Brassica oleracea var. botrytis) by‐products  (CBP) were successfully utilized in muffins as a model system and their feasibility of incorporation was investigated. CBP powder‐based muffin formulations were made by the progressive replacement of wheat flour (WF) with 10%, 20%, and 30% of CBP. The physicochemical, pasting properties, antioxidant potential, textural characteristics, and sensorial attributes were analyzed. Substitution of CBP significantly (p < .05) resulted in an upsurge in crude protein, crude fiber, minerals, total phenolics, and total flavonoid contents, as well as total antioxidant activity values of muffins. The pasting properties were influenced by monitoring an increase in peak, breakdown, final, and setback viscosities. Although the addition of an increasing amount of CBP improved the nutritional characteristics, however, the increased level of replacement (>10%) had significant adverse effects on baking and physical characteristics. The specific loaf volume of the developed muffins decreased the crumb color which became darker, and enriched muffins were hardened in texture. Furthermore, sensory evaluation confirmed the positive effects of CBP incorporation only up to 10%. Overall, present results highlighted that supplementation of wheat muffins with 10% CBP is a beneficial approach to enrich them with nutrients and intensify their antioxidant potential.Cauliflower  were successfully utilized in muffins as a model system and their feasibility of incorporation was investigated. Cauliflower by‐products powder (CBP) based muffin formulations were made by the progressive replacement of wheat flour (WF) that resulted supplementation of wheat muffins with 10% CBP is a beneficial approach to enrich them with nutrients and intensify their antioxidant potential. However, more than 1.3 billion tons of food produced annually ends up in the trash and results in an economic squander of $750 billion. Unfortunately, almost 815 million people in the world are suffering from hunger Boliko, . ConsumpIt has been underlined in various studies that inexpensive and intensively available underutilized by‐products of cauliflower are an exceptional source of proteins, vitamins, minerals, ascorbic acid, carotenoids, antioxidants, and dietary fibers  by‐products, including leaves, stems, and stalks were obtained from the vegetable processing industry in Pakistan.The current research was conducted at the National Institute of Food Science and Technology (NIFSAT), University of Agriculture Faisalabad (UAF), Pakistan. For the investigation, commercial whole wheat flour , vegetable oil , castor sugar , low‐fat milk , baking powder , and whole eggs used in this study were purchased from the local markets in Faisalabad, Punjab, Pakistan. Cauliflower  at 60°C for 16 h till moisture remained at 6%–8%. Then, these by‐products were crushed to a fine powder with a laboratory grinder (Westpoint WF‐7701) and passed through 20 mesh sieves. Afterward, a homogenous mixture of all three by‐products was made by mixing them into equal proportions by weight and packed in airtight containers at ambient temperature till further use.2.3The formulation presented in Table 2.42SO4 by using 5 g of digestion mixture (K2SO4:FeSO4:CuSO4 in 100:5:10 ratio) for 3–4 h until the color was light greenish or transparent. Next, the dilution of the digested material was done in a 250‐mL volumetric flask using distilled water until no residue was left. Then 10 mL of it were taken and 25 mL of alkali, that is, 40% NaOH were added to the distillation apparatus to get a nitrogen‐based sample in the form of NH4OH. Ammonia was liberated and collected in a beaker containing 10 mL of 4% boric acid solution using methyl red as an indicator. The resulting ammonium borate was used for nitrogen determination. Nitrogen was estimated by titrating distillate in the receiver flask against 0.1 N H2SO4 until the light pink color was obtained. Then crude protein was estimated by multiplying nitrogen % with factor 5.7 using the equations Proximate analysis parameters including moisture, fat, protein, fiber, ash, and carbohydrates were analyzed in wheat flour, CBP, and all muffin treatments, using standard methods AACC, . The moin‐hexane, as a solvent, was added to 3 g of dried samples in a flask and connected to the apparatus. Then, tap water circulation was started in the condenser and heat was provided to receiving flask for 14 h. After 6–7 siphons, samples were dried in the hot air oven at 105°C for 1 h and then cooled in desiccators by sequential acid and alkali hydrolysis, and crude fat percentage was determined. Crude fiber content using the Fibertech apparatus  was estimated by digestion of fat‐free sample first in 1.25% H2SO4 and then in 1.25% NaOH solution by the AACC method no. 32‐10 , by following AACC  method n10 AACC, . Additio3:HCLO4 in 7:3 over a hot plate and after digestion samples were diluted with distilled water to make a total volume of 250 mL. Samples were then run on atomic absorption spectrophotometer for determination of the respective minerals.Mineral elements including calcium (Ca), potassium (K), iron (Fe), copper (Cu), magnesium (Mg), and zinc (Zn) were determined by atomic absorption spectroscopy  by using standard protocols AACC, . For thi2.5Rheological properties of dough were determined according to general pasting method no. 76‐21.02 AACC, . Viscogr2.6The total calorific value of subsequent muffins was calculated using the Atwater factor by employing equation 2.7Extracts of cauliflower waste , and prepared muffins, were made by following the protocol of Ansary et al. . The tot2.8For the specific loaf volume of muffins, volume was determined using the rapeseed displacement method mentioned in AACC  and weigL* a* b* (CIELAB) was used to evaluate the color. Colorimeter generally gives three values including the L* value (lightness) that varies from 0 to 100 (corresponding to absolute black and absolute white respectively), a* value that is represented by −a (green at one extremity) to +a (red at the other extremity), and b* value that varies from –b  to +b . From a theoretical perspective, no extreme values exist for a* and b*, however, practically these are frequently numbered from −60 to +60. The total color difference (∆E) was calculated using equation L0*,a0*, and b0* are the color coordinates for the reference sample. A large total color difference corresponds to a greater color change from the reference sample. ∆E values in the range 0.0–2.0 correspond to unrecognizable differences, ∆E values within the interval 2.0–3.5 indicate differences possible to be recognized by an experienced observer, while ∆E values greater than 3.5 specify clear differences in the color of the samples under comparison . Chromatic space of Cartesian coordinates 2.9Sensory evaluation was conducted by 10 trained and 10 untrained panelists recruited (convenient consent‐based participant sampling) from the staff and students at the National Institute of Food Science and Technology, University of Agriculture Faisalabad. Muffins were evaluated based on their appearance, taste, texture, flavor, softness, palatability, color, and overall acceptability on a 9‐point hedonic scale . The control muffin was presented simultaneously with the rest of the muffin samples and was evaluated in random order. Moreover, water was provided to panelists to minimize any residual effects before testing a new sample.2.10p < .05.The experimental data were analyzed using “Statistics 8.1 Statistical Software.” To determine significant differences, ANOVA  with Tukey's multiple comparison test was performed. The level of significance was set at 33.1The proximate, nutritional, and physical properties of wheat flour (WF) and CBP powder are given in Table 3.2p < .05) increase in pasting properties with the incorporation of CBP  from 56.24% to 53.46%, and this was a direct consequence of increasing CBP and decreasing WF concentration in muffins. Ribeiro et al.  increases were obvious in the mineral contents of muffins, which were proportional to increases in the concentration of CBP added to muffin formulations  decreased. Calories in control were 134 kcal, while CBP30 had the lower calories equal to 106 kcal. The high fiber content in low‐caloric muffins with more CBP indicates their high amylose content as compared to control muffins. Amylose gelatinizes at elevated temperatures, so complete gelatinization of amylose is restricted during baking. This results in a more compact structure of muffins after cooling and the resistant starch contributes to a reduction in calories of muffins. Incorporated muffins, thus, will have higher satiety than control muffins due to the impact of CBP  higher total phenolic content, total flavonoid content, and antioxidant activity values than the control wheat muffins  the increase in the hardness value of muffins. Control had a hardness value of 442.3 g and the highest value was 717.2 g for CBP30. Higher hardness values for CBP muffins could also be associated with the gel‐forming properties of CBP . Chewiness and hardness exhibited a similar increasing trend.Texture analysis directly influences consumer acceptability of the developed food products. The texture parameters of the muffins are presented in Table p < .05) were obtained. Results indicated that Hunter L* value ranged from 55 to 74, the a* value ranged from 3.7 in control to −8.4 in CBP30, and the b* values were between 21 and 46. Likewise, Hunter colorimeter analysis was also performed for CBP powder, and results were recorded as L* = 38.29, a* = −1.57, and b* = 11.51. Not surprisingly, the negative value of a* indicated a distinguished greenness. ∆E numerically showed the color difference between the control muffins and CBP muffin treatments. In this case, the ∆E parameter was lowest in CBP10, that is, 19.45, while significantly higher values were reported for CBP20 and CBP30. The darker color of all the CBP muffins, in contrast to the control muffins, was a consequence of a higher substitution of CBP and heating effects. The color of muffins during baking is dependent on the Maillard reaction, sugar caramelization, besides the color of the used ingredients . The poor sensory scores of CBP20 and CBP30 could be due to the higher hardness values and increased viscosity of their batters due to CBP addition. Although muffins with 20% and 30% addition of CBP received lower sensory scores, results of control muffins and CBP10 muffins ranged between 8 and 9 for all attributes except color, which is a good indication. For color, panelists gave similar ratings, given that the visual observation of humans is not highly sensitive as a colorimeter. All CBP‐added muffins were perceived as slightly dark in the sensory evaluation room and were visually seen as having similar hues. In contrast to present findings, Wani et al.  increase in protein, dietary fiber, Ca, K, Mg, Fe, Cu, Zn contents, total phenolic content, total flavonoid content, and total antioxidant activity values of muffins. Significant (p < .05) results were also obtained in color, texture, and other physical properties. A significant increase (p < .05) was observed in colorimeter values and hardness, while a significant (p < .05) decrease was noticed in specific loaf volume, chewiness, springiness, and cohesiveness scores with the addition of CBP. In terms of sensory evaluation scores, control and CBP10 had almost similar scores in terms of appearance, taste, aroma, texture, softness, palatability, and overall acceptability. Substantial contents of health‐promoting substances regard muffins as a novel confectionery product. Overall, muffins with 10% CBP powder were the most acceptable with good physicochemical, nutritional, and organoleptic attributes.In this study, CBP powder, which is recognized as a source of bioactive food components, was used in CBP‐enriched muffins production. This study promotes the preservation and utilization of cost‐effective cauliflower waste and probes its high antioxidant potential with special emphasis to develop nutrient‐rich muffins. The addition of cauliflower powder resulted in significant ; resources ; software ; writing – original draft ; writing – review and editing . Rizwan Shukat: Investigation ; methodology ; validation ; writing – original draft . Iahtisham Ul Haq: Conceptualization ; data curation ; software . Masood Sadiq Butt: Formal analysis ; investigation ; methodology ; validation . Gulzar Ahmad Nayik: Conceptualization ; funding acquisition ; resources ; writing – original draft ; writing – review and editing . Seema Ramniwas: Formal analysis ; investigation ; methodology ; validation . Sami Al Obaid: Funding acquisition ; methodology ; project administration ; resources ; supervision ; writing – original draft . Sulaiman Ali Alharbi: Formal analysis ; funding acquisition ; investigation ; methodology ; supervision . Mohammad Javed Ansari: Formal analysis ; funding acquisition ; software ; writing – original draft . Ioannis K. Karabagias: Data curation ; resources ; validation . Nazmul Sarwar: Conceptualization ; data curation ; validation ; writing – original draft .The authors declare that they have no conflict of interest regarding the publication of this article.


Large hepatocellular carcinoma (LHCC) is prone to short‐term recurrence and poor long‐term survival after hepatectomy, and there is still a lack of effective neoadjuvant treatments to improve recurrence‐free survival (RFS) and overall survival (OS). We retrospectively analyzed the efficacy of preoperative transcatheter arterial chemoembolization (TACE) in solitary LHCC (≥5 cm).A multicenter medical database was used to analyze preoperative TACE's effects on RFS, OS, and perioperative complications in patients with solitary LHCC who received surgical treatment from January 2005 to December 2015. The patients were divided into Group A (5.0–9.9 cm) and Group B (≥10 cm), with 10 cm as the critical value, and the effect of preoperative TACE on RFS, OS and perioperative complications was assessed in each subgroup.In the overall population, patients with preoperative TACE had better RFS and OS than those without preoperative TACE. However, after stratifying the patients into the two HCC groups, preoperative TACE only improved the survival outcomes of patients with Group B (≥10 cm). Multivariate Cox‐regression analysis showed that lack of preoperative TACE was an independent risk factor for RFS and OS in the overall population and in Group B but not in Group A.Preoperative TACE is beneficial for patients with solitary HCC (≥10 cm). First, preoperative imaging evaluation of HCC patients was performed to evaluate tumor size and location.Second, whether or not a patient should receive TACE before surgical resection is determined according to tumor diameter.Finally, with the assistance of interventional equipment, the blood vessels of nutritional tumors are identified and embolized. Inclusion criteria (1) a solitary HCC with tumor diameter ≥5 cm; (2) a postoperative pathological diagnosis of HCC; (3) no extrahepatic metastasis; (4) no radiologic evidence of invasion into the major portal/hepatic vein branches; (5) radical resection of HCC (R0), that is, no residual tumor tissue under direct observation or microscopy; (6) no previous treatment of HCC. Exclusion criteria: (1) younger than 18 years old; (2) poor liver function with Pugh‐Child Class C; (3) missing prognosis and follow‐up information. The ethics committees of the six medical centers approved the study, and the study complied with the Helsinki Declaration and local laws.2.2All patients underwent contrast‐enhanced computed tomography (CT), magnetic resonance imaging (MRI), or chest X‐ray scanning upon admission to the hospital. Preoperative Information on baseline patient characteristics includes age, sex, diabetes mellitus, etiology of liver diseases, cirrhosis, Child‐Pugh grade, platelets count, international normalized ratio (INR), alpha‐fetoprotein (AFP) level, the presence or absence of postoperative adjuvant TACE, maximum tumor size, MVI, satellite nodules, tumor differentiation, and tumor capsule. Continuous variables, such as age, are transformed into binary variables according to recognized cut‐off values or upper and lower lines of normal values.2.32) or oxaliplatin (100 mg/m2) was injected into the proper hepatic artery, and embolization was performed using different embolization materials. The embolization materials used were iodized oil and gelatin sponge cubes, or iodized oil only, which was entirely mixed with these chemotherapeutic drugs as an emulsion and injected. Because TACE was performed at different hospitals, embolization materials varied. Patients were asked to return to the hospital 4–8 weeks after embolization for follow‐up investigations, including routine blood tests, liver, and kidney function, coagulation function, AFP, and imaging. Imaging included abdominal enhanced CT, MRI, or chest X‐ray scans. The above procedures were performed by highly qualified attending physicians who received relevant interventional medicine training.Considering that this was a retrospective study, the decision to use TACE prior to surgery was left to the discretion of the treating surgeon and the patient at that time. The patient was placed supine, locally disinfected, draped, and given local anesthetized. The puncture site was chosen to be 2 cm below the inguinal ligament, and the catheter sheath was placed into the femoral artery using the Seldinger technique. First, the DSA technique helps with abdominal trunk and standard hepatic artery angiography to determine the tumor's location, size, and condition of the tumor. Once the tumor is understood, the catheter sheath is advanced deeper into the left or right hepatic artery or the vessel that feeds the tumor, 5‐fluorouracil , and OS. Postoperative liver failure (PLF) was defined as serum TBIL >50 μoml/L and prothrombin activity (PTA) <50% on day 5 after hepatectomy,2.6n) or percentages of patients (%). Continuous variables were compared by the Student's t‐test or Mann–Whitney U‐test. Categorical variables were compared by the χ2 test or Fisher's exact test. The survival curves of RFS and OS of patients who received or did not receive TACE before surgery were generated by the Kaplan–Meier method, and the log‐rank test was used to compare the differences. The Cox proportional hazard regression analyses were used to adjust for other prognostic factors associated with RFS and OS. All statistical analyses and visualizations of this study were obtained by R version 3.6.1 with the SVA. A p value <0.05 was considered statistically significant.Continuous variables were expressed as median (range) or mean ± standard deviation (SD), categorical variables were reported as number . The baseline characteristics, clinicopathological features, and postoperative complications of each subgroup are listed in Table Out of the 556 HCC patients, 150 27.0%) were treated with preoperative TACE and 406 (73.0%) were not treated with preoperative TACE. For patients who only had one TACE session, the median interval between the TACE and surgery was 5 weeks (range 4–8), and for patients who had multiple preoperative TACE sessions, the median interval between the last TACE and surgery was 4 weeks (range 3–6). There were no significant differences in age, sex, cirrhosis, tumor diameter, Child‐Pugh classification, MVI, pathological grade, postoperative complications, and other variables between the two groups (7.0% were3.2p < 0.05) and recurrence  rates of patients undergoing surgical resection with TACE were lower than those without TACE, showing statistical differences. The median OS and RFS of patients with TACE before surgery were 76 months and 37 months, respectively, longer than those without TACE     Figure . Then, w) Figure . However) Figure .3.3p = 0.006) and prolong the survival . In the study cohort of each subgroup, the univariate and multivariate analysis results were listed in Table p = 0.823) and OS . However, in the Group B, the result reported that preoperative TACE could improve OS .and RFS .Results of univariate and multivariate analyses for the entire study cohort's overall and recurrence‐free survivals are presented in Tables 3.4The comparison of clinicopathological features between Group A and Group B is shown in Table Of the 315 patients in the Group A, 239 (75.9%) were male and 76 (24.1%) were female; 306 patients (97.1%) had chronic HBV infection and two patients (0.6%) were positive for hepatitis C virus RNA.p = 0.211). The proportion of patients with AFP ≥400 μg/L , MVI  and satellite nodules  in Group B was higher than that in Group A, while other clinicopathological indicators were not significantly different.Among 241 patients with a maximum tumor diameter ≥10 cm (Group B), 180 (74.7%) patients were male, and 227 (94.2%) patients had chronic HBV infection. Patients who received preoperative TACE in the Group A less than those with preoperative TACE in the Group B (78/315 [24.8%] vs. 72/241 [29.9%]), but the difference was not statistically significant , and that patients with preoperative TACE had much larger tumor diameters than those without preoperative TACE.Some reports suggest that preoperative TACE may only be significant in patients with an excessively large tumor diameter,The results of this study showed that preoperative TACE benefited HCC patients and improved their RFS and OS in the overall population. However, after stratification, it was clear that the benefit was only significant for patients with tumor diameters ≥10 cm. In recent years, two studies on preoperative TACE from the same medical center, they just will be different in the setting of the inclusion criteria, and then the conclusion is different, and Zhou et al. included the inclusion criterion of tumor diameter ≥5 cm, which concluded that preoperative TACE had no effect on RFS and OS of patient.p < 0.05), reflecting that the tumor biological characteristics of Group B were more aggressive. In other words, preoperative TACE could effectively inhibit the growth and proliferation of highly invasive HCC. Toshiya et al. suggested that preoperative TACE may be more suitable for more aggressive tumor populations.We speculate that the possible reason for this stratification effect is that the corresponding tumor vascularization of Group B is more severe. Since the degree of tumor vascularization is positively correlated with the effectiveness of TACE, preoperative TACE could play a more critical role in Group B and ultimately benefit the survival of patients.In terms of perioperative complications and 30‐day mortality, previous studies have shown that preoperative TACE could increase the intraoperative difficulty and perioperative complications.The results of this study revealed that tumor diameter ≥10 cm, AFP ≥ 400 μg/L, MVI, Edmondson grade, PLT level, and satellite nodules were independent risk factors for postoperative OS and RFS, which were also confirmed by previous studies.Our research has limitations. First, our study was a multicenter retrospective study that did not have a uniform standard for preoperative TACE. Second, considering that this study is a multi‐center study, the embolic materials and chemotherapeutic drugs used in each center are different. Third, most of the people we include are infected with HBV, while the majority of HCC patients in western countries are caused by factors such as HCV or alcohol. This study may not be suitable for western populations.In conclusion, our study demonstrated that preoperative TACE is a safe neoadjuvant that does not increase perioperative complications and mortality. There was a stratification effect on the efficacy of preoperative TACE, and the beneficiary population is HCC patients with tumor diameter ≥10 cm. This study provides further guidance for the treatment of patients with large and huge solitary HCC to avoid unnecessary preoperative TACE.Ali Mo: Conceptualization . Qiao Zhang: Data curation . Feng Xia: Conceptualization ; data curation . Zhiyuan Huang: Resources ; software . Shasha Peng: Supervision ; validation . Wenjing Cao: Supervision ; validation . Hongliang Mei: Data curation ; software . Li Ren: Conceptualization ; resources . Yang Su: Investigation ; methodology . Hengyi Gao: Writing – original draft ; writing – review and editing . Weiqiang Chen: Resources ; software .The ethics committees of the six medical centers approved the study, and the study complied with the Helsinki Declaration and local laws.The patient provided informed consent, which was registered in the medical record.Appendix S1.Click here for additional data file.


Both cachexia and sarcopenia have been considered adverse predictors for prognosis in patients with pancreatic cancer; although sarcopenia and cachexia share some similarities, they are still defined as distinct nutritional conditions. We aimed to explore the differential impacts of sarcopenia and cachexia on prognosis for pancreatic ductal adenocarcinoma (PDAC) patients following radical excision.2/m2 (men) and 38.5 cm2/m2 (women). Cachexia was classified according to the following criteria: involuntary weight loss >5% over the past 6 months, or weight loss >2% and BMI <20 kg/m2, or weight loss >2% and sarcopenia.From January 2015 to May 2022, 614 patients undergoing surgery for PDAC were retrospectively included. Sarcopenia was defined as the L3 total skeletal muscle index below 52.4 cmOf the 614 patients included in the analysis, 62% and 48% were diagnosed with sarcopenia and cachexia, respectively. Kaplan–Meier analysis showed that sarcopenia and/or cachexia were significantly associated with worse overall survival (OS) rather than worse recurrence‐free survival (RFS). Moreover, Cox regression analysis revealed that cachexia rather than sarcopenia was an adverse factor for OS in all PDAC patients. For poorly differentiated PDAC, both cachexia and sarcopenia were significantly associated with shorter OS. However, for moderately/well‐differentiated PADC, cachexia was an independent factor for adverse OS, but not sarcopenia.Sarcopenia and cachexia have different effects on OS for PDAC patients undergoing radical excision. This difference may provide some important information for preoperative management. We found that sarcopenia and cachexia have different effects on OS for PDAC patients undergoing radical excision by studying the data of 614 PDAC patients. In this way, we could understand the differential prognostic values of sarcopenia and cachexia for PDAC patients who receive radical excision, improving the preoperative management for PDAC patients.22.1Patients who underwent radical surgery for PDAC between January 2015 and May 2022 at the Department of Pancreatic Surgery of West China Hospital, Sichuan University were included. The eligibility criteria were as follows: (1) age ≥ 18; (2) radical surgery for PDAC; (3) available abdominal CT scans within 1 week before the operation. The exclusion criteria for this study were as follows: (1) patients undergoing palliative surgery; (2) with liver or other sites metastasis; (3) cases with a history of severe metabolic disease or other cancers within 5 years; (4) patients without any follow‐up information. Follow‐up was performed every 6 months and the last date of follow‐up was July 30, 2022.2.2The following clinical data were collected: patient age, gender, body mass index (BMI), comorbidities, stage of tumor, grade of tumor, length of hospital and intensive care unit (ICU) stay, postoperative complications, surgical procedure, hemoglobin (HGB), albumin (ALB), and adjuvant chemotherapy. Data on postoperative mortality and recurrence were also recorded. The stage of tumor was classified based on the American Joint Committee on Cancer staging system (AJCC) 8th edition and overall postoperative adverse events were evaluated based on the Clavien–Dindo classification system. The primary outcomes of this study were OS and recurrence‐free survival (RFS). OS was defined from the first day after surgery until death or the last follow‐up, RFS was defined from the first day after surgery until recurrence or death or the last follow‐up.2.32, or weight loss >2% and sarcopenia.2)/height (m2). The cut‐offs for L3SMI were 52.4 cm2/m2 for men and 38.5 cm2/m2 for women.Cachexia was classified according to the following criteria: Involuntary weight loss >5% over the past 6 months, or weight loss >2% and BMI <20 kg/m2.4t‐test or Mann–Whitney U‐test for continuous variable and chi‐squared test or Fisher's exact test for categorical variables. Survival curves were plotted using the Kaplan–Meier method and compared using a log‐rank test. Prognostic factors of survival were identified by the Cox proportional hazard model, any factors with a p ≤ 0.10 in the univariate analysis were included in the multivariate analysis. All analyses were performed using SPSS 25. All p values were two‐sided and p < 0.05 were considered statistically significant.Continuous variables were reported as mean values with a standard deviation (SD) and categorical variables were presented as frequency statistics (n and %). Statistical testing was conducted by 3From January 2015 to May 2022, a total of 1543 patients were potentially eligible and 929 patients were excluded who met the exclusion criteria and did not meet the inclusion criteria. Finally, 614 patients were available for this study .According to the sex‐special cut‐offs for L3SMI, 378 patients were diagnosed with sarcopenia, accounting for 62% of all included patients. Sarcopenia was more prevalent among patients who smoked and drank, and males were more likely to suffer from sarcopenia. In addition, sarcopenia was significantly associated with lower BMI and lower ALB, and patients without sarcopenia before surgery were more likely to receive postoperative chemotherapy. In addition, 293 patients were diagnosed with cachexia accounting for 48% of all included patients. Patients with cachexia shared similar clinical characteristics to patients without cachexia, with the exception that they had significantly lower BMI, lower HGB, lower ALB, and longer hospital stay. The overall major adverse events rate (Clavien IIIA‐V) was similar in the sarcopenic group and non‐sarcopenic group as well as in the cachexic group and non‐cachexic group  while sarcopenic patients had comparable OS to non‐sarcopenic patients (log‐rank p = 0.94). Then, we further performed univariate and multivariate Cox regression analysis to identify the risk factors of OS in patients with different stages of PDAC. Cox regression analyses showed that cachexia was an independent risk factor of OS whether in the poorly differentiated group or moderately/well‐differentiated group. However, sarcopenia just was an independent risk factor of OS in the poorly differentiated group not in the moderately/well‐differentiated group ; data curation ; formal analysis ; investigation ; methodology ; software ; writing – original draft . Xing Wang: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; software ; writing – original draft . Zhenjiang Zheng: Resources . Yonghua Chen: Resources . Chunlu Tan: Validation ; visualization ; writing – review and editing . Xu‐bao Liu: Validation ; visualization ; writing – review and editing . Nengwen Ke: Funding acquisition ; project administration ; supervision .This study was supported by the National Natural Science Foundation of China (82002579).The authors declare that there is no conflict of interest.This study conformed to the provisions of the Declaration of Helsinki. All the patients included signed informed consent forms and agreed to data collection. It was approved by the ethics committee of West China Hospital, Sichuan University.Figure S1.Click here for additional data file.Table S1.Click here for additional data file.Table S2.Click here for additional data file.


While the incidence of cholangiocarcinoma is rising, little is known about young‐onset disease. We compared clinical characteristics and outcomes between patients with young‐onset cholangiocarcinoma, diagnosed between the ages of 18 and <50 years, and patients with typical‐onset cholangiocarcinoma, diagnosed at age 50 years or greater.n = 23,826). We compared the frequency of demographic and clinical characteristics between the two groups. We compared overall survival between the two groups using multivariable Cox regression analysis after adjusting for age, gender, race/ethnicity, comorbidity, facility type, tumor location, tumor stage, surgical status, and receipt of radiotherapy, chemotherapy and surgery.We used the National Cancer Database to identify patients with young‐onset cholangiocarcinoma (n = 2520) and typical‐onset cholangiocarcinoma (p < 0.01), and had lower overall comorbidity burden. Patients with young‐onset disease had a greater proportion of intrahepatic cholangiocarcinoma  and stage IV disease . Younger patients were more likely than typical‐onset patients to receive definitive surgery , radiation  and chemotherapy . In adjusted analyses, patients with young‐onset disease had a 15% decreased risk of death, compared with patients with typical‐onset disease .When compared to patients with typical‐onset disease (median age 68 years), patients with young‐onset cholangiocarcinoma (median age 44 years) were more likely to be non‐White (35.0% vs. 27.4%, Patients with young‐onset cholangiocarcinoma may represent a demographically and clinically distinct group from those with more typical‐onset disease. Young‐onset cholangiocarcinoma is associated with distinct demographic and clinical characteristics. Patients with young‐onset cholangiocarcinoma have a 15% decreased risk of death compared to patients with typical‐onset disease after adjusting for demographics, comorbidity, and treatment. The Charlson comorbidity index is a validated measure of overall comorbidity burden composed of 17 disease categories.2.3Baseline covariates were summarized using frequencies and percentages. We used the chi‐square test to compare demographic and clinical characteristics between patients with young‐onset cholangiocarcinoma and typical‐onset cholangiocarcinoma. Survival data were extracted from the NCDB vital status variable, which codes participants as either dead or alive at the date of the last contact. Patients who were alive at the last follow‐up were censored. Overall survival was estimated using the Kaplan–Meier method with log‐rank tests. We used multivariate Cox regression analysis to determine whether young‐onset cholangiocarcinoma was significantly associated with overall survival after controlling the effects of gender, race/ethnicity, comorbidity score, facility type, primary location, receipt of radiotherapy, receipt of chemotherapy, and receipt of surgery. Data were presented using hazard ratios (HR) with a 95% confidence interval (CI).We also performed subgroup analyses for patients with stage I, II, III, and IV disease at diagnosis, and for patients who received definitive surgery, patients who had intrahepatic cholangiocarcinoma, and patients who had extrahepatic cholangiocarcinoma. We used the log‐rank test to compare overall survival in young‐onset cholangiocarcinoma versus typical‐onset cholangiocarcinoma in each of these seven subgroups. Statistical significance was assessed using a two‐sided significance level of 0.05. Analyses were performed using SPSS version 25.0.3p < 0.001). A higher proportion of patients with young‐onset cholangiocarcinoma were diagnosed with stage IV disease . While there was no significant difference in nodal involvement between the two age groups, patients with young‐onset cholangiocarcinoma were more likely to have tumors that were positive for lymphovascular invasion . Patients with young‐onset cholangiocarcinoma were more likely to undergo definitive surgery , receive radiotherapy , and chemotherapy , when compared with patients with typical‐onset cholangiocarcinoma. Younger patients were also more likely to undergo treatment at an academic center .We identified 2520 patients with young‐onset cholangiocarcinoma (median age 44 years) and 23,826 patients with typical‐onset cholangiocarcinoma (median age 68 years). Demographic and clinical characteristics are summarized in Table p < 0.001). In unadjusted analysis, young‐onset cholangiocarcinoma was associated with a 28% decreased risk of death compared to patients with typical‐onset cholangiocarcinoma. After adjusting for gender, race/ethnicity, comorbidity score, facility type, primary location, stage, radiotherapy, chemotherapy, and surgery, patients with young‐onset cholangiocarcinoma had a 15% decreased risk of death compared to patients with typical‐onset cholangiocarcinoma  (Table p < 0.001). The survival difference was present but less pronounced for patients with stage IV disease, in whom young‐onset cholangiocarcinoma was associated with a median survival of 8.6 months versus 5.2 months for typical‐onset cholangiocarcinoma (p = 0.013).Patients with young‐onset cholangiocarcinoma had a median survival of 14.0 months from the time of diagnosis, compared to 9.3 months for typical‐onset cholangiocarcinoma (p < 0.001). Among patients with intrahepatic disease, young‐onset cholangiocarcinoma was associated with longer median survival . This survival benefit was similar in the subgroup of patients with extrahepatic disease . Cumulative survival for subgroups undergoing surgery and for subgroups with intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma are presented in Figure Among the subgroup of patients who underwent definitive surgery, those with young‐onset cholangiocarcinoma had a longer median survival from the time of diagnosis, compared with patients with typical‐onset cholangiocarcinoma  compared with older adults with cholangiocarcinoma.We found that a greater proportion of young‐onset cholangiocarcinoma patients were non‐White compared with typical‐onset cholangiocarcinoma patients. Demographic shifts have also been described in young‐onset colorectal cancer, which prevalence has risen more rapidly among White compared with Black Americans.We found that patients with young‐onset cholangiocarcinoma were more likely to have intrahepatic cholangiocarcinoma, while patients with typical‐onset cholangiocarcinoma were more likely to have extrahepatic cholangiocarcinoma. A relationship between age and tumor location is supported by a US‐based analysis using the SEER database, which found that the mean age of diagnosis for intrahepatic cholangiocarcinoma (67 years) was younger than the mean age of diagnosis of extrahepatic cholangiocarcinoma (72 years).Notably, patients with young‐onset cholangiocarcinoma were more likely to be diagnosed with advanced disease. This parallels findings in young‐onset colorectal,Our study has some notable limitations. We did not have information on several known cholangiocarcinoma risk factors, including primary sclerosing cholangitis, which disproportionately affects younger patients.To our knowledge, the present study is the largest to date analysis of young‐onset cholangiocarcinoma. Our findings suggest that young‐onset cholangiocarcinoma, similar to other young‐onset gastrointestinal malignancies, is associated with distinct demographic and clinical characteristics. These findings may aid clinicians in identifying young‐onset cholangiocarcinoma. Knowledge of these distinct clinical outcomes in younger populations may aid clinicians in guiding patient expectations and in counseling patients with young‐onset cholangiocarcinoma, a condition for which existing information is limited. Further research on the nature of cholangiocarcinoma in the young, including symptom burden, molecular profile, and efficacy and tolerability of specific treatment regimens, is warranted.Sarah Reddy: Conceptualization ; investigation ; methodology ; project administration ; writing – original draft . Suleyman Yasin Goksu: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; resources ; software ; writing – review and editing . Nina N. Sanford: Conceptualization ; writing – review and editing . Radhika Kainthla: Writing – review and editing . David Hsiehchen: Writing – review and editing . Aravind Sanjeevaiah: Writing – review and editing . Amy L. Jones: Writing – review and editing . Georgios Karagkounis: Writing – review and editing . Salwan Al Mutar: Writing – review and editing . Chul Ahn: Writing – review and editing . Muhammad S. Beg: Writing – review and editing . Syed M. Kazmi: Conceptualization ; investigation ; methodology ; project administration ; supervision ; writing – review and editing . Writing assistance: None.None.Reddy, Goksu, Sanford, Kainthla, Hsiechen, Sanjeevaiah, Jones, Karagkounis, Salwan al Mutar, Ahn, Beg: None Reported. Kazmi: Johnson and Johnson.


Climate change is projected to increase global mean annual temperatures as well as the frequency and intensity of extreme heat events. These changes are anticipated to alter the behavior of animals as they seek to thermoregulate in extreme heat. An important area of research is understanding how mutualistic interactions between animals and plants, such as pollination, will be affected by the cascading effects of extreme heat on animal foraging behavior. In this study, we used an experimental and observational approach to quantify the effects of extreme heat on hummingbird foraging preferences for nectar sources in shady versus sunny microsites. We also quantified pollen deposition using artificial stigmas at these sites to quantify potential cascading effects on plant reproduction. We hypothesized that hummingbirds would respond to extreme heat by preferentially foraging in shady microsites, and that this would reduce pollen deposition in sunny microsites on hot days. We found little support for this hypothesis, instead hummingbirds preferred to forage in sunny microsites regardless of ambient temperature. We also found weak evidence for higher pollen deposition in sunny microsites on hot days. Heat waves are predicted to get more intense under global climate change. We used experimental and observational data to determine if hummingbirds prefer to forage in shady microsites during heat waves, and how this might affect pollen deposition. Hummingbirds always preferred to forage in sunny microsites regardless of ambient temperature in both feeder trials and semi‐natural behavioral observation experiments, and there was no effect of ambient temperature or microsite on pollen deposition. A meta‐analysis of plant dependence on vertebrate pollinators found that when birds were excluded from pollinating, fruit or seed production was reduced by 46% . The Anna's hummingbird has experienced an expansion of its geographic range northwards, which may be related to supplemental feeding at backyard feeders, introduced floral species such as Eucalyptus, and climate change  would preferentially forage at experimental feeders in shady microsites versus sunny microsites on hot days (operative temperature in the sun >35°C) when compared to average days over the summer in California. We also test for differences in pollen deposition on artificial stigmas at feeders between sunny and shady microsites on hot days compared to average days. We hypothesized that pollen deposition would be greater at shady versus sunny microsites on hot days as compared to average days.In this study, we test the hypothesis that Anna's hummingbirds , monkeyflower (Diplacus), sage , keckiellas (Keckiella), penstemons (Penstemon), and willowherb (Epilobium)  campus in Hayward, California in the US Figure . The stu2.3Glass feeders with five feeding ports and perches  containing a 30% sucrose solution were placed under a tent canopy with dimensions of 3.05 m × 3.05 m × 2.44 m , a locally abundant hummingbird‐pollinated California native plant. At the end of a feeder trial, artificial stigmas were collected and mounted on glass slides for analysis. Slides were observed at a magnification of 100× using a digital microscope, and any pollen grains present were photographed. Pollen grains deposited per feeder trial were counted manually in slide photos using the program ImageJ  as the feeder trials to determine if they preferred shady microsites to sunny microsites on extremely hot versus average days. Seminatural observations and feeder trials were both spread out evenly during the study period. Birds were observed at 8 different locations on the CSUEB campus . The effect of temperature on number of visits, visits per hour, and visit duration to each category of microsite was analyzed using general linear mixed models (GLMM) using the R package “lme4”  and ambient temperature  as the fixed effect, and a unique session ID, Julian date, human presence , location, and floral abundance as random effects. Observation session duration was included as an offset term. Model selection, quality control, and post‐hoc pairwise comparisons were conducted following the same process as in feeder trial models.33.1N = 164) of all visits were in the shady microsite, while 69% (N = 365) were in the sunny microsite. Feeder trials were approximately balanced between hot (N = 14) and average (N = 16) ambient temperatures.We conducted a total of 30 feeder trials, during which we recorded 529 foraging visits by Anna's hummingbirds; 31% . A model with the same random effects but with temperature as a categorical variable found a similar result (Tables p < .001).The best model for cumulative number of visits included the interaction of microsite and maximum sun temperature as fixed effects, trial ID as the random effect, and an offset term for trial duration Figure  and A1. z = 2.17, p < .05). An identical model with temperature as a categorical variable was the second best model during model selection, and had a similar result, with sunny microsites receiving significantly higher visitation rates regardless of temperature category .When considering visitation rate as the dependent variable, the average visitation rate (visits/hour) was 2.67 ± 1.65 (SD) in shade and 5.75 ± 3.00 (SD) in sun during hot trials, and 2.02 ± 1.08 (SD) in shade and 4.91 ± 2.07 (SD) in sun during average trials. The best model for visitation rate included the interaction of microsite and maximum sun temperature as the fixed effect, trial ID as a random effect, and trial duration as an offset term Figure . The onlt‐value = 1.48, p < .05) and lower visit duration in the shade than in the sun .When considering visit duration during feeder trials, the average visit duration in the shade was 41.41 s ± 25.34 (SD) and 52.85 s ± 12.79 (SD) in the sun during hot trials. The average visit duration in the shade was 28.81 s ± 18.23 (SD) and 45.61 s ± 14.93 (SD) in the sun during average days. The best model for the average visit duration included the interaction of microsite and categorical temperature as fixed effects, Julian day as a random effect, and trial duration as an offset term Figure . No othe3.2Salvia leucantha) and strawberry tree  were the most visited ; of these, 8 were categorized as hot sessions, and 12 were categorized as average. Out of a total of 409 individual observations of behavior across all sessions, 107 were of foraging. Session duration averaged 47 min ± 1.85 (SD), for a total of approximately 17 h of observations. Hummingbird time budgets were apparently different on hot and average days , and post‐hoc tests revealed that this was driven by differences in microsite use on hot sessions, where hot sessions had more foraging in the sun than in the shade , a pattern which differed from their preferences on normal temperature days  as the fixed effect and session ID and floral abundance as random effects Figure  and A5. ys Table .3.3z‐value = 1.94, p = .05), but neither microsite nor categorical temperature was significant individually  and 593.02 ± 950.29 (SD) in the sun during hot trials. The average number of pollen grains deposited in the shade was 404.63 ± 747.27 (SD) and 275.38 ± 747.27 in the sun during average days. The average pollen load  in the shade was 14.90 ± 26.02 (SD) and 58.68 ± 109.22 (SD) in the sun during hot trials. The average pollen load in the shade was 80.37 ± 170.30 (SD) and 20.03 ± 29.25 (SD) in the sun during average days. The best model for average pollen deposition included the interaction of microsite and categorical temperature as fixed effects, Julian day and observation effect as random effects, and session duration as an offset term Tables . We foun4Calypte anna will forage preferentially in shady microsites on hot days was not supported by the feeder trials or seminatural behavioral observations. There were some apparent differences in how hummingbirds spent their time between different behaviors on hot sessions. The most frequently observed behavior in the sunny microsites during hot sessions was foraging . Future studies should control for individual hummingbirds by using color markings to distinguish between individuals if possible, though not all researchers are able to do this given welfare concerns to birds and variation in local regulations on auxiliary color markings  of plants. While this study was conducted in a seminatural environment that consisted primarily of cultivated plants, future studies should consider pollination in more natural environments, as patterns of preference and pollen deposition may differ in those habitats. This is especially relevant considering the potential effects of heat stress and increased evaporation during periods of extremely high temperatures on floral trait expression ; investigation (lead); methodology ; project administration ; validation ; visualization ; writing – original draft ; writing – review and editing . Jenny Hazlehurst: Conceptualization (lead); formal analysis ; investigation (supporting); methodology (supporting); project administration (supporting); resources (lead); software ; supervision (lead); validation ; visualization ; writing – original draft ; writing – review and editing .The authors would like to thank the undergraduate research assistants who helped collect this data, especially Henry Odufalu. We also thank all of the members of the Pollination Ecology & Conservation lab, and members of the graduate thesis committee at CSUEB, including Dr. Erica Wildy and Dr. Nazzy Pakpour, whose early feedback helped shape this research.None.The authors have no conflict of interest to declare.


The human experience is shaped by information from different perceptual channels, but it is still debated whether and how differential experience influences language use. To address this, we compared congenitally blind, blindfolded, and sighted people's descriptions of the same motion events experienced auditorily by all participants  and conveyed in speech and gesture. Comparison of blind and sighted participants to blindfolded participants helped us disentangle the effects of a lifetime experience of being blind versus the task‐specific effects of experiencing a motion event by sound alone. Compared to sighted people, blind people's speech focused more on path and less on manner of motion, and encoded paths in a more segmented fashion using more landmarks and path verbs. Gestures followed the speech, such that blind people pointed to landmarks more and depicted manner less than sighted people. This suggests that visual experience affects how people express spatial events in the multimodal language and that blindness may enhance sensitivity to paths of motion due to changes in event construal. These findings have implications for the claims that language processes are deeply rooted in our sensory experiences. We also express our multimodal experience in language using different modalities, as in speech and gesture. Modern theories of language and cognition, including multimodal language theories, differ in whether they view language as a relatively embodied or disembodied system . Compared to gesture, speech is better suited to represent sequential information coming from auditory and haptic input. Since gesture does not require linearization to the same degree that speech does, it has been described as conveying meaning in a more “holistic” manner that is through analog, iconic, and gradient representations , instead of using external coordinates . Therefore, we also tested whether mentions of landmarks in the blind participants were primarily self‐anchored and those of non‐blind participants were more object‐anchored.Furthermore, previous spatial cognition studies have found that blind people rely mainly on an egocentric rather than allocentric spatial frame of reference , 21 blindfolded , and 21 sighted  native Turkish speakers were paid to participate in the experiment. The sample size was determined by access to the special population with the control groups matched to the number of blind participants recruited. At the time of testing, 12 blind participants had light perception and nine had total blindness  followed by the opening sound.We audio‐recorded locomotion and non‐locomotion events performed by an actress. Locomotion events were the critical items and non‐locomotion events were filler items. We created 12 locomotion events by crossing three manners  with four paths  in relation to a landmark object (door or elevator)—e.g., “In addition, we edited the path azimuth angle using Soundtrack Pro audio editing software to vary the path motion. Five movement angles were created in a semicircular space ranging from 90° left to 90° right with 45° intervals. From the right to the left, these were: 0° (right), 45° (right‐sided), 90° (front), 135° (left‐sided), and 180° (left) motions , and audio was recorded at a fixed distance. We do not examine these items further.SD = 1.9) and non‐locomotion events 8 s (SD = 2.2). The event list and stimuli are available at https://osf.io/qsr7j/.There were 77 trials per person, including a total of 60 locomotion events and 17 non‐locomotion events. Locomotion events lasted on average 9 s  or goal (end point of movement), (b) path (trajectory of motion), and (c) manner (how the action is performed). We also coded whether landmarks reference either: (i) external objects  or (ii) self‐anchored —see Table 2.4.2path‐only gestures depict the trajectory of movement without representing mannermanner‐only gestures show the style of movement without representing trajectorypath+manner gestures depict both trajectory and manner of motion simultaneouslyParticipants’ spontaneous representational gestures (pointing and iconic) were identified for each target motion event description Kita, . We codeWe calculated the ICC between two coders to measure the strength of inter‐coder agreement for identifying a gesture and coding each type of gesture. Agreement between coders was .88 for identifying gestures and between .82–.93 for type of gesture—i.e., .89 for coding pointing gestures, .89 for coding path only, .93 for manner only, and .82 for path+manner gestures.3lme4  = .91, p = .635.First, we tested whether participants differed in the speech they produced for motion events. We ran a glmer model with the fixed factor of group  on binary values for mention of motion event description in speech  as a dependent variable. It revealed no effect of group on motion event description, 3.1.2χ2 (2) = 15.41, p < .001. Blind participants mentioned landmarks more than blindfolded  and sighted  participants, and there was no difference between blindfolded and sighted participants, β = .032, SE = .012, t = 0.26, p = .97.We predicted that blind participants would segment descriptions using more mention of landmarks than blindfolded and sighted participants. To account for baseline differences in the number of motion event descriptions produced, we calculated the ratio of landmark  per motion event description for each participant and item. We ran an lmer model with the fixed factor of group using the ratio of mention of landmark per motion event description as the dependent variable Fig. . The modχ2 (2) = 14.98, p < .001, showing that blind participants mentioned more landmarks in their speech than non‐blind participants, and an effect of landmark category, χ2 (2) = 160.33, p < .001, showing that object‐anchored landmarks were mentioned more than self‐anchored landmarks. Yet, the model also revealed an interaction between group and landmark category, χ2 (2) = 161.03, p < .001. To follow‐up the interaction, we compared the effect of group separately by landmark category. As expected, blind participants referred to self‐anchored landmarks more than blindfolded  and sighted  participants, and there was no difference between blindfolded and sighted participants . But, the groups did not differ in terms of reference to object‐anchored landmarks, all ps > .10.We further predicted that if blind people rely more on an egocentric frame of reference, they would use more self‐anchored landmarks than blindfolded and sighted participants. In contrast, blindfolded and sighted people would use more object‐anchored landmarks than blind participants. To test this, we calculated the ratio of mention of self‐anchored and object‐anchored landmark per motion event description for each participant and item. Then, we ran an lmer model with the fixed factors of group and landmark reference (object‐ or self‐anchored) using the number of mention of landmark per motion event description as the dependent variable Fig. . The mod3.1.3χ2 (2) = 0.68, p = .71, no effect of type of expression, χ2 (2) = 0.004, p = .95, but an interaction between group and type of expression, χ2 (2) = 16.31, p < .001. To follow‐up the interaction, we used the emmeans function to compare the groups for path and manner use separately.Next, we examined whether participants differed in how they expressed path and manner in speech. For this, we calculated the ratio of mention of path and manner per motion event description for each participant and item. We ran an lmer model with the fixed factors of group and type of expression (path vs. manner) and their interaction term using the ratio of mention of path and manner per motion event description as the dependent variable Fig. . The modβ = .224, SE = .103, z = 2.18, p = .075), blind and blindfolded , or blindfolded and sighted . However, the difference between blind and sighted participants  was significant when we did not use the conservative p‐adjustment in emmeans: blind participants mentioned path more than sighted participants.For path, although the interaction was significant in the model, there was no difference in the mention of path between blind and sighted  but not blindfolded  participants. There was no difference between blindfolded and sighted participants β = –.162, SE = .104, t = –1.56, p = .12). The interaction between group and type of expression can be seen in Fig. For manner, blind participants mentioned manner less often than sighted  and iconic  gestures per motion event description for each participant and item. For these calculations, total counts of pointing gestures, path‐only, manner‐only, and path+manner gestures were divided by the number of motion event descriptions for each trial. Hand gestures constitute 81.5% of pointing gestures. The data were analyzed in the same way as speech.3.2.1M = 0.44, SD = 0.48), blindfolded , and sighted  participants; F = 3.18, p = .049. A post‐hoc Tukey test showed that blindfolded participants had more gestures than blind participants (p = .041), but there was no difference between sighted and blind (p = .25) or blindfolded and sighted participants (p = .65).We compared the groups in terms of their overall gesture ratio using a one‐way between‐participants ANOVA. There was a significant difference in the gesture ratio between blind (3.2.2χ2 (2) = 5.81, p = .055. Blind participants produced more pointing gestures than sighted  but not blindfolded participants . There was no difference between blindfolded and sighted participants .We predicted that if blind participants would use more landmarks in their speech than non‐blind participants, this might be reflected in more pointing gestures to landmarks Fig. , and Sec3.2.3χ2 (2) = 10.39, p = .006, an effect of type of expression, χ2 (2) = 1354.7, p < .001, and an interaction effect of group and type of expression, χ2 (2) = 52.67, p < .001.To compare iconic gestures, we ran an lmer model with the fixed factors of group and type of expression  using the ratio of path and manner gestures per motion event description as the dependent variable Fig. . The modβ = .227, SE = .007, t = 30.99, p < .001) or path+manner gestures . To follow‐up the interaction, we used the emmeans function to compare the groups for each gesture type separately. Blind participants produced fewer path‐only gestures than blindfolded  and sighted  participants. Also, blind participants produced fewer manner‐only gestures than blindfolded  and sighted participants . Blind participants also produced fewer path+manner gestures than blindfolded  but not sighted  participants. There was no difference between blindfolded and sighted participants in terms of path‐only , manner‐only , or path+manner  gestures.All groups produced more path‐only gestures than manner‐only  speakers, blind speakers were more likely to use landmarks and, in particular, more self‐anchored landmarks. In addition, blind speakers tended to talk more about path and less about manner of motion events than sighted speakers. With regard to co‐speech gesture, we observed a similar gesture rate between blind and sighted speakers. However, speakers’ gesture frequency differed depending on the gesture type: blind speakers produced more pointing gestures with landmarks than sighted speakers, but had fewer path and manner gestures than non‐blind speakers (blindfolded and sighted). Even though all speakers’ gesture patterns were consistent with the Turkish motion typology , blind speakers produced fewer iconic gestures than non‐blind speakers overall. We contextualize and discuss each of these points in more detail.The fact that Turkish blind and non‐blind (blindfolded and sighted) individuals did not differ in the overall amount of verbal descriptions produced is perhaps not surprising given that blind people are good at processing auditory information . We also found that blind speakers had more pointing gestures to posited landmarks in gesture space than sighted speakers. Taken together, this is in line with previous studies that find blind people rely more on egocentric than allocentric frames of reference when learning spatial layouts  speakers. In particular, when landmarks were mentioned, blind speakers were more likely to refer to them in relation to their own position . So, this path segmentation is a result of more mentions of landmarks . Together with the increased landmark use, increased mention of path in speech suggests that blindness may enhance sensitivity to paths due to changes in event construal that arise from altered spatial cognition  speakers. Even though there was a mismatch in the frequencies of path in speech and path in gesture, speech and gesture type were still coupled with respect to motion event depictions in Turkish—i.e., separated path and manner use in both speech and gesture . This could suggest an additional role of momentary lack of vision in the expression of spatial language (see also Mamus et al., Finally, the gestures of congenitally blind speakers offer fresh insights into multimodal language production theories. Our results showed that both blind and sighted speakers’ gesture patterns were in line with what we would expect considering the typology of a verb‐framed language, i.e., Turkish (e.g., Kita & Özyürek, 5Theories of embodied cognition propose that multimodal language processes are rooted in sensory and motor experience (Barsalou, Overall, we found that blind people were more likely to mention landmarks, especially those in relation to themselves, than both sighted and blindfolded people. They were also more likely to mention path of motion in speech than sighted people while omitting manner in both speech and gesture. However, based on our current data, we cannot rule out the possibility that blind speakers of a satellite‐framed language may show more resilience in extracting manner information from the sound. While the verbal encoding of path and manner did not differ between blind and blindfolded people, the differences in the gestural encoding of path and manner distinguished blind people from both sighted and blindfolded people. This suggests that beyond merely a temporary lack of sight, a lifetime of blindness changes how these components are represented in gesture. This may be because iconic gestures are more difficult to build upon non‐visual information alone.Although the current data illustrate differences between blind and sighted people, it remains unclear whether the differences in language use occur because blind people's lifetime of perceptual experience influences their conceptualization of spatial events or because blind people extract event information from auditory input for linguistic expressions differently than sighted people. Further research on blind people's language use is needed to uncover precisely how perceptual experience shapes multimodal language.Taken together, our study illustrates that a lack of visual experience affects how people encode spatial events for multimodal language production.https://osf.io/qsr7j/.This article has earned Open Data and Open Materials badges. Data and materials are available at


Spread through air spaces (STAS) is a pattern of invasion recently identified in non‐small cell lung cancer (NSCLC), with a poor prognosis. However, the predictive impact of STAS in stage IB NSCLC is not well understood. This investigation aims to assess the prognostic influence of STAS in stage IB NSCLC.We reviewed 130 resected stage IB NSCLC between 2010 and 2015. Beyond the central tumor edge, lung parenchymal air gaps containing cancer cells were identified as STAS. In order to estimate recurrence‐free survival (RFS) and overall survival (OS), Cox models and Kaplan–Meier techniques were utilized. Logistic regression analysis was employed to define the factors influencing STAS.p = 0.02; 5‐year RFS, 59.5% vs. 89.7%, p = 0.004) In a semiquantitative assessment, the RFS and OS were shorter in survival analysis when STAS increased . The association between STAS and poor differentiation, adenocarcinoma, and vascular invasion  was statistically significant.Of 130 patients, 72 (55.4%) had STAS. STAS was a significant prognosticator. Kaplan–Meier method showed that STAS‐positive patients had a significantly lower OS and RFS than STAS‐negative patients (5‐year OS, 66.5% vs. 90.4%, The STAS is an aggressive pathological feature. RFS and OS could be significantly reduced by STAS, while it also serves as an independent predictor. Furthermore, STAS was discovered to be a substantial risk factor for recurrence with limited resection.22.1Institutional ethic review boards of The First Affiliated Hospital of Ningbo University and Taizhou Hospital of Zhejiang Province have approved this retrospective study , which also included the clinical databases that we reviewed from 2010 to 2015 for lung cancer patients with surgical resection. American Joint Committee on Cancer (AJCC) staging 8th version guidelines was used to identify the pathologic phase.2.2Typically, 130 Eosin‐stained tumor and hematoxylin slides were examined by two pathologists blinded to the participant's medical outcome using a Leica DM2000 microscope  and a typical 22‐mm diameter eyepiece.Pathological evaluations were depending on the 2015 World Health Organization (WHO) categorization of pulmonary cancers and the AJCC TNM Classification, 8th edition. The STAS was identified as per the procedure by Kadota et al.2.3The primary observation indicators of this study were OS and RFS.t‐test, while qualitative data were examined using chi‐square and Fisher's exact tests. Log‐rank testing was performed to assess statistical variations, and Kaplan–Meier analysis was utilized to determine OS and RFS rates. Employing multivariate and univariate Cox proportional hazards regression models, the predictive factors were examined. The risk factors associated with STAS were predicted using the binary logistic regression model. Statistical significance is demonstrated by a p value less than 0.05.The statistical software SPSS 21.0 was utilized for analysis. Quantitative data were evaluated using a 33.1From 2010 to 2015, 1149 NSCLC patients received surgical treatment at The First Affiliated Hospital of Ningbo University and Taizhou Hospital of Zhejiang Province. There were 142 patients with stage IB based on postoperative pathological staging. Finally, 130 patients with stage IB lung cancer (88 ADC and 42 SCC) met the criteria. All patients underwent VATS including uniport (55 cases) and multiport (75 cases) thoracoscopic surgery. One hundred and twenty patients underwent radical lobectomy, while 10 patients received limited resection. There were 30 patients received postoperative adjuvant chemotherapy, and 100 patients did not received postoperative adjuvant chemotherapy. Typically, 86 males and 44 females were included, with a mean age of 64.12 years, ranging from 31 to 80 years. Sixty‐three patients had never smoked while 67 patients were past or current smokers. There were 72 STAS‐positive cases , and 58 negative cases. STAS was found to be low in 33 cases and high in 39 cases. After surgery, the enrolled lung cancer patients received regular outpatient or telephone follow‐ups. The last follow‐up was on August 31, 2018, whereas the average follow‐up period was 46.89 (0.5–98) months. Twenty‐four patients died due to various reasons. In addition, 26 patients had tumor recurrence. Table 3.2p value was 0.041, <0.001, 0.047, respectively) . (Table p = 0.002)  Table . Univari). Table  Besides,2) Table .3.3p = 0.02) , and could lower the postoperative survival time (Table The 130 patients were monitored for an average of 46.89 months (ranging from 0.5 to 98 months), and 24 patients were died. Survival analysis showed that STAS‐positive patients had a significantly lower overall survival than STAS‐negative patients  Figure . Univarip = 0.002, see Figure Participants in the high STAS group had a significantly poorer prognosis than those in the low STAS and STAS‐negative groups, according to the survival analysis of the STAS quantitative stratified group. There was a significant variation in the prognosis between the groups , poor differentiation (p = 0.021), and STAS (p = 0.007) were important factors for RFS; while multivariate survival analysis, STAS (p = 0.041) and vascular invasion (p = 0.041) were significant risk factors for RFS (Table Twenty‐six (20%) of the 130 patients had tumor recurrences after surgery, twenty‐one of whom were STAS positive and five of whom were STAS negative. The STAS‐positive group had a recurrence rate of 29.2% (21/72) while the negative group had 8.6% 5/58). The STAS‐positive patients had a significantly lower 5‐year RFS than STAS‐negative patients   Figure .4Surgical resection is the recommended form of treatment for lung cancer, which is the primary reason for cancer‐related deaths globally.In 2015, Kadota et al. defined STAS.The STAS is a newly discovered pathomorphological feature in NSCLC with a positivity rate ranging from 14.8% to 55.4%.In their 2015 research report, Kadota et al. pointed out that in early‐stage ADC, STAS was a substantial risk factor for subjects with limited resection prognosis, but had no significant effect on patients who received lobectomy.In 2015, the adoption of STAS for ADC in the WHO categorization for lung cancer after verification in two large independent cohorts from Germany and the United States.n = 130) retrospective study. Secondly, operations in the process of surgery and pathological section may affect STAS incidence. Further investigation is required since there is still disagreement about whether STAS is an in vivo impact in any instance or possibly an artifact.In this study, there are some limitations. Firstly, this is a single‐center, small sample size ; formal analysis ; investigation ; validation ; writing – original draft ; writing – review and editing . Xianqiao Wu: Data curation ; formal analysis ; investigation ; validation ; writing – original draft ; writing – review and editing . Tianzheng Fang: Data curation ; formal analysis ; investigation ; validation ; writing – original draft ; writing – review and editing . Zhen Ge: Investigation ; methodology ; validation ; writing – review and editing . Jiayuan Liu: Investigation ; methodology ; validation ; writing – review and editing . Qinglong Wu: Investigation ; software ; validation ; visualization ; writing – review and editing . Lin Zhou: Investigation ; software ; validation ; visualization ; writing – review and editing . jianfei Shen: Conceptualization ; project administration ; resources ; supervision ; validation ; writing – review and editing . Chengwei Zhou: Conceptualization ; project administration ; resources ; supervision ; validation ; writing – review and editing .There are no declared conflicts of interest.This investigation has been authorized by the medical ethics committees of The First Affiliated Hospital of Ningbo University, and Taizhou Hospital of Zhejiang Province.Project batch number is KS20232003. Project acceptance number is 2023KS0009.


Despite the prevalent expression of freezing behavior following Pavlovian fear conditioning, a growing body of literature suggests potential sex differences in defensive responses. Our study investigated how female defensive behaviors are expressed in different threat situations and modulated by the estrous cycle. We aimed to compare freezing and flight-like responses during the acquisition and retrieval of fear conditioning using two distinct unconditioned stimuli (US) in two different spatial configurations: (1) electrical footshock (FUS) in a small, conventional enclosure with a grid floor, and (2) a predator-like robot (PUS) in a spacious, open arena. Fear conditioning with FUS showed no substantial differences between male and female rats of two different estrous cycles (proestrus and diestrus) in the levels of freezing and flight. However, when PUS was employed, proestrus female rats showed significantly more flight responses to the CS during both acquisition and the retrieval compared to the male and diestrus female rats. Taken together, our findings suggest that hormonal influences on the choice of defensive strategies in threat situations are significantly modulated by both the type of US and the spatial configuration of the environment. Therefore, it is crucial to understand the fundamental disparities underlying fear and anxiety to develop more comprehensive and effective treatments for both sexes4. However, a substantial gap in our understanding stems from the fact that the majority of basic research in this field has predominantly involved male animals, resulting in a scarce data from female rats5. This is further complicated by mixed results from studies involving female rodents. Some studies suggest that females exhibit more active defensive behaviors such as flight responses7, while other studies have observed the opposite, where females demonstrate more passive defensive behaviors than males8. Adding to this complexity, a number of studies have reported no sex differences in the levels of either freezing or flight responses10. Therefore, despite the growing inclusion of female rats in recent research, there is a clear need for more systematic analyses that specifically investigate sex differences in animal research12.Anxiety and fear-related disorders are more common in women than in men, with a lifetime prevalence nearly twice as high15. In this paradigm, animals are repeatedly presented with a neutral conditioned stimulus (CS)—typically a tone—and an aversive unconditioned stimulus (US), such as an electrical footshock. The footshock immediately produces reflexive jumping followed by freezing, a common defensive response characterized by a robust state of immobility18. Interestingly, the choice of defensive strategies, including freezing, depends on contextual factors, one of which is perceived distance from the threat19. Although the footshock, delivered through a metal grid, simulates a painful encounter with a predator20, it remains invisible to the animals, making the distance to the threat indeterminable. As animals cannot predict the distance from this invisible threat, freezing becomes the most probable defensive behavior in the fear conditioning paradigm. However, survival in natural environments requires more flexible defensive strategies in the face of dynamic threat situations. Given this, recent studies have incorporated ecological threat paradigms, including real predators22 or predator-like robots25, to explore defensive behaviors beyond freezing. Thus, these ecological paradigms could potentially resolve the ongoing controversy surrounding the existence of sex differences in defensive behavior.Pavlovian fear conditioning serves as a standardized laboratory procedure for investigating threat processingTo address this issue, this study aimed to probe the potential influence of sex on defensive behaviors, with an emphasis on how the type of US and the extensive environment of a naturalistic setting shape these variations. For this purpose, we initially employed conventional fear conditioning using an electrical footshock as US (FUS). We then employed a fast-approaching predator-like robot as a new type of US (PUS), allowing animals to visually and audibly detect the impending threat, and observed any changes in defensive behaviors. In addition, to investigate the role of hormonal influences, we monitored the naturally cycling estrous stages in females. With an aim to clarify the discrepancies in existing findings about female defensive behavior, our experiments suggest that there are sex differences in defensive behavior in response to CS and the choice of defensive behaviors is influenced by the type of US and the spatial configuration of the environment where the US is presented. These findings highlight the necessity for careful consideration of these factors when studying sex differences in defensive behaviors.26. Proestrus was characterized by a predominance of nucleated epithelial cells, which are round or oval-shaped with centrally located nuclei within the cytoplasm  peak during proestrus, while these hormones reach the lowest point during diestrus27, we monitored the ratio of epithelial cells and leukocytes for a minimum of 10 days. Figure For accurate classification, rats were assigned to either proestrus or diestrus stages when at least 80% of cells in the smear tests corresponded to the specific cell type. To confirm at least two complete regular estrous cycles, considering the typical rat estrous cycle length of 4–5 daysF = 3.83, p < 0.05). Bonferroni pairwise comparisons revealed that proestrus female rats (n = 9) traveled significantly greater distance compared to male . Although the distance traveled by proestrus females was greater than that of diestrus females (n = 9), this difference was marginal  = 296.37, p < 0.001), indicating that all three groups showed progressive increase in freezing during the acquisition of the CS–FUS association  = 2.29, p = 0.11), and there was no significant interaction between these factors  = 0.39, p = 0.82). We then analyzed the flight score, which encompasses rapid forward movements akin to running and vertical movements such as jumping  = 12.65, p < 0.001) and trial  = 9.21, p < 0.001) on the levels of flight score, but no interaction between these factors was observed  = 1.25; p = 0.30). Planned comparison demonstrated significant differences between proestrus female rats and both male (p < 0.001) and diestrus female (p < 0.001) rats, but these differences were observed only on the first trial , and the protocol is illustrated in Figs. F,81 = 296ing Fig. A and S1BF = 0.92, p = 0.40; Fig. Fear memory was tested 24 h after the conditioning by presenting three CSs. Recognizing the potential shift in the estrous cycle within a 24-h period, we collected and assessed vaginal samples 1 h before the retention test. Although there was a decrease in the specific cellular markers compared to the conditioning day, approximately 70% of these markers on the retention day aligned with their respective group  = 5.56, p < 0.01), with proestrus female rats moving a greater distance compared to both male (p < 0.05) and diestrus female (p < 0.05) rats  and expanded the testing environment to allow more room for escape Fig. B. The saF2,5 = 5.56,25. There were significant main effects in both group  = 85.02, p < 0.001) and trial  = 23.58, p < 0.001) in freezing, as well as a significant interaction between these variables  = 4.50, p < 0.001; Supplementary Video p < 0.001; Fig. F = 161.50, p < 0.001) and trial  = 2.46, p < 0.05), with a significant interaction between them  = 5.46, p < 0.001) on flight scores. Subsequent post hoc comparison revealed that flight scores from proestrus female rats were significantly higher compared to both male and diestrus female rats on trial 2–5  = 9.21, p < 0.001), a marginal effect of trial  = 2.27, p = 0.06), and a significant interaction between them  = 4.35, p < 0.001). During trials 2–5, proestrus females exhibited a higher frequency of fleeing compared to other groups  = 51.93, p < 0.001), a marginal trial effect  = 2.25, p = 0.07) and a significant interaction effect  = 2.19, p < 0.05). Notably, jumping behavior was exclusive to proestrus females, demonstrating a higher frequency during trials 2–5 compared to other groups  = 8.46, p < 0.001). However, there was no significant effect observed for trial  = 1.10, p = 0.36), nor was there an interaction between group and trial  = 1.69, p = 0.103). Planned comparison showed that while no group differences emerged until trial 3, the proestrus group had significantly fewer collisions on trials 4 and 5 (p < 0.01) compared to both male and diestrus female rats, which showed no difference between them  or flight  in each group. The results suggest that collision had little contribution to shaping the defensive behavior. Therefore, the different number of collisions in proestrus rats might indicate that they preferred flight response in the same threat situation, reflecting the difference in motivation-to-action mapping due to the cycle difference.Subsequently, when the female rats reached their next cytological peak Fig. C, male B. The prroup  = 53.95, p < 0.001) and trial  = 4.27, p < 0.05), with no interaction between these factors detected  = 0.54, p = 0.71). Planned comparisons showed that both male and diestrus female rats displayed significantly higher levels of freezing compared to proestrus female rats across all three trials  = 110.11, p < 0.001) and trial  = 4.09, p < 0.05) effects were significant, without a detected interaction  = 0.55, p = 0.70). Subsequent planned comparisons revealed that proestrus female rats displayed significantly higher flight responses than both male and diestrus female rats on every trial . Consistent with a large number of previous studies, the conventional fear conditioning protocol with FUS elicited predominantly freezing CR19, an influential theory of defensive behavior, states that the choice of defensive behavior is largely determined by the perceived proximity of the threat which includes both spatial and temporal distance. Interestingly, there was a 6.5-s difference in the interstimulus interval between the two threat conditions: 9 s for FUS compared to 2.5 s for PUS. This discrepancy hints at the potential for altered threat perception and subsequent choice of defensive behavior due to differing levels of temporal imminence. While our study provides initial insights, the influence of this temporal variation on threat perception warrants further investigation.Our study demonstrated that a defensive flight response was reliably observed in proestrus female rats during both the acquisition and retrieval phases of fear conditioning with PUS. However, no such difference was found in conditioning with FUS. One obvious difference between the two experiments is the type of the US used, which might have played a critical role in differentiating the expression of the defensive response to the learned threat cue. According to the Predatory Imminence Continuum theory33. The conditioning box, where FUS was presented, was mainly a trap-like space surrounded by walls as opposed to the spacious arena where PUS was utilized. From the rat’s perspective, the spacious arena offers an escape route, thereby promoting the choice of running as a viable defensive option. Therefore, when a threat is uncertain or an escape route is unavailable in a confined space, it is highly probable for rats to lean towards a more passive defensive form of response18. This might have been the case for FUS-based fear conditioning where dermal pain is not readily recognized as a threat by the innate defensive system. In such a context, the animal’s choice of defensive response is primarily immobility. In contrast, PUS-based fear conditioning, where threat imminence is perceptible, flight responses are observed more frequently during the early trials in all animals. Yet, as the trials progressed, the flight responses of male and diestrus female rats significantly decreased, shifting to the more passive freezing behavior. However, proestrus female rats maintained a high level of flight responses throughout the conditioning sessions. This suggests that the proestrus rats inherently prefer active defensive behavior in threat situations, possibly influenced by cyclical hormonal changes, which subsequently impact their motivation-to-action mapping.Another key determinant might be the spatial configuration of the environment where the animal is exposed to the threat34, even in environments with potential dangers. For example, previous study reports that proestrus female rats exhibit reduced freezing and engage in active risk assessment when exposed to cat odor, while diestrus female rats display more passive freezing behavior35. In line with this, we observed that proestrus female rats traveled significantly more distance than diestrus female rats in the open field test  male and female Sprague Dawley rats obtained from Orient Bio and Koatech, Korea were used in this study. The rats were acclimated to the laboratory for at least two weeks and underwent daily examination of vaginal lavage for at least 10 days prior to the behavioral tests. Rats were group housed (two/cage) in a climate (21 ± 2°) and humidity (50%) controlled vivarium, and they were maintained on a 12-h light–dark cycle with access to food and water ad libitum. All the behavioral experiments were conducted during the dark phase of the cycle. Due to the absence of any surgical process, no anesthesia was used. At the end of the experiment, the animals were euthanized with CO41 for 10 days prior to the behavioral tests. During behavioral tests, examinations of vaginal lavage were performed 1 h before each behavioral test. The identification of each phase was based on the proportion of nucleated epithelial cells and leukocytes observed in the vaginal smear. To quantify these types of cells, an image of the collected vaginal cells on a glass slide was captured using a light microscope . The captured image was then utilized for cell counting analysis, and female rats were categorized as either proestrus or diestrus based on an open field test and a fear conditioning day. One female rat was excluded due to an unstable estrous cycle. Male rats underwent similar procedure daily except pipette insertion to match the level of stress. To match the level of stress the females experienced, male rats underwent similar daily procedure as the vaginal smear, but without undergoing the pipette insertion.To ensure appropriate estrous cycles for behavioral tests, females underwent daily examination of vaginal lavage42.Proestrus and diestrus female rats along with male rats were subjected to an open field test to assess their general activity levels. Rats were individually placed in an open arena (80 × 80 × 30 cm) and allowed to freely move for 10 min. After the free exploration, rats were returned to their home cages. The arena was cleaned with a 70% ethanol solution between animals. General activity levels including distance traveled and velocity were recorded and analyzed offline by a video tracking software Ethovision 39. Animals underwent habituation, fear conditioning, and retention in two identical clear Plexiglas boxes (25 × 30 × 40 cm) enclosed within a sound-attenuating cubicle with background noise (45 dB SPL) provided by a ventilation fan. During habituation and retention test, the boxes were covered with bedding materials and lit with white light. For conditioning, each box was illuminated with green light and floored with 16 stainless steel rods  connected to a scrambled shock generator . A tone  as a conditioned stimulus (CS) was presented from a small speaker on the side of each box connected to a tone generator  through an amplifier . For unconditioned stimulus (US), a footshock  was delivered through the grid.The fear conditioning paradigm employed in this study was adapted and modified from previous studiesOn the first day of conditioning, we conducted habituation where animals were allowed to freely explore the chamber for 10 min. On the next day, following a 2-min acclimation period, rats received 3 CS trials which co-terminated with a 1-s footshock, with an average inter-trial interval (ITI) of 120 s ranging from 110 to 130 s. Then 24 h later, to test retention of fear memory, the rats received 3 CS presentations with a 20-s ITI in the same context as on the habituation. A video camera was mounted beside each box to monitor and analyze behaviors of rats, and the chamber was thoroughly cleaned with 70% ethanol at the end of each experiment.25, was used as a US (PUS) to simulate the experience of being chased by a predator. Briefly, the robot used in this experiment was a box-shaped vehicle (15 × 26 × 35 cm) with four high traction rubber tires. The speed (0.7 m/s) was controlled by a Bluetooth®-based microcontroller with a custom-written program in Arduino , and the CS  was generated by two 8-Ω speakers mounted in the front and back of the robot. The direction of the robot’s movement (clockwise/counterclockwise) was randomly determined with equal probability. Fear conditioning with chasing robot was conducted in an apparatus consisted of an acrylic donut-shaped structure, comprising a clear acrylic cylinder (24 cm in diameter) nested within a larger black acrylic cylinder (60 cm in diameter). This configuration formed a circular track with a width of approximately 18 cm and a wall height of 45 cm t tests. When significant interactions were detected, post-hoc pairwise comparisons were conducted using Bonferroni correction. To compare the cell types among female rats, Student’s t-tests were performed. Pearson’s correlation coefficient was employed to analyze the relationship between behavioral indices . Two-tailed p-values of < 0.05 were considered statistically significant. Data were expressed as mean ± SEM.Statistical analyses were performed using a statistics software package . Behavioral results were analyzed using two-way repeated measures analysis of variance (ANOVA) that contained within-subjects variables  and between-subjects factors (group). Group comparisons were also conducted with one-way ANOVA or Student’s Supplementary Information.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.