Coptis chinensis were extracted by ultrasound-assisted technology, and the contents of palmatine and berberine were used as indexes by using Coptis chinensis as raw material and eutectic solvent as extractant. In addition, the effects of hydrogen bond donor type, molar ratio of hydrogen bond donor to acceptor, material-liquid ratio, water content of eutectic solvent system, sonication time, power, and ultrasonic temperature on the extraction rate of palmatine and berberine were studied. The optimum extraction technology of palmatine and berberine from Coptis chinensis was determined by single-factor experiment and response surface optimization test. As a result, it showed that the eutectic solvent system was constructed with choline chloride as hydrogen bond acceptor and phenol as hydrogen bond donor, with a molar ratio of 1 : 3. In addition, water content of the eutectic solvent system was 30%, ratio of material to liquid was 30 g/mL, ultrasonic time was 30 min, ultrasonic power was 200 W, and ultrasonic temperature was 60°C. At this time, the contents of palmatine and berberine in Coptis chinensis were 16.7145 mg/g and 57.4013 mg/g, respectively, which were predicted to be the same as the value, and the extraction effect was better than that of traditional extraction solvent method.The effective components of  Coptis chinensis, C. deltoidea C. Y. Chenget Hsiao, and C. teeta Wall of Ranunculaceae, Coptis chinensis are commonly known as Weilian, Yalian, Yunlian [Coptis chinensis was first published in the Classic of Shennong Materia Medica and was listed as a top-grade medicine [Coptis chinensis include alkaloids, lignans, flavonoids, and acidic components [As the dry rhizome of  Yunlian , bitter  Yunlian , which c Yunlian . Besides Yunlian , 4. In amedicine : “the smmponents . The maimponents , of whicmponents .As first reported by Abbott et al. in 2003 , deep euCoptis chinensis based on the ultrasound-assisted eutectic solvent. Besides, the effects of different DESs and other extraction factors on the extraction of effective components of Coptis chinensis were investigated through single-factor experiment. In addition, we combined with response surface method to optimize the extraction process of effective components of Coptis chinensis and compared with traditional water extraction and alcohol extraction. This study is committed to providing a greener, efficient, economical, and environmentally friendly new method for the extraction of alkaloids from Coptis chinensis. It can better promote the in-depth development of Coptis chinensis and provide data support for the development of green chemistry and the research of natural products of traditional Chinese medicine.In this study, we performed the isolation and extraction method of palmatine and berberine from Firstly, the chromatographic analysis was carried out by using a high-performance liquid chromatography system . In addition, a JP-300G ultrasonic cleaner was purchased from Wuhan Jiapeng Electronics Co., Ltd. Moreover, QL-901 Vortex instrument was purchased from Haimen Qilinbell Instrument Manufacturing Co., Ltd. Besides, TGL-16 freezing centrifuge was purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd.Coptis chinensis was produced in Sichuan Province, China, purchased from Jilin Pharmaceutical Store (Changchun); these samples meet the requirements of Coptis chinensis in Chinese Pharmacopoeia (2020 edition). Berberine hydrochloride (≥85.9%) and palmatine hydrochloride (≥85.7%) standards were used for HPLC determinations which were purchased from China Food and Drug Identification Institute. Chromatographic grade methanol and acetonitrile were purchased from Fisher Company of the United States. All reagents were of analytical grade. Phosphoric acid was purchased from Tianjin Guangfu Technology Development Co., Ltd., and ultrapure water was purchased from Hangzhou Wa Co., Ltd. Choline chloride was purchased from Shanghai Zhanyun Chemical Co., Ltd. Maltose, malic acid, lactic acid, fructose, xylitol, and citric acid were purchased from Zhengzhou Kangyuan Chemical Products Co., Ltd. Phenol and acetic acid were purchased from Xilong Science Co., Ltd. Glycerin was purchased from Guangzhou Miya Cosmetics Co., Ltd. Propylene glycol was purchased from Tianjin Zhonghe Shengtai Chemical Co., Ltd. Urea was purchased from Tianjin Ruiji Chemical Co., Ltd.In addition, the TM C18 column . Mobile phase included acetonitrile, 0.1%, phosphoric acid aqueous solution . The selected wavelength was 345 nm, the injection volume was 20 μL, the flow rate was 1.0 mL/min, and the column temperature was 30°C.The chromatographic column was an Alltimaμm pore-size filter. In addition, the reference solution was obtained for further HPLC analysis.The reference substance of palmatine and berberine was precisely weighed and dissolved with methanol to obtain a mixed reference solution containing 0.572 mg/mL palmatine and 1.140 mg/mL berberine. The solution was precisely absorbed and diluted 1 mL 1 : 10 with methanol and filtered with a 0.22 The hydrogen bond donor and hydrogen bond acceptor were mixed according to the molar ratio of Coptis chinensis was extracted (the powder passed through a 100-mesh sieve) and was added to eutectic solvent according to the ratio of material to liquid at 1 : 30 (g/mL). Besides, it was placed in 60°C warm bath for 5 min and was vortex oscillated for 5 min, and then ultrasonic extraction of 30 min (200 W) at 60°C was conducted and it was vortex oscillated for 1 min and centrifugation of 3000 rpm/min for 5 min was conducted.50 mg Coptis chinensis were investigated, including DESs type, molar ratio, water content, material-liquid ratio, sonication time, ultrasonic temperature, and ultrasonic power. Several factors which had great influence on the extraction efficiency were selected as the independent variables of the response surface through the experimental design. The content of palmatine and berberine in Coptis chinensis was analyzed and optimized by response surface method.Critical variables in the extraction process of Coptis chinensis extract was adequate. In addition, the number of theoretical plates was more than 3000  as ordinate (y) and reference substance concentration (mg/mL) as abscissa (x). The regression equations of palmatine and berberine were obtained as y = 33054x − 11.777 (r = 0.9997), the linear range was 0.011∼0.572 mg/mL; y = 36898x − 352.3 (r = 0.9996) and the linear range was 0.023∼1.140 mg/mL, respectively.The mixed reference substance reserve solution was diluted 50, 25, 10, 5, 2.5, and 0 times, respectively. In addition, the peak area value was recorded by HPLC analysis. The calibration curve was drawn with peak area . In addition, 1.5 mL of DES-5 (water content 30%) was added, and Coptis chinensis was extracted according to the method described in Six samples of Coptis chinensis powder was added, and DES-5 (water content 30%) 1.5 mL was added after steaming and drying the solvent in water bath. Coptis chinensis extract was extracted according to the method described in Six samples of Rhizoma Coptidis with known content, about 25 mg each, were precisely removed from palmatine reference solution (concentration 0.745 mg/mL) 0.5 mL and berberine reference solution (concentration 0.555 mg/mL) 2.5 mL in the same centrifuge tube. The well-proportioned Coptis chinensis.The composition of DESs determines its physical and chemical properties, which affects its extraction efficiency of natural compounds. The effects of 11 kinds of DESs  on the cCoptis chinensis is the most when the molar ratio of choline chloride to phenol is 1 : 3. Less intramolecular hydrogen bonds may be formed, and the viscosity is very high when the ratio of choline chloride is low. It is not conducive to full contact with Coptis chinensis powder. The viscosity decreases and the extraction amount increases with the increase of phenol. The extraction amount of palmatine and berberine decreases when the molar ratio is more than 1 : 4. It may be caused by the instability of eutectic solvent and the decrease of hydrogen bond. Therefore, the molar ratio of 1 : 3 resulted more beneficial to the extraction of effective components of Coptis chinensis.The viscosity of most DES decreases with the increase of temperature and the increase of hydrogen-bonded donors . As can Coptis chinensis in a certain range. However, if we continue to increase the water content; it may affect the intermolecular hydrogen bond of DESs, change the structure of DESs, and reduce the solubility of effective components of Coptis chinensis. In this study, the effects of choline chloride-phenol eutectic solvents with different amounts of water on the extraction of palmatine and berberine from Coptis chinensis were investigated. As shown in Coptis chinensis also increased gradually with the increase of water content. However, it changed when the water content was more than 40%, and the extraction efficiency of Coptis chinensis decreased. This indicated that the water content of DES has a great influence on the extraction efficiency of Coptis chinensis.The addition of water reduces the viscosity of DESs. Moreover, the decrease of viscosity is beneficial to improve the cavitation effect of ultrasonic wave and reduce the loss of ultrasonic wave in the process of propagation . ConsequCoptis chinensis.The amount of solvent determines the efficiency to a certain extent of the whole extraction process. It can be seen from Coptis chinensis increases with the extension of time. The extraction efficiency of DESs reached the higher value with 30 min of sonication time. Despite this if time is too long, it may lead to the decomposition of active components of Coptis chinensis or other chemical reactions may occur. Consequently, the extraction value of palmatine and berberine decreases after 40 min.Ultrasonication is an important step in the whole extraction process; sonication time directly affects the results. It can be concluded from Coptis chinensis was the highest when the ultrasonic power reached 200W. Besides, more than 200W had almost no effect on the extraction effect, so the power of ultrasonic extraction was set at 200W , berberine , and total content  were selected as evaluation indexes. The central point was tested in quintuplicate, the response surface analysis model was of 29 test sites with 4 factors and 3 levels. Response surface establishes optimum values for the control variables that will result in maximum activity or yield over the region evaluated in a specific experimental design [According to the single-factor experimental results and Box-Behnken design principles, the test conditions are selected using the software Design-Expert 8.0.6. Moreover, the influencing factors such as moisture content (A/%), molar ratio (B), ultrasonic temperature (C/°C), and sonication time (D/min) were selected as independent variables, and palmatine ;  (mg/g) = 16.24 – 0.053A – 0.031 B + 0.016C –0.082C 0.0035D+0.026AB + 0.054AC – 0.042 AD – 0.034BC – 0.13BD – 0.011CD – 1.28AY2  2 – 4.50B2 – 2.339C2 – 2.79D2 ;  (mg/g) = 57.31 + 0.0049A + 0.016B + 0.082C + 0.078D + 0.004AB – 0.0039AC+0.0073AD – 0.017BC – 0.026BD – 0.0083CD – 0.17AY3 2 – 8.21B2 – 4.15C2 – 4.05D2 .  (mg/g) = 73.55−0.048A – 0.015 B + 0.088 C + 0.084D + 0.031AB + 0.051AC – 0.034AD – 0.052BC – 0.16BD – 0.050CD – 1.46AThe quadratic polynomial equations of the response values p < 0.001), and the misfit items were all >0.05, and the correlation coefficient R2 > 0.98. This indicated that the fitting degree of the model was high, and the equation could better reflect the relationship between the factors and the response surface.According to the results, the three models were extremely significant , and the ratio of material to liquid was 1 : 30 g/mL. Moreover, the extraction rate of berberine and palmatine was the highest with a sonication time of 30 min. The extraction rate of berberine and palmatine was affected by the ratio of material to liquid, temperature, and time in ultrasonic-assisted eutectic solvent extraction. DESs possess many preferable characteristics, including safety, nontoxicity, biodegradability, sustainability, low cost, and easy preparation, and it avoids the harm to the extraction personnel and environmental pollution.In this study, Coptis chinensis but also provides a reference for the extraction of active components of traditional Chinese medicine.The extract can be used in food, medicine, and other fields. It reflects the advantages of high yield and high speed of extracting active compounds by low eutectic solvent. This study not only provides a new method for the extraction of alkaloids from


Tisagenlecleucel (tisa‐cel) is a second‐generation autologous CD19‐targeted chimeric antigen receptor (CAR) T‐cell therapy approved for relapsed/refractory (R/R) large B‐cell lymphoma (LBCL). The approval was based on the results of phase II JULIET trial, with a best overall response rate (ORR) and complete response (CR) rate in infused patients of 52% and 40%, respectively. We report outcomes with tisa‐cel in the standard‐of‐care (SOC) setting for R/R LBCL. Data from all patients with R/R LBCL who underwent leukapheresis from December 2018 until June 2020 with the intent to receive SOC tisa‐cel were retrospectively collected at 10 Spanish institutions. Toxicities were graded according to ASTCT criteria and responses were assessed as per Lugano 2014 classification. Of 91 patients who underwent leukapheresis, 75 (82%) received tisa‐cel therapy. Grade 3 or higher cytokine release syndrome and neurotoxicity occurred in 5% and 1%, respectively; non‐relapse mortality was 4%. Among the infused patients, best ORR and CR were 60% and 32%, respectively, with a median duration of response of 8.9 months. With a median follow‐up of 14.1 months from CAR T‐cell infusion, median progression‐free survival and overall survival were 3 months and 10.7 months, respectively. At 12 months, patients in CR at first disease evaluation had a PFS of 87% and OS of 93%. Patients with an elevated lactate dehydrogenase showed a shorter PFS and OS on multivariate analysis. Treatment with tisa‐cel for patients with relapsed/refractory LBCL in a European SOC setting showed a manageable safety profile and durable complete responses. This article provides real‐world European data on the results of relapsed/refractory large B‐cell lymphoma patients treated with tisagenlecleucel. The results of two pivotal phase 2 clinical trials led to the approval of axicabtagene ciloleucel (axi‐cel) and tisagenlecleucel (tisa‐cel) by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for patients with R/R LBCL after 2 or more lines of systemic therapy. These trials had strict inclusion criteria and infused around 100 patients each, mainly in United States (US).Several single‐center and registry‐based studies have shown that treatment with axi‐cel is feasible outside the clinical trial setting with a similar safety and efficacy profile to the pivotal trial.To provide valuable information on patient outcomes with commercial tisa‐cel in LBCL, we performed a national, multicenter, retrospective study evaluating the safety and efficacy of tisa‐cel in a European real‐life setting.22.1Data were collected retrospectively on all consecutive patients with R/R LBCL who underwent leukapheresis with the intent to manufacture commercial tisa‐cel at 10 Spanish institutions from December 1st, 2018, until June 1st, 2020.For the safety analysis, we included all patients who received a tisa‐cel infusion and had a minimum follow‐up of 1 month. Efficacy‐evaluable patients included those who met the prior criteria and had an imaging response assessment. Survival outcomes were assessed in all patients who underwent leukapheresis  and in patients who received a CAR T‐cell infusion. The study was approved by the ethics committee of the Vall d’Hebron Hospital Board.2.2Patients were selected by hematologists around the country when they met technical data sheet criteria. A checklist with the usual screening tests  was forwarded to the Spanish Ministry of Health, who reviewed the proposal. Once it received approval, apheresis was performed. Bridging treatment was usually carried out at the local hospital.2/day) and cyclophosphamide (250 mg/m2/day) in all cases and started once tisa‐cel had arrived on site; if the CAR T‐cell product did not meet commercial release criteria according to EMA requirements  but it was considered acceptable by the physician, patients were offered treatment through an expanded access protocol and their results were included. After 2–4 days of chemotherapy washout, patients received the CAR T‐cell infusion in a hospitalization regimen to guarantee a close monitoring of adverse events, such as cytokine release syndrome (CRS) and immune effector cell‐associated neurotoxicity syndrome (ICANS). Management of these adverse events was carried out according to the institutional guidelines in each center. Infectious complications were managed homogenously according to the Spanish consensus guidelines.Lymphodepleting (LD) chemotherapy included three consecutive days of fludarabine  scan immediately before the start of LD chemotherapy (after the last bridging regimen) at the infusing center. Disease evaluation after CAR T‐cell therapy was scheduled at 1, 3, 6, 12, 18, and 24 months after infusion. The imaging reports were based on the Lugano recommendation for response assessment,2.3Disease status at leukapheresis was defined as one of three possibilities: (a) primary refractory if never achieving end‐of‐treatment CR; (b) refractory to last therapy if not primary refractory but not achieving a complete response to the most recent therapy, (c) or relapsed. Bridging therapy was defined as any lymphoma‐specific treatment administered after leukapheresis and before lymphodepleting chemotherapy.Grading of CRS and ICANS was performed following the American Society for Transplantation and Cellular Therapy (ASTCT) criteria.Objective response rate (ORR) was defined as the percentage of patients who achieved a partial remission (PR) or complete remission (CR) after CAR T‐cell infusion. Progression‐free survival (PFS) was defined as the time from apheresis (ITT population) or CAR T‐cell infusion until relapse, progression, or death from any cause. Overall survival (OS) was defined as the time from apheresis (ITT) or CAR T‐cell infusion until death of any cause. The duration of response (DOR) was defined as the time from CR or PR to relapse, progression, or death from any cause, whichever occurred first.2.4A descriptive analysis of all included variables in the study was performed. Continuous variables were expressed as median and interquartile range (IQR), and categorical variables were expressed as absolute values and percentages. Univariate logistic regression model was carried out to estimate the association between ORR and baseline factors. Survival analysis (PFS and OS) was calculated using the Kaplan–Meier method and the log‐rank test was used for statistical comparison. Cox proportional hazard models were used to obtain hazard ratios (HRs) with 95% CIs. For variable selection in multivariate analysis, we used the least absolute shrinkage and selection operator (LASSO) method to construct the most parsimonious model.The data analyses were carried out using R statistical software version 3.6.2.33.1Ninety‐one patients with R/R LBCL underwent leukapheresis for tisa‐cel. Seventy‐five (82%) patients received the CAR T‐cell infusion, whereas 16 (18%) did not. Reasons to not receive the infusion were: progressive disease , manufacturing failure , and psychiatric disorder . All 75 patients had at least the first disease response evaluation at 1‐month post‐infusion.8 CAR positive viable T‐cells (IQR 1.5–4.2). Eight products were considered OOS according to EMA requirements and four could not be manufactured. Reasons for OOS were low cellularity  and low viability . Six of the eight OOS products were infused  developed any grade of CRS; 21 (28%) and four (5%) patients developed grade ≥2 and grade ≥3 CRS, respectively. Eleven (15%) patients developed any grade of ICANS, whereas five (7%) and one (1%) developed grade ≥2 and grade ≥3 ICANS, respectively. The median time from infusion to the onset of symptoms of CRS and ICANS was 2 days (IQR 1–4) and 7 days (IQR 5–9), respectively. Tocilizumab and steroids were administered to 24 (32%) and 16 (21%) patients, respectively. Ten (13%) patients required admission to the Intensive Care Unit. By day 90 post‐infusion, three (4%) patients experienced treatment‐related mortality: two from bacterial infection, specifically one case of Klebsiella pneumoniae BLEE sepsis and another of multiresistant Pseudomonas aeruginosa soft tissue infection  and one from macrophage activation syndrome (MAS)The univariate analysis of risk factors for the development of adverse events is summarized in Table 3.33.3.1Among the 75 infused patients, the best response achieved was CR in 24 (32%) patients and PR in 21 (28%), with an ORR of 60%. In the ITT analysis, the best response achieved was CR in 26% (24/91) with an ORR of 49% (45/91). Patients who achieved a response (CR or PR) had a median duration of response of 8.9 months (95%CI 2.2–NA). Stable disease and progressive disease were the best response in 6 (8%) and 24 (32%) patients, respectively. Of the six infused OOS products, two patients achieved a CR, one patient achieved a stable disease and eventually progressed, whereas three patients progressed at the first disease assessment Table .Regarding the patients who achieved an initial PR at the 1‐month disease assessment, five  converted to CR at 3 (2 patients), 6, 12, and 18 months, respectively; the other patients in PR progressed in the following 3 to 6 months , or were in a maintained PR at data cutoff . Regarding the nine patients who achieved an initial SD, two patients converted to a CR at 6‐ and 18‐months post‐infusion, respectively, and one patient improved to a PR at 3 months post‐infusion; one patient remained in SD at 6 months post‐infusion and the remaining five patients had progressed at data cutoff.p = 0.05]) and a history of previous indolent lymphoma   score  Figure . In the ) Figure . There wp = 0.54) and OS in comparison to patients who did not develop grade 2 or higher adverse events. There was no significant impact of tocilizumab or steroid use on the ORR .Patients who developed grade 2 or higher CRS or ICANS had a similar ORR  and OS of 93% and 39% (p < 0.001), respectively  and 10.7 months (95%CI 7.4–NA), respectively  and 11.1 months (95%CI 7.9–NA), respectively Figure .p = 0.01] and high LDH levels  maintained the independent statistical significance in the multivariate model  had a significantly lower PFS in the univariate analysis Table . Primaryp = 0.03] and LDH levels  remained significantly associated with OS  patients received an infusion. The median time from apheresis to infusion was similar to the registration trialEight products were considered OOS in our study, mainly due to low cellularity. Low viability, a larger problem in the US, was not as frequent, probably due to the lower EMA limit in comparison with the FDA (70% vs. 80% respectively).per kg of body weight, an ECOG ≥1 and elevated LDH levels showed an increased risk of developing grade 2 or higher adverse events. In line with the pivotal CAR T‐cell trials,Focusing on the safety analysis, the incidence of severe adverse events, including both CRS (5%) and ICANS (1%), was lower than the JULIET trial , taking into account the usual caveats derived from different grading systems.Efficacy results in our study were similar to the pivotal trial. Interestingly, 12‐month PFS for patients who achieved a CR at first disease assessment was 87%, confirming that most patients in this subgroup will maintain their response over time. However, the PR to CR conversion rate was lower in our study than in the pivotal trialPatients with high LDH levels had a worse outcome after treatment with tisa‐cel. Other studies also identified LDHThere are some limitations to this study. The data were collected retrospectively and many of the baseline characteristics were captured before apheresis. Also, disease evaluation was only available previous to lymphodepleting chemotherapy; thus, it was not possible to assess the impact of disease response to bridging treatment as a prognostic factor for the efficacy of CAR T‐cell therapy. Longer follow‐up is needed to confirm the long‐term duration of complete remissions and safety after tisa‐cel therapy.To the best of our knowledge, this is the largest standard‐of‐care cohort of patients reported to date with tisa‐cel for R/R LBCL in a European country. Our results confirm that treatment with tisa‐cel in Europe is feasible and has similar results to the pivotal trial.Ethical approval: All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Ethical approval was granted by the Vall d’Hebron Hospital Ethical Committee, study identified with code PR(AG)404/2020.G.I. declares having received honoraria from BMS/Celgene, Gilead, Novartis, Janssen, and Roche, not related with this article. G.V. reported receiving honoraria for speaker activities from Merck Sharp & Dohme and an advisory role from Astrazeneca. A.M. declares having received Gilead research fundings and honoraria from Novartis and Takeda. JM. S. declares having received honoraria from Roche, Novartis, Gilead, Celgene, Janssen, Takeda, and Incyte; also consulting for Roche, Novartis, Gilead, Celgene, Janssen, Incyte, Celltrion, and Sandoz. P.A. has received honorarium for advisory and speaker faculty from Janssen, Roche, Celgene, Abbvie, Gilead, and Astrazeneca. A.M.G‐S. declares having received honoraria from Roche, Celgene, Janssen, Servier, Gilead; consulting fees from Roche, Celgene/BMS, Morphosys, Kyowa Kirin, Clinigen, Eusa Pharma, Novartis, Gilead, Servier; research funding from Janssen. JL. R‐O. declares having received honoraria from Novartis, Kite/Gilead, BMS/Celgene. P.B. declares having received honoraria from Amgen, Celgene, Gilead, Incyte, Jazz Pharmaceuticals, MSD, Novartis, Pfizer, and Roche, not related with this article. P.B. received funding from the Carlos III FIS16/01433 Health Institute, Asociación Española contra el Cáncer (Ideas Semilla 2019) and a PERIS 2018–2020 grant from the Generalitat de Catalunya (BDNS357800), not related to this study.Concept and design were undertaken by PB and GI. Data analysis and interpretation were performed by GV, PB, and GI. Collection and assembly of data were performed by all authors. All authors contributed to manuscript writing and final approval of the manuscript, and are accountable for all aspects of the work (ensuring questions related to accuracy or integrity of the work are appropriately investigated and resolved.Supplementary MaterialClick here for additional data file.


There is little evidence on adult benefits from early childhood interventions in low and middle‐income countries. We assessed adult cognition, psychosocial skills and behaviour from a stimulation trial conducted in Jamaica.SD of references) aged 9–24 months were enrolled in a two‐year randomised‐controlled trial of nutritional supplementation and/or stimulation. At mean age 31.79 (SD 0.40) years, 95 of 127 participants  were assessed. Children without stunted growth were also followed as a comparison group . Measurements included IQ, executive function, mental health, psychosocial skills, personality traits and risk behaviours. A block permutation test, valid for small sample sizes, was used. Analyses accounted for the randomisation protocol, multiple hypothesis testing and attrition.Children with stunted growth (height‐for age <−2n = 48) had significantly greater IQ  and cognitive flexibility  compared with no‐treatment . They also had reduced depressive symptoms , increased grit  and conscientiousness , lower substance use  and risk taking related to health and work . There were 18 significant outcomes of 33 assessed. Comparison participants had higher IQ than no‐treatment  and treatment groups ; and better executive function, lower social inhibition and risk taking than the no‐treatment group.Treatment group participants (stimulation intervention with or without supplementation, The wide‐ranging benefits at 31 years from the stimulation intervention supports investment in larger scale programmes to promote early childhood development in disadvantaged children. The lower IQ in the treatment group compared with comparison participants, emphasises the need for continued efforts to prevent early childhood growth retardation. There is increasing global attention to investment during early childhood as a strategy to reduce inequality  and gender. The order of group assignment was determined randomly and children in each of the four strata were assigned to: supplementation only, stimulation only, both interventions or no‐intervention. Participants were enrolled by a researcher unaware of the assignment protocol, which was done by another researcher.SD) from the same neighbourhoods were enrolled as a comparison group. Recruitment was conducted in January–November 1987 and interventions were provided for 2 years. All groups received weekly home visits from community health workers (CHW) and free health care. CHWs had at least complete primary education and had worked in the primary health system. CHWs who delivered stimulation were given 4 weeks training.Children without stunted growth , giving three composite scores: Perceptual reasoning, Verbal comprehension and Full‐scale IQ , ICC for Verbal Fluency category switching was 0.55, Card Sorting total correct 0.71 and Tower score 0.56.Scales not used previously in Jamaica were piloted, and test‐retest reliability determined (no. of respondents = 19). Intra‐class correlation coefficients (ICC) were ≥0.88. For executive function tests  was 0.71 for social inhibition, 0.59 for grit, 0.53 for self‐control and ≥ 0.83 for anxiety, depression and self‐esteem.Participants were contacted using previous locator information. Following informed consent, participants residing in Jamaica were assessed at our research unit. IQ and executive function tests were administered followed by questionnaires. We attempted to relocate migrants measured at age 22 and participants who migrated subsequently. Migrants lived in the United Kingdom, the United States and Canada. The majority were measured in their homes and a few at locations convenient to them. Measurements were conducted by three persons, blind to participants’ group assignment, between May and December 2017.Ethical approval was obtained from the University of the West Indies Ethics Committee. Written informed consent was obtained. US $50 or equivalent was given to each participant. Assessments were conducted in private and participants identified by ID number only on all tests/questionnaires.The trial used stratified randomisation based on child age on enrolment ≤16 months or >16 months and gender. These variables, as well as whether child received supplementation, and whether the mother had secondary education (which differed between treatment and no‐treatment groups) are used to partition the data into subsets that contain both treatment and no‐treatment participants Appendix . The estWe account for multiple outcomes using the stepdown procedure  and 64 of 84 comparison participants (76.2%). Attrition in the trial was 27.7% in the no‐treatment group and 22.6% in the treatment group. Reasons for loss are shown in Figure The only differences in enrolment characteristics were child weight‐for‐height and mother’s education Table . There wTable The treatment group had significant gains in full scale IQ scores, verbal and perceptual sub‐scales with moderate effect sizes.Significant gains in the card sort scores indicate greater cognitive flexibility. The rank mean score indicates an overall gain for executive function tests.The treatment group had reduced depressive symptoms. Anxiety, and social inhibition were not significantly different. Rank mean analysis showed overall better mental health.Treated participants had higher grit scores. Self‐esteem and self‐control were not significantly different. Rank mean analysis showed an overall effect.The treatment group had significantly higher conscientiousness. There were no other differences in personality.The treatment group had reduced alcohol use but no significant difference in marijuana use, the rank mean score indicates reduced overall substance use.The treatment group were less likely to take risks related to health, work and trust in others. Rank scores also indicate reduced risk taking.There were no significant differences in fights and use of weapons, gun use and gang membership, or in overall rank scores.The pattern of treatment effects for non‐migrants was similar to the total sample Tables .Exploratory analyses by gender showed no differences in treatment effects other than reduced risk taking in women only Tables .Correction for attrition produced qualitatively equivalent results Tables .SD. No significant difference in IQ was detected at 7 years, however, the impact increased at age 11 years and was also significant at 17, 22 and 31 years. The treatment group had significantly reduced depressive symptoms at 17, and this remained lower at 22 and 31 years.Figure Differences between the comparison group and the no‐treatment and treatment groups were examined Tables . CompariParticipants from the Jamaica stimulation trial were followed to age 31 years and showed sustained intervention benefits to IQ and depressive symptoms. Benefits to executive function, grit and conscientiousness, lower substance use and risk taking are shown for the first time. This is the longest follow up of a randomised controlled trial of an early childhood stimulation intervention in LMIC and included a wide range of measurements. Retention was reasonable for follow‐up of this duration.The initial impact of the intervention on cognition declined and was no longer significant at age 7 years  and similar to that with emotional stability (r = .26). One adult follow‐up that measured grit found no benefits (Heckman & Karapakula, The treated group also had higher levels of grit. Most literature on grit is from high income countries and questions whether grit is a separate trait or part of conscientiousness as grit correlated highly with conscientiousness and moderately with emotional stability (Credé, Tynan, & Harms, Alcohol and overall substance use were lower in the treated group. Reduced marijuana use (Campbell et al., The mechanisms for sustained intervention benefits are unknown, however, the intervention aimed to improve task‐orientation and sustained attention and included many problem‐solving activities. Children’s efforts and success in completing tasks also received much positive feedback and celebration. These may have contributed to benefits in grit, executive function and possibly conscientiousness. We also focused on building the mothers’ self‐esteem and child‐rearing skills which may have contributed to sustaining benefits. Finally, the initial cognitive benefits were large and may have contributed to gains in other domains.SD, using a one‐sided test. We adjusted for multiple comparisons within domains and significant findings were of moderate to large effect size. Nonetheless, multiple testing is still a concern given the number of outcomes and subgroup analyses. For two of the executive function tests, the test‐retest reliability was low. There were few statistically significant differences on enrolment, with lower maternal education and child weight‐for‐height in the treatment group. Mothers with less education, and their children, may respond differently to interventions and maternal education was included in the conditional variables.Small sample size is a study limitation. We used a permutation test appropriate for small sample inference. The current sample size has 80% power at 5% significance test to detect a difference of 0.5The study provides evidence of life‐course benefits from a relatively low input intervention across cognitive, psychosocial and behavioural variables that may reduce intergenerational poverty transmission. The participants were extremely disadvantaged, and the level of disadvantage in untreated groups increases the likelihood of long‐term gains (Bailey, Duncan, Odgers, & Yu, Analysis with the comparison group showed that the no‐treatment group had large deficits in IQ and cognitive flexibility. Despite substantial gains, IQ also remained lower in the treatment group. Stunting affects 149 million children under 5 years UNICEF,  and thesThis low‐cost, feasible early childhood intervention had life changing effects, similar to higher input interventions (Campbell et al., Appendix S1. Attrition at 31 years old.Appendix S2. Sampling variation and permutation blocks.Appendix S3. Description of empirical evaluations.Table S1. Conditional block permutation inference on participants characteristics using t‐statistic – all data.Table S2. Treatment effects on cognitive, psychosocial and personality outcomes for non‐migrants and by gender.Table S3. Treatment effects on risk taking and violence behaviors for non‐migrants and by gender.Table S4. AIPW inference on cognitive, psychosocial and personality outcomes conditional all data.Table S5. AIPW inference on risk taking and violence behaviors conditional all data.Table S6. AIPW inference on cognitive, psychosocial and personality outcomes conditional summary.Table S7. AIPW inference on risk taking and violence behaviors conditional summary.Table S8. Comparison vs. no‐treatment conditional inference on cognitive, psychosocial and personality outcomes.Table S9. Comparison vs. no‐treatment conditional inference on risk taking and violence behaviors.Table S10. Comparison vs. treatment conditional inference on cognitive, psychosocial and personality outcomes.Table S11. Comparison vs. treatment conditional inference on risk taking and violence behaviors.Click here for additional data file.


Labs as guide dogs or sniffer dogs in usage have been introduced into China for more than 20 years. These two types of working dogs own blunt or acute olfactory senses, which have been obtained by artificial selection in relatively closed populations. In order to attain stable olfactory attributes and meet use-oriented demands, Chinese breeders keep doing the same artificial selection. Though olfactory behavior is canine genetic behavior, genotypes of OR genes formed by breeding schemes are largely unknown. Here, we characterized 26 SNPs, 2 deletions, and 2 insertions of 7 OR genes between sniffer dogs and guide dogs in order to find out the candidate alleles associated with working specific traits. The results showed that there were candidate functional SNP alleles in one locus that had statistically severely significant differences between the two subpopulations. Furthermore, the levels of polymorphism were not high in all loci and linkage disequilibrium only happened within one OR gene. Hardy–Weinberg equilibrium (HWE) tests showed that there was a higher ratio not in HWE and lower FST within the two working dog populations. We conclude that artificial selection in working capacities has acted on SNP alleles of OR genes in a dog breed and driven the evolution in compliance with people's intentions though the changes are limited in decades of strategic breeding. Artificial selection has been largely responsible for breed-specific and strain-specific traits of modern dogs . CompareThe offsprings of pure breed Labs as guide dogs or sniffer dogs abroad were purchased from Japan by guide dogs training organizations and from Germany by police departments into China two decades ago. The same as foreign ones did, Chinese working dog organizations continued to breed Labs following the principle of closed-flock reproduction and occasionally adopted some excellent bloodlines of Labs from amateurs in the civilian clubs into Lab working dogs populations. Based on Labs' health, a temperament test of scent interest and a search test divide Labs into candidates of guide dogs with no scent interest or sniffer dogs displaying great scent interest and eager searching during early training –12. ThenOlfactory capacity and sensitivity are related to canine olfactory receptor gene polymorphism in dog breeds and individuals , 13. SelThis study was carried out in Police-Dog Technology Department, Criminal Investigation Police University of China. The protocol for the animal experiment was reviewed and approved by the Ethics Committee of Police-Dog Technology Department (JQJS2019-0001), and all applicable institutional and governmental regulations concerning the ethical use of animals were followed.n = 36; females, n = 13) and 43 sniffer dogs  of Labs, whose ages ranged from 2 to 4 years old. The Labs were reproduced by China Guide Dog Training Center and Police-Dog Technology Department, Criminal Investigation Police University of China. According to the certificates of the dogs' pedigree, the two groups sampled were unrelated. All the dogs treated in a humane manner passed the qualification assessments of working dogs and had been competent for the tasks assigned. Blood samples were obtained from each dog, and then Universal Genomic DNA Extraction Kit  was used to extract DNAs.The trial included two subpopulations, 49 guide dogs , and 1 μM  of each specific primer and water up to 50 μL. PCR products were loaded onto a 2% agarose gel stained with GeneFinder  and DNAs were extracted from the gel. The purified products of PCR were cloned into pMD18-T vector  and positive clones specific for each of the seven amplicons were sequenced in both directions. Nucleotide BLAST of the sequences was done. Then the purified products of PCR were massively directly sequenced by the forward or reverse primers.Polymerase chain reaction (PCR) was conducted using a Veriti thermal cycler  in a total volume of 50 μl reaction system that consisted of Premix 4 μL, the purified PCR products 0.5 μL, primer 1 μL (1 μM), and 4.5 μL ddH2O, starting with 30 cycles of denaturation at 96°C for 20 secs, annealing at 55°C for 20 secs, and extension at 60°C for 4 mins, followed by an extra stop reaction initiated by adding 0.5 μL of glycogen, 1 μL of 3M sodium acetate (pH5.2), and 1 μL of 100 mM Na2-EDTA (pH8.0) per reaction. 40 μL of sample loading solution was added into DNA precipitates with ethanol in individual tubes to resuspend the samples. The capillary electrophoresis was analyzed on Beckman GeXP Genetic Analysis System according to Genome-Lab Dye Terminator Cycle Sequencing with Quick Start Kit .Sequencing PCR reaction was performed in a 10 F-value of an inbreeding coefficient was derived by [1- (Ho/He)] by EXCEL software too [r2) and the chi-square test about alleles on single locus in a pairwise study were analyzed by SHEsis online tool. A logistic regression analysis of alleles and genotypes was performed on IBM SPSS Statistics 25 software. Fst values were calculated by GenAlEx 6.5 software [http://provean.jcvi.org/index.php [http://agvgd.hci.utah.edu/ [https://sift.bii.astar.edu.sg/ [Allele frequency, Genotype frequency, observed heterozygosity (Ho), and polymorphism information content (PIC) were calculated by PowerStats software v1.2 . Expecteware too . Hardy–Wware too . Linkagesoftware . The toondex.php , Align-G.edu.sg/  were useP < 0.05), and highly significant differences (P < 0.001) were shown in the four SNP loci of which the three SNP loci were breed-specific between the two working groups given in P < 0.05) given in Allele frequencies and genotype frequencies of 30 SNP loci are given in In this study, except for 12 homozygous monogenotype loci, PIC values of 18 SNPs ranged from 0 to 0.375. Among the 18 SNPs, 10 SNPs belonged to low polymorphism (PIC<0.25) and 5 SNPs belonged to moderate polymorphism (0.25 < PIC<0.5) for all Labs. As far as the other three SNPs, OR0007:c.331 T >C, OR0007:c.430 G > A, and OR7215:c.251 C >T, were concerned, they belonged to either low or moderate polymorphism, which differed between guide dogs and sniffer dogs seen in . As coulP values of HWE tests for 18 SNP loci were given in P = 0.0005 emphasized the need for multiple comparisons by applying Bonferroni correction (the significant level = 0.05/92). Except 12 homozygous monogenotype loci, the results revealed that 5 SNP loci were out of HWE (P < 0.0005) and 5 SNP loci were in the state of Hardy–Weinberg balance (P > 0.0005) for all Labs. The other eight SNP loci showed either significant departure or no significant deviations from HWE between working populations, while six of the eight SNP loci were in HWE in the collective. After the two working groups and the collective were analyzed, respectively, there was a higher ratio 73.3% not in HWE in sniffer dogs than 66.7% in guide dogs and 63.3% in the collective. Furthermore, the value of genetic differentiation between the two working populations was 0.054 (0.05﹤FST﹤0.15) when the loci OR0006:c.524 G > T, OR0007:c.331 T >C, OR0007:c.430 G > A, OR0007:c.793 G > C, and OR16C11:c.294 G > A in HWE were considered.F-values of average inbreeding coefficient were −0.226 for guide dogs and −0.241 for sniffer dogs given in r2 value that was more than 0.80 was shown only within OR0007 gene for the two working dog groups (see The oups see .Nonsynonymous mutations are divided into two kinds of being tolerated (>0.05) and deleterious (≤ 0.05) with SIFT. The biological impact effects of amino acid substitution induced by nonsynonymous SNPs were considered “deleterious” (<−2.5) and “neutral” (>−2.5) by PROVEAN v.1.1.3. We estimated 24 nonsynonymous SNPs using SIFT, PROVEAN, and Align GVGD. 6 SNPs were predicted to be “untolerated” with a score below 0.05 by SIFT, 7 SNPs are predicted to be “deleterious” by PROVEAN and 10 SNPs were classified as a class C65 change, the first highest pathogenicity category by Align GVGD see . OR0006:The aim of this study was to explain how use-oriented selection could lead to phenotypic changes of olfactory behavior in Labs. Olfactory traits of guide Labs and sniffer Labs are expected to play a role in canine behavioral genetics . We focu>T in any Labs group showed that selective breeding in working dog populations had tried to gain popular hereditary features and avoid inbreeding in a limited degree. Furthermore, the mean F-values of inbreeding coefficients verified the negative regression in the two working dog groups. Low levels of linkage disequilibrium obtained for every working dog subpopulation were consistent with homogeneity within a dog breed [Meanwhile, the loss of genomic diversity (PIC<0.375) resulted from strong selective pressures of olfactory behavior through gene pathways . In addiog breed , 33. ThiThe analysis of 26 SNPs by the collective and two working Labs groups showed that the proportions of being in HWE were lower than 52.6% in a former study of Labs that was a lower proportion among dog breeds . It was In silico prediction tools, SIFT, PROVEAN, and Align GVGD evaluated the pathogenicity of missense variants of 24 SNPs because bioinformatics analysis alone was usually not sufficient . In ordeA number of analyses were conducted to reveal canine breed phylogeny or relation with some complicated qualities due to SNP variation. However, few studies aimed at subpopulation differentiations within a same dog breed. The strong artificial selection was only performed urgently to gain working attributes. Labs working in different fields acted as the sample model. SNP loci in OR genes presented indexes on the positive and negative selection, namely, a preference for acute sniffing and being numb about odor. Our analysis demonstrated that possibly functional alleles were quickly evolving in compliance with people's intentions though the changes were limited in decades of artificial selection.


Vicia faba L.; FB) seeds relative to their nutritional composition, their content of antinutritional factors, and their impact on animal performance. The literature indicates that FB plant is a cool‐season, annual grain legume that grows the best in cool and humid conditions. Its seeds are rich in protein, energy, and mineral compounds and have particularly high unsaturated fatty acid levels. However, FB seeds also contain various proportions of antinutritional factors (ANFs) that can interfere with nutrient utilization in nonruminants. The various processing methods are efficient in either reducing or inactivating the ANFs of FB seeds, with extrusion treatment offering the most effective method of improving apparent nutrient and energy digestibility of nonruminants. In vivo studies on ruminants, pigs, poultry, and fishes reveal that FB seeds have the potential to be used as a substitute for soybean meal and/or cereal seeds in livestock diets in order to support milk, meat, and/or egg production.The review evaluates faba bean ( Faba bean plant is a cool‐season, annual grain legume that grows the best in cool and humid conditions. Its seeds are rich in protein, energy, and mineral compounds and have particularly high unsaturated fatty acid levels. Its seeds have the potential to be used as a substitute for soybean meal and/or cereal seeds in livestock diets in order to support milk, meat, and/or egg production. These APisum sativum L.), FB seeds contain similar amounts of tannins and polyphenols, but relatively less TIA, genistein, and daidzein content  and 0.810 in cows and heifers Table , respectIn ram diets containing FB seeds 162 g/kg), the apparent digestibility of DM, OM, and CP was somewhat lower than that of the SBM control diet  diet (500 g/kg), the apparent ileal DM and OM digestibility of male broilers fed a raw FB diet (500 g/kg) was markedly higher, while the apparent ileal CP and amino acid digestibility in raw FB diets were similar to or somewhat lower than the lupin diet. In contrast, 6‐ to 32‐day‐old male broilers fed raw FB seed diets had markedly higher apparent ileal DM and OM digestibility than broilers fed the SBM control diet (170 g/kg), but apparent ileal digestibility of DM, OM, and CP were generally unaffected in broilers fed with FB seed diets with inclusion levels of 80–160 g/kg  of FB seed only change slightly when subjected to cooking, autoclaving, flaking, and germination Table  Ferruzz. The extCooking, autoclaving, dehulling, flaking, extrusion, and germination changed the in vitro digestibility of the nutrients in FB seeds. In vitro CP digestibility of FB seeds significantly increased by 10.2%, 14.1%, and 11.8% after cooking in tap water (3 ml/g dry seeds), autoclaving at 121°C for 30 min, and germinating at room temperature for 3 days, respectively, because of the reduction or inactivation of ANFs  seeds with raw FB seeds on milk yield and composition of dairy goats produced no differences in milk yield, or milk fat, protein, and lactose contents  or a FB seed diet (538 g/kg) had similar final body weight  and ADG (0.226 kg/day) [60]. In another study, Lestingi et al.  studied longissimus dorsi muscle when fed with a SBM diet (160 g/kg of diet) or a diet containing 242 g/kg of raw FB seeds  , and fish diets at appropriate levels of up to 300, 110, 160, and 420 g/kg, respectively. The inclusion of higher levels of FB seeds in pig, poultry, and fish diets is possible after the ANFs have either been eradicated or reduced with the use of preprocessing treatments to improve the nutritional value of the seeds. Of the preprocessing treatments, extrusion treatment facilitates the inclusion of higher FB seed levels in the diets of nonruminants without any adverse effects on their performance.The authors declare that they have no competing interests.Zhu Meng: Software ; Writing‐original draft ; Writing‐review & editing . Qingqing Liu: Data curation ; Investigation ; Methodology ; Writing‐original draft ; Writing‐review & editing . Yan Zhang: Data curation ; Software . Jiahong Chen: Data curation ; Investigation ; Software . Zhipeng Sun: Data curation ; Investigation ; Software . Chunhuan Ren: Data curation ; Funding acquisition . Zijun Zhang: Funding acquisition ; Writing‐review & editing . Xiao Cheng: Data curation ; Writing‐original draft . Yafeng Huang: Data curation ; Formal analysis ; Software ; Writing‐original draft ; Writing‐review & editing .No ethical approval was required, as this is a review article with no original research data.


We report a case of fever of unknown origin in a patient with MDS associated with IgM‐MGUS. The patient was positive for MYD88 mutation, and chemotherapy for LPL/WM improved the fever. Analysis of MYD88 and the effect of chemotherapy on LPL/WM finally revealed the latent LPL/WM in this case. We report a case of fever of unknown origin in a patient with MDS associated with IgM‐MGUS whose IgM level and rate of tumor cells were low. A positive MYD88 mutation and the effect of chemotherapy on LPL/WM finally revealed the latent LPL/WM. Approximately 10%–30% of MDS cases have immune system disorders leading to fever.Lymphoplasmacytic lymphoma (LPL) is a lymphoid malignancy with immunoglobulin M (IgM) monoclonal gammopathy comprising minute monoclonal B cells, including mature B cells, differentiating into plasma cells. LPL with bone marrow invasion is defined as Waldenström's macroglobulinemia (WM).We have encountered an MDS case with recurring fever of unknown origin. No evidence of infection, tumor fever, or immunological diseases was found via various examinations. Bone marrow aspiration was consistent with low‐risk MDS as observed at diagnosis. Monoclonal IgM was positive but at low levels, consistent with monoclonal gammopathy of undetermined significance (MGUS).The positive MYD88L265P mutation and good response to LPL/WM‐targeted therapy were useful for diagnosing underlying LPL/WM, a rare complication of MDS.2A 55‐year‐old man was diagnosed with classical Hodgkin's lymphoma and achieved complete remission by chemotherapy. At the age of 60, he was diagnosed with left renal cell carcinoma and achieved remission by left kidney resection. In November of 2016, at the age of 73, the patient was hospitalized for pneumonia and determined to have anemia. During the same hospital stay, he was also diagnosed with treatment‐related MDS  by bone marrow aspiration, and with IgM‐MGUS by serum protein electrophoresis and immunoelectrophoresis  for MYD88L265P using marrow cells was positive Figure ,2, 3Based on the history of IgM‐MGUS, monoclonal B cells in the bone marrow, and positive MYD88L265P mutation, we considered that the tumor fever was due to LPL/WM developed from MGUS as a differential diagnosis.Although MYD88 mutation has a high sensitivity for LPL/WM, its specificity is low. A search of the literature Table 4, 5,2 on days 1, 4, 8, and 11; dexamethasone [40 mg] /body on days 1, 4, 8, and 11; and rituximab 375 mg/m2 on day 11The MYD88‐positive rate is also high in IgM‐MGUS. We thus could not rule out IgM‐MGUS from LPL/WM. Therefore, in August of 2018, we administered bortezomib, dexamethasone, and rituximab (BDR) .Many rituximab‐containing regimens are effective for WM/LPL based on phase II trials. In this case, BDR was selectively administered because of the renal impairment (creatinine clearance rate: 18 ml/min/1.73 mThe prevalence of MGUS in patients with MDS ranges between 2% and 10%. Moreover, in a study on 1,198 patients with MDS, 6 patients had complications of LPL.Clinicians should watch for symptomatic LPL/WM in their patients with MDS, as in the present patient, whose IgM level and rate of tumor cells were low. In this patient, MYD88 mutation analysis and BDR therapy played a significant role in diagnosing LPL/WM and informing the treatment strategy.Not applicable.The authors declare no conflict of interest.Yotaro Motomura drafted the article and provided and cared for study patients. Yoshihiro Umezawa and Osamu Miura served as scientific advisors. Tomoyuki Arimatsu and Keigo Okada provided and cared for study patients. Takashi Kumagai critically revised the article for important intellectual content.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


The optimized formulation was characterized by particle size, drug entrapment efficiency, Fourier transform infrared, Transmission electron microscopy analysis and drug release behavior. Pure doxycycline hydrochloride  and optimized doxycycline hydrochloride encapsulated Tween 80 coated chitosan nanoparticles (DCNPopt)  were explored against ketamine induced psychosis in mice. The experimental studies for DCNPopt, with mean particle size 237 nm and entrapment efficiency 78.16%, elucidated that the formulation successfully passed through blood brain barrier and exhibited significant antipsychotic activity. The underlying mechanism of action was further confirmed by behavioral, biochemical, neurochemical estimations and histopathological study. Significantly enhanced GABA and GSH level and diminished MDA, TNF-α and dopamine levels were observed after administration of DCNPopt at just half the dose of pure doxycycline hydrochloride, showing better penetration of doxycyline hydrochloride in the form of Tween 80 coated nanoparticles through blood brain barrier. This study demonstrates the hydrophilic drug doxycycline hydrochloride, loaded in Tween 80 coated chitosan nanoparticles, can be effectively brain targeted through oral delivery and therefore represents a suitable approach for the treatment of psychotic symptoms.To develop statistically optimized brain targeted Tween 80 coated chitosan nanoparticulate formulation for oral delivery of doxycycline hydrochloride for the treatment of psychosis and to evaluate its protective effect on ketamine induced behavioral, biochemical, neurochemical and histological alterations in mice. 3 Psychosis, a debilitating neuropsychiatric disorder, is characterized by positive, negative and cognitive symptoms. Complex pathophysiological mechanisms evolved in the progression of psychosis include GABAergic, dopaminergic, cholinergic dysfunction, hypofunctioning of NMDA receptors along with neuroinflammation and oxidative stress impedes its treatment  Laboratories Private Limited, India. Chitosan and sodium tripolyphosphate (TPP) were purchased from Hi-Media, Mumbai, India. Olanzapine and ketamine were obtained from Ranbaxy Laboratories Limited and Neon Pharmaceutical Private Limited, India, respectively. Other chemicals used in the present study were of suitable analytical reagent grade.Swiss albino male mice  were purchased from Disease Free Small Animal House, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar (Haryana), India. Since, estrogens  have neuroprotective effect  and X2 , were adopted for as required by the design and the factor levels were suitably coded. Doxycycline hydrochloride loaded chitosan nanoparticles (DCNP) were prepared using modified ionotropic gelation method  of the formulated DCNP was determined by dynamic light scattering technique, while zeta potential was measured using electrophoretic light scattering technique .y = 0.025x − 0.055, (R2 = 0.999), where y represents absorbance and x represents concentration (mg/ml). Amount of drug present in the supernatant was subtracted from the total amount of drug added and accordingly DEE was calculated.The supernatant, after centrifugation of DCNP , was collected, filtered through 0.22 μm membrane filter and amount of drug present was measured at 347 nm by UV–Visible spectrophotometer . The amount of drug in supernatant was calculated using the equation in vitro and in vivo studies.Design Expert Software, version 10.0.6.0  was employed to fit polynomial equations with attached interaction terms for the correlation of studied responses with chosen variables. Particles size and DEE were selected as response variables for systematic optimization. Optimized formulation was found by locating feasible space as well as exhaustive grid search was done for tracing the possible solution. Optimum solution was also provided by the software using the overlay plots. The optimized formulation (Tween 80 coated DCNP) was utilized for all the in vitro drug release from optimized batch of DCNP in a USP Type II (Paddle type) apparatus. Optimized DCNP  were dispersed in 5 ml phosphate buffer saline (PBS) (pH 7.4) and placed in a dialysis membrane bag , previously soaked in distilled water for 1 hour. The bag was placed in 300 ml PBS (37 ± 1 °C) at 50 rpm in dissolution apparatus. Aliquot of 5 ml was withdrawal at regular time intervals and replenished with equal volume of fresh dissolution media. The samples were analyzed, in triplicate, for the concentration of doxycycline hydrochloride using UV–Visible spectrophotometer  at 347 nm against suitable blank and the amount of drug released at various time intervals was calculated. Mechanism of release was determined by fitting in vitro drug release data in different drug release kinetic models like zero order, first order, Hixson-Crowell, Higuchi and Korsemeyer Peppas.Equilibrium dialysis technique was employed for the study of High resolution TEM  was used for the size and morphological characterization of the optimized DCNP batch. Formulated nanoparticles were suspended in HPLC grade water and sonicated (4 min) for disaggregation of nanoparticles. The sample was placed on a carbon coated grid (300 mesh), negatively stained with 2% phosphotungstic acid (pH 7.0) and analyzed using HR TEM at 200 kV.opt) by KBr pellet method.FTIR technique  was used to rule out any chemical interaction between chitosan, doxycycline hydrochloride and optimized DCNP  only, group 3 received olanzapine  followed by administration of ketamine  after 30 minute of olanzapine, group 4 received placebo  followed by administration of ketamine  after 30 minute of placebo, group 5 received doxycycline hydrochloride  followed by administration of ketamine  after 30 min of doxycycline hydrochloride, group 6 received doxycycline hydrochloride  followed by administration of ketamine  after 30 min of doxycycline hydrochloride, group 7 received doxycycline hydrochloride  followed by administration of ketamine  after 30 minute of DCNPAcute administration of ketamine produces hyperlocomotor activity, whereas on chronic administration, it produces depressive and cognitive symptoms of psychosis  containing water up to the height of 15 cm (23 ± 2 °C). Acquisition of the state of immobility with minimum floating is considered as behavioral despair akin to depressive symptoms of psychosis. Mouse was placed for 2 min in the chamber for habituation and afterwards immobility period was recorded for the next 4 min  was used to measure the effect of DCNPPassive avoidance test is commonly used to measure the SDL which is a parameter to check the memory of small animals. The model consists of a chamber (27 × 27 × 27 cm) whose one wall is made of plexiglas, three walls made of wood, besides a grid floor with wooden platform. A 15 W bulb was used to illuminate the chamber and 20 V AC electric shocks were given to floor during the experiment. Experiment was performed into two learning sessions followed by one memory session. During the first learning session, animal was placed on wooden platform and when it stepped down on grid floor with all its paws before 60 s, shocks were delivered for 15 s. Next learning session was performed after an interval of 90 min, in which animals were free from shock. SDL is defined as the time taken by the animal to step down with all its paws from the wooden platform to the grid floor. Memory session was performed on next day and the animal was observed for 300 s to evaluate the cognitive function of animals. Animals were not provided shocks during the memory session .Animals in groups 1–7 were sacrificed using cervical decapitation and immediately brain was removed, washed, weighed and divided into two equal parts. Homogenate of one part was prepared in 0.1 M phosphate buffer (pH 7.4) and used for the estimation of GSH, MDA and total protein level. Other part of brain was homogenized in phosphate buffer (pH 8) and then centrifuged at 3000 rpm for 10 min. The supernatant so obtained was used for the estimation of AChE activity.4 M−1 cm−1 and results expressed as µmole/mg brain protein  and centrifuged at 5000 rpm for 10 min. Then, 2 ml disodium hydrogen phosphate buffer  and double distilled water (0.4 ml) were added into 0.5 ml of the obtained supernatant. 0.25 ml of 0.001 M DTNB  was added into above mixture, followed by incubation at room temperature for 10 min. Developed yellow color was read at 412 nm using UV–Visible spectrophotometer. GSH level was calculated using molar extinction coefficient of 1.36 × 10 Ellman, .5 M−1 cm−1 and results are expressed as nmole of MDA/mg brain protein  was estimated in the form of thiobarbituric acid reactive substances (TBARS). About 0.5 ml brain homogenate and 0.5 ml tris–HCl buffer (pH 7.4) was mixed and incubated at 37 °C for 2 h. After incubation, 1 ml of TCA was added and centrifuged at 1000 rpm for 10 min. About 1 ml of supernatant so obtained was added with 1 ml of 0.67% thiobarbituric acid (TBA) and kept on boiling water bath for 10 minute. After cooling, 1 ml double distilled water was added and absorbance was read at 532 nm. TBARS level was calculated using extinction coefficient of 1.56 × 10n Wills, .The reaction mixture consists of 0.4 ml separated supernatant, 0.1 ml DTNB  and 2.6 ml of 0.01 M sodium phosphate buffer (pH 8) and its absorbance was taken on UV–Visible Spectrophotometer at 412 nm. Further, 0.02 ml acetylthiocholine iodide solution added, stand for 15 min and absorbance was again taken at 412 nm. The change in absorbance was calculated and results expressed as mmol/minute/gram of brain weight Ellman, .Animals in groups 8–14 were sacrificed and brain was isolated, washed, weighed and divided into two equal parts. One part was homogenized in 3 ml of HCl-butanol (0.1 M HCl in butanol) for the estimation of dopamine level while another part was homogenized in 5 ml of 0.01 M HCl for the estimation of GABA level.Schlumpf et al.  method wAbout 0.1 ml of brain homogenate was mixed with 0.2 ml 0.14 M ninhydrin solution (in 0.5 M carbonate bicarbonate buffer of pH 9.95) and heated on water bath at 60 °C for 30 min After cooling, 5 ml of copper tartrate reagent  was mixed and stand for 10 min followed by reading of absorbance at 377/455 nm using spectrofluorimeter  was used to preserve the isolated brains, which were fixed into paraffin. Paraffin sections were prepared and stained with hematoxylin and eosin for histopathological studies.n = 6). The difference between the different groups were analyzed using analysis of variance (ANOVA) followed by Tukey’s test using GraphPad Instat. The difference more than p < .05 was consider as statistically significant.All the data are observed as mean ± SEM  and amount of Tween 80 (B) was investigated on the two response variables viz. particle size and DEE. Average particle size of all the batches ranges between 198 and 257 nm, while the DEE was found between 72.38 and 86.62%. The generated polynomial equations were used for the analysis of the responses and analysis of variance was done using the statistical parameters like sum of squares, degree of freedom, mean sum of squares and F value using the software Design Expert. The significance of the developed model was judged based on p value, F value, correlation coefficient (R2) and adjusted correlation coefficient (adj R2).3The polynomial equations obtained using regression analysis for the responses (particle size and DEE) are as follows:F-value and p-value; in our case, it is less than .05 at 95% confidence level. The values for particle size are Df = 5, F = 7.91, p = .0085, R2 = 0.8496, Adjusted R2 = 0.7421, while for DEE Df = 2, F = 23.60, p = .0002, R2 = 0.8252, Adjusted R2 = 0.7902. Significance of the developed model is further represented by sufficiently high values of R2 and adjusted R2. The large F-value obtained with Y3, indicates that the effect variance is much higher than the error variance.In the above regression equations, the positive and negative symbols before the coefficients (main and interaction terms) represent their synergistic or antagonistic effect on the responses, respectively. The significance of the developed models was established on the basis of the calculated Using the desirability approach for numerical optimization and desired responses, solution for optimized batch was obtained by setting the parameters viz. minimum particle size (198–250 nm) with maximum entrapment efficiency (70–90%). More importance was given to particle size than DEE. The parameters as suggested by Design Expert software for optimum formulation were chitosan 0.4765  and Tween 80 0.4212  which resulted in the formulation of nanoparticles with a particle size of 212.86 nm and DEE 80.449%. The percent mean error between predicted values and experimental values was found to be 9.69% (for particle size) and 2.84% (for DEE) indicating the success of optimization process for DCNP.The 3D surface plots showing the effect of formulation factors on the response variables, have been depicted in opt, shown in TEM analysis of DCNP−1 due to the O–H stretching vibrations. The presence of aromatic ring in the structure of doxycycline hydrochloride is shown by aromatic C–H stretching at 3001 cm−1 and strong absorption band at 923 cm−1 due to out of plane bending of ring C–H bonds. The band at 1702 cm−1 is due to presence of carbonyl group (C = O stretching), while the C–C–C stretching and C–C(=O)–C bending is shown at 1248 cm−1. Amide I band due to carbonyl absorption at 1645 cm−1 and amide II band due to N–H bending at 1572 cm−1 confirms presence of amide group in the structure of doxycycline hydrochloride, however, N–H stretching bands overlaps with the O–H stretching bands in the region 3500–3100 cm−1. Strong absorption at 1093 cm−1 is attributed to C–N stretching of amine group. On the other hand, FTIR spectra of chitosan shows intense broad band in the region 3600–3150 cm−1 due to O–H stretching vibrations. The C–H stetching vibrations are present at 2925 cm−1 and N–H stretching vibrations merges with O–H stretching vibrations in the region 3600–3150 cm−1. N–H bending (scissoring) of amine is present at 1645 cm−1, while C–N stretching is present at 1078 cm−1. Peak of C–O–C in the glucopyranose ring is present at 1030 cm−1, while the specific bands of β (1 → 4) glycosidic bridge at 1154 and 898 cm−1  resulted in correlation coefficients (R2) 0.854, 0.593, 0.999, 0.962, and 0.691 for zero order, first order, Higuchi, Korsemeyer-Peppas and Hixson Crowell models, respectively. Higuchi model was depicted as best fit model with R2 value of 0.999, which represents drug release through matrix system. Here drug release is as a diffusion process based on Fick’s law  decreased the hyperlocomotor activity induced by ketamine. Pure doxycycline hydrochloride  (p < .001) and DCNPopt  (p < .001), significantly [F  = 31.412] decreased the ketamine induced hyperlocomotor activity. Furthermore, DCNPopt  was also remarkable to diminish the locomotor counts in comparison to pure doxycycline hydrochloride even at 50 mg/kg, p.o. The effect of DCNPopt  on locomotor activity was statistically equivalent to olanzapine   significantly increased locomotor activity after administration for seven days. Olanzapine  significantly  .p < .001) decreased the stereotyped behaviors induced by ketamine. Treatment with pure doxycycline hydrochloride   and DCNPopt  (p < .001) was significant [F  = 64.646] to decrease the ketamine produced stereotyped behaviors. Moreover, DCNPopt  was also found to be remarkable to lessen the stereotyped behaviors as compared to pure doxycycline hydrochloride at the dose level of 50 mg/kg, p.o. The effect of DCNPopt  on stereotyped behaviors was not significant in comparison to olanzapine  which reveals effect of DCNPopt is statistically equivalent to olanzapine for the treatment of ketamine induced stereotyped behaviors  administration for 10 successive days induced stereotyped behaviors in mice. Olanzapine  significantly (ehaviors .p < .001) and DCNPopt  (p < .001) [F  = 126.09]. However, DCNPopt even at the dose of 25 mg/kg was more effective than pure drug at the dose of 50 mg/kg in alleviating the immobility duration in FST. Olanzapine  significantly (p < .001) decreased the immobility duration increased by ketamine. The effect of DCNPopt  on immobility duration was statistically equivalent to olanzapine   for 14 successive days, which was effectively decreased by the treatment of pure doxycycline hydrochloride   .opt  did not show any significant difference in step down latency in comparison to ketamine. Olanzapine  significantly (p < .001) [F=  = 101.82] increased the step-down latency decreased by ketamine. However, DCNPopt  did not produced increase in step-down latency in comparison to olanzapine  (p < .001)  for 20 successive days, which indicates memory loss. Pure doxycycline hydrochloride  and DCNP < .001) .opt  significantly  decreased TNF-α level as compared to ketamine treated animals. DCNPopt  was also remarkable to reduce TNF-α level as compared to pure doxycycline hydrochloride  indicating better penetration into systemic circulation. Olanzapine  significantly (p < .001) decreased the serum TNF-α level increased by ketamine. Also, the effect of DCNPopt  on serum TNF-α level was statistically equivalent to olanzapine   administration for 21 successive days. Treatment with pure doxycycline hydrochloride  and DCNPg, i.p.) .opt  . DCNPopt was found to be more effective to increase GSH level than pure drug as DCNPopt showed pronounced brain targeted effect at the dose of 25 mg/kg than the pure drug at the dose of 50 mg/kg. Olanzapine  significantly (p < .001) increased the brain GSH level decreased by ketamine. The effect of DCNPopt  on brain GSH level was found to be statistically equivalent to olanzapine   for 21 successive days, which was significantly increased with the treatment of pure doxycycline hydrochloride  and DCNPg, i.p.) .opt  was significant  to decrease MDA level in mice brain as compared to ketamine treated animals. Brain targeting efficiency of DCNPopt is further illustrated by remarkable decrease in MDA level at a dose of 25 mg/kg, which was even lower than pure doxycycline hydrochloride at the dose of 50 mg/kg. Olanzapine  significantly (p < .001) decreased the MDA level increased by ketamine. The effect of DCNPopt  on MDA level was found to be statistically equivalent to olanzapine   for 21 successive days as indicated by increased MDA level in brain. However, treatment with pure doxycycline hydrochloride  and DCNPg, i.p.) .opt  for 21 successive days was not significant to reduce AChE activity as compared to ketamine. Olanzapine (5 mg/kg i.p.) significantly (p < .001) decreased the AChE activity increased by ketamine. However, the effect of DCNPopt  on AChE activity was statistically significant as compared to olanzapine  (p < .001)  for 21 successive days, brain AChE activity was increased. However, treatment with pure doxycycline hydrochloride  and DCNP < .001) .opt  . In addition, DCNPopt  significantly decreased dopamine level as compared to pure doxycycline hydrochloride  showing added bioavailability in brain. Olanzapine  significantly (p < .001) decreased the brain dopamine level increased by ketamine. The effect of DCNPopt  on brain dopamine level was statistically equivalent to olanzapine   for 21 successive days, which was significantly reversed by treatment with pure doxycycline hydrochloride  and DCNPg, i.p.) .p < .001) increased the GABA level decreased by ketamine. Also, treatment with pure doxycycline hydrochloride  and DCNPopt  significantly  increased GABA level in mice brain as compared to ketamine treated animals. Additionally, DCNPopt (25 mg/kg) was also significant to increase GABA level as compared to pure doxycycline hydrochloride (even at 50 mg/kg), indicating improved bioavailability and penetration in brain through blood brain barrier after coating of nanoparticulate doxycycline hydrochloride with Tween 80. The effect of DCNPopt  on GABA level was statistically equivalent to olanzapine   for 21 successive days. Olanzapine  significantly  .opt  for 21 successive days was significant to reverse ketamine  produced histopathlogical alterations such as perinuclear vacuolization, dilated vascular channels and hyperchromatic nuclei in brain as shown in Above findings were also confirmed by histopathological examinations. Chronic treatment with DCNPopt batch revealed their sub-spherical and spherical shape. The particle size of DCNPopt obtained from TEM imaging was lesser than zetasizer, as zetasizer measures hydrodynamic volume, which is not the case with TEM. The average poly dispersity index was below 1.0 showing monodisperse, homogenous particle size distribution. Further, zeta potential value of DCNPopt was highly positive indicating good physical stability, which may be due to the unreacted amino acid residual on chitosan backbone. DEE of optimized formulation was 78.16%, which was almost closer to the predicted value. Increasing the concentration of chitosan leads to increase in DEE which may be attributed to better polymer network formation and lesser leaching of the drug. Fitting of different kinetics models indicated that drug release from nanoparticles follows Higuchi model  represents drug release through matrix system based on the principle of diffusion, hence, the release of doxycline from DCNP followed diffusion controlled release.The present study was designed to develop novel, optimized, biodegradable, biocompatible, nontoxic and economical brain targeting drug delivery system consisting of Tween 80 coated doxycycline hydrochloride nanoparticles and studied against ketamine induced psychosis in animal models for their possible behavioral, biochemical, neurochemical and histological alterations. Being reported as a stable, nontoxic, biodegradable, biocompatible and inexpensive biopolymer potentiating brain uptake of several drugs for the management of psychiatric disorders  promote inflammatory signaling. An increase in free radicals causes overproduction of MDA, which is the final product of polyunsaturated fatty acids peroxidation in the cells. Reduced GSH molecule neutralizes free radicals by bonding through its extra electron to the ROS molecules capturing free radical electron. In biochemical studies, DCNPopt was significant to diminish the level of TNF-α and lipid peroxidation, while increased GSH level as compared to pure doxycycline hydrochloride and ketamine resulting in improved anti-inflammatory and antioxidant effects; thus, better pharmacodynamic effects due to improved bioavailability in brain. Moreover, available literature on anti-inflammatory  and hyperactivated microglial cell are related with negative symptoms of psychosis along with other psychiatric disorders. Ketamine is also connected with oxidative damage by producing ROS and increasing the release of inflammatory cytokines (TNF-α) by the activation of microglial cells (Zugno et al., opt and pure doxycycline hydrochloride treatment showed non-significant effect revealed by behavioral and AChE activity assessments.Acetylcholine is a one of the main neurotransmitter involved in formation of memory, which is degraded by AChE enzyme (Soni & Parle, opt significantly decreased ketamine produced histopathlogical changes like perinuclear vacuolization, dilated vascular channels and hyperchromatic nuclei in brain, demonstrating the efficacy of DCNPopt at much lower dose than pure drug. These observations again support the enhanced penetration of doxycycline hydrochloride through blood brain barrier when given in the form of Tween 80 coated nanoparticles. The results of DCNPopt against positive and negative symptoms of psychosis are comparable to olanzapine, an established antipsychotic agent used clinically, which has been taken as a standard drug in the present study. Collectively, behavioral, biochemical and histopathlogical results showed pure doxycycline hydrochloride as well as its nanoformulations were able to diminish the ketamine induced positive and negative symptoms of psychosis; the effects produced by DCNPopt at dose of 25 mg/kg are even better than the effects produced by pure doxycycline hydrochloride even at 50 mg/kg dose, showing significant reduction in dose by its brain targeting through nanoparticulate formulation.Furthermore, administration of DCNPopt were found to be significant as compared to software predicted values. The in vitro drug release was found to be 95.44% in 24 h, indicating controlled release of doxycycline hydrochloride from DCNPopt. Doxycycline hydrochloride has showed noticeable antipsychotic activity, otherwise used for its antibiotic activity; increased GABA and GSH level and decreased MDA, TNF-α and dopamine level could probably be involved fort its protective mechanism in the treatment of ketamine induced psychotic symptoms. Observed antipsychotic effects of doxycycline hydrochloride in the form of DCNPopt at significantly lower dose than its pure form indicates better brain uptake of doxycycline hydrochloride through blood brain barrier achieved through brain targeting by Tween 80 coating of nanoparticles. This indicates the effectiveness of nanoparticulate drug delivery system in targeting of hydrophilic drugs by incorporating in Tween 80 coated chitosan nanoparticles for the potential treatment of neuropsychiatric disorders.The present study suggested a novel nanoparticulate formulation of doxycycline hydrochloride for the treatment of ketamine induced psychotic symptoms in animal models. The optimization was performed to evaluate the effect of concentration of chitosan and Tween 80 on DEE and particle size. The observed parameters for DCNP


Even clinically “asymptomatic” carotid stenosis is associated with multidomain cognitive impairment, gray matter (GM) atrophy, and silent lesion. However, the links between them remain unclear. Using structural MRI data, we examined GM asymmetry index (AI) and white matter hyperintensity (WMH) in 24 patients with severe asymptomatic carotid stenosis (SACS), 24 comorbidity‐matched controls, and independent samples of 84 elderly controls and 22 young adults. As compared to controls, SACS patients showed worse verbal memories, higher WMH burden, and right‐lateralized GM in posterior middle temporal and mouth‐somatomotor regions. These clusters extended to pars triangularis, lateral temporal, and cerebellar regions, when compared with young adults. Further, a full‐path of WMH burden (X), GM volume , AI , and neuropsychological variables (Y) through a serial mediation model was analyzed. This analysis identified that left‐dominated GM atrophy and right‐lateralized asymmetry in the posterior middle temporal cortex mediated the relationship between WMH burden and recall memory in SACS patients. These results suggest that the unbalanced hemispheric atrophy in the posterior middle temporal cortex is crucial to mediating relationship between WMH burden and verbal recall memories, which may underlie accelerated aging and cognitive deterioration in patients with SACS and other vascular cognitive impairment. Using a high‐dimensional voxel‐wise gray matter (GM) asymmetry algorithm, we identified rightward GM shifts in a language/speech pathway in severe asymptomatic carotid stenosis (SACS) patients. Further, the mediation analysis indicated that atrophy in the left posterior middle temporal gyrus (pMTG) and asymmetry in the right pMTG serially mediated the relationship between white matter hyperintensity (WMH) and verbal memory. Using metanalytical decoding and functional connectivity gradient map, we showed that the observed differences between SACS and controls in asymmetry index spanned across orofacial somatosensory/motor primary and temporal association cortices  and were in a potential “recall memory” network. These findings may facilitate a better understanding of the neuroanatomical basis of cognitive impairment in carotid stenosis. Recent evidence suggests that WMH burden predicts verbal memory deficits and is mediated by lateral temporal atrophy in AD  who were matched on comorbidities  and demographics . These data have been partially reported previously  and 22 healthy young adults aged 26.95 ± 3.37 years . These independent datasets were collected using the same MRI scan protocols. Participants' demographic and clinical characteristics are summarized in Table Additionally, to trace the aging trajectories of the GM asymmetry, we analyzed publicly available data in the Southwest University Adult Lifespan Dataset  completed a comprehensive cognitive battery within 7 days of the MRI scan, including (a) general cognitive assessment and screening using the MMSE  using a MAGNETOM Trio 3.0 T MR scanner , including T1 anatomical  and T2‐weighted fluid attenuated inversion recovery  images covering the whole brain. Young adults were scanned using a dedicated 3.0 T Siemens PRISMA scanner , with a 64‐channel head coil and similar parameters. Detailed descriptions of the MRI scan protocols have been reported in previous studies , Computational Anatomy Toolbox , and MATLAB 2013a (https://www.mathworks.com). The anatomical images were first visually inspected and then preprocessed with procedures including inhomogeneity correction, skull stripping, and registering to a diffeomorphic anatomical registration using lie algebra . The normalized images were modulated with a Jacobian determinant and smoothed with an 8 mm full‐width‐at‐half‐maximum (FWMH) Gaussian kernel.Voxel‐based morphometry (VBM) was performed using a conventional method with Statistical Parametric Mapping . The Z‐map of each patient was then thresholded (|Z| ≥ 3.1 and cluster size ≥10 voxels) within the right hemispheric mask, which resulted in an AI probability map (superimposed Z map).Given the side of stenosis likely affects the direction of AI, we obtained individual signed 2.6www.applied-statistics.de/lst; Schmidt et al., κ = 0.3; Schmidt et al., WMH size was calculated based on T1 and 3D T2‐FLAIR images using the Lesion Segmentation Tool  in SPSS 16.0 , with a significance level of p < .05. Imaging data were contrasted between groups using two‐sample t‐tests with total intracranial volume (TIV), gender, and age as covariates of no interest. The results were corrected for spatial extent across the GM (VBM) or right side hemisphere mask (AI) using a voxel‐wise threshold of p < .001 with a cluster‐wise family wise error (FWE) rate correction at p < .05.Five SACS patients were excluded due to artifacts or low weight scores. Demographic and clinical data were contrasted between groups using two‐sample independent p < .05.We also examined Pearson's linear correlations between mean AI within each significant cluster, behavioral measurements, and WMH burden in SACS patients. All correlation results were corrected for multiple comparisons using the Bonferroni approach at a significance of 2.8d' and three indirect pathways including (2) a2 × c, (3) a × b2, and (4) a × b × c  from the Additionally, to delineate the developmental trajectory of AI over a broad age range of 18–80 years, we applied the same methodology in the SALD dataset and examined both linear and quadratic age effects.3p < .005) and delayed recall memories  but higher WMH loads . The two groups were comparable in gender, age, education, underlying diseases  and smoking (ps > .05). For details, please see Table As compared to HC, SACS patients showed significantly worse immediate , and cerebellar regions in SACS patients vs. young adults , but the pMTG did not show a trend of age‐related effect , SACS patients showed significantly different AI distributions  and delayed recall memories  in patients with SACS. Additionally, we found significantly positive associations between adjusted WMH size and AI in several regions, including the operculum insula , the precentral gyrus , the pMTG , and the thalamus  from the 3.63.6.1d', p = .003), suggesting that increased WMH predicts decreased delayed recall memory  but positively affected the right pMTG AI . The left pMTG volume significantly negatively impacted pMTG AI  and positively associated with delayed recall . The pMTG AI significantly negatively related to delayed recall . After controlling for the GM atrophy and AI, the direct effect of WMH on delayed recall was no longer significant . Finally, the bootstrap procedure revealed a significant indirect effect that was significantly different from zero . Additionally, the indirect effects of WMH burden through GM asymmetry alone or together with atrophy were significantly different from zero  or 35% (path a2c) variances of total effects in delayed recall, and 18% (path abc) or 37% (path a2c) in immediate recall memory, respectively. The covariates of no interest, i.e., TIV, age, and education, differently interact with the mediators.Our data demonstrated that atrophy and asymmetry in bilateral pMTG serially mediate the relationship between WMH and verbal memory, which provides insight into the link between neuroanatomical path and both carotid stenosis and verbal memory impairment. Similar to findings in AD patients .Our data identified a link between the AI differences and the principal gradient, that is, the pMTG on the top end and auditory/orofacial motor regions on the lower end. Remarkably, bilateral pMTG serve as mediators in mediating the relationship between WMH and verbal memory and are also anchored to the default mode and somatomotor/sensory networks  with metanalysis and seed‐based functional connectivity. Similarly, the metanalytical activation map with the term “recall” (244 studies) in An exception is that we did not find a significant mediating role of hippocampus in recall memory. It is possible due to the fact that hippocampus is relatively small and cannot survive after multiple corrections. On the other hand, from a network perspective Fox, , the pMTConsistent with a recent report (Kong et al., 4.4Considering the focal alterations in the semantic and speech circuit, our findings have some potential clinical applications. For example, intensive training (e.g., speech, aerobic exercises, and attentional training; Kurth, Gaser, & Luders, 4.5The present study has a few potential limitations that are worth noting. First, we analyzed changes in neuroanatomical pathways, a determinant in cognitive impairment of asymptomatic carotid stenosis and also changes in hemodynamics (Lattanzi et al., 5In conclusion, SACS is associated with left‐dominated atrophy and consequently right‐lateralized GM shifts in the lateral temporal regions. Furthermore, unbalanced hemispheric atrophy in the lateral temporal region is crucial in mediating relationship between WMH burden and recall memory, which may underlie accelerated aging and cognitive deterioration in SACS and other VCI.Lei Gao: Collected data, designed the methods, performed analysis, and wrote the manuscript. Yaqiong Xiao: Revised the manuscript, performed the analysis, interpreted, and discussed the results. Haibo Xu: Conceived the study, interpreted and discussed the results, and revised the manuscript. All the authors contributed to the revision of the manuscript.AppendixS1. Supporting Information.Click here for additional data file.