help, denouncing, and leaving the relationship.Once the variables were collected and prior to develop the analysis, data cleaning process was carried out to ensure that numerical variables were within the range established by the researchers. It is important to note that at the beginning of the study, 49 independent variables were considered, but 16 variables with a significant number of missing values ). Therefore, these 16 variables were excluded from the analysis and not explained in this section. Thus, only the dependent variable and 33 independent variables included in this section were considered in the study.The first group consists of 23 independent variables. On the one hand, there are 11 variables (number 1\u201311) related to past criminal history. These variables correspond to past criminal behaviours proven and convicted by court judgements. Variable number 1 contains criminal history not related to intimate partner violence against women by males. Variable number 2 involves exclusively prior criminal behaviours related to this intimate partner violence. Variables number 3\u201311 detail which specific crimes against women by male intimate partners were perpetrated . The remaining 12 variables are related to sanctions imposed for the perpetration of these criminal behaviours towards women in the relationship. The variables 12\u201317 specify the types of sanctions imposed, and variables 18\u201323 the length of said sanctions. The literature revealed that prior criminal behavior is a strong and reliable predictor of future criminal behavior46.In this experimental study, we have explored the performance of 14 classifiers belonging to diverse families of models. These classifiers were implemented in Weka, a popular machine learning toolkit. The models have been defined based on the distinct families considered, encompassing Bayesian classifiers, function-based classifiers, instance-based classifiers, tree-based classifiers, and rule-based classifiersBayesNet. BayesNet is a classification algorithm that implements a Bayesian network model. It uses probabilistic dependencies between features to make predictions. It is based on the Bayes\u2019 theorem and employs a network structure to represent conditional dependencies among variables, allowing for efficient and accurate classification tasks.NaivesBayes. NaiveBayes is another classification algorithm that is based on the Bayes\u2019 theorem. It assumes that all features are independent of each other given the class variable. This \u201cnaive\u201d assumption simplifies the computation, making it computationally efficient. Unlike BayesNet, NaiveBayes does not consider the dependencies between features, which can lead to less accurate predictions in scenarios where feature interdependencies exist.Functions-based classifiers (5 classifiers). Function-based classifiers, such as logistic regression, neural networks, and support vector machines (SVM), are powerful algorithms that learn a mathematical function to map input features to class labels. In this study, we have considered five examples of function-based classifiers: 3.Logistic regression. The Logistic regression classifier is a popular algorithm used for binary classification tasks. It is based on the logistic regression model and uses a sigmoid function to estimate the probability of an instance belonging to a particular class. It fits a linear decision boundary by maximizing the likelihood function, making it suitable for problems where the relationship between features and the target variable is assumed to be linear.4.SimpleLogistic. The SimpleLogistic classifier is an extension of the Logistic classifier. It combines logistic regression with feature selection by using a backward elimination process. It starts with a full set of features and removes them one by one based on their contribution to the model\u2019s performance, resulting in a simpler and potentially more interpretable model. SimpleLogistic can be useful when dealing with datasets with a large number of features and aiming to find a compact yet accurate model.5.MultilayerPerceptron. The MultilayerPerceptron classifier is a versatile and powerful algorithm for both classification and regression tasks. It is based on artificial neural networks and uses multiple layers of interconnected nodes (neurons) to learn complex relationships between features and the target variable. With adjustable hidden layers, activation functions, and learning parameters, it can effectively capture non-linear patterns in data and adapt to different problem domains. It is especially useful when dealing with large-scale datasets and complex decision boundaries.6.RBFClassifier. The RBFClassifier is a classifier based on radial basis function (RBF) networks. It uses a set of radial basis functions to transform the input data into a higher-dimensional space. Each function represents a prototype or center point, and the classifier assigns instances to the class associated with the nearest prototype. RBFClassifier is effective for nonlinear classification tasks and can handle complex decision boundaries, making it suitable for datasets with intricate relationships between features and the target variable.7.SMO. The SMO  classifier is a fast and efficient algorithm for training support vector machines (SVMs). It solves the quadratic optimization problem by decomposing it into a series of smaller sub-problems. SMO is known for its ability to handle large datasets and high-dimensional feature spaces effectively. It is a popular choice for binary classification tasks and can handle both linearly separable and non-linearly separable data using different kernel functions.Instance-based classifiers (2 classifiers). Instance-based classifiers are algorithms that make predictions based on the similarity between instances in the training data and the test instance. In the study, we implemented two classifiers from this family of models: 8.IBk. The Ibk classifier is an instance-based learning algorithm that utilizes the k-nearest neighbors (k-NN) approach for classification. It assigns a class label to an instance based on the majority vote of its k nearest neighbors in the training set. Ibk is a versatile classifier that can handle both numerical and categorical attributes. It is particularly useful for datasets with local patterns or where instances with similar attribute values tend to belong to the same class.9.KStar. Kstar is a classifier that belongs to the family of instance-based learning algorithms. The classification of a test instance is determined by examining the class labels of similar training instances, as measured by a similarity function. What sets Kstar apart from other instance-based learners is its utilization of an entropy-based distance function for calculating the similarity between instances.Tree-based classifiers (4 classifiers). Tree-based classifiers are a group of algorithms that construct decision trees to make predictions. These classifiers partition the feature space into smaller regions based on feature values and use a tree structure to navigate through the decision-making process. In the study, we have evaluated the performance of four classifiers from this family of models: 10.LMT. The LMT (Logistic Model Trees) classifier is a hybrid model that combines decision trees with logistic regression. It constructs a decision tree where each leaf node contains a logistic regression model. This approach allows LMT to capture both linear and non-linear relationships between features and the target variable. LMT is particularly useful when dealing with datasets that have complex interactions among variables, offering a balance between interpretability and predictive accuracy.11.RandomTree. The RandomTree classifier is designed to construct a tree by considering a subset of K randomly chosen attributes at each node. It does not perform any pruning techniques. Additionally, it offers the option to estimate class probabilities (or target mean for regression) using a hold-out set, a process known as backfitting. This feature allows for improved estimation of class probabilities or target values during the training process.12.RandomForest. The RandomForest classifier is an ensemble learning algorithm that combines multiple decision trees to make predictions. Each tree is constructed using a random subset of features and a bootstrap sample of the training data. The final prediction is determined by aggregating the predictions of all trees, resulting in improved accuracy and robustness against overfitting. RandomForest is widely used for classification and regression tasks, particularly for handling complex datasets with high-dimensional feature spaces.13.J48. The J48 classifier is an implementation of the C4.5 algorithm, which constructs decision trees based on information gain. It recursively splits the data based on the best attribute at each node, aiming to maximize the separation of classes. J48 handles both categorical and numerical attributes and supports pruning to avoid overfitting. It is a popular and widely used classifier due to its simplicity, interpretability, and ability to handle both binary and multi-class classification problems.Rule-based classifier (1 classifier). Rule-based classifiers are a category of algorithms that generate if-then rules to make predictions. These classifiers use a set of rules that describe the relationships between features and class labels. Within this group of models, we have focused our attention on a single model: 14.JRip. The JRip classifier is a rule-based classifier that generates a set of if-then rules to make predictions. It uses a modified version of the RIPPER algorithm and applies a two-phase optimization process to construct accurate and compact rule sets. JRip is particularly useful when interpretability is important, as the generated rules can provide insights into the decision-making process. It is suitable for both binary and multi-class classification tasks and performs well on datasets with discrete or categorical attributes.In the implementation of the algorithms, cost-sensitive learning was utilized to incorporate the cost of misclassifications. A weight of 2 was assigned to false positives (cases of LV incorrectly classified as N-LV), while a weight of 1 was assigned to false negatives (cases of N-LV incorrectly classified as LV). This weighting scheme ensured that the penalty for false positives was more severe than for false negatives. This approach was necessary due to the inherent class imbalance between LV and N-LV classes. Without employing this process, the algorithms tended to prioritize the recognition of the majority class (N-LV class). However, it is crucial to accurately identify the minority class as well, achieving a balanced classification of both classes.Bayesian classifiers (2 classifiers). Bayesian classifiers are a family of classification algorithms based on the application of Bayes\u2019 theorem. They calculate the probability of an instance belonging to a particular class by considering the probabilities of its features given each class. In this study, we have implemented two examples of Bayesian classifiers to explore their effectiveness in classifying the real-world problem of the research study: 47. RFE iteratively removes the least significant features, ranking them based on their contribution to the model\u2019s performance. This technique helped us focus on the most relevant variables and avoid redundancy in our analysis, resulting in a more streamlined and interpretable model.To ensure that only informative variables were included in the analysis, and not redundant factors, a straightforward feature selection algorithm was applied, Recursive Feature Elimination (RFE)To ensure the reliability of the outcomes, a hold-out as a specific form of resampling technique was applied to the final sample (491 cases), dividing the data into two sets. The first set, which included 66% of the total sample (324 cases), was used for a training classification model. The second set consisted of 34% of the sample and was used for the model testing (167 cases). This splitting was conducted 30 times, randomizing by altering the seed of the set partitioner.49, have shown that a 66\u201334% split strikes a reasonable balance between model training and testing while avoiding overfitting. This choice is consistent with industry standards and helps ensure the model\u2019s performance generalizes well to unseen data. The decision to repeat the experiment 30 times, rather than 50 or 100, was influenced by practical considerations and computational resources. While a larger number of repetitions can provide more stable results, 30 repetitions have been found to yield reliable outcomes, as demonstrated in studies like Perales-Gonz\u00e1lez et al. and Dur\u00e1n-Rosal et al.51. Additionally, this number allowed us to balance computational efficiency and robustness in the analysis.As for the specific split of 66% for training and 34% for testing, this choice was made based on a combination of prior research findings and best practices in machine learning. Numerous studies, such as Fern\u00e1ndez-Navarro et al. and Guti\u00e9rrez et al.An incremental approach is adopted in analyzing the independent variables to comprehensively examine the LV and N-LV, considering their multifaceted nature. The study examines whether considering more variables can help differentiate between LV and N-LV. Accordingly, the study examines whether the detection of LV and N-LV is more accurate when a broad set of variables is considered. This incremental progression is based on the need to unravel the complex factors associated with LV and N-LV and ultimately obtain a more holistic understanding of the phenomenon under study. Thus, all the aforementioned algorithms were utilized in three distinct phases, with each phase repeated 30 times using the hold-out process. During Phase 1 of the study, the algorithms were applied solely to the independent variables of Group 1. Phase 2 extended the analysis to include the independent variables from both Group 1 and Group 2. Lastly, in Phase 3, the algorithms incorporated the independent variables from Group 1, Group 2, and Group 3, as outlined in detail in \u201c52. This approach ensures a consistent and comprehensive evaluation across all classifiers, facilitating meaningful comparisons and providing valuable insights into their performance.All the metrics considered in our evaluation are derived from the confusion matrix. The reason for this choice is the diversity of families of classifiers included in our study. While these classifiers may have varying output formats, they all provide information about the predicted category for each instance. By utilizing this information, we can construct the confusion matrix, which enables us to calculate metrics such as accuracy, precision, recall, and F-scoreA confusion matrix affords a comprehensive overview of a classification model\u2019s performance. This matrix illustrates in a table the connection between predicted and actual classifications within a specific data set, allowing an evaluation of the precision of the model and the errors it produce. The confusion matrix used in the study is shown in Table 52 for more detailled information): 52. Mathematically, it is defined as: Correct classification for N-LV class or class 1. It is also called as the sensitivity of the first class in52. In this case, the percentage of correctly classified LV patterns, which is defined as: Sensitivity for LV class or class 2CCRThe correct classification rate (CCR) often referred to as accuracy, is a metric used in classification tasks to measure the proportion of correctly classified instances out of the total number of instances in a dataset. It is a fundamental performance measure in machine learning and is typically represented as a percentage. Formally, the correct classification rate (CCR) can be defined as: F-scoreP is the precision, defined as: R is the recall, defined as: The F-score, also known as the AA53. The AA (macro average) is a metric commonly used in imbalanced classification scenarios. In imbalanced datasets, where one class significantly outnumbers the others, accuracy alone may not provide an accurate representation of a model\u2019s performance. In such cases, macro-averaging the accuracy can offer valuable insights, since by using macro-averaged accuracy, you give equal importance to each class, regardless of its size. Mathematically, it is defined as: The Average Accuracy (AA) is the arithmetic average per-class effectiveness of a classifier. It is usually referred as macro-averageGMThe Geometric Mean corresponds to the geometric average of the partial accuracies for each class. GM is another metric applicable in imbalanced classification scenarios, and it exhibits greater sensitivity to unbalanced classifications within the baseline classes (N-LV and LV) when compared to macro-average accuracy (AA). GM is mathematically defined as follows: The specific metrics derived from the confusion matrix considered in the study are the following  in extracting information from court decisions and accurately predicting Intimate Partner Femicide (IPF). From a data-focused standpoint, this study perceives legal text as a valuable source of information that can be assessed and analyzed using AI methods such as Natural Language Processing (NLP) and Machine Learning (ML) algorithms. Leveraging these techniques, patterns and connections within legal texts were identified, facilitating the prediction of IPF. This novel application of AI in the criminological field specifically focuses on utilizing legal texts within the domain of law.59.The findings of this study suggest that the information extracted from court decision texts proves valuable in identifying risk indicators for IPF. The different algorithms employed in the study exhibited competitive performance in distinguishing between lethal and non-lethal violence in male-to-female intimate partner relationships. Specifically, it was observed that high accuracy in differentiating these groups can be achieved by considering a comprehensive set of variables, including criminal history, prior sanctions, characteristics of violence against women perpetrated by their intimate partners, as well as the environmental and situational context in which this violence occurs. These results align with existing literature emphasizing the significance of considering a combination of variables for effective crime detection30. Court judgments provides documented history of individuals\u2019 involvement in criminal activities as well as their sanctions, and this information provides insights into recidivism. A first analysis was conducted with AI analyzing data of criminal records and sentences imposed on repeat offenders of violence against women in intimate partner relationships, and it detected patterns of repeat criminal behaviors related to IPF and the effectiveness of sentencing strategies on this behavior. However, this information was only able to detect about half of the cases of lethal and non-lethal violence suffered by women in intimate partner relationships. It indicates that criminal records and sanctions are relevant information to detect both types of violence, but there are cases where it is not sufficient to identify them. Some studies reveal that criminal records are a risk factor for IPF33, but no previous studies on the effect of sanctions on IPF have been identified, and the current study contributes in this regard. Our study reveals that the imposition of prior non-custodial sanctions is more related to LV than N-LV. In particular, the sanction of prohibition to approximate the victim is the one most associated with LV.According with the literature, past criminal behavior is considered a strong predictor of future criminal behavior35. Previous research consistently demonstrates that the geographic location matters in the perpetration of IPF. Studies found higher rates of IPF in rural areas compared to urban areas38. This geographical disparity may be attributed to factors such as limited availability of resources, and reduced seeking help or intervention in situations of intimate partner violence62.The aforementioned factors motivated the development of a second analysis, which incorporated environment and situational information pertaining to the occurrence of crimes against women. This expanded analysis enabled the detection of a higher number of cases involving both lethal and non-lethal violence. Analyzing crime incidents targeting women by their intimate partners across time and space yields crucial insights into the modus operandi and behavior of perpetrators involved in both types of violence. Specific geographic and temporal characteristics are associated with these criminal activities, distinguishing between lethal and non-lethal violence against women. Thus, IPF is influenced by context characteristics, and not only by individual factors of aggressors and victims40. This is consistent with the findings of the current study. It found that people in the offender\u2019s and victim\u2019s immediate context have an influence on crime. Additionally, the study revealed that not only place and situational characteristics but also specific time periods play a crucial role in Intimate Partner Femicide (IPF) perpetration. The study identified distinct temporal patterns that differentiate IPF from non-lethal violence. Recognizing these temporal and geographical patterns is essential for implementing targeted interventions in specific areas and moments. This information facilitates the strategic allocation of resources, focusing not only on individuals at risk of experiencing IPF but also on specific locations and time periods associated with IPF.Additionally, prior research revealed that characteristics of the neighborhood also relates to IPF. For instance, limited community cohesion increases the risk of crime. By contrast, supportive social networks and strong social ties can act as protective factors in crime, including IPF64. This could explain why variables related to severity, frequency, and escalation of violence lead to the detection of a greater number of IPF cases. Although physical violence has received the most attention in terms of predicting IPF63, the present study, as well as a few others43, demonstrates that psychological violence is also a significant determinant.The third analysis, in addition to incorporating the variables from the first and second analyses, included specific variables related to the characteristics of violence against women by their intimate partners. This comprehensive approach facilitated the detection of over three-quarters of both lethal and non-lethal violence cases. By considering factors such as the severity level, frequency, escalation, type  of violence experienced by victims, and their coping strategies to address violence. This third analysis accurately identified a significant majority of both lethal and non-lethal violence cases. According to the scientific literature, in most cases, IPF is not an isolated event but rather the culmination of a pattern of escalating violence of abuse65. The RBFClassifier was selected for this analysis because it is the algorithm that shows the most competitive performance on a single model, in contrast with the RandomForest, which is competitive but is an ensemble of models. The findings revealed that the discriminative power between both classes emerged when considering all variables collectively. Past criminal behaviors and sanctions imposed for their commission factors alone are insufficient to correctly classify LV and N-LV, as information is required on how violence against women occurs and the environment and situation in which it occurs. It has been identified that a specific criminal history of intimate partner violence  is related to LV when physical and psychological violence is present, it becomes more frequent and severe, there were disputes prior to violence, and violence was exercised in places where there is commonly no nobody and social control is absent. In these cases, most of the victims have adopted disengagement coping strategies. This highlights the need to consider the overall characteristics to correctly identify LV and N-LV and not to draw general conclusions from a couple of factors.An additional local sensitive analysis of the RBFClassifier was conducted in order to further deeper insights into the specific variables among all ones analyzed that contribute to the correct classification of LV to N-LVThe study not only offers valuable insights into IPF risk indicators but also extends the application of this knowledge to law enforcement professionals and related stakeholders, such as police and victim support services, who have access to the analyzed data. These professionals play a crucial role in identifying risk signals given the vast amount of data they handle, and our findings could assist them in this task. Consequently, these professionals would be able to promptly screen cases at risk of IPF, based on the validated risk indicators from our study, as soon as they obtain the case information. This information provides them with a foundation for urgent interventions by referring the cases to specialized services, implementing enhanced safety measures, and connecting victims with specialized support organizations, among other preventive actions.The study has certain limitations that should be taken into account when interpreting the results. Firstly, the findings are based on specific Spanish legal documents, potentially limiting the generalizability of the findings to different legal contexts or document types. Secondly, the absence of long-term follow-up for the analyzed cases means that it is unknown whether instances of nonlethal violence identified during the study may escalate to lethal violence in the future. The study\u2019s third limitation is the lack of qualitative information. Further longitudinal studies will be necessary to address this limitation and complement the preliminary findings of these analyses. It is important to note that the nature of the study made it impossible to extract information from interviews or other qualitative sources involving women who have experienced lethal violence. Therefore, the study relied solely on the analysis of available legal documents.This study pioneers the use of Artificial Intelligence (AI) to highlight the critical information present in legal documents for crime prediction, specifically focusing on male-to-female intimate partner violence. However, the approach employed has the potential to be extended to other types of crimes, marking new research inroads within the field of criminology. The study utilizes Natural Language Processing (NLP), a sub-field of AI, to extract court judgments related to male-to-female intimate partner violence from the Vlex legal database. Additionally, Machine Learning (ML), another branch of AI, is employed to identify specific elements in legal texts associated with Intimate Partner Femicide (IPF), which refers to lethal violence against women by male intimate partners.The findings demonstrate that information regarding past criminal behaviors, imposed sanctions, the severity and frequency of violence against women, as well as the environmental and situational factors, successfully distinguish over three-quarters of both lethal and non-lethal violence within male-to-female intimate partner relationships. Integrating these findings into professional practices holds the potential to enhance the detection and prevention of IPF cases. Consequently, this study makes a valuable contribution to the field of criminology by expanding knowledge in the area of IPF and supporting effective prevention efforts."}
+{"text": "Drosophila melanogaster, including the oldest extant specimens of this species. By comparing historical samples ranging from the early 1800s to 1933 against modern-day genomes, we document evolution across thousands of generations, including time periods that encompass the species\u2019 initial occupation of northern Europe and an era of rapidly increasing human activity. We also find that the Lund, Sweden population underwent local genetic differentiation during the early 1800s to 1933 interval  but then became more similar to other European populations thereafter . Within each century-scale time period, our temporal sampling allows us to document compelling candidates for recent natural selection. In some cases, we gain insights regarding previously implicated selection candidates, such as ChKov1, for which our inferred timing of selection favors the hypothesis of antiviral resistance over insecticide resistance. Other candidates are novel, such as the circadian-related gene Ahcy, which yields a selection signal that rivals that of the DDT resistance gene Cyp6g1. These insights deepen our understanding of recent evolution in a model system, and highlight the potential of future museomic studies.The ability to perform genomic sequencing on long-dead organisms is opening new frontiers in evolutionary research. These opportunities are especially notable in the case of museum collections, from which countless documented specimens may now be suitable for genomic analysis\u2014if data of sufficient quality can be obtained. Here, we report 25 newly sequenced genomes from museum specimens of the model organism  Genomes from 25 museum specimens of Drosophila melanogaster (some more than 200 years old) reveal that small populations occupying northern Europe gave way to well-connected fly populations across the continent, and also illuminate targets of recent selection, including genes that may have helped this species adapt to novel climates, viruses, and insecticides. Museum collections around the world contain billions of specimens collected over the last centuries. These collections serve as a window back in time\u2014to an era before industrialization and modern agricultural practices\u2014and could as such provide insights into issues ranging from insecticide resistance to climate change. The analysis of so-called historical DNA (hDNA), including from museum samples, accordingly holds much promise, but obtaining high-quality genomic data from older specimens remains a significant challenge ,2.A handful of prior studies have targeted whole-genome sequences from museum insect specimens. These have included proof-of-concept sequencing studies ,4 and inDrosophila melanogaster is a primary model system for population genetics. Recently, the availability of data from multiple time points has begun to improve the prospects for distinguishing natural selection from neutral evolution in this system. Genome sequencing of wild-caught and laboratory-maintained D. melanogaster individuals has enabled the study of genomic evolution across seasonal  and  and Drosl . Hence, museomic study of European D. melanogaster offers the potential to reveal both demographic and adaptive changes during this dynamic time interval, against the backdrop of a well-annotated genome with detailed knowledge of gene function.Here, we obtain and analyze genomes from historical ironment , along wDrosophila  and multiple early 1800s specimens of D. melanogaster. These include 4 specimens from the collection of Carl Fredrik Fall\u00e9n of Lund University . From the same time period, 3 specimens exist from the collection of Johan Wilhelm Zetterstedt , a student of Fall\u00e9n, and from 1822 professor at Lund University. This material comprises the lectotype (not examined here) and 2 paralectotypes of Drosophila approximata, a junior synonym of D. melanogaster.The insect collections held in Lund and Stockholm are among the oldest in the world and contain many important samples, including the specimens used to erect the genus Imago and larvae in sawdust [unintelligible] can of raisins.\u201d If the flies were caught by Fall\u00e9n himself, a potential locality would then be his lodgings in central Lund , and that the material was collected before 1811, when Fall\u00e9n began to spend considerable time elsewhere. His first mention of Drosophila can be found in a letter to Zetterstedt dated 14 September 1810 . The paralectotype samples examined from Zetterstedt\u2019s collection are labeled Lund and Smol  respectively, and they are likewise difficult to precisely date. Based on Zetterstedt\u2019s Diptera Scandinaviae disposita et descripta, he collected D. approximata in Lund . The specimen examined here from Sm\u00e5land (and likewise the lectotype) was attributed to Sven Ingemar Ljungh (1757 to 1828), civil servant and prominent naturalist, who resided at Sk\u00e4rsj\u00f6 manor . It is unclear whether these specimens are the ones mentioned in his treatise on Scandinavian Diptera, but if so, the flies would likely have been caught within a decade or so of Fall\u00e9n\u2019s samples. However, the owner of a given specimen may or may not have been its collector . These Swedish specimens are, to the best of our knowledge, the earliest D. melanogaster available in any collection, potentially predating the Meigen holotypes kept in Paris by 2 decades.The precise timing, location, and collector for the 6 examined specimens is not easy to decipher. The Fall\u00e9n samples, collected in Lund, are accompanied by an original note, stating (translated) \u201cFig 1F) collected by Joseph Waltl (1805 to 1888), undated but likely collected in the 1850s. Also from Copenhagen, there is 1 specimen collected by Rasmus William Traugott Schlick (1839 to 1916) on northeast Zealand, Denmark , undated but likely collected in the 1880s. Lastly, we have a set of 15 specimens from the collection in Lund , collector unknown, labeled \u201cZootis 1933\u201d\u2014the informal name of the former Zoological Department at Lund University .From the collection in Copenhagen, there are 2 specimens from Passau, Germany . The roughly 200 year time frame of this analysis may encompass 3,000 fly generations )S2 Table). Because the inclusion of related individuals in estimates of allele frequency generates artifactual nonindependent sampling, we sought to identify and mask individual chromosomal intervals displaying \u201cidentity by descent\u201d (IBD) from downstream population genetic analyses .A preliminary analysis of pairwise genetic distances among the genomes indicated that some specific pairs of individuals from within the early 1800s and 1933 Sweden samples had genetic similarity implying relatedness . Results indicated varying levels of relatedness among individuals within the 1800s and 1933 Sweden samples, up to levels expected for first or second order relatives, and a much lower level of relatedness between the 2 specimens from 1840s Germany . In addition to IBD between genomes, we found that a few of the 1933 Lund samples had higher levels of within-genome IBD due to inbreeding\u2014several samples from 1933 had long regions of chromosomes with depleted heterozygosity , consistent with mating between close relatives.We identified IBD regions between a given pair of individuals based on windows in which they had unusually low genetic distance between them . We inferred IBD windows spanning various lengths, up to whole chromosome arms  in Lund appeared to show a decline with time , which could reflect the action of genetic drift. However, we note that damage-induced errors may inflate diversity estimates, especially for the 1800s sample . In addition, the modern Lund sample represents a published pool-seq data set, analyzed via a distinct pipeline including the masking of rare variants, which may deflate its diversity estimate.We then sought to examine how within-population genetic diversity and between-population genetic differentiation have changed across more than 2 centuries. We focused initially on the X chromosome in order to avoid the influence of inversions . PatterFST) revealed a curious temporal trajectory . When Lund samples are compared to modern samples from around Europe, between the 1800s and 1900s sampling points, Lund became more differentiated from other populations. In contrast, between the 1900s and 2000s sampling points, Lund became more similar to other modern European populations. Concordant patterns were observed from principal components analysis (PCA) based on X-linked variation . Here, 1800s Lund genomes were seen to cluster with modern European genomes, whereas the 1933 Lund genomes form a distinct cluster. This unexpected pattern of transient local differentiation could indicate that distinct evolutionary forces had predominant influences on genomic diversity between these time points.Patterns of genetic differentiation , which may have also limited local population sizes. Consistent with this hypothesis, Zetterstedt [Increased genetic differentiation during the 1800s to 1933 period could reflect an elevated influence of genetic drift. This interval likely represents the earliest days of n Europe , perhapsterstedt  describeterstedt ,28 and Fterstedt .Fig 1A), both of which may have been conducive to population growth in this human commensal insect (and hence reduced drift). In parallel, increased human transportation, particularly the expanded shipping of fruit and other commodities, would be expected to facilitate increased long-distance dispersal of D. melanogaster. We note that such temporal shifts in demography would not be detected by conventional demographic inference based on modern samples, and it is unclear how well simple demographic models can recapitulate the effects of more complex histories on genomic diversity.In contrast, the genomic homogenization between Lund and other European regions that occurred between 1933 and the 2010s could reflect increased migration through time, in conjunction with reduced genetic drift in fly populations that may have grown with time. Both of those demographic changes might be predicted based on concurrent changes in human activity during the 20th century. This time period featured a 5-fold increase in the human population of Lund and the surrounding region, along with a warming climate ((2L)t from one 1933 Lund genome, and no inversions among the 1800s genomes (S6 Table). In the modern Lund sample, (2L)t is at 13% frequency, while other tested inversions appear to be absent (S6 Table). Hence, although we cannot rule out changes in inversion frequencies through time, inversions do not appear to be a likely driver of genomic differentiation between time points.We also examined whether changes in the frequencies of common polymorphic inversions may have influenced genetic diversity between time points see . We founNe) for each time interval. For each chromosome arm separately, we used simulations to identify the value of Ne that best matched the empirical mean change in allele frequency at non-singleton SNPs between time points . These values are of the same order of magnitude as an estimate of 9,500 from a northeast United States population between 1975 and 2015 [\u03c0 from coalescent simulations based on our estimated Ne values to those from the empirical data, using a demographic model previously estimated for a French population [\u03c0 reasonably well, our low estimates of Ne yielded simulated values of \u03c0 that were less than half of those actually observed in our empirical data (S7 Table). These results indicated that drift-only models did not provide an accurate approximation of the Lund population\u2019s demographic history and that true Ne values were probably greater than our estimates. Instead, migration may have played an important role in shifting allele frequencies without decreasing \u03c0, as suggested above. Estimating a reasonable spatiotemporal demographic model that incorporates both local population sizes and geographic patterns of genetic structure and gene flow may require more extensive sampling of population genomic data across space and time.We further investigated a simple model of the demographic history of Lund by using a Bayesian approach to identify the best-fitting effective population size (ints see . In conton (e.g. ), our poand 2015 . EstimatFST-based population branch statistic . Overall, we identified 190 outlier regions for the 1800s to 1933 time interval (referred to below as scan A) and 173 for the 1933 to 2010s interval (scan B), with some of these regions incorporating multiple outlier windows (S8 Table). Since not all outliers may represent true positive targets of recent natural selection, we only discuss loci that had among the most extreme frequency changes across each century, referring to these outlier regions based on their ranked maximal window PBE values. This restricted focus is motivated by uncertainty regarding the actual number of loci under selection in each time interval, which we do not attempt to estimate due to limitations in the data and in our ability to identify a realistic neutral model.One principal goal of this study is to identify instances of elevated genetic differentiation between time points that may reflect the action of recent positive selection. Our SNP-based genome scan focused on population branch excess (PBE ), a stattic (PBS ) but is CHKov1 , which is thought to encode an ecdysteroid kinase [D. melanogaster populations, which was reported to correlate with resistance to an organophosphate pesticide [CHKov1/CHKov2 locus has, however, also been found to correlate with resistance to infection by the D. melanogaster sigmavirus (DmelSV) [D. melanogaster acquired this rhabdovirus around 200 years ago [refractory to Sigma P (ref(2)p) within outlier region A20. Ref2p activates the Toll pathway and has been shown to confer resistance to DmelSV [In both time intervals, the top window PBE value was also associated with the widest outlier region, which is consistent with relatively strong positive selection. For the 1800s to 1933 interval, the top outlier (labeled A1) was a 31 kb region that included the gene esticide . Transpo(DmelSV) . Given tears ago , which po DmelSV .CHKov1 and ref(2)p are of particular interest because mutational variants associated with viral resistance have been previously identified [CHKov1, several SNPs have alleles in linkage disequilibrium with a transposon insertion in the gene [D. melanogaster population samples [entified ,38. In Cthe gene  associatthe gene . Based o samples .ref(2)p for the 1800s time interval appeared not to be driven by the short, complex deletion identified by [The outlier PBE score for ified by . Based oCytochrome P450 6g1 . This gene is associated with resistance to dichlorodiphenyl-trichloroethane (DDT) and other insecticides in D. melanogaster. As with ChKov1, there is prior evidence for positive selection associated with transposon insertions and gene duplication [The top outlier for the 1933 to 2010s interval (denoted region B1) spanned 75 kb and included a known target of insecticide resistance evolution, lication \u201342. HereFig 6A\u20136D depicts 4 narrow SNP-level patterns of allele frequency change focused on the 1800s to 1933 time interval, whereas Fig 7A\u20137D depicts 4 such examples for the 1933 to 2010s interval . Focusing on the earlier period, a few SNPs upstream of hector , a calcitonin-like neuropeptide receptor involved in male courtship behavior [Fig 6A). In region A8, a cluster of SNPs within a 5\u2032 UTR intron showed the highest PBE values at rutabaga , a calcium-responsive adenyl cyclase that influences learning, memory, lifespan, circadian rhythm, ethanol tolerance, and response to heat and oxidative stress [LysC and LysD , a locus for which a gene duplication and inversion polymorphisms have been reported [D. melanogaster populations [CG17032 , for which the highest PBE value was at a synonymous variant (at R5 position 3L:15953092). Among top 1800s to 1933 outliers with more diffuse SNP-level signals, the A2 region encompassed sturkopf, a lipid droplet-associated protein that regulates growth and stress response [cabut (cbt), a transcription factor that regulates metabolic and circadian responses to nutrition [Both of the above outlier regions were relatively broad, and some of the other top outliers also showed somewhat less-broad plateaus of elevated genetic differentiation between temporal samples. In contrast, several other top outliers showed more narrowly localized signals of elevated genetic differentiation. At least in spatial analyses of local adaptation, broader intervals of genetic differentiation are more likely to be associated with selection favoring a single initial haplotype (resulting in a hard sweep), whereas narrow signals of frequency change may result from selection favoring multiple initial haplotypes, resulting in a \u201csoft sweep\u201d . Narrow behavior , showed e stress \u201349. We areported , and whiulations . In contresponse  and is kutrition .Adenosylhomocysteinase  yielded a PBE value nearly as extreme as that observed at Cyp6g1, but with a far narrower genomic scale . Curiously, the top SNP PBE value occurs at a synonymous variant (at R5 position 3L:15953092). Ahcy regulates methionine metabolism, histone methylation, and lifespan [Ahcy does not appear to have been detected as a top selection candidate by previous studies. The next-highest region , Lysine demethylase 4B (Kdm4B) also impacts histone methylation and circadian rhythm [Prosap from this same 1933 to 2010s time interval (B6), along with cbt (A4) and rut (A8) from the 1800s to 1933 scan, also represent top outliers with circadian functions. The evolution of circadian behavior in high latitude D. melanogaster populations is well known . The non , involved in phototransduction, olfaction, and phospholipid transport [Fig 7C) centered on a gene that was recently detected as a top outlier in the 1975 to 2015 US temporal population genomic study cited above [Hexosaminidase 2 (Hexo2) encodes a sperm-associated protein that may play a role in fertilization [CR43835 , for which no function is known.For the narrow signal at region B8, the highest PBE SNPs were observed at the upstream end of ransport \u201367. Notaed above ; Hexosamlization ,69. WherFig 7, for the 1933 to 2010s scan specifically, there was an abundance of X-linked loci among the top outliers: although the X chromosome constitutes less than one fifth of the analyzed genome, 12 of the top 15 outlier regions were X-linked (S8 Table). Explanations of this enrichment could include greater X chromosome effects of sampling variance , genetic drift, or positive selection . Howevezygosity ) and thezygosity . HoweverAmong the top outliers discussed from each period, none appeared within the full list of 1% PBE outliers from the alternate time period. At least for genes promoting adaptation to local environmental conditions (such as temperature or day length), we might predict that alleles driving adaptation in the 1933 to 2010s time interval would have been adaptive in the 1800s to 1933 time interval as well. It is possible that each local population initially possessed only a subset of adaptive variants, and others arrived later via migration (or mutation). Alternatively, in the 1800s to 1933 interval, the frequencies of causative variants at some genes may have still been too early in their rise to generate extreme outlier PBE signals, but poising them to rise more quickly in the 1933 to 2010s interval. Another possibility is contingency\u2014some variants may have only become beneficial after other adaptive changes had occurred due to epistatic interactions among circadian genes. Alternatively, this lack of outlier overlap could indicate that selective pressures differed between eras or that adaptive events tended to complete within a given time period rather than spanning between them.D. melanogaster. Prosap (in region B6) is thought to encode a structural component of the postsynaptic density [D. melanogaster populations [phantom (phm), which encodes a cytochrome P450 protein involved in ecdysteroid biosynthesis [D. melanogaster population [polyhomeotic-proximal (ph-p), a developmental and chromatin-modifying gene , whi, whiD. me . Enrichment was also observed for membrane (and vesicle) organization, compatible with the existence of population differences in membrane lipid composition [Cyp6g1 in separate outlier regions, potentially reflecting pre-insecticide roles of insecticide-related genes (as with the ChKov locus described above). For the 1933 to 2010s interval, insecticide-related GO categories were not enriched among outliers. Instead, metabolic processes dominated the top results , and categories related to cell division were enriched as well (S9 Table). As with membrane composition, metabolism is also known to vary significantly between D. melanogaster populations from contrasting thermal environments  enrichment analysis to the broader sets of top 1% PBE outlier regions, using the permutation approach initially described in , separatposition  and thisposition . Curiouss e.g., ,85)..S9 TableTo our knowledge, our samples include the oldest pinned insects from which genome sequences have been reported . Overall, 96% of our specimens yielded analyzable genomes, and 73% yielded genomes of relatively high quality. Although data quality remains an important concern, these results are encouraging for future museomic studies of small invertebrates.D. melanogaster is the smallest animal from which historical genomes have been obtained. Surprisingly, that small size may have even been an advantage. Male D. melanogaster are smaller than females , and yeD. melanogaster, but also the specimens themselves. Beyond confirming the sex of each fly, we inferred from relatedness that the Swedish early 1800s flies, which had been linked to 3 different scientists and 2 different localities , were actually from the same time and place. These results highlight important challenges when working with very old specimens, namely the lack of detailed collection data and potentially imperfect record keeping through time. Intriguingly, the unsequenced lectotype specimen  is, however, mounted with a distinctly different pin. The lectotype may accordingly be one of Ljungh\u2019s flies, as described in Zetterstedt\u2019s Diptera Scandinaviae. The exact collection date of the sequenced flies is unknown, although it may have occurred by 1810 . Hence, our study provides a wealth of candidates for functional evolution that can be investigated in subsequent laboratory studies, through leverage of the molecular, genetic, and transgenic tools available in this model system. Although museum specimens for this (or any) species are finite, further historical genomic studies in D. melanogaster have the potential to greatly inform the spatial and temporal dynamics of adaptive evolution and population history, broadening the range of insights that can be obtained from genomic variation in this population genetics model species.The expanse of existing knowledge regarding the biology and genome of Drosophila melanogaster were sampled from the entomological collections of Biological Museum , Swedish Museum of Natural History , and National History Museum Denmark  . The library prep followed mainly the protocol of . BrieflyDrosophila Genome Nexus (DGN) pipeline as described in [D. melanogaster reference genome allowed newly assembled museum genomes to be compared against published data from modern population samples of individual genomes. Modern genomes from France and Egypt were reported in the subsequent DGN publication [The procedures for alignments and variant calling used to process the museum genomes largely followed the ribed in . Using tlication . A sampllication . Populatlication  and asselication  is descrFollowing read trimming using fastp (v0.21.0 ), we folDiploid variant calling was initially performed at all sites, except for the X chromosomes from male flies. The sex of the flies in the museum samples was not identified prior to sequencing. It was identified by calculating the ratios of average read depth in the X chromosome versus the autosomes, which is expected to be approximately 1:1 in females and 1:2 in males. All perl scripts referenced in this section and elsewhere were executed on perl v5.34.0, all python scripts were executed on python 3.8.6 , and the R scripts were run using R v.4.2.2.For those museum genomes with a total coverage of <80% and average per-site read depth of <10, we concluded that diploid variant calls could not be confidently made. These were instead treated as \u201cpseudo-haploid\u201d with only a single nucleotide called per site; UnifiedGenotyper (GATK ) was runPublished pool-seq data  was obtaj unique lineages at a site given nr reads and nc the number of chromosomes in the sample pool . The effective count j follows a distribution based on Stirling numbers of the second kind S, i.e., the number of ways in which nr reads can be partitioned into j lineages. The probability of j independent lineages in the sample is then:Pool-seq data introduces several statistical sampling artifacts that are not an issue with individual sequencing. Specifically, the number of reads contributed by each individual varies, so that some genomes are overrepresented and others are underrepresented. To deal with this, we calculate an effective allele (lineage) count , i.e., tThe effective number of distinct lineages  was estimated from the expectation of this distribution. We found a maximum pool size of 183 for the autosomes (median = 145) and of 95 for ChrX (median = 70). Allele frequencies at each site were multiplied by the pool size to generate the appropriate downsampled counts for population genetic analysis.The total number of reads and the average read length was assessed from each bam file using CollectInsertSizeMetrics from the Picard suite of tools. Based on the depth and coverage criteria given above, the low-quality genomes that were treated as pseudo-haploid in the analysis pipeline were 18SL4, 18SL5, 18SL6, 18SL12 (from 1800s), 19SL3, and 19SL7 (from 1933). One sample from 1933, 19SL2, was of such low quality that it was excluded from any further processing or analysis.To determine whether low-quality sequences contributed a significant number of false positive variants in each population sample, we tallied the number of \u201csingletons\u201d\u2014instances where a given genome had an allele otherwise not observed among any other analyzed genome across populations. The fraction of singleton alleles that were A or T bases was calculated to determine if cytosine deamination accounted for many of the singletons, and whether this phenomenon was correlated with sample quality. Singleton minor alleles were excluded from certain downstream analyses, such as demographic inference, while a total allele count of at least 4 was required for all analyses unless otherwise indicated. We also calculated the Pearson correlations between read depth and coverage  to quantify the association between these quality metrics.Drosophila Genome Nexus [IBD among certain genomes in both the 1800s and 1933 Lund samples was initially implicated based on highly variable pairwise differences among genomes. We therefore set out to identify and mask redundantly sampled sequences in as targeted a manner as possible, while leaving maximal sample sizes of unrelated chromosomes at each locus. We implemented an IBD identification and masking framework similar to that employed by the me Nexus  to mask me Nexus , averagiith genome, the background genetic distance di is the median Hamming distance between the ith genome and unrelated modern Lyon samples. This genome-specific individual distance estimate was used to obtain a distance correction factor mi that accounts for its tendency to yield higher or lower distances than others from its population sample. The baseline population-specific distances D for 1800s and 1933 Lund were estimated as the median pairwise distance among individuals with no obvious IBD based on the Hamming distance matrix. Pairwise distances were compared to this baseline, scaled with respect to the Lyon outgroup distance, as follows.We then sought to account for genome-specific differences in genetic distances, such as due to sample quality. For each chromosome window of the i,j collected in the same location and time point, the chromosome window Hamming distance is d, while mi, mj respectively are the scaling factors specific to sequences i,j\u2014calculated from di,dj divided by the median pairwise distance between all of the Lund sequences (of a given time point) and the Lyon sequences.For 2 genomes D\u2019 = dmimj, a chromosome window is considered to be IBD between 2 diploid sequences  if D\u2019 < (7/8)D, i.e., closer to the predicted IBD distance of (3/4)D than to the outbred background distance D. The reasoning is that if 1 allele from each individual is IBD, then \u00bc of pairwise allelic comparisons will yield zero distance due to this IBD, and hence, the overall expected distance is \u00be of that from a non-IBD pair. For haploid to haploid , the same rule with a factor of \u00bd is applied, while for haploid to diploid  a factor of \u00be is used.For the rescaled distance When pairwise IBD regions are identified, this window is masked in 1 genome of the pair. In a pairing of a low-quality and high-quality genomes, the masking is prioritized so that low-quality genomes are preferentially masked in order to minimize data loss.To avoid false negatives due to IBD regions covering partial windows, when a window was masked, the proximate half of the \u201cupstream\u201d and the \u201cdownstream\u201d window was also masked. While this practice may mask some non-IBD regions, it errs on the conservative side for avoiding oversampling IBD alleles .\u03c0). Therefore, to identify enrichment of homozygosity in inbred genomes, we compared the observed heterozygosity per window to the median pairwise distance among non-IBD pairs from the same population in that window. If the within-window heterozygosity of a sample was less than \u03c0/2, the window was flagged as IBD and a randomly selected allele of the 2 in the window was masked.In addition to IBD among genomes, there was also evidence for depleted heterozygosity as a consequence of inbreeding in a subset of genomes from both the 1800s and  the 1933 Lund populations, i.e., intragenomic IBD. In non-inbred genomes, the heterozygosity should approximately equal the population nucleotide diversity  in the IBD analyses, because the latter showed low divergence when compared to some of the 1800s Lund samples. There was unambiguous evidence of close relatedness between 18SL6 and 18SL10, suggesting that the former was in fact from Lund and thus could be grouped with this population in subsequent analyses. In contrast, 18DZ5 showed roughly 1% of its genome with apparent IBD with 1 Lund genome, and lesser levels with 2 others. As these could be false positives due to conservative filtering criteria, we continued to treat this sample as being from Zealand rather than Lund in demographic analyses based on location, while at the same time masking that small region of the genome (18SL12) that showed low, IBD-like divergence with respect to 18DZ5.Drosophila, and only considered polymorphic sites without missing data in any of the analyzed samples).Temporal and spatial divergence among different populations (defined by time and location) was assessed with analyses of population genetic distances. Our analysis focused on X chromosome sequences (to the exclusion of low recombination regions defined by  because Dxy) and FST [To avoid distortions due to sample sizes, the number of alleles taken from each population  was downsampled to match the 4 alleles from the IBD-masked 1800s Lund allele counts. This entailed selecting 4 random independently sampled chromosomes at each site for the 1933 Lund population, the 2010s Lund pool-seq population, and the other modern populations, sampling independently at each analyzed site. This downsampled data was used to calculate nucleotide diversity within each population, as well as between-population genetic distance . For each of the 25,094 polymorphic sites with no missing data across all 35 samples in the analyzed regions of the X, the frequency of the minor allele at each site was calculated. PCA was then used to compute the 35 eigenvectors of the correlation matrix.In(2L)t, In(2R)Ns, In(3L)P, In(3R)K, In(3R)Mo, and In(3R)P. The frequency of each inversion was estimated from the median frequency of the inversion-associated alleles among their respective loci. For the Lund pool-seq data, the requirement of more than 4 minor alleles per site to call minor alleles was relaxed to identify low-frequency inversions, i.e., even singleton minor alleles were included.To assay for inversions, we used the list of inversion-linked SNP alleles identified by ,96 for tIn(3R)P, 4 of the 20 inversion-linked alleles were present at low frequency in the modern Lund pool-seq data), while the absence of inversion-linked alleles at the majority of sites suggested that the inversion itself is absent. In such cases, the use of mean allele frequency as a proxy for inversions would result in a nonzero estimated inversion frequency that is much lower than the reciprocal of the number of chromosomes sampled in the population. The estimated frequencies for each inversion in modern Lund versus the historical populations were compared with Fisher exact tests on karyotype counts, using R.We used the median rather than the mean allele frequency because some alleles may be absent due to missing data. Additionally, not all of the alleles identified previously  as inverNe) for the 1809 to 1933 and 1933 to 2015 intervals by implementing a simple drift-only neutral model of evolution . To estimate Ne for these 2 time intervals, we compared the mean changes in allele frequencies across observed segregating sites to changes in allele frequencies simulated under a Fisher\u2013Wright (FW) model of random genetic drift in discrete, non-overlapping generations. Specifically, we implemented a Bayesian approach to optimize Ne that minimizes the difference between observed Using allele frequency data from 1800s, 1933, and 2010s Lund, we estimated local effective population size  at the ith locus were used to approximate the initial allele frequencies fi in the population using Bayesian estimation. The prior of fi was based on a mutation-drift equilibrium with a site mutation rate of 5.21 \u00d7 10\u221210 per generation [N*. The posterior probabilities of fi were calculated using Bayes\u2019 Theorem from the prior probability distribution and the observed frequencies at each site. The initial allele frequencies in each simulation replicate were generated by drawing n alleles equal to the number in each sample. Using a uniform prior instead of mutation-drift equilibrium distributions resulted in very similar posterior distributions of fi.We initialized the simulations by calculating the allele frequencies at polymorphic loci at each time point in the Lund populations. Regions of low recombination, as defined by  were excneration  and a cafi(0) at simulation start time, representing 1809 and 1933 for the first and second time intervals, respectively. The frequencies in generation t+1 were generated by drawing a binomial sample ~ bin(fi(t), N*) at each site, where fi = xi/N for xi count of what was the minor allele at t = 0. The model assumes a sufficiently large gene pool for sampling with replacement and treating each locus as statistically independent . This sampling is repeated for 1860 (non-overlapping) generations for 1809 to 1933 and 1230 generations for 1933 to 2015, i.e., for 15 generations per year [n = 14 (n = 9 for ChrX) was drawn to represent the 1933 Lund sample, giving terminal sample allele frequencies fi*(T) based on ~ bin(fi(T), n).Population allele frequencies were initialized to per year ,17. In tNe for the 1933 to 2015 time interval, with an important exception. Because the 2010s Lund samples contributed pool-seq data, the additional sample variance that is introduced by sampling of both individuals and reads must be incorporated into the simulation. In the terminal (census) generation, the simulations implemented a two-step sampling model consistent with the nature of pool-seq data. Namely, we sampled 240 alleles (corresponding to the 120 flies) in the census generation ~ bin(fi(T), 240). This created a pool of 240 alleles where the frequency of the  minor allele frequency was fi\u2019(T), with an expectation equal to fi(T). To simulate the final sample set, we drew n alleles from this pool-seq subset of individuals , i.e., ~ bin(fi\u2019(T), n). This final draw gave terminal sample allele frequencies fi*(T).Much the same method was used for estimating the best fit ip = |fi*(T)\u2013pi(0)| were averaged across polymorphic sites, giving Ne by running the FW simulations over 1,000 replicates over a range of N* = {1,000\u202620,000} in increments of 1,000 and selecting the value minimizing the difference between simulated and observed N* in 5 increments of 100 on either side of the coarse-grained optimum.The absolute differences between final and initial sample allele frequencies in the simulations \u0394N1, N2) for the time intervals 1809 to 1933 and 1933 to 2015 by comparing \u03c0 observed in the 1800s, 1933, and 2010s Lund samples to that of simulated populations of sizes N1, N2 at these time points. The simulations were initiated by sampling N1 alleles from a source European population based on the inferred demographic history of D. melanogaster [N1 Lund sample then experienced genetic drift  for 1860 generations  until 1933, at which point the population size was set to N2 and the simulations continued for a further 1230 generations. This neutral demographic model was implemented using the program ms [\u22128, a gene conversion rate of 6.25 \u00d7 10\u22128, and mean gene conversion tract length of 518 bp [We evaluated the accuracy our estimates of effective population size  between each pair of populations. Here as in other studies, window FST was calculated by summing the numerator terms and summing the denominator terms of the FST estimator [These statistics are based on log-transformed window stimator , and obtGiven that a viable demographic model was not available to provide a distribution of PBE values under a null hypothesis, outlier PBE values were defined based on the upper 1% quantile of the window distribution (which contained 240 windows genome wide). Because strong selection can create linkage disequilibrium that extends across multiple windows, we aggregated adjacent or near-adjacent PBE-significant windows (separated by no more than 2 non-outlier windows). Although SNP-level genetic differentiation can sometimes allow the detection of soft sweeps that do not register as outlier in window-level scans, our sample sizes of museum genomes were not sufficient to enable this approach . Insteadp-values were computed from 10,000 random permutations, based on the proportion of replicates with an equal or greater number of outlier regions associated with a given GO category as observed in the empirical data.We performed a GO enrichment analysis on genes located PBE outlier regions from the 1800s- and 1900s-focused genome scans in order to establish whether certain functional categories  were disproportionately overrepresented among genes that may differ between the historical and modern populations. The GO analysis methods followed the approach of , which iS1 TableAvailable collection information is given for each museum specimen. All early 1800s Sweden flies (including the 1 labeled Sm\u00e5land) were ultimately inferred to have come from the same collection event. Sequencing statistics including read count and lengths, depth of coverage, genomic coverage, singleton rate, and proportion of singletons with A or T are also given. Sample 19SL2 was excluded from most analyses because of its low sequencing depth and genomic coverage. The sex of each specimen was inferred from the relative levels of sequencing depth on the X chromosome and the autosomes. Genomic characteristics of a representative set of genomes from modern material ) are provided, as depicted in (XLSX)Click here for additional data file.S2 TableTab A. Mean pairwise genetic distance between X chromosomes from museum and modern genomes . Unusually low values are highlighted. Mean distance against a panel of modern genomes is indicated at the bottom. No genomes were found to have anomolously high distances to all others, suggesting that they all reflect D. melanogaster specimens. Tab B. Proportion of each genome masked due to pairwise IBD with other genomes, given as a fraction of the total genome and each chromosome arm. Tab C. Proportion of each genome masked due to inbreeding IBD, as indicated by runs of minimal heterozygosity, for each full genome and each chromosome arm.(XLSX)Click here for additional data file.S3 TableRelease 5 positions are given in separate tabs for each chromosome arm.(XLSX)Click here for additional data file.S4 TableRelease 5 positions are given in separate tabs for each chromosome arm.(XLSX)Click here for additional data file.S5 TableLoadings for each genome are given for all 35 principle components. The X chromosome was analyzed to avoid the influence of autosomal inversions. Up to 5 apparently unrelated individuals were included per population sample.(XLSX)Click here for additional data file.S6 TableInversion frequency estimates and results from each SNP reported to be associated with each inversion (by Kapun and colleagues (2016)) are given in separate tabs. Estimation of inversion frequency was based on the median of SNP frequencies, in order to avoid spurious nonzero estimates.(XLSX)Click here for additional data file.S7 TableTop: Mean frequency differences and haploid effective population size estimates from drift-only models for each Lund time interval. Bottom: Comparison of nucleotide diversity from simulations (using estimated Ne) versus observed data, for arm 3L.(XLSX)Click here for additional data file.S8 TableTab A. For the 1800s time interval , PBE outlier regions based on the upper 1% quantile and overlapping genes are given. Tab B. For the 1900s time interval , PBE outlier regions based on the upper 1% quantile and overlapping genes are given. Tab C. For the 1800s time interval, PBS and PBE for all genomic windows are given. In addition to window values, the maximum SNP PBE and PBS are given. Nucleotide diversity, Dxy, and FST values are provided as well. Tab D. For the 1900s time interval, PBS and PBE for all genomic windows are given. In addition to window values, the maximum SNP PBE and PBS are given. Nucleotide diversity, Dxy, and FST values are provided as well.(XLSX)Click here for additional data file.S9 TableTab A. Gene ontology enrichment for the 1800s-focused PBE outliers. Tab B. Gene ontology enrichment for the 1900s-focused PBE outliers. For each GO category, the total number of genes in the analyzed regions is given, along with the number of outlier regions associated with these genes, and the identities of these outlier-overlapping genes. A permutation P-value is also given for each GO category, which does not attempt to correct for the number of GO categories tested.(XLSX)Click here for additional data file."}
+{"text": "Worldwide, intracranial aneurysms are associated with a high mortality rate. While endovascular management has proven to be the choice of treatment in selected patients, patient demographics and aneurysm characteristics differ between study populations.This study aimed to investigate the profile of patients with intracranial aneurysms who underwent endovascular management in the Interventional Neuroradiology Unit at Chris Hani Baragwanath Academic Hospital. Patient demographics, risk factors, indications, aneurysm characteristics and intra-operative complications were studied.This was a 3-year retrospective study of all adult patients between 01 January 2018 and 31 January 2021. The Chi-square test was used to compare categorical variables.p = 0.020), neck size dimensions less than 4 mm (p = 0.010), and aneurysms located in the internal cerebral artery (ICA) circulation (p = 0.001).A total of 77 patients were included in this study. The mean age of the patients was 47 \u00b1 11.6 with a male-to-female ratio of 1:1.8. Hypertension was the most reported risk factor in 27% of patients. There was no statistical correlation between the gender groups according to presentation, multiplicity, aneurysmal size dimensions and locations. According to the presentation, there was statistical significance in ruptured intracranial aneurysms (The study findings support known parameters including females and anterior circulation aneurysm preponderance, and the low complication risk of endovascular management. Interestingly, intracranial aneurysms presented with rupture at smaller size dimensions.This study provides valuable insights into intracranial aneurysm characteristics and endovascular management efficacy in a resource-limited setting. Several risk factors have been linked to the development of intracranial aneurysms such as female predilection, hypertension, smoking, high alcohol consumption, familial or genetic syndromes and certain aneurysmal morphological features.4 These risk factors are not universal as there remains variation in the literature across demographics. Intracranial aneurysms in pregnancy are reported to occur in approximately 1.8% of women, with rupture occurring in the range of 1\u201310 per 100 000 pregnancies.11 Furthermore, human immunodeficiency virus (HIV) infection remains a leading cause of mortality and morbidity in South Africa.8 In a local study, HIV-associated intracranial aneurysms were shown to occur in patients at a younger age and often with complex morphological features.12 The prevalence of multiple intracranial aneurysms is 17% \u2013 33.4% in patients suffering from subarachnoid haemorrhage.13 Multiple aneurysms as a risk factor are, however, not yet well understood. A South African audit found that patients with multiple aneurysms had a predominance for patients older than 40 years.14According to the World Health Organization (WHO), non-communicable diseases (NCDs) are the leading cause of death globally, with low- and middle-income countries carrying the highest burden,15 Intracranial aneurysms are reported to have an 85% anterior circulation predominance.5 In an African study, variations in the location of aneurysms differed in population groups.16 Aneurysm size categorisation into small, medium and large differs in the literature and is also controversial in the decision to treat; several references refer to an aneurysm size of 7 mm as one of the criteria for treatment.19 International literature recommends intervention only for aneurysms greater than 7 mm or aneurysms in the posterior circulation; however, in a South African study, it was found that ruptured aneurysms occurred with aneurysm sizes less than 7 mm.18 The mean aneurysm size dimensions also appeared to rupture at smaller sizes in the HIV-infected population.12Intracranial aneurysm rupture depends on certain characteristics such as size, location and shape.20 Endovascular coiling of intracranial aneurysms has been shown to significantly improve outcomes compared with surgical clipping.22 In a recent meta-analysis, surgical clipping attracted a poorer outcome compared with endovascular management; no significant difference was observed in mortality and re-bleeding.23 Although endovascular management is becoming more widely adopted as the first-line management of ruptured and unruptured aneurysms, the scope and implementation in Africa are still considered suboptimal.25 In a South African-based study, a 16% reduction in mortality and major morbidity was achieved through endovascular management.26Endovascular management of intracranial aneurysms is an evolving field with new technological advancements.27 Thrombo-embolic events and intra-operative rupture are the most common complications experienced with endovascular coiling of intracranial aneurysms.28 In a recent multicentre cohort, thrombo-embolic events occurred more frequently, with a female and middle cerebral artery (MCA) predominance. In addition, it was found that aneurysms that are small and located in the anterior cerebral and communicating arteries were more frequently associated with intraoperative rupture.28Endovascular coiling was also shown to be a more durable treatment method compared with surgical clipping in terms of patients\u2019 long-term outcomes.This study aimed at assessing the profile of patients with intracranial aneurysms who underwent endovascular management at Chris Hani Baragwanath Academic Hospital (CHBAH) in Soweto, South Africa, investigating the patient demographics, risk factors, aneurysm characteristics and intraoperative complications.This was a retrospective study of patients who underwent endovascular management of ruptured and unruptured intracranial aneurysms in the Interventional Neuroradiology Unit at CHBAH over a period of 3 years, from 01 January 2018 to 31 January 2021. The data were recorded on an electronic data recording sheet, and included patient demographics (age and gender), risk factors , presentation on admission (ruptured or unruptured), aneurysm location , aneurysm size (neck and maximum diameter dimensions), aneurysm multiplicity (single or multiple), and intraoperative complications (aneurysm perforation and thromboembolic event). Aneurysm size definitions were adopted from Pierot et al. where maximum diameter (dome width) was dichotomised into < 5 mm and \u2265 5 mm and a neck size \u2265 4 mm was defined as a wide-neck aneurysm, rendering a narrow-neck aneurysm as < 4 mm.p-value < 0.05 was used to determine statistical significance by utilising the Chi-square test.All patients 19 years of age or older who underwent endovascular management for index ruptured or unruptured intracranial aneurysms with complete records (images and angiogram reports) were included. Angiogram reports and accompanying images were obtained from the Picture Archiving and Communication System (PACS) in the Radiology Department at CHBAH. The principal investigator was primarily responsible for data collection and data analysis supported by a biostatistician. A The study was approved by the Human Research Ethics Committee of the University of the Witwatersrand (certificate number M211038). Participants\u2019 consent was not sought as this was a retrospective record review and to maintain strict anonymity, no personally identifiable information was recorded.p = 0.217). The angiogram reports yielded 21 (27%) patients with documented hypertension, one patient with hypercholesterolaemia (1%), and two (4%) patients from the female group who presented with ruptured intracranial aneurysms during pregnancy.A final sample of 77 patients who underwent endovascular management for intracranial aneurysms were included. A total of 49 were female and 28 were male patients  with a mean age of 47 years (Sixty six patients (87%) presented with ruptured intracranial aneurysms. One patient\u2019s mode of presentation was unspecified on the angiogram report and there was no supporting imaging on PACS as the patient was referred from a regional hospital; therefore, the patient was excluded from the statistical analysis according to presentation. A total of 89 aneurysms were detected among the 77 patients. The majority of patients had a single aneurysm (88%), while nine (12%) presented with multiple aneurysms. Most intracranial aneurysms were narrow neck aneurysms (83%). Similarly, the maximum diameter dimensions were less than 5 mm in 83%. There was no statistical significance in presentation, multiplicity, neck and maximum diameter dimensions regardless of gender .p = 0.01)  constituted a significant proportion (83%) of aneurysms on presentation ( = 0.01) .In total, AComA (22%) aneurysms were the most common. Basilar tip aneurysms (2%) accounted for the least common location. Males were mostly affected by ICA (26%) and AComA (24%) aneurysms. Anterior cerebral artery (22%) and AComA (22%) aneurysm locations were equally distributed among females. No statistical significance was demonstrated in intracranial aneurysm location according to gender .p = 0.02) and of these, AComA aneurysms were the most common (23%). All patients who underwent intervention for AComA aneurysms were ruptured on presentation; however, there was no statistical significance according to location (p = 0.064). The ICA aneurysms demonstrated statistical significance (p = 0.001) according to location (In total, 88% of the study population presented with ruptured intracranial aneurysms (location .Referring to 25 There was poor yield of patient risk factors in the angiogram reports. Hypertension was the most reported risk factor consistent with the literature.15 Intracranial aneurysms in pregnancy are rare and it is postulated that normal haemodynamic changes during pregnancy may play a role in the risk for complications in patients harbouring intracranial aneurysms.11 Although not included in this study as a risk factor, HIV-associated aneurysms have been shown to present in younger patients and at smaller sizes.12Consistent with local and international studies, this South African-based population study of patients who underwent endovascular management for intracranial aneurysms demonstrated a female predominance with a male: female ratio of 1:1.8.26 This study population demonstrated an AComA predominance, regardless of gender or presentation. Internal cerebral artery aneurysms demonstrated significance according to presentation and had a near equal distribution between the ruptured and unruptured groups.There is a high population variance regarding intracranial aneurysm location in the literature, some with a posterior circulation predominance and some with an anterior predominance.2 A patient presenting with an unruptured aneurysm may be asymptomatic or present with isolated cranial nerve palsies or with non-specific symptoms such as headaches. There was a statistical difference between patients presenting with ruptured intracranial aneurysms on presentation in this study. This is consistent with the literature where it has been shown in a systematic review and meta-analysis that the prevalence of unruptured intracranial aneurysms ranged between 0% and 41.8% with an overall mean prevalence of 2.8% between studies.29Patients with ruptured aneurysms often present with subarachnoid haemorrhage on initial diagnostic imaging.19 This study demonstrated that most intracranial aneurysms presenting with rupture were less than 4 mm and less than 5 mm for aneurysmal neck and maximum diameter size dimensions, respectively. Although there are known independent risk factors increasing the risk of developing intracranial aneurysms, because of the poor reporting yield in angiogram reports in this study, comparisons could not be sought.According to the literature, larger aneurysms are an independent risk factor for the risk to rupture. Although the literature is not clear as to the exact dimensions, some studies have reported maximum aneurysm diameters of 6 mm \u2013 7 mm as a guide for the decision to treat.24 The two most common complications in endovascular neuro-intervention are perioperative intracranial aneurysm perforation and acute thromboembolic phenomenon. This study population had a 4% complication rate that correlates with the literature documenting 2% \u2013 8%, while surgical clipping complications can range between 15% and 50%.28 In this study, endovascular intervention for intracranial aneurysms showed a low complication risk and was managed actively to minimise postoperative long-term complications.Endovascular neuro-intervention has been shown to reduce morbidity and, in some studies mortality, compared to surgical clipping.This was a retrospective, single institutional study with a small sample size limiting comparison of the location, aneurysm morphology and larger-sized aneurysm subcategories. In addition, angiogram reports did not include comprehensive documentation of patient risk factors and were primarily dependent on information provided on the patient\u2019s request forms for endovascular management. Finally, this study did not investigate long-term clinical outcomes on subsequent follow-up cerebral angiography.Overall, this study supports known parameters including a female and anterior circulation aneurysm preponderance, and the low complication risk of endovascular management. Interestingly, intracranial aneurysms presented with rupture at smaller size dimensions. Multi-institutional prospective studies are recommended to further investigate aneurysm morphology and risk factor stratification. The findings may influence the decision threshold to treat smaller-sized intracranial aneurysm dimensions and anterior circulation aneurysms in our population."
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