Accurate diagnoses of CSF leaks are imperative in the context of preventing meningitis, and delays in diagnosis and treatment are associated with worse outcomes. Improved characterization of rare, spontaneous CSF leaks may prove beneficial in correctly diagnosing affected patients.Primary, spontaneous cerebrospinal fluid (CSF) leaks secondary to defects in the clivus are exceedingly rare. Additionally, primary, spontaneous CSF leaks are typically present in obese women with idiopathic intracranial hypertension (IIH). In the present study, we report the first case of a primary, spontaneous CSF leak in the inferior-posterior wall of the clivus in an atypical patient with a BMI of 18.9 kg/m Accumul2O ,9. This 2O ,10.2.There are conflicting reports regarding the location of the bony defect causing spontaneous CSF leak. In a case series of six patients with clival CSF leaks, all defects were found to be in the superior, midline area of the clivus . SeveralA 54-year-old female with no prior history of surgery or trauma presented to the emergency department with a chief complaint of severe headache for the past two days. She never experienced an episode like this in the past, but did report intermittent clear, thin liquid leaking through the right side of her nose for the past 15 months. She reported that the rhinorrhea was worse in the morning and that she could feel the fluid spread down her throat while upright. Additionally, tipping her head forward would elicit a significant amount of clear fluid discharge from her nose. The prior year, the discharge became thicker, and she subsequently visited an otolaryngologist. The right-sided nasal discharge was collected and analyzed, and the β2-transferrin assay was negative for CSF. She was given a diagnosis of chronic sphenoid sinusitis versus CSF leak at this time.The initial computerized tomography (CT) scan ordered in the emergency department revealed a small air-fluid interface in the right dominant sphenoid sinus, suggestive of sphenoid sinusitis Figure .9/L) and hyponatremia (127 mmol/L). The patient’s lumbar puncture yielded a colorless, hazy fluid with elevated protein (86 mg/dL) and total nucleated cells . These findings in conjunction with the patient’s co-existing acute sinusitis were suggestive of bacterial meningitis, and she was subsequently admitted to the inpatient floor, receiving antimicrobial coverage with IV vancomycin, ceftriaxone, and acyclovir.The patient was discharged home on a regimen of Augmentin, Percocet, Sudafed, and Zofran. However, the patient’s condition failed to improve the following day, and she continued to exhibit symptoms of waxing and waning confusion, delirium, and hallucinations. She was brought back to the emergency department, for further workup and management. Laboratory findings were significant for a neutrophil-predominant leukocytosis , canalis basilaris medianus (CBM), and craniopharyngeal canal are rare anatomical variants of the clivus . These vEarly detection is essential in preventing the severe sequelae of CSF fistulas, including bacterial meningitis and intracranial abscess . The patTo our knowledge, this is the first case reported of a patient presenting with a spontaneous posterior-inferior clival wall CSF leak. With higher rates of meningitis associated with spontaneous CSF leaks in the clivus compared to other locations, the importance of early intervention and the push for clinicians to perform aggressive workups in suspected patients is prudent. A patient’s medical history of diseases associated with decreased bone density should also be taken into consideration as a potential factor that might contribute to spontaneous clival CSF leaks.


The objective of this single-center retrospective cohort study was to investigate the relationship between blood transfusion and persistent inflammation, immunosuppression, and catabolism syndrome (PIICS). The study was conducted at the Critical Care Center at Toho University Omori Medical Center, Japan. We included 391 patients in the PIICS group  and 762 patients in the non-PIICS group . We performed univariate and multivariate logistic regression analyses using PIICS as the objective variable and red blood cell (RBC) or fresh frozen plasma or platelet (PLT) transfusion and other confounding factors as explanatory variables. In addition, we conducted a sensitivity analysis using propensity score matching analysis. The multivariate and propensity score analyses showed that RBC and PLT transfusions were significantly associated with PIICS. This is the first study to report an association between RBC and PLT transfusions and PIICS. Our findings have contributed to better understanding the risk factors of PIICS and suggest that physicians should consider the risk of PIICS occurrence when administering blood transfusions in intensive care unit (ICU) patients. Once PIICS develops, the patient's immunity weakens, activities of daily living worsen, and eventually, long-term mortality increases3. Therefore, it is imperative to predict and diagnose PIICS as early as possible. The original diagnostic criteria were length of hospital stay > 14 days, C-reactive protein (CRP) > 0.15 mg/dL, lymphocyte count < 800/μL, weight loss > 10% or body mass index < 18 during hospital stay, creatinine height index < 80%, albumin < 3.0 mg/dL, pre-albumin < 10 mg/dL, and retinol-binding protein < 10 μg/dL3. However, in 2020, Nakamura et al. proposed that CRP > 3.0 mg/dL was an additional appropriate criterion4. Nakamura et al. also identified the presence of disseminated intravascular coagulation (DIC) at admission as a factor associated with PIICS5.Persistent inflammation, immunosuppression, and catabolism syndrome (PIICS) has been recently described in critically ill adults and has since been receiving increasing attention. PIICS refers to a condition in which a serious condition persists after intensive care in the acute phase. PIICS is mainly characterized by persistent inflammation, which is accompanied by immunosuppression and catabolism10. The causative factors underlying anemia progression in ICU patients are not only acute bleeding but also blood dilution, dysregulation of iron metabolism, and blunting of erythropoietin response by inflammatory cytokines9. Blood products can cause transfusion-related immune modification (TRIM) that suppresses immunity13. Furthermore, the transfusion of red blood cell (RBC) products and platelets (PLT) are risk factors for nosocomial infections in various environments17. Moreover, Péju et al. reported that the administration of fresh frozen plasma (FFP) and PLT products in patients with sepsis increases the risk of infections after transfusion18. Péju et al. concluded that TRIM might also be present in FFP and PLT. Additionally, blood transfusion affects the immune system, especially in ICU patients, where blood transfusion is a frequently performed procedure.Nevertheless, further evidence is required to identify the exact risk factors of PIICS. One potential risk factor of PIICS is blood transfusion; however, no study published to date has investigated the relationship between blood transfusion and PIICS. Trauma and surgery patients in the ICU often receive blood transfusions. In addition, anemia progresses in ICU patients without obvious bleedingWe hypothesized that blood transfusion is associated with the onset of PIICS in ICU patients. Therefore, the aim of this study was to investigate the relationship between blood transfusions and PIICS.This is a single-center, retrospective cohort study, planned and conducted at the Critical Care Center, Toho University Omori Medical Center, in accordance with the Declaration of Helsinki as revised in 2013. It was approved by the Ethics Committee of the Toho University Omori Medical Center . Due to the retrospective nature of the study, the need for written informed consent was waived.The subjects were tertiary emergency patients admitted to the ICU between January 2019 and March 2021. Patients with acute deterioration in the hospital or those scheduled to undergo surgery were not included. The exclusion criteria were age < 18 years and death within 14 days of admission to the ICU. It should be noted that patients who were admitted to the ICU and moved to the general ward within 14 days were not excluded.19); acute physiology and chronic health evaluation (APACHE) II score; DIC 20 at admission; the presence of renal replacement therapy (RRT); received extracorporeal membrane oxygenation ; duration of mechanical ventilation (MV); presence of shock ; immunosuppressant use (yes or no); vasopressor use (yes or no); received blood transfusion ; the storage time of all transfusion products; and if patients were administered steroids (yes or no) on day 14. The following data from blood test results were extracted: CRP and albumin levels, lymphocyte count on admission and on day 14, creatinine (Cr) and hemoglobin (Hb) levels, PLT count, prothrombin time, fibrinogen, fibrin or fibrinogen degradation products (FDP), and lactate and glycated hemoglobin (HbA1c) levels. If there were no data available on day 14, the nearest values within days 12–16 were used instead.At this hospital, the profile, physical examination findings, laboratory findings, and treatment details of patients are recorded electronically. These electronic medical records were used as the data resource, and data retrieval began in July 2021. The following information was extracted from the medical records: patient profile ; primary disease; presence of sepsis (yes or no); surgical history; comorbidity  for > 15 days and met the criteria of CRP > 3.0 mg/dL or albumin < 3.0 mg/dL or lymph < 800/μL on day 14 were assigned to the PIICS groupThe primary outcome was PIICS onset and the secondary outcome was 90-day mortality.U test; if it was a normal distribution, it was expressed as mean ± standard deviation and tested using Student's t test. Categorical variables were expressed as percentages and tested using the chi-square test.We used the Kolmogorov–Smirnov test to test the normality assumption of our data; the variable was treated as following an abnormal distribution when the null hypothesis was rejected at 5% and as following a normal distribution when the null hypothesis was accepted. If the continuous variable had an abnormal distribution, it was expressed as the median (interquartile range) and tested using Mann–Whitney’s 5; steroid use, underwent surgery, duration of mechanical ventilation, presence of shock, vasopressor use, and immunosuppressant use on day 14; and blood transfusion . Next, a multivariate logistic regression analysis was performed with PIICS as the objective variable and all the above variables as explanatory variables. Variance inflation factor was calculated for each factor, and if it was < 10, the factors were confirmed to have no multicollinearity. In addition, a receiver operating characteristic (ROC) curve was drawn to evaluate the diagnostic value of the volume of RBC transfused in predicting PIICS in ICU patients and the area under the curve (AUC) was calculated.To analyze the factors related to the onset of PIICS, univariate analysis was performed with PIICS as the objective variable and with each of the following factors as explanatory variables: patient profile ; factors involved in the diagnosis of PIICS ; factors reported as potential risk factors in previous studies ® version 7 . Statistical significance was set at p < 0.05.Next, sensitivity analysis using propensity score matching was performed. Propensity scores were calculated using a logistic regression model with the presence or absence of RBC transfusion as the objective variable and the following factors as explanatory variables that may have been considered in performing RBC transfusion: age; sex; height; weight; presence of sepsis; underwent surgery; CCI; Hb level; PLT counts; prothrombin time; fibrinogen, FDP, and lactate concentrations; APACHE II score; overt DIC; RRT; and ECMO. After calculating the propensity score, a caliper width of 0.2 was used for nearest neighbor matching. Multivariate logistic regression analysis was performed with a matched cohort using PIICS as the objective variable and the following factors as the explanatory variables: CRP, albumin, and Cr levels; lymphocyte counts, HbA1c levels; received steroids; duration of mechanical ventilation, presence of shock, vasopressor use, and immunosuppressant use on day 14 and blood transfusion . The explanatory factors used in the multivariate analysis were selected as a result of excluding the factors used to calculate the propensity score. Statistical analyses were performed using StatFlexDuring the observation period, 1426 patients were admitted to the ICU from the emergency department. Forty-two patients were excluded because they were < 18 years old. Out of the remaining 1384 patients, 231 patients died within 14 days and were also excluded. As a result, 762 patients were grouped into the non-PIICS group and 391 patients into the PIICS group , and the RBC + FFP + PLT combination had a positive predictive value for PIICS of 92.5% (62/67).In the matched cohort, there were 144 patients in the non-PIICS group and 162 in the PIICS group. Compared with the patients in the non-PIICS group, those in the PIICS group had a significantly different distribution of primary diseases, higher incidence of surgery, higher CRP and Hb levels, HbA1c level, APACHE II scores, and lower albumin levels (Table In the multivariate analysis with matched cohorts, CRP and HbA1c levels, duration of MV, and RBC and PLT transfusions were significantly associated with PIICS Table .Table 4MThe present study results showed that PIICS is significantly associated with RBC and PLT transfusions. This result was consistent even after adjusting by propensity score matching. In addition to RBC and PLT transfusion, PIICS was associated with age, sepsis, surgery, CRP and albumin levels, lymphocyte count, Hb and HbA1c levels, APACHE II score, and duration of MV on day 14.5. Thus, the results of the present study are consistent with their findings. However, in contrast to the report by Nakamura et al.5, which summarized the risk factors for patients admitted to the emergency ward and ICU regardless of in-hospital or outpatient settings; the present study included patients admitted to the ICU from the emergency department. The difference in patient cohorts between the two studies may explain the differences in findings. Specifically, the patients in this study were younger and mostly male. Furthermore, the number of patients with sepsis was smaller and most had a higher APACHE II score than those in the study reported by Nakamura et al.5. In addition, the overt DIC was surprisingly small in this study. Furthermore, a new analysis was conducted regarding blood transfusion, which may have caused the difference in the results.Nakamura et al. reported that age, sex, sepsis, albumin level, lymphocyte count, Hb and HbA1c levels, Cr level, and overt DIC were significantly associated with PIICS13. In addition, PLT-derived factors may cause immunosuppression, suggesting that PLT and residual-PLT in the RBC products may cause TRIM11. This study is the first study to investigate the relationship between the onset of PIICS and transfusion products. In addition, most of the transfusion products in this study were administered shortly after admission to the patients in the ICU. From the results of this study, it is hypothesized that the immunosuppressive effect and inflammation-inducing effect of blood transfusion last for about 14 days. It is also suggested that the administration of transfusion products could prolong high CRP levels and reduce lymphocyte counts. The mechanism of TRIM is not clear, but there may be a mechanism by which transfusion products reduce the number of lymphocytes. Critically ill patients, such as those admitted to the ICU, have anemia, thrombocytopenia, and are affected by various drugs. In such patients, the immunosuppressive effect of blood transfusions may be greatest. This study deals with the most severely ill patients in the PIICS studies, and TRIM may have been prominent. In the future, it is desirable to verify the effects of TRIM on critically ill patients in terms of cytokines and mediators.It has been suggested that TRIM induces inflammation as well as immunosuppression via allogeneic mononuclear cells, leukocyte-derived soluble mediators, and soluble human leukocyte antigen peptideFurthermore, RBC and PLT transfusions were associated with PIICS, suggesting that it would be important to consider transfusion criteria according to various diseases and stages in future studies.This study has several limitations. First, the characteristics of the patient population may have influenced the results as this was a single-institution retrospective study; thus, the results cannot be generalized. Nevertheless, a relatively large number of samples were collected and analyzed. Second, the effects of confounding factors that were not considered in this study were not removed. Third, patient factors that required RBC transfusion may have influenced the outcome, and the effects were incompletely removed. However, as a countermeasure, propensity score matching analysis was performed. Fourth, there may be cases where the diagnosis of PIICS was inaccurate as there were no data available for day 14; in such cases, we included data from the nearest date. Fifth, It was not possible due to the nature of the study to collect donor information for each blood product administered. In addition, since all blood products used in this study were leukocyte-removed prior to storage, most leukocytes and platelets were removed. However, the presence of cytokines released from the cells of blood products cannot be ruled out, and data on cytokines could not be collected. These factors can also affect TRIM and PIICS.In conclusion, this is the first study to report an association between RBC and PLT transfusions and PIICS. RBC + PLT transfusion showed a high positive predictive value for PIICS. However, it is suggested that PIICS does not increase even if FFP is added to the combination of RBC + PLT. Our findings have contributed to better understanding the risk factors of PIICS and suggest that physicians should consider the risk of occurrence of PIICS when administering blood transfusions in ICU patients.


Tadehagi triquetrum (Linn.) Ohashi (Fabaceae) (TT), is a traditional herbal medicine used especially in China’s ethnic-minority communities, such as the Zhuang, Dai, Li and Wa aeras. As an ethnic medicine, it has long been used to treat various diseases.TT from 1979 to October, 2021 by highlighting its chemical classification, structural features, pharmacological applications and folk applications to provide inspirations and suggestions for accelerating further research of this traditional phytomedicine.This review summarised the phytochemical and pharmacological progress on TT in this article has been obtained using these multiple scientific databases including Scifinder, Web of Science, ScienceDirect, Wiley, ACS publications, Springer, PubMed, China Knowledge Resource Integrated Database from the China National Knowledge Infrastructure (CNKI), Google Scholar and Baidu Scholar. Some information was also collected from classic literature on traditional Chinese medicines.The information on TT to date by the comprehensive analysis of the current literature. A large number of traditional uses and pharmacological studies have exhibited diversified bioactivities of various TT extracts and its metabolites, including anti-inflammatory, antimicrobial, anti-hepatitis B virus, hepatoprotective, insecticidal, etc.More than 70 compounds have been isolated and reported from TT has various medicinal uses and some of them have been supported by modern pharmacological researches. Further detailed studies on the action mechanisms, pharmacodynamics and structure-function relationships of single compounds or active constituents from TT are also required.As a famous traditional medicine with a long history,  Tadehagi (Fabaceae), which consists of 6 species in the world, is mainly distributed in tropical Asia, Pacific Islands and Australia. Two of these plants growing only in China, namely Tadehagi triquetrum (Linn.) Ohashi and Tadehagi pseudotriquetrum (DC.) Ohashi, distributed widely in the southern tropical and subtropical regions of China, such as Guangxi, Guangdong, Hainan, Yunnan, and Hunan province , ‘Bai-Lao-She’ (Guangdong province), ‘Niu-Chong-Cao’ (Hainan province), ‘Lan-Gou-She’ (Jiangxi province), ‘Cha-Bao’ (Zhuang name) in Chinese. So far, according to ‘The Plant List’, T. triquetrum has 7 accepted synonyms , it has bitter taste and the property of cool, which possesses the effects of clearing heat, detoxifying, draining dampness, eliminating phlegm, expelling parasites and dispersing blood stasis. It has long been used to treat colds, acute tonsillitis, acute pharyngitis, hookworm disease, lung abscess, nephritis edoema, dysentery, jaundice, rheumatic arthritis and scabies in the form of a single herb or herbal preparation formula . Plant taxonomy was verified by ‘iPlant’ database (http://www.iPlant.cn). Additional information was also collected from Chinese herbal medicine books (in our school library), PhD and MSc theses (downloaded from CNKI), read manually and analysed in groups. Key words used in the literature search were: Tadehagi triquetrum, phytochemistry, pharmacology, pharmaceutical activities, chemical constituents, and other related search terms. Relevant reports and articles that appeared in some news media or newspapers, as well as literature that was not published in formal professional magazines and periodicals, have been excluded.This literature review of TT have led to the isolation of flavonoids (1-30), phenylpropanoids (31-49), phenolic compounds (50-57), triterpenoids (58-62), steroids (63-66) and other miscellaneous compounds (67-73)  . The che1-30) (1-2), flavonols and their glycosides (1-14), flavanonols (15-17), flavanone , one flavan-3-ol (18), isoflavonoids (21-30). Some of them exist as flavonoid glycosides and the aglycones in all of these glycosides are derived from flavonols and flavanones, which are mainly formed by the glycosylation of glucose, rhamnose and galactose. The C-5 and C-7 of TT flavonoids are oxidised in the form of hydroxylation or glycosylation. Investigation on the ethanol extract of the whole plant using bioactivity-guided isolation method led to the discovery of anthelminthic activity of three isoflavonoids    have bee1-30)  includinTT. Nineteen phenylpropanoids (31-49) have been isolated and identified from TT  from TT′s aerial part are rare dimeric tadehaginoside derivatives, which have a unusual bicyclo [2.2.2] octene skeleton or cyclobutene ring  was determined by NOESY correlations and optical rotation values compared with the reported literature  have been identified from TT  from TT′s stems part are lipid-soluble component and they can be crystallised in solvents with low polarity such as petroleum ether mixed with chloroform  have increased their hydrophilicity due to the presence of carboxyl groups which could be converted from methyl oxidation at C-4 or C-28  have been isolated from TT , one roseoside (70), one fatty acid (71) and two alkaloids (72-73).In addition, several miscellaneous compounds  have beeTT had extensive range of biological activities.Modern pharmacological studies and plentiful clinical research literature have indicated that various extracts and purified compounds from TT was evaluated with measuring the paw volumes at 1 h intervals for 3 h and comparing with control groups. The results suggested it at all the doses  could significantly decrease paw volume compared to control (p < 0.05) and the maximum inhibition of paw edoema was found at 60 min in the 300 mg/kg dose group, which were comparable with standard drug diclofenac sodium. In addition, over-production of nitric oxide (NO) indicates tissue damages. It from 25 to 75 μg/mL exhibited moderate inhibitory of NO in a dose dependent manner. Besides, the carrageenan administration process also could result in increasing in cyclic adenosine monophosphate (AMP)-phosphodiesterase. In a phosphodiesterase activity test, It showed better inhibitory effect against cyclic AMP phosphodiesterase than the positive control, mefloquine.  including a large number of ethnologic herbs, play an important role in the treatment of various diseases. 4) is considered as a hepatotoxic revulsant by generating reactive species, such as trichloromethyl radical (CCl3−), to destroy hepatocellular cells and undermine the liver functions. The hepatoprotective effect of tadehaginoside (38) isolated from TT′s aerial part was investigated using the hepatic injury model of CCl4-lesioned rats. The findings showed that it dose-dependently suppressed the cell proliferation of HepG2 cells in vitro, and it significantly lowered the serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), immunoglobulin E (IgE), and leukotriene (LT). The pathological examination suggested the hepatocellular damage was effectively mitigated by its treatment. Moreover, cytochrome c oxidase (COX) mRNA expression in hepatocytes was upregulated  and Kelch-like ECH-associated protein 1 (Keapl) were progressively increased, and the downstream enzymes consisting of γ-glutamylcysteine synthetase (γ-GCS), glutathione (GSH), and catalase (CAT), were activated and their contents in hepatocytes were gradually elevated, which may be involved with the downregulation of tumour necrosis factor alpha (TNF-α) and nuclear factor-kappa B (NF-κB)-expressed protein  of Plumbago zeylanica L. and Carapax trionycis were the monarch drugs, Astragalus propinquus Schischkin, Atractylodes macrocephala Koidz, Paeonia lactiflora Pall. and Angelica sinensis (Oliv.) Diels were the minister drugs, TT and Lycopus lucidus Turcz. Ex Benth. were adjuvant drugs  , the ethyl acetate fraction, n-butanol to extract the 70% ethanol extract of TT′s stems and leaves to prepare the polyphenol-rich extract (PRE). It could lower hyperinsulinemia, improve oral glucose tolerance, and reduce hyperlipidaemia and liver fat content (p < 0.05). PRE treatment could increase the content of liver glycogen, and the activity of hepatic glucokinase and pyruvate kinase (p < 0.05). The eight compounds were isolated from PRE based on the bioactivity-guided isolation method. Six polyphenols , particularly rutin (14), increased glucose consumption by hepatocytes, suggesting that polyphenols accounted for the antidiabetic effect of PRE  on consumption of glucose was evaluated on HepG2 cells. In the NBD-glucose (2-NBDG) uptake assay, its activity (10 mM) was comparable to insulin (0.1 mM), indicating a potent activity in stimulating glucose uptake by myotubes. Very interestingly, tadehaginosin (49) showed stronger activity than compound 48, since they belonged to two structurally related phenylpropanoids, one lignan and one cumarin. The results suggested tadehaginosin could be a potential therapeutic strategy for stimulating glucose consumption and be meaningful in treatment of diabetes  are main characteristics of type 2 diabetic (T2DM). The activity of tadehaginosin , a key regulator of glucose homeostasis. A series experiments of molecular biology showed that 34 significantly enhanced the transcriptional activity of PPARγ and increased the level of glucose transporter-4 (GLUT-4) protein whose expression was regulated by PPARγ. Therefore, the above results indicate compound 34 is regarded as a most prominent compound for the treatment of diabetes.In another study, compound TT, has been used clinically in treatment of gout for decades and has achieved good clinical efficacy. Pharmacological studies in vitro have shown that the decoction could reduce the degree of toe swelling in mice with sodium urate injection and inhibit the synthesis of TNF-α and IL-1. Furthermore, it can significantly decrease the level of uric acid in hyperuricaemia mice of yeast intragastric administration, and suppress the activities of xanthine oxidase (XOD) and adenosine deaminase (ADA) which are key enzymes for purine metabolism to produce uric acid and a key link in the regulation of uric acid production   prevented foam cells formation in RAW264.7 cells. And above three compounds could up-regulate of cholesterol efflux-related genes such as ABCA1 and ABCG1, and inhibit the expression of CD36 and SR-1, which are cholesterol influx-related genes. The structure-activity relationship study showed cinnamyl is the most pivotal group of these compounds  can decrease oleic acid (OA)-elicited neutral lipid accumulation and intracellular contents of total cholesterol and triglyceride in the HepG2 cell model  Rchb. f. The clinical observation found that it can relieve the burning pain, promote the blister subside and swelling decrease, prevent re-contamination and promote wound repair. The wounds of first-degree burns cured by this lotion generally did not leave paralysis marks, but only slight changes in the skin’s pigment. The lotion can also be used as an oil agent, which has little irritation on the wound surface and is suitable for children burns  had remarkable effects on inhibiting or killing coccidian eggs  with high pressure liquid chromatography and the label-free method. HPLC-TOF-MS was utilised to analyse the ingredients of its active fractions and identify twelve compounds   could kill all these molluscs within 17 hours  and kaempferol (3) with the concentration from 0.005-0.01% had the activity within 24 hours. Thus, it was suggested that they might be effective components of TT (Li et al. Compounds TT is rich in polyphenolic compounds which have been considered to possess antioxidant activities. Because of the availability and the presence of flavonoids and phenolic compounds, 95% ethanol extract of TT’s leaves was evaluated for its antioxidant activity in vitro. The extract could decrease significantly the concentration of DPPH-radical and H2O2, whose effect on scavenging of free radicals was close to the control ascorbic acid. This extract at the concentration of 50 μg/mL had the maximum H2O2 scavenging effect (Kalyani et al. TT (Wang and Zhang Infantile anorexia is a common disease in paediatrics, which usually occurs in infants under one year old. The patients mainly have symptoms such as anorexia and fatigue. ‘Xiaoer Kang’ granule is a kind of Chinese patent medicine including TT to treat various genitourinary diseases (Zhang et al. Gao and Chen used the ‘Gongying Hulu’ decoction to treat prostate hyperplasia with the syndrome type of moist-heat on bladder in TCM Gao . Sun hasTo verify the traditional record of deworming, its insect extermination effect was investigated towards the larvaes of Tenthredinidae and Arctiidae using its different dosage forms (Li et al. TT for Artemisia capillaris Thunb., a commonly used traditional Chinese medicine with the effect of reducing dampness and removing jaundice (Jin et al. TT’s clinical applications as the main herb, such as treating carbuncle, heat dysentery and malnutrition (Ge and Li Deng often substitute TT has many medicinal applications. It can be used as a single medicine or in combination with other TCMs in multiple ethnic groups to treat various ailments. Some medicinal uses are clearly recorded in traditional medicine classics and official ethnic literature, while others have been directly used in folk life through generations of oral transmission. Just because of its clinical efficacy and history, more and more researchers are interested in it. So, extensive phytochemistry and pharmacology investigations on TT have been carried out. Therefore, this review described and summarised the detailed chemical constituents and their biological activities of TT, which could provide the foundation for future research. To the best of our knowledge, 73 compounds are present in this plant and mainly include flavonoids, phenylpropanoids, phenolic compounds, triterpenoids and steroids. Among them, some compounds show good pharmacological activities, especially anti-atherosclerotic and antidiabetic activities. Studies indicated the phenylpropanoids in TT had the potential in treatment of diabetes disease, but their structure-activity relationship and the mechanism of action required deeper research. Some studies have found single compounds with novel skeletons, but their activities seemed to be unsatisfactory.As a famous traditional medicine with a long history, TT, collection time, processing methods, extraction conditions. In particular, the extraction method of TT is decoction in folk clinical application, but in most reports, it is reflux extraction by ethanol. So, it is very necessary to go into the clinic and study its preparation process in advance. Besides, in many cases, its extracts performed better than single compounds, suggesting that these single pure constituents could not well explain the traditional medicinal activities of TT. This may indicate its mechanism of action is related to multiple components. Hence, it is necessary to develop a variety of pharmacological models and methods, such as network pharmacology strategies, to promote the research of multiple components and multiple targets of TCMs.It is particularly worth mentioning that the results of modern pharmacological research do not seem to be as good as it is used in real life. This may involve multiple factors, such as the authenticity of in vivo studies. But many of the early experiments were very superficial. A relatively large amount of research has focussed on its extracts and even powders, without really finding its effective ingredients. Therefore, the studies based on monomeric compounds are still lacking, especially in terms of activity screening, action mechanism and pharmacodynamics. Furthermore, more clinical trials should be conducted to verify its functions. On the whole, the research is not systematic and in-depth enough, specifically for one therapeutic effect. Of course, suitable pharmacological experimental models in vitro and in vivo, are also very vital and urgent.To be sure, many of pharmacological studies have been conducted with its extract or its formulations, and many of them are based on TT.In addition, studies on its composition analysis and quality control, such as component fingerprint, have not yet established clear and reliable strategies. In the future, further research is necessary to investigate phytochemical and pharmacological activities, including the deeper molecular mechanism, in order to discover active compounds from


Synapse degeneration is an early event in pathological frontotemporal lobar degeneration (FTLD). Consequently, a surrogate marker of synapse loss could be used to monitor early pathologic changes in patients with underlying FTLD. The aim of this study was to evaluate the relationship of antemortem cerebrospinal fluid (CSF) levels of 8 synaptic proteins with postmortem global tau and TDP-43 burden and cognitive performance and to assess their diagnostic capacity in a neuropathological FTLD cohort.n = 24, mean age-at-CSF 67 years ± 11), FTLD-TDP  or AD  as well as cognitively normal controls  from the Penn FTD Center and ADRC. We used a semi-quantitative measure of tau and TDP-43 inclusions to quantify pathological burden across 16 brain regions. Statistical methods included Spearman rank correlations, one-way analysis of covariance, ordinal regression, step-wise multiple linear regression and receiver-operating characteristic curves.We included patients with a neuropathological confirmation of FTLD-Tau (rs = .55 to .88). In FTLD-TDP, we observed low antemortem CSF levels of calsyntenin-1 and neurexin-2a compared to AD  and controls , which were inversely associated with post-mortem global TDP-43 burden . A multimarker panel including calsyntenin-1 was associated with TDP-43 burden  and MMSE score  in FTLD. A second multimarker synaptic panel, also including calsyntenin-1, was associated with MMSE score in FTLD-tau  and improved diagnostic performance to discriminate FTLD-Tau and FTLD-TDP neuropathologic subtypes (AUC = .83).CSF calsyntenin-1 and neurexin-2a were correlated in all patient groups (These synaptic panels have potential in the differential diagnosis of FTLD neuropathologic subtypes and as surrogate markers of cognitive performance in future clinical trials targeting TDP-43 or tau.The online version contains supplementary material available at 10.1186/s13024-022-00534-y. A prominent feature of frontotemporal lobar degeneration (FTLD) is a region-specific degeneration and loss of synapses –4, whichThe aim of this study is to a) compare the cerebrospinal fluid (CSF) profile of a panel of synaptic proteins in a neuropathological cohort of confirmed FTLD-Tau and FTLD-TDP, b) evaluate the relationship of antemortem CSF levels with postmortem Tau and TDP-43 burden and cognitive performance and c) determine their diagnostic accuracy. As reference groups we include cognitively normal controls where synapse degeneration is absent and Alzheimer’s disease (AD) where synapse degeneration is widespread. The proteins included in the study  were brought forward from our previous study in clinical AD cohorts . These pn = 24) or FTLD-TDP (n = 25) and patients with a clinical and neuropathological diagnosis of AD (n = 25). Patients included for study had prospective CSF collection as part of ongoing observational research programs at The Penn FTD Center aimed at developing biological markers that reflect pathophysiological changes in FTLD proteinopathies to improve antemortem diagnosis and prognostication. Patients underwent a Mini-Mental State Examination (MMSE) by neurologists masked to synaptic biomarker data within 5 months of the lumbar puncture. We also analyzed CSF samples from cognitively normal controls (n = 35) that were recruited from the community and screened by revising their medical and medication history. Controls self-reported a negative neurological and psychiatric history and were within the normal range of cognitive performance . CSF samples from patients and controls were processed in the same way. Pre-analytical processing details for these CSF samples can be found elsewhere ). When controlling for the other variables, lower age-at-death (p = 0.01), lower CSF calsyntenin-1 (p = 0.0006), elevated CSF VAMP-2(p = 0.02) and being female (p = 0.01) were associated with elevated global TDP-43 protein. Additional File To determine whether a combination of synaptic proteins could better explain postmortem tau and TDP-43 burden, we performed stepwise mixed entry linear regression starting with the synaptic proteins and covariates. No multimarker model was associated with global Tau  but not in FTLD-Tau  or FTLD-TDP . For comparison, no individual synaptic protein was associated with MMSE in FTLD  or in either neuropathologic subtype . Neurexin-3a and VAMP-2 were the significant contributors to this model. When controlling for the other variables, lower CSF VAMP-2 (p = 0.02) and elevated CSF neurexin-3a (p = 0.02) were associated with worse MMSE score. Additional file We first tested the association of the synaptic proteins from Panel A[CLSTN1-VAMP2] with MMSE score in FTLD patients including sex, age-at-CSF, and years of education as covariates. The panel was associated with MMSE in the total FTLD group . Age-at-CSF, calsyntenin-1, VAMP-2 and gluA4 were the significant contributors to the model (henceforth termed Panel B[CLSTN1-VAMP2-GLUA4]). When controlling for the other variables, elevated age (p = 0.03), elevated CSF calsyntenin-1 (p = 0.005), lower CSF VAMP-2 (p = 0.01) and lower CSF gluA4 (p = 0.03) were associated with worse MMSE score. Additional file r2 = 0.38, p = 0.02) and in all FTLD . GluA4, neurexin-3a and syntaxin-1b, age-at-CSF and years of education were the significant contributors to this model (henceforth termed Panel C[GLUA4-NRX3A-STX1B]). When controlling for the other variables, less education (p = 0.02), elevated age (p = 0.005), lower CSF gluA4 (p = 0.002), elevated neurexin-3a (p = 0.003) and elevated CSF syntaxin-1b (p = 0.03) were associated with worse MMSE score. Additional file To determine whether other combinations of synaptic proteins could improve the association of Panel A, we performed stepwise linear regression starting with the synaptic proteins and covariates. Compared to Panel A, a 6 variable model including calsyntenin-1, gluA4, syntaxin-1b, VAMP-2, age-at-CSF and education showed a stronger association with MMSE in FTLD-Tau .Thus, several multimarker panels of synaptic proteins are associated with cognitive performance in FTLD. Panels B and C were not associated with postmortem global TDP-43 or global Tau burden . This panel was statistically comparable to tau CSF biomarkers, pTau, tTau and pTau/tTau ratio . When distinguishing FTLD-Tau from AD, neurexin-3a gave nominally the highest AUC . Panel A[CLSTN1-VAMP2] showed a nominal improvement (Table p = 0.61) but still performed worse than pTau and the ptau/Aβ42 ratio .The synaptic proteins showed modest diagnostic accuracy to distinguish FTLD-Tau from controls (highest AUC = 0.67 for neurexin-2a). The best multimarker panel was Panel B[CLSTN1-VAMP2-GLUA4], which showed only a nominal improvement (Table p = 0.41) but performed better than CSF tau markers . When distinguishing FTLD-TDP from AD, calsyntenin-1 gave nominally the highest AUC . Panel B showed only a nominal improvement (Table p = 0.56) and was significantly worse than the pTau/Aβ42 ratio .Neurexin-2a gave nominally the highest AUC  for distinguishing FTLD-TDP from controls. Panel B[CLSTN1-VAMP2-GLUA4] showed only a nominal improvement (Table p < 0.05 vs Panel B) and the ptau/aβ42 ratio .None of the synaptic proteins alone could discriminate FTLD-TDP from FTLD-Tau subtypes . However, Panel B performed significantly better , AD and cognitively normal controls. CSF levels of calsyntenin-1 and neurexin-2a were correlated in all patient groups and were lower in the FTLD-TDP neuropathological subtype compared to neuropathologically confirmed AD and cognitively normal controls. Furthermore, antemortem CSF levels of calsyntenin-1 and neurexin-2a inversely correlated with global TDP-43 burden post-mortem. Combining calsyntenin-1 with VAMP-2 and neurexin-3a improved this association with TDP-43 burden and was also correlated with cognitive performance in FTLD. Combining calsyntenin-1 with VAMP-2, gluA4 and syntaxin-1b showed high accuracy in the differential diagnosis of FTLD-TDP and FTLD-Tau neuropathologic subtypes and was associated with cognitive performance in FTLD-Tau. Thus, when measured in CSF, combinations of multiple synaptic proteins were better predictors of global TDP burden and cognitive performance and showed better diagnostic accuracy compared to individual synaptic proteins. To our knowledge, this is the first study to relate CSF levels of synaptic proteins to FTLD neuropathological subtypes.NRXN1, NRXN2, NRXN3) with alternative promoters  and extensive alternative splicing resulting in over 1000 distinct neurexin isoforms, each associated with specific neuronal cell types [Calsyntenin-1 is abundant in most neurons of the CNS [36] where it has been shown to modulate postsynaptic calcium signaling and promote dendritic spine assembly . VAMP-2 ll types . The forll types , 22. Thell types .C9orf72, GRN or VCP mutations ) was present in 17% of the FTLD-Tau and 24% of the FTLD-TDP patients, stepwise regression showed that AD comorbidity was not a good predictor of TDP-43 burden and was therefore excluded from the final model. On the other hand, AD comorbidity was a predictor of MMSE and was included in Panel C. Thus, AD comorbidity may influence cognitive performance in FTLD. This is consistent with a recent study showing that neurodegenerative comorbidities contribute to transition from mild cognitive impairment to dementia .A limitation of this work is the relatively small study size compared to other CSF biomarker studies. However, CSF samples from well-annotated autopsy cases of these uncommon conditions are scarce and the neuropathological confirmation reduces the substantial heterogeneity inherent in clinical FTD cohorts. It should also be noted that the mean time from CSF to death was 5 years in FTLD-Tau and 4 years in the FTLD-TDP subtypes, reaching up to 10 years in some cases. This long interval and variability could influence the relationship between participants’ CSF biochemical signature and their final neuropathological findings. Finally, our control participants lack neuropathological confirmation. However, complete clinical and neuropsychological evaluations were performed to exclude significant medical  conditions in these participants.A biomarker that can distinguish FTLD neuropathologic subtypes in clinical cohorts would be a valuable addition to the biomarker arsenal. In this regard, Panel B[CLSTN1-VAMP2-GLUA4] identified in this study provides proof-of-concept that these synaptic proteins are differentially altered and can distinguish between the two most common neuropathologic subtypes. Moreover, all panels described here were associated with MMSE score and therefore show proof-of-concept as surrogate measures of cognitive performance. These panels may be relevant for future clinical trials targeting TDP-43 or tau pathology, where there will be a need for a surrogate measure of cognitive performance, independent of the drug target. In addition, the postsynaptic modulator, calsyntenin-1 both alone and in combination with other synaptic proteins, has potential as an objective measure of TDP-43-mediated degeneration in FTLD. Validation of these findings in independent cohorts is needed to fully determine their clinical potential.Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.


Lumbar spine surgery can be complicated by perioperative cerebrospinal fluid (CSF) leak. However, development of brain herniation secondary to CSF leak following lumbar spine surgery has not been previously reported in the current literature. This case report describes a 48-year-old woman who, after a revision lumbar decompression and fusion, experienced CSF leak followed by development of brain herniation, which resulted in patient demise. The postoperative period was complicated by patient nonadherence to conservative management of CSF leak. A cerebrospinal fluid (CSF) leak can arise from various etiologies, including trauma, hydrocephalus, infection, iatrogenic, and spontaneous ,2. UnforA 48-year-old female with a history of multiple previous spine surgeries presented with continued complaints of back and right lower extremity radicular pain. Physical examination was remarkable for tenderness to palpation along the lower lumbar spine but otherwise was neurologically intact without obvious provocative maneuvers. A magnetic resonance imaging (MRI) was obtained showing significant scar tissue but otherwise stable laminectomies at L4-5 and L5-S1 with known anterior interbody at the L5-S1 level. There was evidence of anterolisthesis at L4-5 due to bilateral pars defects of L5, as well as an anterolisthesis at L5-S1  scan of head was obtained. At this time, the CT scan was interpreted as normal. The patient was then intubated and later transferred to a tertiary care hospital, where the CT scan was again reviewed by another provider and interpreted as a brain herniation Figure . The patProcedure-related complications following lumbar spine surgery have been widely reported in the literature, including wound infection, neurologic deficit, hardware malposition, durotomy, urinary tract infection, and deep venous thrombosis ,7. AlthoVarious incidence rates of CSF leak following lumbar spine surgery have been reported, ranging from 5.5% to 21%, with higher rates observed for revision surgeries compared to primary surgeries . CurrentAnother rare complication associated with CSF leaks is foramen magnum syndrome, a constellation of neurologic findings in the setting of cerebellar tonsillar ectopia. Badve et al. present a series of two cases of foramen magnum syndrome seen after iatrogenic CSF leaks during lumbar spine surgery . In thesGiven the possibility of these fatal complications, early detection and management of postoperative CSF leaks are crucial. Management of a patient with CSF leak after spinal surgery may involve conservative measures such as bedrest, analgesics, and hydration or require surgery, including primary repair or epidural patching ,14. ThisThe possible mechanism of the brain herniation in the present case may be related to changes in intracranial pressure secondary to CSF leak. Intracranial hypotension typically results from loss of cerebrospinal fluid, and CSF leak is the prevailing theory for the etiology of spontaneous intracranial hypotension -17. SignAlternatively, the patient may have had an underlying congenital abnormality which predisposed her to developing a brain herniation. Chiari malformations are characterized by herniation of the cerebellar vermis and/or tonsils. However, this possibility could not be thoroughly explored in the present case as post-mortem MRI would be necessary. Furthermore, while late presentations are possible, congenital malformations would be more likely to present with symptoms at an earlier age. The incidence of asymptomatic Chiari malformation is reported to be about 2% .We illustrate the case of a CSF leak and consequential brain herniation in a patient following elective lumbar spine surgery. These events arose despite the intraoperative identification and repair of the CSF leak. Postoperatively, the patient developed postural headaches and did not adhere to the repeated recommendations to remain supine. The patient ultimately expired one week after the surgery. CT scan of the head revealed transtentorial brain herniation.This represents the first described case in the literature of a brain herniation secondary to CSF leak following spine surgery. Spine surgeons should be aware of this rare, but potentially life-threatening complication, and patients should be appropriately counseled, especially in the setting of patient noncompliance.


All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates.The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins  The YidC/Oxa1/Alb3 proteins are found in bacteria, mitochondria, and chloroplast where they play a pivotal role in membrane protein biogenesis . More reBacillus halodurans (Escherichia coli (E. coli YidC) with an unusual hydrophilic cavity located within the inner leaflet of the membrane that is accessible from the cytoplasm and lipid bilayer, but not from the periplasm. In addition, YidC possesses a conserved coiled-coil region in the cytoplasm comprised of two α-helices CH1 and CH2 that most likely is involved in substrate binding of YidC substrates such as Pf3 coat (E. coli YidC), which is kinked by two conserved prolines, one in TM1 of the core (TM2 of E. coli YidC) and one in front of CH1. These prolines along with the disordered loop (Pro-Leu-Gly-Gly-Cys-Phe-Pro in E. coli YidC) connecting CH2 to the second TM core segment (TM3 of E. coli YidC) likely function to move the substrate from the coiled-coil domain into the hydrophilic groove.In 2014, the structures of YidC from lodurans  and Eschhia coli  were solPf3 coat . InteresE. coli YidC residues that have been determined as contacting the TM segment of substrates during insertion were found to cluster on the same face of TM3 and TM5 that forms a ‘greasy slide’  phospholipid bilayer showed the protein to be more compact than the crystal structure and reveals significant thinning in the vicinity of the protein (E. coli YidC is in proximity to tyrosine 516 and 517 at the top of the groove (via water molecules. These tyrosine residues are part of an aromatic cluster at the interface between the aqueous environment in the groove and the embedded hydrophobic portion of YidC in the outer leaflet of the membrane.A molecular dynamics (MD) simulation of  protein . The thi protein . The stre groove  Fig. 1)E. coli YE. coli YidC and Streptococcus mutans YidC2. We found that the positive charge (R366) is important for the E. coli YidC when a nearby tyrosine at position 517 is changed to an amino acid with an apolar side chain. However, it is not essential when the tyrosine is changed to a hydrophilic or aromatic amino acid. Similar results were also found with S. mutans YidC2. Solvent accessibility assays show that the arginine is required to keep the upper part of the groove solvent accessible when Y517 is replaced with an apolar residue. Strikingly, both E. coli YidC and S. mutans YidC2 can function in membrane protein insertion when the arginine is replaced with a negatively charged glutamic acid residue, provided that a suppressor mutation is also present, which most likely stabilizes the groove region. Taken together, these results support the hypothesis that the positively charged residue is employed to keep the groove hydrophilic and water exposed rather than to attract the negative charge region of the substrate to be translocated.To gain further insights into the role of R366 in the YidC insertase activity, we investigated the function of this conserved positively charged residue in the hydrophilic groove of the E. coli YidC are located above arginine 366 at the top of the hydrophilic groove (B). Our first question concerned the necessity of these two aromatic residues. Y516, Y517, or both Y516 and Y517 were mutated to alanines. The activities of these mutants were determined utilizing the YidC depletion strain JS7131, with the endogenous yidC knocked out and a new copy of yidC gene under the control of the araBAD promoter introduced at the lambda attachment site  and double R366A/Y516C did complement (C). Procoat-Lep corresponds to PC with its C-terminal cytoplasmic region extended by 101 amino acids of the periplasmic domain of leader peptidase (Lep) . Similar results were seen with the Pf3-23Lep substrate, which has the Lep residues 23 to 323 fused after the TM segment of Pf3 coat and an arginine introduced after the TM of Pf3-23Lep to prevent translocation of the Lep C-terminal domain . When Pf3-Lep (P) inserts across the membrane, proteinase K (PK) digests the N-tail of Pf3-Lep and converts it to a smaller fragment (F). Figure 2B shows that Pf3-23Lep was inserted efficiently with either the R366A or Y517A single mutant, while insertion was blocked with the R366A/Y517A double mutant . It is worth noting that unlike Pf3-23Lep, which showed almost complete loss of insertion with the double mutant, PC-Lep was only partially inhibited. This has also been observed previously  . If PC-Ll domain  . In contrast, when the residue at position 517 had a hydrophilic  or aromatic side chain (517W and 517F), we observed full complementation under YidC depletion conditions (A and C). For the 366 residue, no difference was observed if there was a slightly hydrophobic alanine or a hydrophilic asparagine residue (data not shown). As a second test of YidC activity, we performed membrane insertion assays using PC-Lep and Pf3-23Lep (D). Membrane insertion of PC-Lep and Pf3-23Lep was inhibited when the 517 tyrosine was changed to an apolar, nonaromatic amino acid, and arginine 366 was changed to either an Ala or Asn residue corroborating the complementation results  is removed. Complementation studies revealed that the R366 neutral mutant (R366A or R366N) did not complement the YidC depletion strain when combined with an apolar substitution for Y517 , while very poor complementation was observed for the Y517T and Y517M mutants , A and Cnditions , A and Cf3-23Lep  and detef3-23Lep D. Membra results .Figure 3S. mutans 247YidC2  is essential as the protein was inactive when the R73 residue in the hydrophilic groove was substituted with a neutral or negatively charged residue  in the S. mutans YidC2 could affect the requirement of R73 (corresponding to R366 in E. coli). Remarkably, when the W234 residue at the top of the groove of 247YidC2 was substituted with amino acids with hydrophilic sidechains (Asn or Ser), YidC2 could complement the YidC depletion strain even without the positively charged residue in the groove (R73Q), while it did not complement with an apolar amino acid at 234  (B). As expected, the membrane-insertion activities of the S. mutans R73Q/W234V and R73Q/W234I 247YidC2 mutants were inhibited for the PC-Lep substrate and was completely abolished for the Pf3-Lep substrate (3D).Previously, we found that the strictly conserved arginine R73 in  or Ile) B. As expubstrate  and 3D.E. coli YidC and S. mutans YidC2: the conserved positively charged residue is required for activity when there is an apolar residue at position 517 in the E. coli YidC or at position 234 in the S. mutans YidC2, but not when there is a hydrophilic residue at these sites.Taken together, these results reveal a pattern that applies to both E. coli YidC and S. mutans YidC2. We hypothesize that the positively charged residue is required to maintain a hydrophilic environment in the groove.The results reported above show a clear correlation between the requirement of the conserved positive charge and the hydrophobicity near the top of the hydrophilic groove in both A) and refers to the circumstance where Y517 and R366 are left unmutated except when their own solvent accessibility is studied (top panels). Under the mutated condition, Y517 is exchanged to an apolar isoleucine residue and R366 is substituted with a neutral asparagine amino acid simultaneously.To test this hypothesis, we examined the solvent accessibility of nine previously determined groove residues under WT and mutated conditions in the intact bacterial cells at 30 °C using a well-developed cysteine alkylation assay . Using tFig. 4A shows that the solvent exposure of Y517C is decreased considerably when the R366 is changed to a neutral asparagine. Similarly, R366C is much less water accessible when Y517 is mutated to apolar isoleucine residue. Likewise, the solvent accessibility is decreased for 432C when Y517 and R366 are substituted with an Ile and Asn, respectively. A less dramatic effect on water exposure is observed further down in the groove. With the Y517I and R366N mutations introduced, the solvent exposures of S520C and I428C residues are decreased, but to a lesser extent, than the cysteine mutants in the upper region of the groove. A negligible effect was observed when the water accessibility was examined toward the cytoplasmic side of the groove at positions M475, Y370, M498, and T524 when the Y517I and R366N mutations were made. A summary of the results is shown in Figure 4B, with the top panels representing the WT and the bottom panels the mutated construct. Cyan indicates residues that are completely water exposed, yellow moderate accessibility, and red residues that are not solvent accessible. The top panel shows that the groove is mostly aqueous under WT condition, while a cluster of red and yellow residues can be observed at the dome of the groove under mutated condition as revealed by the bottom panel, implying that the top part of the groove is not hydrated in these cases.Figure 4C were purified, spin-labeled with  methanethiosulfonate (MTSL), reconstituted into 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes, and subjected to EPR power saturation experiments at 22 °C as described in “Figure 4C, from which the saturation parameters P1/2 were obtained and the immersion depth parameters Φ were calculated using Equation To confirm these results, we also performed electron paramagnetic resonance (EPR) power saturation studies to examine solvent accessibility of a spin label incorporated into the hydrophilic groove. His-tagged YidC mutants listed in Figure 4Φ value (−0.8). When Y517 was substituted with a hydrophilic residue (Y517N), position 366 remained accessible (Φ = −0.8). However, the spin label at 366 was no longer water accessible (Φ = 0.9) when the apolar residue isoleucine was introduced at position 517 (C).As a control, a spin label was introduced at position 366 and determined to be solvent accessible as indicated by the highly negative tion 517 C.C shows that in all cases studied , position 517 was accessible to solvent. However, a closer investigation of the Φ values suggested that when the positive charge was removed (R366N/Y517C or R366I/Y517C), 517 became much less solvent accessible (−0.2) than when it was present (−0.9), indicating that the positive charge at 366 contributes to keeping the groove hydrated.A spin label was also attached to the top of the hydrophilic groove (Y517C) and studied with different mutations at R366. Figure 4E. coli YidC and the Y517I/R366N mutant at ∼27 °C (300 K) embedded in a POPE:POPG (75:25) lipid bilayer. To evaluate the level of hydration at various locations within the groove, the number of water molecules within 6 Å of Cα atoms of various groove residues during the last 40 ns was counted . The new serine side chain (F433S) is located above site 366 in the hydrophilic groove and may interact with the tyrosine 517 residue (see A). As expected, the R366E/F433S YidC mutant promotes membrane insertion of PC-Lep and of Pf3-23Lep . To fluorescently label YidC, a cysteine was introduced in the cytoplasmic loop C1 at position 405 in the cysteine-less WT, R366E, and R366E/F433S YidC proteins. The mutants were purified and Atto647N was attached to residue 405C. The fluorescently labeled YidC proteins were then reconstituted with 1,2-dioleoyl-sn-glycero-3-phosphocholine to generate proteoliposomes. After the purified Pf3 coat protein was labeled at residue 48 with the Atto520 dye in the cytoplasmic tail region, the Pf3 coat protein substrate was added to the proteoliposomes containing the YidC proteins in increasing amounts (see Kd of 241 ± 75 nM (SD of 10 independent measurements) for the WT and 349 nM ± 26 nM for R366E/F433S (experiments performed at room temperature). No Kd could be determined for the R366E mutant. Clearly, the R366E mutant was defective in the binding of the Pf3 coat protein, whereas WT YidC and the double mutant R366E/F433S were capable of efficient substrate binding.If the arginine in the groove serves to make the groove sufficiently hydrated, then a negative charge should also function in this capacity. However, previous studies showed that the negative charge inactivated YidC . To deteondition A. The neidue see A. As expubstrate D. To fluunts see . The binS. mutans YidC2 (R73D) in which R73 corresponds to R366 in E. coli . This S. mutans double mutant R73D/W234M had a mutation of the W234 residue corresponding to the E. coli Y517 YidC residue. Interestingly, when we made the corresponding Ser mutant in the S. mutans YidC2 R73D background, we found that this R73D/F151S YidC2 mutant  was also fully functional at room temperature but inactive at 37 °C . Moreover, a mutant with a substitution of the nearby W234 residue, namely R73D/W234Y, was also found to be temperature-sensitive with little growth at 37 °C but functional at room temperature. The temperature-sensitive phenotype of these mutants suggests that the negatively charged residue in the groove destabilizes the protein at higher temperature. B and C summarizes the membrane insertion results of PC-Lep and Pf3-23Lep  that rescued the activity of the  E. coli  . Remarkably, the F433S suppressor mutation hydrogen bonds to the tyrosine 517 most of the time in the MD simulations (C) predicts that the interior of the groove of the R366E/F433S mutant is indeed negatively charged (red color), while for the WT YidC (B), the groove is slightly positively charged (blue color).To explore the molecular mechanism that maintains the functionality of R366E/F433S YidC mutant, we performed a 100 ns simulation at 27 °C A. Remarkulations , which mulations C predict WT YidC B, the grWe conclude from these results that YidC is functional with a negatively charged residue in the groove when there is a suppressor mutation, indicating that the electrostatic attraction between the arginine in the groove and the negatively charged residues in the substrate region is not required for YidC-mediated insertion.B. subtilis SpoIIIJ and the acidic residues in the N-tail of the substrate MifM are required for insertion . Similar results were observed for the Gram-positive S. mutans YidC2. By substituting W234  with a more hydrophilic (serine or asparagine) residue, we were able to make the originally essential R73 dispensable  of l or not . If an al or not C. Similapensable . Note thE. coli YidC shows that it is located at the dome of the hydrophilic groove and is part of an aromatic cluster at the interface between the aqueous environment in the groove and the hydrophobic outer leaflet embedded portion of YidC. Interestingly, the water accessibility in the top one third of the groove is affected when Y517 is substituted with an apolar residue and is significantly decreased when the positive charge at 366 in the middle of the groove is simultaneously removed (A–C). This was determined by examining the water accessibility in the groove by the reactivity of introduced cysteine residues to NEM and by EPR power saturation studies with a cysteine-incorporated spin label in the groove and monitoring whether the residue is lipid exposed or solvent exposed using paramagnetic O2 and NiEDDA quenchers, respectively. Finally, a decrease in the exposure of water surrounding the groove residues R366, S520, N521, and Q527 was corroborated by examining the number of waters in a fully equilibrated YidC in a lipid bilayer by MD simulations. We cannot rule out that the changes in solvation are caused by small conformational changes in the groove due to the interruption of the aromatic cluster. Alternatively, the water molecules may just fail to reach the top of the groove because of the additional hydrophobicity introduced with the hydrophobic sidechains at 517.A closer look at the position of Y517 in the structure of  removed , A–C. Thet al.  that allow YidC to be functional with a negatively charged residue in the groove. In addition, S. mutans YidC2 variants with an aspartic acid substituting for R73 in combination with W234Y or F151S are fully active at room temperature, although impaired at 37 °C. Interestingly, in all cases, the suppressor mutations are part of the aromatic cluster, which we believe stabilizes the negatively charged residue in the YidC groove through indirect interactions. In the case of the R366E/F433S YidC mutant, MD simulations show that F433S forms a hydrogen bond to tyrosine 517  was from our own laboratory collection. Anti-6X His tag antibody (horseradish peroxidase) was purchased from Abcam.Lysozyme, EDTA, L-(+)-Arabinose, D-(+)-Glucose, and Mal-PEG were purchased from Sigma-Aldrich. PEG6000 was purchased from Hampton Research. Proteinase K was from Qiagen. Isopropyl 1-thio-β-D-galactopyranoside was from Research Products International Corp. Phenylmethylsulphonyl fluoride was purchased from United States Biochemical (Affymetrix). Phosphate Buffered Saline, pH 7.2 and NEM were purchased from Thermo Scientific. Tran-methionine (70 μCi/mL) for 1 min.35S]-labeling at 37 °C, an equal volume of 20% TCA was added to precipitate total proteins. The precipitate was washed with ice-cold acetone and resuspended in Tris–SDS (pH 8.0) buffer. The samples were then subjected to immunoprecipitation with antiserum against Lep to examine membrane insertion by signal peptide processing of PC-Lep to coat-Lep.To analyze the membrane insertion of PC-Lep, a signal peptide cleavage assay was used. After , 3 mg of the spin label MTSL was added to the YidC sample on the column and shaken for 24 h at 4 °C. YidC was eluted from the column with high imidazole buffer  and collected in 1 mL fractions. The samples were analyzed by 15% SDS-PAGE and the fractions containing pure YidC were pooled and dialyzed with buffer  overnight to remove glycerol, imidazole, and any free spin label. The YidC protein was concentrated by centrifugation using a 50 kDa Amicon spin concentrator.Cell pellets were thawed on ice and resuspended in PBS buffer pH 7.2 containing Lysozyme (1 mg/mL). The cells were sonicated on ice for several cycles at an output of 65% power to break open the membranes. The samples were centrifuged at 40,000et al.  and lipid vesicles prepared. The dry lipid film was resuspended in HEPES buffer (pH 8.0) containing 50 mM KCl, 0.02% (w/v) DDM, and incubated at 37 °C for 1 h. The sample was vortexed at a low speed to form multilamellar vesicles. Unilamellar vesicles were generated by the extrusion technique . Specifically, 1 mL of the lipid suspension was extruded 7 to 11 times through a membrane with a pore size of 0.4 μm until a semi-clear solution is achieved. For preparing proteoliposomes, a concentrated spin-labeled YidC sample  was added to the lipid sample and extruded as described above. The final protein:lipid molar ratio was set to 1:400. SM2-Biobeads were prepared according to the manufacturers protocol and added to the proteoliposomes to remove excess DDM as described in Kusters et al.  The remo−3. Langevin dynamics was utilized to enforce constant temperature T = 300 K with a damping coefficient of 0.1 ps−1. Constant pressure simulations (NpT) at 1 atm were conducted using the hybrid Nosé-Hoover Langevin piston method with a 200 fs decay period and a 100 fs damping time constant. Each system was energy-minimized and equilibrated in the NpT ensemble  to backbone atoms were applied when indicated. Coordinates of all atoms in the system were saved every 2 ps. Computation of the electrostatic potential was carried out using the VMD PMEpot plugin with an Ewald factor of 0.25 Å−1 using conformations saved every 80 ps for simulations in which YidC’s backbone was constrained. Molecular images were created with the molecular graphics program VMD (A system with an equilibrated (∼185 ns), crystallography-based model of the WT YidC protein embedded in a lipid bilayer (75% POPE and 25% POPG)  plugin tensemble . Harmonigram VMD .Table 1SPower saturation experiments were performed on a Bruker EMX X-band CW-EPR spectrometer consisting of an ER 041XG microwave bridge coupled with an ER 4123D CW-Resonator (Bruker BioSpin)  is the difference in the P1/2 values for air- and nitrogen-exposed samples, and ΔP1/2(Ni-EDDA) is the difference in the P1/2 values for NiEDDA and nitrogen-exposed samples  equilibrated with nitrogen as a control; (2) equilibrated with a lipid-soluble paramagnetic reagent: air (20% oxygen); and (3) equilibrated with nitrogen in the presence of a water-soluble paramagnetic reagent NiEDDA chelate (2 mM), as described . The sam equilibr samples .Acceptor signal:A cysteine was first incorporated at position 405 of a cysteine-less YidC at the cytoplasmic face of the membrane into the WT and the mutants 366E and 366E/433S. The 405C residues of the YidC mutants were used to attach a fluorescent label with the maleimide dye Atto520 (donor). Residue 48 of the substrate Pf3 was labeled with Atto647N (acceptor). For the labeling reaction, 1.3 fold excess of the dye was added to the protein and incubated for 1.5 h at room temperature. The free dye was separated from the protein by size-exclusion chromatography with a Superdex 200 10/30 column, and the YidC proteins were reconstituted in 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes. The fluorescence measurements to examine binding were performed on a Fluorolog 2.3 (Jobin Yvon-Spex). The extinction wavelength was set at 500 nm to excite the donor dye (Atto520), and the emission wavelength was scanned from 510 to 700 nm. The fluorescence signal of the acceptor was monitored at 669 nm. The Atto647N-Pf3 coat protein was added in steps, each after 1 min, to the proteoliposomes containing 300 nM Atto520-YidC. The concentrations in each of the steps were 60, 60, 150, 150, 150, 300, and 300 nM, reaching a final concentration of 1.17 μM. The measurement started after an incubation period of 1 min after the addition of the Pf3 coat substrate. For our analysis of the binding of Pf3 coat to YidC, we plot the ΔFThe measurements were background-corrected by a buffer and labeled Pf3 only spectrum. The values of the fluorescence signal were corrected by a titration factor of the added Pf3.KD, the binding curve was fitted against the following equation:For the calculation of the All data generated for this study are included within this article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Macaca mulatta) exhibit pronounced individual differences in social traits as measured by the macaque Social Responsiveness Scale-Revised. The macaque Social Responsiveness Scale was previously adapted from the Social Responsiveness Scale, an instrument designed to assess social and autistic trait variation in humans. To better understand potential biological underpinnings of this behavioral variation, we evaluated the trait-like consistency of several biological measures previously implicated in autism  from the RAS-MAPK and PI3K-AKT pathways). We also tested which biological measures predicted macaque Social Responsiveness Scale-Revised scores.Rhesus monkeys , samples were collected thrice over a 10-month period. The following statistical tests were used: “Case 2A” intra-class correlation coefficients of consistency, principal component analysis, and general linear modeling.Cerebrospinal fluid and blood samples were collected from n = 57), to explore potential correlations between the reliable biological measures and their relationship to macaque Social Responsiveness Scale-Revised score; a three-component solution was found. Follow-up analyses revealed that cerebrospinal fluid arginine vasopressin concentration, but no other biological measure, robustly predicted individual differences in macaque Social Responsiveness Scale-Revised scores, such that monkeys with the lowest cerebrospinal fluid arginine vasopressin concentration exhibited the greatest social impairment. Finally, we confirmed that this result held in the larger study sample .All biological measures (except AKT) showed significant test–retest reliability within individuals across time points. We next performed principal component analysis on data from monkeys with complete biological measurement sets at the first time point (These findings indicate that cerebrospinal fluid arginine vasopressin concentration is a stable trait-like measure and that it is linked to quantitative social trait variation in male rhesus monkeys.The online version contains supplementary material available at 10.1186/s13229-021-00442-w. All primate species are social for at least some portion of their lifespan . SocialiIntroduction of the Social Responsiveness Scale (SRS) facilitated rapid and large-scale assessment of social trait variation in humans , 7. ThisThe SRS has proven to be a useful measure of social variation in multiple and diverse human societies –17. GiveVery little is known at present about the biological underpinnings of quantitative social trait variation in human and non-human primates. This is because prior biological investigations have typically focused on the development of species-typical social behavior  in rodent species . NeverthThe first class of biological candidates includes the hypothalamic neuropeptides oxytocin (OXT) and arginine vasopressin (AVP) , and theThe second class of biological candidates includes two main kinase signaling pathways, RAS-MAPK and PI3K-AKT, which include ERK1/2, PTEN and AKT(1–3). ASD is a common comorbidity in rare syndromes  caused by mutations in signaling molecules immediately upstream of ERK  , 33. SimTo better understand the biological basis of social trait variation in primates, here, we measured mSRS scores in a sample of socially housed rhesus monkeys using a refined and highly reliable version of the mSRS , as descN = 76 rhesus monkeys. Subjects were studied in two cohorts to accommodate project workload, and to ensure that all behavioral observations were conducted during the non-breeding season.  Cerebrospinal fluid (CSF) and blood samples were collected from all N = 76 subjects. In a subset of subjects (n = 43), biological samples were collected thrice over a 10-month period. This enabled us to assess the trait-like consistency of our CSF and blood biological measures across multiple time points in a manner not readily achievable in human participants. Collecting biological samples over much of the year also enabled us to examine the stability of these biological measures across the breeding and non-breeding seasons. Finally, experimenters were blinded to monkeys’ mSRS scores during biological sample collection and quantification.A detailed study timeline is provided in Table N = 76 male rhesus monkeys (Macaca mulatta), born and reared at the California National Primate Research Center (CNPRC). All subjects lived in mixed age and sex groups of 58 to 145 individuals, with 98.22 ± 24.14 (Mean ± SD) animals per group. Groups comprised 2–18 matrilines (Median: 14 matrilines per group). Each group was housed in a large, outdoor, half-acre (0.19 ha) field corral (30.5 m wide X 61 m deep X 9 m high). Subjects were housed among 16 of the 24 field corrals.Subjects were Subjects were tattooed as infants and dye-marked periodically to facilitate easy identification for husbandry- and research-related procedures. Monkeys had ad libitum access to Lixit-dispensed water. Primate laboratory chow was provided twice daily, and fruit and vegetable supplements were provided once weekly. Various toys, swinging perches, and other forms of enrichment in each corral, along with outdoor and social housing, provided a stimulating environment.Subjects were 3.73 ± 1.17 (Mean ± SD) years old and ranged in age from 1.25 to 6.27 years at the time of study enrollment. Subjects underwent quantitative behavioral data collection as part of a larger research program focused on the biology of macaque social functioning . EligibiRank was assessed in each corral by behavioral management personnel who recorded aggressive and submissive interactions following food provisioning. Because each corral contained a different number of males, using the raw rank was ineffective as a direct measure that could be compared across all subjects. Thus, rank was calculated as the proportion of males in the group that the focal individual outranked, such that the highest-ranked individual had a value of 1 and the lowest-ranked individual had a value of 0 . Ranks aSubjects were observed unobtrusively in their home field corrals. Inter-observer reliabilities of > 85% agreement were established prior to commencing experimental data collection. Each animal was observed for two 10-min focal samples per day (0800–1030 and 1045–1300) over a 2-week period . A maximum of eight subjects (range: six to eight), residing in one or two corrals, were observed per biweek per observer. Behavior was recorded at 15-s intervals using instantaneous sampling and time-ruled check-sheets . At the N = 76 monkeys. A complete biological measurement set was available from n = 57 of these subjects. A further subset of these monkeys (n = 43) underwent sample collection at two additional time points to enable determination of within-individual consistency in the biological measures for the present study. Sampling procedures were the same as those employed in our prior research [Subjects in the present study underwent biological sample collection as part of the larger research program. CSF and blood samples were first collected 79.37 ± 33.42 days (range: 21 to 157 days) after mSRS-R score ascertainment in all research , and ideSamples were collected between 0800 and 1100 to minimize any potential circadian effects on the biological measurements. Collection of both CSF and blood samples was accomplished within 10–15 min of initial cage entry; only one monkey per day was sampled from the same corral. Briefly, each subject was captured from his home corral, rapidly immobilized with telazol (5–8 mg/kg), and moved to an indoor procedure room. Supplementary ketamine (5–8 mg/kg) was used as needed to facilitate complete immobilization. Immediately following relocation, CSF (2 mL) was drawn from the cisterna magna using standard sterile procedure. CSF samples were immediately aliquoted into 1.5-mL siliconized polypropylene tubes and flash-frozen on dry ice.Next, whole blood samples (up to 25 mL) were drawn from the femoral vein. Blood was collected at room temperature into EDTA-treated vacutainer tubes for kinase quantification. Samples were spun over a Ficoll-hypaque gradient and mononuclear cells collected from the interface were washed in PBS 2x, pelleted, and solubilized . Samples were spun to remove insoluable material and then aliquoted. Whole blood was also collected into PAXgene tubes for neuropeptide receptor gene expression. Samples were left at room temperature for 2 h, subsequently transferred to − 20 °C for 24 h, and then transferred to − 80 °C per manufacturer’s guidelines. All biological samples were stored at − 80 °C until quantification.After sample collection, each subject was prophylactically administered metoclopramide and ketoprofen. Additionally, replacement fluids were given if needed per veterinary guidelines. Subjects were placed in a standard laboratory cage located in a hospital/transition room for recovery overnight, and then returned to their home corrals the next day.CSF OXT and AVP concentrations were quantified using commercially available enzyme immunoassay kits  –44. Thes2 qPCR Primer Assays for Rhesus Macaque OXTR and AVPR1A , and endogenous control  was used for normalization. qPCR was performed on the QuantStudio 3 Real-Time PCR System  with SYBR Green . cDNA was PCR amplified in triplicate, and Ct values from each sample were obtained using QuantStudio 3 qPCR software. Analyses were conducted using the comparative Ct method (2−ΔΔCt) [Measurement of OXTR and AVPR1A mRNA levels was conducted using protocols developed for rhesus monkeys . Total R(2−ΔΔCt) .Blood kinase activation levels were determined using previously published protocols , which hn = 43 monkeys met this inclusion threshold.Data were analyzed using JMP Pro 14 . We first calculated the test–retest reliability of the neuropeptide and kinase signaling pathway data as a “Case 2A” intra-class correlation coefficient of consistency estimated by a Restricted Maximum Likelihood Mixed Model following . This yin = 57 subjects which had a complete set of biological measurements at the first collection time point. Eigenvalues and scree plots suggested a 3-component solution, accounting for 69.1% of the total variance. This was extracted by varimax rotation for principal components. PCA loadings are effect sizes equivalent to the correlation coefficient between the component and the variable [We next performed principal component analysis (PCA) to better understand potential collinearities between the reliable biological measures and their relationship to mSRS-R score. This is critical to prevent collinearities from producing false negatives  in our final General Linear Model (GLM) analysis (see below). This approach is an example of best-practice “sensitivity analysis” . Since avariable .n = 57 subjects had complete biological sample sets available at the first collection time point for analysis. However, CSF AVP concentration had been quantified on all N = 76 subjects, and an AVP value was available from n = 75 of them. This enabled us to run a follow-up analysis in the entire study sample excluding all biological measures except CSF AVP concentration, but including all of the aforementioned blocking factors, in the model.This PCA suggested that CSF AVP concentration and differential neuropeptide receptor gene expression might predict mSRS-R score, whereas the other biological measures appeared unrelated (as assessed by variable loadings). In order to test this hypothesis, we performed a GLM, predicting mSRS-R score, given the reliable biological measures. The analysis controlled for corral, cohort, age, and rank. Blood OXTR and AVPR1A gene expression were highly correlated, and collinear with total and differential neuropeptide receptor gene expression. We therefore included the total neuropeptide receptor gene expression as the sum of the OXTR and AVPR1A gene expression to capture correlated expression of the two genes, and differential neuropeptide receptor gene expression as the difference between OXTR and AVPR1A gene expression to capture relative up or down regulation of these receptors. All of the other reliable biological measures were included in the analysis. This approach conservatively tests each biological measure controlling for all others, and thus identifies variables driving a relationship while excluding variables that are only secondarily correlated. The assumptions of GLM  were tested graphically. No transformations were required. As noted above, ηp2), which is the square of the partial correlation coefficient rp for continuous predictors, following best practice for GLMs [The advanced methods employed here greatly increase power and reduce sample size (often by 5- or 10-fold) over analyses such as T-Tests or simple regression, while simultaneously also reducing false positives –54. Howefor GLMs .All neuropeptide and kinase signaling measures  showed significant test–retest reliability within individuals across multiple collection time points Table . The tesScores on the mSRS-R in this sample ranged from 26 to 88. As observed in past research studies of SRS score distribution in the general human population , 58, mSRPCA extracted a three-component solution. Only one component loaded mSRS-R score and did so along with two biological measures: CSF AVP concentration and blood differential neuropeptide receptor gene expression. The other biological measures loaded onto two separate components Table .Table 3PF1,34 = 6.629; P = 0.0146; ηp2 = 16.3%; rp = − 40.4%; Fig. F1,55 = 4.555; P = 0.0373; ηp2 = 7.65%; rp = − 27.7%).We next used GLM to test the contribution of the biological measures to mSRS-R score. Only CSF AVP concentration significantly predicted mSRS-R score, with lower CSF AVP concentrations predicting higher mSRS-R scores  showed significant test–retest reliability within individuals across time points. PCA revealed a potential association between CSF AVP concentration and mSRS-R score; this relationship was subsequently confirmed by GLM . Specifically, CSF AVP concentration, but no other biological measure that we tested, robustly predicted individual differences in mSRS-R score, such that monkeys with the lowest CSF AVP concentration exhibited the greatest social impairment. These findings buttress those of a prior study reporting stable within-individual consistency of CSF AVP concentration in a modest sample of  monkeys , and add monkeys  and Auti monkeys .It has been known for nearly three decades that AVP signaling plays a critical role in mammalian prosocial behavior. AVP was first implicated in social bond formation and paternal care in voles , 60, 61.These findings implicating AVP in mammalian prosocial functioning led us recently to investigate whether CSF AVP concentration is lower in ASD cases vs. controls. This was indeed the case, as in two independent ASD cohorts, CSF AVP concentration accurately differentiated pediatric ASD cases and controls (aged 1.5–19 years). Specifically, across the range of CSF AVP concentrations, the likelihood of ASD increases over 1,000-fold, corresponding to nearly a 500-fold increase in risk with each 10-fold decrease in CSF AVP concentration , 42. We It is well established that ASD is a principally polygenic inherited brain disorder , and appThis study had several limitations that warrant comment. First, although we used a sophisticated primate model that enabled reverse-translation of a scale that measures social and autistic trait variation in humans, we acknowledge that animal models nevertheless are approximations for examining human neurodevelopmental disease. Second, the relationship between CSF AVP concentration and mSRS-R score in this study was correlational, not causal. Moreover, measurement of AVP in cisternal CSF precluded a more mechanistic understanding of AVP’s role in primate social functioning. Future research using viral vector, optogenetic, gene editing, and/or positron emission tomography radiotracer tools will be required to address this issue in a more tractable manner. Third, unlike the biological measures in this study which were assessed three times, mSRS-R scores were only assessed once per subject, using a relatively new instrument. However, we previously assessed the psychometric properties of the 36-item mSRS, and in the process omitted all items that did not show robust test–retest and inter-rater reliability, resulting in the highly reliable 17-item mSRS-R. We also note that mSRS-R scores are closely related to multiple other measures of social functioning . Fourth,In conclusion, this study marked a first step toward better understanding the biological basis of quantitative social trait variation in primates. Findings from this study established the trait-like consistency of nearly all of the biological “readouts” from the AVP, OXT, RAS-MAPK, and PI3K-AKT signaling pathways in rhesus monkeys. Finally, CSF AVP concentration robustly predicted quantitative social trait variation in this species. Research is now required to better understand the role of the AVP signaling pathway in species typical and atypical primate social functioning, and in human primates, AVP’s involvement at the extreme of the social trait distribution, particularly in the pathogenesis of ASD.Additional file 1.. macaque Social Responsiveness Scale-Revised Instrument.