In addition, correlation analyses between GFAP/S100B levels and CSF routine parameters were performed within the patient group. Patients with unipolar depression had significantly higher levels of GFAP than controls . These results remained significant in a sub-analysis in which all controls were compared with patients suffering from depression matched 1:1 by age and sex . Levels of S100B did not differ significantly between patients and controls . GFAP levels correlated positively with albumin quotients (p < 0.050), S100B levels correlated positively with white blood cell counts (p = 0.001), total protein concentrations (p < 0.001), and albumin quotients (p = 0.001) in the CSF. The significance of the study is limited by its retrospective and open design, methodological aspects, and the control group with idiopathic intracranial hypertension. In conclusion, higher GFAP levels in patients with depression may be indicative of altered microglia activity, especially in astrocytes, in patients with unipolar depression. In addition, correlation analyses support the idea that S100B levels could be related to the integrity of the blood–brain/CSF barrier. Further multimodal and longitudinal studies are necessary to validate these findings and clarify the underlying biological processes.Inflammatory processes involving altered microglial activity may play a relevant role in the pathophysiology of depressive disorders. Glial fibrillary acidic protein (GFAP) and calcium-binding protein S100B are considered microglial markers. To date, their role has been studied in the serum and tissue material of patients with unipolar depression but not in the cerebrospinal fluid (CSF). Therefore, the aim of the current study was to examine GFAP and S100B levels in the CSF of patients with major depression to better understand their role in affective disorders. In this retrospective study, 102 patients with unipolar depression and 39 mentally healthy controls with idiopathic intracranial hypertension were investigated. GFAP and S100B levels were measured using commercially available ELISA kits. CSF routine parameters were collected during routine clinical care. The mean values of GFAP and S100B were compared using age (and sex) corrected ANOVAs. Matched subgroups were analyzed by using an independent sample  The most common pathophysiological explanation of depression relies on the monoamine hypothesis, which relates depressive symptoms to a lack of neurotransmitters such as serotonin, increased degradation of monoamines, or impaired monoamine sensing2. However, in recent years, an increasing number of studies have suggested that inflammatory processes may also be responsible for depressive symptomatology10. As a result, the role of glial cells and their specific markers in body fluids have been investigated increasingly. The concentrations of glial fibrillary acidic protein (GFAP) in the cerebellum, prefrontal cortex, and anterior cingulate cortex have been found to be lower in patients with depression than in healthy controls13. In addition, decreased numbers of astrocytes have been found in hippocampal regions of patients with major depressive disorder14. Among other functions, GFAP is a crucial factor for an intact blood–brain barrier15. Moreover, proteins in the S100 protein family can influence inflammatory processes. They regulate a variety of cell types, such as astrocytes, lymphocytes, and smooth muscle cells16. Elevated serum concentrations of S100B have been found in patients with depression and may prove valuable for evaluating both diagnosis and treatment response23. Most studies that have examined these inflammatory markers in the context of major depression relied on serum or tissue samples. The concentration, origin, and role of inflammatory markers in the cerebrospinal fluid (CSF) of patients with depression have received considerably less attention in psychiatry . Furthermore, the correlative relationship of GFAP/S100B with CSF routine parameters was investigated.The study was part of a larger retrospective project that was approved by the local ethics committee . Lumbar punctures were performed after carefully gathering information and after obtaining written informed consent either as a part of routine clinical care to rule out organic causes of depressive syndromes or—regarding the control group—as a diagnostic investigation or treatment approach for IIH.N = 102) were compared to controls with IIH (N = 39). Only patients aged from 18 to 65 years were included. Patients diagnosed with unipolar depressive syndromes were treated as inpatients at the Department of Psychiatry and Psychotherapy, University of Freiburg. They were diagnosed by experienced professional psychiatrists based on the criteria in the International Statistical Classification of Diseases and Related Health Problems, 10th revision. Patients diagnosed with schizophrenia, bipolar disorder, or underlying dementia were excluded. Previous neurological illnesses were not a fundamental exclusion criterion, but only patients who received a lumbar puncture during inpatient psychiatric treatment to clarify the depressive syndrome were included. The control group has already been described in previous studies24. Control subjects were also between 18 and 65 years old, and they additionally had to be free of any psychiatric diagnosis and not have taken psychotropic medication. No matching between patients and controls regarding sex or age was performed for the whole cohort of study participants (for details see Table https://cran.r-project.org/web/packages/MatchIt/MatchIt.pdf) by age and sex was implemented using the MatchIt package25 in R26, which accesses functions from the optmatch package27. Thus, the calculations were performed using logistic regression (distances as propensity scores). From 134 study participants (96 patients and 38 controls) with available GFAP findings, 38 optimal matching patients were assigned to the 38 control subjects .In this study, consecutive patients diagnosed with unipolar depression N = 10 were com28. The routine analyses were carried out in the CSF laboratory, Department of Neurology and Neurophysiology, University of Freiburg (https://www.uniklinik-freiburg.de/neurologie/klinik/diagnostische-einrichtungen/liquor-labor.html). The residual CSF material was then deep-frozen at −80 °C. Only patients and controls with sufficient residual material were included for GFAP and S100B analyses. The mean storage period of the CSF material was 3.60 ± 2.86 years for the patient group and 3.53 ± 2.33 years for the control group. Therefore, the mean storage time did not differ significantly between the groups .The routine CSF analysis included the determination of the following dimensional values : white blood cell (WBC) count, protein concentration, albumin quotient (AQ), and immunoglobulin (Ig)G index. The measuring was performed according to an established methodologyGFAP measurements were performed using commercially available high-sensitivity ELISA kits from Aviscera Bioscience , and S100B analyses were performed using high-sensitivity ELISA kits from Cloud-Clone Corp. . The ELISA kits were used in accordance with the manufacturer’s specifications, with the exception of using CSF samples. Both kits have not yet been validated for CSF testing.t-tests, differences in sex ratio were calculated with χ2. Univariate ANOVAs with age as a covariate were performed for comparison of S100B and GFAP. In a post hoc analysis, we added univariate ANOVAs with age and sex correction for comparison of S100B and GFAP concentrations. Levels of routine CSF parameters  were analyzed using univariate ANOVAs with age and sex correction. S100B and GFAP concentrations were correlated with routine CSF parameters  and different clinical/psychometric scores  score, Global Assessment of Functioning (GAF) score, and psychopathological scores following the German Association for Methodology and Documentation in Psychiatry (AMDP scores)) in the patient group using Pearson correlation coefficients. Significance was set to p values of <0.05. Given the exploratory approach, no correction for multiple testing was performed.Statistical analyses were performed using Statistical Package for the Social Sciences, version 25 . Age and GFAP levels (only for age and sex matched subgroups) were compared using independent sample p = 0.001), with 54% of patients and 85% of controls being female. Likewise, a significant difference in age was found between patients and controls (p < 0.001). Most patients (75%) experienced a recurrent or chronic depressive episode, and 25% of patients had experienced their first episode. Five patients had moderate depression (5%) and 97 patients (95%) had severe depression. A total of 99 patients (97%) received psychotropic medication. Neurologic comorbidities occurred in 15% of patients.Tables F = 31.057, p < 0.001), and S100B levels still showed no significant differences between the groups . When comparing all controls (N = 38) with available GFAP results and a matched group of patients with depression (N = 38), differences in age  and sex  were no longer evident. The differences in GFAP levels between patients suffering from depression (632.26 ± 309.18) and controls (245.56 ± 176.25) remained highly significant .The concentrations of GFAP and S100B in CSF are presented in Table The routine CSF findings are presented in Table r = 0.323, p = 0.001), total protein , and AQs . GFAP levels in the patient group correlated positively with AQs . No significant correlations between S100B or GFAP levels and suicide attempts, number of earlier inpatient stays, CGI, GAF, or AMDP scores were detected.S100B levels in the group of patients with unipolar depression correlated positively with WBC count  was found in the patient group.30. GFAP may also be involved in long-term potentiation31. So far, only few studies have investigated the concentrations of GFAP in the CSF of patients with major depression32. In the only other comparable study, GFAP in CSF was not found to be related to major depression in elderly women33. However, in contrast to the current study, this was a much older patient group, which could possibly explain the differences to our study results (with higher GFAP levels). As outlined above, several other studies have found lower GFAP levels and decreased numbers of astrocytes in different cerebral regions of patients suffering from depression compared to healthy controls14. In contrast to the present investigation, these studies utilized postmortem tissue samples instead of CSF and may therefore not be directly comparable as CSF samples exclusively measure extracellular concentrations of GFAP, while in tissue samples, intracellular and membrane-bound proteins add to the signal intensity in concentration measurements. Moreover, secretion of GFAP into CSF with subsequent distribution in the whole CSF spaces may have a diluting effect regarding cerebral tissue and lead to a relative enrichment in CSF. Still, the discrepancy between elevated concentrations of GFAP in CSF and decreased GFAP levels in brain tissue samples needs to be further addressed, ideally in studies combining both analyses for the same patients.GFAP levels are known to be altered in brain damage—for example, as a result of traumatic injuries or in the course of neurodegenerative or neuroinflammatory processes34 and bipolar disorder35. In a group of Japanese patients with major depression, S100B was positively correlated with the Hamilton sleep subscale36, no similar correlation with the AMDP score for sleep dysfunction was detected in the present study. A meta-analysis of inflammatory biomarkers in the CSF of patients with mood disorders found that total protein, albumin, and AQs were higher in patients with mood disorders than in healthy controls38. The basic CSF findings from the current study indicating increased protein levels and AQs are in line with these previous results.The lack of a significant difference in the concentrations of S100B in CSF between patients and controls in the present study is in agreement with earlier findings in patients with major depression39. GFAP is widely regarded as an established marker for astrocytic pathologies, and a decreased expression level of GFAP and reduced density of GFAP-immunoreactive astrocytes in tissue samples from various cerebral areas have been associated with major depression42. Additionally, there seems to be a difference in glial changes between early- and late-life depression, with rather subtle glial changes in elderly patients44. For the first time, the current study produces evidence of astrocytic pathology in major depression in terms of increased GFAP signals based on CSF measurements. In a recent prospective study comprising 12 patients with spinal cord injury, GFAP was found to be elevated in CSF possibly reflecting neuronal injury45. Accordingly, elevated levels of GFAP in CSF could also be a consequence of neuronal damage. Moreover, based on the current data, it is not possible to determine if GFAP is released by glial cells as part of a response to neuronal damage or if it is the result of glial cell loss. Further investigations are needed to connect the CSF findings to possible pathophysiological pathways that may precede GFAP release, such as the chronic mild stress response46.Pathologic changes in glial cells and glial metabolism have long been suspected to play a key role in major depression47. The involvement of S100B would provide a link to inflammatory processes, as S100B can bind to the receptor for advanced glycation end-products, which is a known regulator of immunologic and inflammatory pathways48. In addition, in a rat model, chronic stress was found to influence S100B expression in tissue samples, thus yielding a possible connection between the stress response and depression49. In the current study, no significant difference in S100B levels in CSF was identified between patients with depression and controls. Therefore, the results of our study do not support the previously reported alterations in serum and animal models. However, the detected correlations between S100B levels and AQs and protein levels support the long-held hypothesis that S100B levels could be markers for disrupted blood–brain/CSF function50.Extensive studies on S100B in depression have been conducted in the last two decades. Elevated concentrations of S100B in serum samples have been associated with major depression, and a decrease in S100B in the course of therapy may be an indicator of therapy response, although the latter is under dispute10. Certainly, data indicating a possible role of GFAP in the pathogenesis or progression of major depression will add to the growing knowledge of the possible involvement of inflammatory processes in depression52. Based on our data, it is not clear if inflammatory processes are the cause of depression or if they occur as a secondary effect in patients with depression. Hence, future longitudinal studies involving CSF examinations, including the measurement of GFAP, in combination with additional immunological markers are necessary.As only very limited data about the relation of GFAP in CSF to unipolar depression are currently available, this study can provide a starting point for further investigations. Given that GFAP is elevated in the CSF of patients with major depression, it may serve as an additional state or trait biomarker in depression. However, the results of previous studies to date tend to indicate a transdiagnostic phenomenon  corrected ANOVAs were performed in this study, and the results remained stable. Finally, for a sub-analysis, 1:1 matching by age and sex was performed. Again, GFAP levels were identified to be significantly elevated in patients with unipolar depression compared with matched controls. Thus, the authors concluded that the age (and sex) differences alone did not sufficiently explain the group differences. The effects of other influencing factors, such as medication, psychiatric comorbidity, or body mass index, cannot be excluded53. Furthermore, the presence of neurological comorbidities within the group of patients may have a confounding influence on the results. In our department, lumbar punctures are only performed in severe cases with major depression to rule out organic forms. Therefore, the results are not generalizable to all patients with unipolar depression. Because of the invasive nature of lumbar punctures, healthy controls were not recruited for ethical reasons. Additionally, this study was intended as a hypothesis-generating investigation to serve as a potential starting point for further research on GFAP and S100B levels in CSF as potential biomarkers in unipolar depression, limiting the justification for a possible control group of healthy volunteers even further. Thus, given these restrictions, patients with IIH and without psychiatric comorbidities were used as a mentally healthy control group. This approach has been established in previous studies28, but nevertheless, pathological processes in the IIH group may have influenced the results54. Preanalytical handling of the samples was primarily performed to assure measurement of the respective routine CSF parameters. Therefore, the samples were not deep-frozen directly after lumbar punctures, which would have been optimal57. However, at room temperature GFAP and S100B have been reported to be stable in CSF for up to 24 h (GFAP) and 2 days (S100B), respectively60. Storage time also did not differ between the patient and control group. Therefore, preanalytical effects are unlikely to have influenced the results.Although a large number of patients underwent CSF examinations, the current study has some relevant limitations. Because of the retrospective nature of the study, only limited psychometric data were available. In addition, the study groups were not matched for age and sex. For age, a relevant influence has been reported in previous studies. Si et al. reported a decrease in GFAP levels in postmortem brain tissue with age in 15 patients with major depression and 15 controlsThis study is one of the first to measure S100B and GFAP in the CSF of patients with unipolar depression. Elevated GFAP levels were detected; therefore, GFAP levels may serve as an additional biomarker for major depression in the future, but further investigations, including multimodal longitudinal studies, are needed to clarify the exact role and reliability of intrathecal GFAP alterations in depression.


Currently no reliable tools are available for predicting the risk of central nervous system (CNS) infections in patients with intracerebral hemorrhage after undergoing ventriculostomy drainage. The current study sought to develop and validate a nomogram to identify high-risk factors of CNS infection after ventriculomegaly drain placement for intracerebral hemorrhage.A total of 185 patients with intracerebral hemorrhage who underwent ventriculoperitoneal drainage were enrolled to the current study. Patients were divided into a CNS infection group (20 patients) and a non-CNS infection group (165 patients). The baseline data from both groups was used to develop and evaluate a model for predicting the likelihood of developing CNS infection after ventriculoperitoneal drain placement for intracerebral hemorrhage.The finding showed that operative time, intraventricular drainage duration, postoperative temperature, white blood cell count in cerebrospinal fluid (CSF), neutrophils ratio in CSF, Red blood cell count in CSF, and glucose content in CSF were correlated with CNS infection. A nomogram for predicting the risk of CNS infection was constructed based on these variables. The c-index and the AUC of the ROC curve was 0.961, showing good discrimination. Clinical decision curve analysis indicated that the nomogram clinical application ranged between 1 and 100%. The clinical impact curve was generated to set with a threshold probability of 0.5.The nomogram reported in the current study can be used by clinicians to identify patients likely to have secondary CNS infections, so that clinicians can better treat these patients at earlier stages. Neurosurgery central nervous system infections (NCNSIs) refer to infections either secondary to neurosurgical illness or intracranial and intraspinal disorders that require management by neurosurgery . CNS infSurgery of external ventricular drainage is a common treatment for intracerebral hemorrhage. However, the patient is at risk of developing a CNS infection because the drainage tube passes through the patient in and out of the skull. CNS infections can be severe, thus affecting the daily life of the patient, or can be fatal . Early dA nomogram model is a tool for integrating disease-related risk factors and for determining numerical probabilities of target events with individualized ability to predict the risk of developing disease through an intuitive model display . PreviouIn the current study, risk factors for secondary CNS infections such as operative time, postoperative temperature, intraventricular drainage duration, CSF white blood cell count, CSF neutrophils ratio, CSF red blood cell count, and CSF sugar content were identified through regression analysis. A nomogram model was established based on the above risk factors for personalized prediction of the likelihood of secondary CNS infection in patients with intracerebral hemorrhage undergoing ventriculostomy.The current study retrospectively included patients who underwent ventriculostomy due to intracerebral hemorrhage from January 2016 to August 2020 at the Meizhou People’s Hospital. Basic clinical information of the patients, including gender, age, length of surgery, duration of drain retention, postoperative temperature, and whether CNS infection occurred secondarily, were obtained from medical records. Laboratory test results of patients in the CSF were obtained, including white blood cell count, neutrophil ratio, number of red blood cells, glucose content, protein content, and chloride content in the CSF. Patients gave informed consent prior to the study. The study was approved by the ethics committee of Meizhou People’s Hospital.9/L and neutrophil ratio > 0.8. (3) Intracranial pressure and cerebrospinal fluid related examinations including intracranial pressure [Diagnosis of neurosurgery CNS infection is divided into clinical diagnosis and etiologically confirmed diagnosis. Compliance with 1–4 of the following criteria indicates a clinical diagnosis , compliapressure , CSF chaPatient sex, age, postoperative temperature, operation time, Intraventricular drainage duration, Intraventricular irrigation, diabetes, pneumonia were obtained. CSF samples were obtained through ventricular drainage. All samples included the last cerebrospinal fluid from which the drainage tube was removed, and the test items included CSF bacteria culture, white blood cell count in CSF, neutrophil ratio in CSF, number of red blood cells in CSF, glucose content in CSF, protein content, and chloride content in CSF. All biochemical tests were performed using standard automated laboratory methods.P < 0.05.Differences in baseline characteristics between the two groups were determined using spss22.0 for intergroup contrast. Lasso regression was performed for risk factor selection and selected risk factors were used in multivariate analysis. A risk prediction model of secondary CNS infection after ventriculomegaly drainage for intracerebral hemorrhage was established using the RMS R package based on findings from logistic regression analysis. Concordance index (c-index) was used to evaluate the accuracy of the model. Bootstrap method (1000 random samplings) was used to internally validate the model and a calibration curve was generated. The accuracy of the nomogram was explored using Hosmer–Lemeshow test [All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors. For this type of study, formal consent is not required.The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. All patients gave their full consent to participate in this study, and a written consent form was obtained from each patient.6/L, neutrophil ratio was 87.05 ± 8.42%, RBC count was 216045.00 ± 314093.96/L, CSF glucose content was 1.26 ± 1.12 mmol/L, CSF protein content was 4.59 ± 3.58 g/L, and CSF chloride content was 119.26 ± 9.24 mmol/L. The non-CNS infection group comprised 97 males and 68 females with an age of 60.95 ± 12.37 years. In addition, the mean operative time of the non-CNS group was 1.78 ± 1.27 h, mean Intraventricular drainage duration was 13.16 ± 9.71 days, mean postoperative temperature was 38.13 ± 0.76 °C, WBC count was 1551.23 ± 3912.15 × 106/L, neutrophils ratio was 69.49 ± 24.11%, RBC count was 145736.48 ± 404144.48/L, CSF glucose content was 3.53 ± 1.61 mmol/L, CSF protein content was 4.35 ± 5.36 g/L, and CSF chloride content was 121.46 ± 6.82 mmol/l  and non-CNS infection group (165 patients). CNS infection group comprised 14 males and 6 females, with a mean age of 59.35 ± 9.89 years. The mean operative time of the CNS infection group was 2.18 ± 1.33 h, mean Intraventricular drainage duration was 22.50 ± 14.18 days, mean postoperative temperature was 38.77 ± 0.4964 °C, WBC count was 18009.30 ± 24567.19 × 10λ values for optimal λ value, which has the lowest model error at this time. The simplest model was obtained within one standard deviation of the dotted left line, corresponding to the dotted right line. A total of 7 variables including operative time, Intraventricular drainage duration, postoperative temperature, CSF WBC count, CSF neutrophils ratio, CSF RBC, and CSF glucose content were included in the analysis.Lasso regression was used to screen risk factors that are potential predictors of CNS infection. Nine variables, including operative time, Intraventricular drainage duration, postoperative temperature, CSF WBC, CSF neutrophils ratio, CSF RBC, CSF glucose content, CSF protein content, and CSF chloride content, were used in constructing the lasso regression curve using R glmnet package Fig. a. The fiP = 0.0032) and CSF glucose content (P = 0.01) were independent risk factors for predicting secondary CNS infection in patients who underwent ventriculostomy drainage for ICH. Notably, the risk of CNS infection increased with increase in the Intraventricular drainage duration and with decrease in CSF glucose level. Lasso regression findings were combined with clinical characteristics to construct a nomogram model based on logistic regression model. The nomogram comprised 7 variables including operative time, intraventricular drainage duration, postoperative temperature, CSF WBC count, CSF neutrophils ratio, CSF RBC count, and CSF glucose content  was used to internally validate the model and for construction of the calibration curve  was an independent risk factor for predicting secondary CNS infection in patients with ICH who had undergone ventriculostomy drainage. Notably, the risk of secondary CNS infection increased with increase in intraventricular drainage duration. This indicates that surgeons should have good control over operative time when performing ventriculoperitoneal drainage. Normal concentration of CSF glucose is 2.5–4.5 mmol/L, which is 2/3 of the serum glucose level [P = 0.0075) was an independent risk factor for predicting secondary CNS infection. This is consistent with the findings by Namani et al. that reduced CSF sugar levels indicate a poor prognosis in patients with septic meningitis [CNS infections include meningitis and (or) encephalitis, mainly caused by abnormal cerebrospinal fluid such as an elevated white blood cell count . CSF in iagnosis . Furtheriagnosis . The finiagnosis . A previiagnosis . Surgeryiagnosis . The finse level . The finningitis . The bacIn summary, the findings of the study showed that operative time, intraventricular drainage duration, postoperative temperature, CSF white blood cell count, CSF neutrophils ratio, CSF red blood cell count, and CSF sugar content were risk factors for secondary CNS infection. A nomogram was established based on the above-mentioned risk factors to predict the risk of secondary CNS infection after ventriculomegaly catheterization for cerebral hemorrhage with good discrimination and accuracy. Notably, the nomogram is simple and easy to perform, and the indicators used are all clinically available items. However, this study is a single center study with a relatively small sample size. Therefore, a large sample multicenter study to provide more information for risk prediction of CNS infections should be conducted. However, this model provides a basis for clinical management of secondary CNS infections after ventriculomegaly drainage. In addition, it helps guide clinicians in screening high-risk patients.


Stroke is a massive public health problem. The rupture of vulnerable carotid atherosclerotic plaques is the most common cause of acute ischemic stroke (AIS) across the world. Currently, vessel wall high-resolution magnetic resonance imaging (VW-HRMRI) is the most appropriate and cost-effective imaging technique to characterize carotid plaque vulnerability and plays an important role in promoting early diagnosis and guiding aggressive clinical therapy to reduce the risk of plaque rupture and AIS. In recent years, great progress has been made in imaging research on vulnerable carotid plaques. This review summarizes developments in the imaging and hemodynamic characteristics of vulnerable carotid plaques on the basis of VW-HRMRI and four-dimensional (4D) flow MRI, and it discusses the relationship between these characteristics and ischemic stroke. In addition, the applications of artificial intelligence in plaque classification and segmentation are reviewed. AIS is the second leading cause of death to humans after cancer. High recurrence, disability and mortality pose heavy economic and disease burdens on patients and their families . EmbolisVulnerable carotid plaques have specific morphological and hemodynamic characteristics. However, conventional angiography techniques, such as digital subtraction angiography (DSA), computed tomography angiography (CTA) and magnetic resonance angiography (MRA), can assess only the degree of vascular stenosis. They cannot provide specific characteristics of vulnerable carotid plaques. However, guidelines from the American Society of Neuroradiology (ASNR) have highlighted that not only the degree of vascular stenosis but also plaque composition is attributable to the risk of stroke associated with carotid plaques . DevelopThis review summarizes developments in the imaging and hemodynamic characteristics of vulnerable carotid plaques based on VW-HRMRI and 4D flow, and it discusses the relationship between these characteristics and ischemic stroke. In addition, the applications of artificial intelligence in plaque classification and segmentation are reviewed.Vulnerable carotid plaques have the following histopathologic features: lipid-rich necrotic cores , thin fibrous cap (<165 μm), intraplaque hemorrhage (IPH), intraplaque inflammation and neovascularization, plaque surface ulceration and positive vascular remodeling. The ability of VW-HRMRI to classify plaques has been demonstrated by evidence of high consistency between VW-HRMRI and histopathology in detecting vulnerable plaques . Plaque Plaque vulnerability is associated with the LRNC and thin fibrous cap. LRNC is composed mainly of cholesterol crystals, apoptotic cell debris and calcium, showing isointensity on time-of-flight (TOF) images, T1-weighted images (T1WIs) and T2-weighted images (T2WIs), with no or mild enhancement ,9,10. ThLRNC has been shown to be an independent imaging predictor of vulnerable carotid plaques, with the area under the receiver operating characteristic curve (AUC) used for identifying vulnerable plaques being 0.690 . The lipThe fibrous cap is a layer of fibrous connective tissue covering the lipid core. An intact fibrous cap presents as a continuous hypointensity band on TOF, often isointensity on T1WIs, hypointensity or hyperintensity on T2WIs and slight hyperintensity on proton density-weighted images (PDWIs). Fibrous cap thinning or discontinuity is an important characteristic of vulnerable plaque. Gadolinium-based contrast enhancement VW-HRMRI is the imaging technique of choice for imaging this characteristic .IPH is one of the key characteristics of vulnerable plaques and is mainly attributable to vascular endothelial damage and neovascular dilation and leakage . The AUCThe signal of IPH varies because it depends on the oxidative state of hemoglobin. VW-HRMRI is the best imaging technique for the detection of IPH. It shows up as hyperintensity on T1WIs and isointensity or hypointensity on T2WIs around week 1; hyperintensity on both T1WIs and T2WIs during weeks 1–6; and hypointensity on both T1WIs and T2WIs after week 6. However, no evidence is available to connect the time of IPH with occurrence of AIS; thus, further studies are needed.Other important characteristics of vulnerable plaques are intraplaque inflammation and neovascularization. Studies have shown that ¹⁸F-sodium fluoride PET and molecular imaging are innovative techniques for the detection of plaque inflammation, and contrast-enhanced ultrasound (US) and computed tomography (CT) can also be applied to detect intraplaque neovascularization ,20. HoweA cross-sectional study of 62 patients with carotid atherosclerotic plaques reported that intraplaque macrophage infiltration is associated with plaque rupture and AIS , but theThe surface of carotid plaques can be classified as smooth or irregular. Irregularity in the plaque surface—particularly ulceration—is considered as a characteristic of vulnerable plaques. The definition of carotid plaque ulceration varies depending on the modality used or even among different research groups. In terms of histology, ulceration describes an endothelial defect of at least 1000 μm in width, resulting in the exposure of the plaque’s necrotic core to circulation. On an image, ulceration is reserved for cavities measuring at least 1 mm or 2 mm, according to different studies and proposed risk stratification systems ,25. The One study demonstrated that ulceration was associated not only with the presence of IPH but also with LRNC. It is a precursor of stroke events, and ulcerated plaques are related to a sevenfold increase in ipsilateral stroke risk . Animal Vulnerable carotid plaques can also be identified on the basis of the pattern of vascular remodeling. During the slow formation of atherosclerotic plaques, the vascular lumen can develop positive remodeling  and negative remodeling . Plaques with negative remodeling typically have small lipid cores with calcification and fibrosis, indicating a stable state of plaques. Those with positive remodeling are usually characterized by LRNC and intraplaque inflammation, which are vulnerable plaques prone to rupture . One stuIn addition to the above qualitative imaging characteristics, the identification of vulnerable carotid plaques also includes quantitative imaging characteristics. Common quantitative parameters include plaque volume and thickness, the normalized wall index (NWI), the remodeling index and the vascular stenosis ratio, as shown in Larger plaque volume is a useful characteristic for identifying vulnerable carotid plaques. Similar to plaque volume, the large maximum plaque thickness is a characteristic of vulnerable carotid plaques because it is linked to the large plaque volume. MRI possesses superior soft-tissue contrast, so VW-HRMRI is highly useful for quantifying the thickness and volume of carotid plaques.A cross-sectional study of 1072 patients showed that larger maximum carotid plaque thickness (quantified by MRI) was strongly associated with cerebral ischemic symptoms and the occurrence of stroke . One retNWI based on VW-HRMRI is a better indicator to evaluate carotid plaque burden than the degree of vascular stenosis, with high accuracy and repeatability of measurements. In a study of middle cerebral artery atherosclerosis, measures of plaque burden significantly improved the assessment of vulnerable plaques compared with the degree of vascular stenosis . NWI = wThe plaque remodeling index is a quantitative indicator to evaluate the pattern of vascular remodeling. Its calculation formula is as follows: remodeling index = vascular area at the slice with the narrowest lumen/vascular area at the adjacent slice with normal vascular wall. A remodeling index ≥1.0 indicates positive remodeling, while a remodeling index <1.0 indicates negative remodeling.The degree of vascular stenosis is assessed by the stenosis ratio defined by NASCET  criteria. Stenosis ratio =  × 100%. Numerous studies have shown that the degree of vascular stenosis is an influencing factor for AIS but is insufficient to predict the occurrence of AIS in patients with carotid atherosclerosis, because in most cases, vulnerable carotid plaques are accompanied by positive vascular remodeling . A growiMorphology and hemodynamics are the focuses of the study of carotid atherosclerotic plaques. As mentioned above, carotid plaque morphology studies have been considerably advanced with the application of VW-HRMRI. However, by morphology alone, it still cannot completely distinguish vulnerable plaques from stable plaques, especially in the case of atypical plaques . TherefoHemodynamics characterizes the interaction between blood and the vascular wall. Sufficient evidence has shown that hemodynamic factors are involved in the formation and progression of carotid atherosclerotic plaques by causing intimal injury and inducing physiological and morphological changes in vascular endothelial cells and smooth muscle cells . CommonlFirst, Zhang et al.  measuredPDGF-A) and vascular cell adhesion molecule-1 (VCAM-1); the transfer of atherosclerotic substances; and the adhesion of monocytes to the endothelium. The low WSS alters endothelial flow patterns at the molecular and cellular levels; a decrease in WSS led to the 18-fold proliferation of endothelial cells within 48 h [WSS has been confirmed by numerous studies as a critical hemodynamic parameter in the formation and progression of carotid atherosclerotic plaques. WSS refers to the frictional force between blood flow and the endothelium exerted on the unit area of the vascular wall. It is closely related to blood characteristics, blood velocity and vascular morphology. It is now generally accepted that low WSS is an important factor in promoting carotid plaque formation ,52. Artehin 48 h . Moreovehin 48 h ,56, and hin 48 h .However, few studies have explored the relationship between PG values and carotid plaque formation. In one investigation , the PG Similarly, there are few studies on the relationship between carotid plaque formation and EL values. Sia et al.  discoverIn addition to carotid plaque formation, WSS may also affect plaque composition and its stability. A growing amount of evidence has demonstrated that low WSS promotes carotid plaque formation, but after which, high WSS advances it into a vulnerable plaque. WSS >40 Pa can directly damage the vascular endothelium, aggravate inflammatory response and spur lipid core progression and IPH in carotid atherosclerotic plaques. The location of increased WSS frequently matches the sites of carotid plaque ulceration, inflammation and rupture . Groen eZhang et al.  studied Huang et al. found that the mean WSS at the sites of IPH was higher than that at nonbleeding sites. The reason may be that high WSS spurs the expression of vascular endothelial growth factor, which induces the formation of new vessels and damages the vascular endothelial barrier in plaques, thereby leading to IPH . TuenterIn addition, longitudinal and cross-sectional studies have demonstrated a link between high WSS and lipid deposition in plaques. Plaque regions of high baseline WSS become “softer” over time, i.e., lipid deposition; high WSS is related to LRNC .In larger plaques (plaque burden ≥46%), a significant positive linear relationship between WSS and plaque burden was observed, but it does not indicate a causal relationship between high WSS and a higher plaque burden .There is controversy over the relationship between high WSS and positive vascular remodeling. In a 6-month follow-up study, Stone et al. observed that regions of low baseline WSS exhibited positive vascular remodeling . HoweverPlaque structural stress (PSS) is deemed to play a potential role in carotid plaque rupture. When PSS exceeds the strength of the fibrous cap covering the plaque, plaque rupture occurs. Histology research revealed higher PSS in the area of plaque rupture . In addiAlthough VW-HRMRI and 4D flow can provide accurate qualitative and quantitative assessments of the morphology and hemodynamic of carotid atherosclerotic plaques, both the qualitative and quantitative characteristics are subject to the subjectivity of observers and the heterogeneity of imaging protocols. At the same time, substantial microcosmic information is missing. Machine learning based on radiomic analysis provides a new method to quantify the heterogeneity of lesions through high-throughput extraction and an analysis of digital features from medical images by using computer software. It has been used in the qualitative diagnosis of lesions, clinical grading and staging, tumor genetic biomarker identification, efficacy evaluation and prognosis. The radiomics research includes the following steps: (1) image acquisition and preprocessing; (2) regions of interest (ROIs) segmentation; (3) feature extraction and screening; and (4) model establishment and model performance evaluation. Initial radiomic studies on atherosclerotic plaques were based on a texture analysis of CT or US images ,79. Compp < 0.05) and the AUCs >0.65 were set as the inputs for random forest training features. The results showed that seven radiomic features from T1WIs and three from T1WI-CE images were independently associated with plaque stability. The diagnostic performance parameter AUC for plaque classification was 0.833 with VW-HRMRI features, 0.893 with T1WI radiomic features and 0.918 with T1WI-CE radiomic features. The combination of T1WI and T1WI-CE radiomic features improved the AUC to 0.936, significantly higher than those of the imaging feature model and single-sequence radiomic feature model.Shi et al.  conducteSubsequently, Shi et al.  performeZhang et al.  establisThese results indicate that compared with the traditional subjective qualitative and quantitative imaging characteristics, the radiomic method enables finding more differential features, showing its higher value in determining plaque vulnerability. However, the ROIs in the above studies are all based on manual segmentation, which is time-consuming and subject to observer variability. Furthermore, complex vascular curvature, too-small plaques and surrounding tissue disturbance pose more challenges to the manual segmentation of plaques.At present, artificial intelligence has played an indispensable role in the medical industry. Plaque automatic segmentation based on artificial intelligence can improve the work efficiency of physicians. So far, various segmentation methods have been proposed at home and abroad, which are predominantly divided into those based on traditional image processing and those based on deep learning. The segmentation results based on traditional image processing are not accurate enough to achieve the requirements of clinical diagnosis.Deep learning was first proposed in 2006 by Hinton, who then gradually applied it in image processing . Deep-leFew studies have been reported on VW-HRMRI plaque segmentation. In 2017, Dong et al.  used FCNAlthough the above automatic segmentation accuracies are not satisfactory, these studies have proven that it is feasible to apply neural networks to automatically segment carotid plaques and their components. It lays the foundation for future research and provides new research directions.In summary, VW-HRMRI, 4D flow and artificial intelligence improve the efficiency and accuracy of vulnerable plaque assessment, which are significant for guiding clinical aggressive therapy to reduce the risk of plaque rupture and stroke. However, VW-HRMRI has high requirements for hardware and software, and the inspection time is long. At present, the research and application of 4D flow are still few, and the major hardware manufacturers have not yet fully commercialized VW-HRMRI. Radiomics studies on atherosclerotic plaques are retrospective at present, which cannot determine the ability to predict the risk of stroke. Therefore, large-sample multicenter prospective studies are needed to provide more valuable information in future and ultimately promote these advanced technologies so that they may be used in efficient clinical applications.


There are two types of abnormal hematopoiesis in solid tumor occurrence and treatment: pathological hematopoiesis, and myelosuppression induced by radiotherapy and chemotherapy. In this review we primarily focus on the abnormal pathological hematopoietic differentiation in cancer induced by tumor‐released granulocyte colony‐stimulating factor (G‐CSF) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). As key factors in hematopoietic development, G‐CSF/GM‐CSF are well‐known facilitators of myelopoiesis and mobilization of hematopoietic stem cells (HSCs). In addition, these two cytokines can also promote or inhibit tumors, dependent on tumor type. In multiple cancer types, hematopoiesis is greatly enhanced and abnormal lineage differentiation is induced by these two cytokines. Here, dysregulated hematopoiesis induced by G‐CSF/GM‐CSF in solid tumors and its mechanism are summarized, and the prognostic value of G‐CSF/GM‐CSF‐associated dysregulated hematopoiesis for tumor metastasis is also briefly highlighted. G‐CSF/GM‐CSF are well‐known facilitators of myelopoiesis and mobilization of hematopoietic stem cells. G‐CSF/GM‐CSF‐induced hematopoietic dysregulation has a critical role in the progression and metastasis of solid tumors, characterized by myeloproliferative phenomena, extramedullary hematopoiesis, and bone marrow stem/progenitor compartment destruction. EMHextramedullary hematopoiesisG‐CSFgranulocyte colony stimulating factorGM‐CSFgranulocyte‐macrophage colony stimulating factorHSChematopoietic stem celliMCimmature myeloid cellMDSCmyeloid‐derived suppressor cellM‐MDSCmonocytic myeloid‐derived suppressor cellPMN‐MDSCpolymorphonuclear myeloid‐derived suppressor cellTAMtumor‐associated macrophageTANtumor‐associated neutrophilTMEtumor microenvironmentHematopoiesis is the production of blood cells and immune cells from pluripotent hematopoietic stem cells (HSCs) in hematopoietic organs. This process is precisely controlled by the level of endogenous hematopoietic growth factor and the interplay of transcriptional and epigenetic networks . In geneIn recent years, hematopoietic abnormalities in solid tumors characterized by myeloproliferative phenomena, extramedullary hematopoiesis (EMH), and bone marrow stem/progenitor compartment destruction have attracted more and more attention. It is considered to play an important role in the tumor immunosuppressive milieu, metastasis, and prognosis.lo/−Sca‐1+c‐Kithi (LSK) cell population. Evidence suggests that hyperactivation of the CCL2/CCR2 pathway in the spleen of tumor‐bearing mice contributes to the recruitment of LSK HSPCs [The spleen, one of the major sites of EMH induced by solid tumors, is characterized by an accumulation of various hematopoietic stem/progenitor cell (HSPC) types and is associated with a significant myeloid skew within the LinIn vitro, LSECs have been confirmed to support differentiation, proliferation, and survival of HSCs [The liver is one of the primary organs where EMH can take place. A study suggested that an extramedullary niche suitable for HSCs migration and differentiation consist of liver sinusoidal endothelial cells (LSECs) characterized by high expression of stromal‐derived factor (SDF)‐1 . In vitr of HSCs . Evidenc of HSCs . Notably of HSCs .Research suggests that the serum levels of cytokines, including G‐CSF, GM‐CSF, and proinflammatory cytokines produced by malignant cells and stromal cells are usually elevated in patients with solid tumor cancers . SubsequTumor‐derived factors regulate the differentiation of HSCs in bone marrow, subsequently contributing to hematopoietic dysregulation in cancer patients. The different hematopoietic processes in patients with malignant tumors are shown in Fig. et al. [As summarized in Table , tumor‐det al.  discoverThe regulation of the proliferation and differentiation of hematopoietic progenitor cells (HPCs) is generally controlled by hematopoietic growth factors (HGFs), a type of glycoprotein hormone, in the bone marrow. Although not very common, cancers, especially the most rapidly advancing ones, secrete HGFs like G‐CSF and GM‐CSF. For example, tumor cells from acute myeloid leukemia (AML) patients expressed transcripts for HGFs including GM‐CSF and G‐CSF . FurtherG‐CSF and GM‐CSF are well‐known cytokines secreted by immune cells, fibroblasts, and endothelium. In physiological conditions, G‐CSF and GM‐CSF can induce the differentiation of HPCs into different immune cells, including granulocytes, monocyte‐macrophages, T‐cells, and natural killer (NK) cells in the bone marrow. Therefore, they are widely used as potent factors to control radiation‐ and chemotherapy‐induced neutropenia in cancer therapies , 18, 19.et al. [et al. found a 4–7‐fold increase of circulating GMPs in peripheral blood in patients with seven different types of tumors (n = 133), which positively correlated with disease progression. These reports reveal the vital role of circulating HSCs in patients with cancer [Giles et al.  found inet al. . Furtherh cancer .+ suppressive regulatory T‐cells (Tregs), which are critically involved in immune tolerance and homeostasis. The production and immunosuppressive effect of MDSCs are well described in many diseases including, but not limited to, obesity, cancer, autoimmune disorders, and infection. Indeed, it has been previously proven that MDSCs can be recruited by tumor‐derived chemotactic factors, enter tumor microenvironments, and differentiate into mature immunosuppressive cells represented by TAMs.MDSCs represent a heterogeneous population of largely immature myeloid cells whose production is induced by tumor‐derived factors, such as G‐CSF, GM‐CSF, and IL‐6. MDSCs have suppressive properties in T‐cell proliferation, cytotoxic and NK cell activation, and also promote the differentiation and expansion of Foxp3+Ly6G−Ly6Chi) and polymorphonuclear MDSCs (PMN‐MDSCs) (CD11b+Ly6G+Ly6Clo). In humans, there is another subtype called early‐stage MDSCs (e‐MDSCs), which express the myeloid markers CD33 and CD11b but lack the myeloid lineage markers CD14 and CD15/CD66b [In mice, two major subtypes of MDSCs have been identified, including monocytic MDSCs (M‐MDSCs) (CD11b15/CD66b .+ in the tumor microenvironment (TME) [MDSCs inhibit antitumor immune response through various mechanisms. L‐Arginine is important for T lymphocyte proliferation and production of interferon‐gamma (IFN‐γ) by NK cells. MDSCs promote the depletion of L‐arginine by arginase 1 (Arg‐1) hypersecretion, thereby suppressing the antitumor effect of T and NK cells , 29. Indnt (TME) .TAMs and MDSCs are the most myeloid cell populations in TME . Monocyt+CD14−CD66b+CD15hi [et al. [et al. [Neutrophils play a key role in the defense against infection and activation of both the innate and adaptive immunity. In malignant tumors, TANs have been regarded as an important component of TME . In humab+CD15hi . In 2009b+CD15hi . N1 neutb+CD15hi . Furtherb+CD15hi . Interesb+CD15hi . Within  [et al.  demonstr [et al. . However [et al.  have shoet al. [In contrast, N2 neutrophils accelerate tumor progression through various mechanisms. N2 neutrophils release multiple enzymes, including myeloperoxidase (MPO), neutrophil elastase (NE), neutrophil collagenase (MMP8), and gelatinase B (MMP9). MMPs facilitate extracellular matrix remodeling and angiogenesis . NE and et al.  found th+CD68+) and alternatively activated M2 macrophages (CD163+CD206+) [+ T‐cells by producing Arg‐1, iNOS, ROS, and RNS, but also inhibit antitumor immune response by recruiting Tregs [TAMs represent one of the primary tumor‐infiltrating immune cell types, displaying functional plasticity and adapting to change within the microenvironment . In gene+CD206+) . Contras+CD206+) . In glio+CD206+) . M2 macr+CD206+) . Further+CD206+) . Thus, Mng Tregs . Therefoin vitro. G‐CSF has been widely used to mobilize HSCs in the bloodstream for transplantation for over two decades. It efficiently mobilizes HSCs into the blood by targeting HSCs niche function and bone formation via specific bone marrow macrophages.The first identified HGFs were called CSFs because they could stimulate growth and clone formation of various bone marrow HSCs −Gr1− bone marrow progenitor cells, subsequently inducing them to differentiate into MDSCs [GM‐CSF is one of the main tumor‐derived soluble factors that can induce the differentiation of monocytic/granulocytic bone marrow cells into CD11bto MDSCs . Many stto MDSCs . High doto MDSCs .lyn and p56/59hck, STAT1, STAT3, and STAT5, as well as components of the p21ras/Raf/mitogen‐activated protein kinase pathway [G‐CSF/GM‐CSF, two recognized cytokines involved in the regulation of hematopoiesis, can control the self‐renewal of HSCs and regulate the differentiation of HSCs into specific lineages. What’s more, G‐CSF and GM‐CSF were identified as “lineage‐specific” cytokines regulating progenitors in the granulocyte, granulocyte/macrophage lineages, respectively . In gene pathway . However pathway , finally+HLA−DR−/low MDSCs in the peripheral blood [+CD11b+ MDSCs and Tregs in the peripheral lymphoid organs [MDSCs are characterized by their myeloid origin, immature state, and immunosuppressive ability, mainly including M‐MDSCs and PMN‐MDSCs . They plal blood . In addid organs . In solid organs . A noveld organs . Moreoved organs . Evidencd organs .Neutrophils are widely present in the human peripheral blood circulation. Especially when the host is injured or infected, G‐CSF will mobilize and expand neutrophils. Therefore, G‐CSF has been used to support patients receiving chemotherapy . But cliTumor‐derived G/GM‐CSF play a critical role in the activation and differentiation of TANs. G‐CSF could induce TANs to generate NETs, which has been confirmed to possess a dual effect on tumor growth . MoreoveIn vivo and in vitro data confirmed that the activation of the GM‐CSF/STAT5 signaling pathway contributes to the progression of tumor and M2 phenotype polarization of TAMs [It has long been assumed that TAMs originate from monocytes/macrophages generated from HSCs; these cells are heterogeneous and versatile that could undergo their phenotypic/function dynamics in response to microenvironmental signals . TAMs ar of TAMs . In a mo of TAMs . However of TAMs . These rNormally, the development of blood cell lineages is tightly controlled by endogenous signals that drive the differentiation of HSCs to highly proliferative lineage‐committed progenitors , 97, 98.+CD11b+ MDSCs are mainly responsible for the secretion of these cytokines [+ T lymphocytes by producing high levels of Arg‐1 and inducible iNOS [+ CD15+ CD11b+ CD33+ HLA‐DR− Lin− was correlated with poor prognosis in patients with NSCLC [Lung cancer is the most common malignant cancer driven by ectopic secretion of G‐CSF/GM‐CSF, including primary and metastatic types , 107. Inytokines , 111. A ytokines . There wble iNOS , 114. Hoth NSCLC . Furtherth NSCLC . Smokingth NSCLC , 118. Itth NSCLC . Given t+HLA‐DR−) in their blood that are composed of neutrophilic , lineage‐negative , and monocytic  subsets. And these MDSCs promote the progression of GBM by suppressing T‐cell IFN‐γ generation [Glioma is the most frequently occurring type of malignant brain tumor, including glioblastoma (GBM), the highest‐grade primary central nervous system cancer . An incrneration . A studyneration .+ Foxp3+ Tregs [in vitro study suggested that M2 macrophages could promote human urothelial bladder cancer (UBC) T24 cells survival [G‐CSF was initially extracted from the human bladder carcinoma cell line 5637, suggesting an important role in progression of bladder malignancies . An auto3+ Tregs . Furthersurvival . These rVarious studies suggest an increased frequency of M2 macrophages in colorectal cancer (CRC) patients, which then induced the progression and liver metastasis of CRC through promoting tissue remodeling, angiogenesis, and immune dysfunction , 142. WiIn vitro, although melanoma cells were found to express the G‐CSFR transcript, the enhancement of cell proliferation and invasion do not occur on G‐CSF stimulation, suggesting an absence of the G‐CSFR protein [Melanoma is a highly malignant cancer with a tendency to metastasize early. A clinical case study reports the presence of severe G‐CSF secretory melanoma . In vitr protein . However protein . Congrue protein . But oth protein . Multipl protein . One stu protein . Moreove protein . TAMs ma protein . There w protein , 158. Ev protein .G‐/GM‐CSF can enhance the dissemination and bone metastasis in prostate cancer. G‐CSF increases a cancer stem cell phenotype through upregulation of octamer‐binding transcription factor 3/4 (Oct3/4), nanog homeobox pseudogene 8 (NANOGP8), and ATP‐binding cassette transporter G2 (ABCG2) . GM‐CSF + T lymphocyte in the spleen were decreased, while the percentages of M‐MDSCs, CD11b+ F4/80+ macrophages and PMN‐MDSCs at day 21 after tumor cell inoculation [Hepatocellular carcinoma (HCC) is a primary liver malignancy with a high global prevalence and a dismal prognosis. Immune system instability is a major clinical performance of HCC . Bone maculation . Moreoveculation . Recent culation . Furtherculation . These rAML is a complex, heterogeneous hematological malignancy caused by mutations in immature myeloid cells differentiation and proliferation . EMH in Tumor‐associated hematopoietic disorders work through a feedback mechanism within the TME and participate in the formation of the tumor immunosuppressive microenvironment. Based on a series of data highlighting the interplay of tumor growth, angiogenesis, and hematopoietic dysregulation, it is generally believed that abnormal hematopoiesis can accelerate tumor progression by inducing the generation and activation of tumor‐associated immunosuppressive cells. G‐CSF/GM‐CSF‐induced hematopoietic dysregulation can be used as a marker of poor prognosis in various tumors. However, it should be noted that many different local, systemic mechanisms and regulatory pathways have been implicated in hematopoietic dysregulation during tumor evolution, and the exact mechanism of this regulation is still not fully understood. Therefore, improving dysregulated hematopoietic environments based on the elimination of tumor‐induced abnormal immunosuppressive cells may be a valuable therapeutic approach.The authors declare no conflicts of interest.J‐LG and G‐Y Lv designed and revised the review, K He, R Hoffman, X Liu and R‐Z Shi wrote the manuscript.) and G‐YL (lv.gy@263.net) designed the project. KH, RDH, XL, and R‐ZS wrote the article.


A wearable triboelectric nanogenerator (denoted as CSF-TENG) is designed using biodegradable and carboxymethyl chitosan-silk fibroin (CSF) film.In vitro biodegradation of CSF film is performed through trypsin and lysozyme. 63.1% of CSF film is removed by trypsin and lysozyme after degrading for 11 days.An intuitive writing system is designed by CSF-TENGs-based human-machine interface to promptly track writing steps, highlight the stroke in advance, and access the accuracy of letters.The online version contains supplementary material available at 10.1007/s40820-022-00965-8. Voc ≈ 165 V, Isc ≈ 1.4 μA, and Qsc ≈ 72 mW cm−2. Further, in vitro biodegradation of CSF film is performed through trypsin and lysozyme. The results show that trypsin and lysozyme have stable and favorable biodegradation properties, removing 63.1% of CSF film after degrading for 11 days. Further, the CSF-TENG-based human–machine interface (HMI) is designed to promptly track writing steps and access the accuracy of letters, resulting in a straightforward communication media of human and machine. The CSF-TENG-based HMI can automatically recognize and correct three representative letters , which is benefited by HMI system for data processing and analysis. The CSF-TENG-based HMI can make decisions for the next stroke, highlighting the stroke in advance by replacing it with red, which can be a candidate for calligraphy practice and correction. Finally, various demonstrations are done in real-time to achieve virtual and real-world controls including writing, vehicle movements, and healthcare.Letter handwriting, especially stroke correction, is of great importance for recording languages and expressing and exchanging ideas for individual behavior and the public. In this study, a biodegradable and conductive carboxymethyl chitosan-silk fibroin (CSF) film is prepared to design wearable triboelectric nanogenerator (denoted as CSF-TENG), which outputs of The online version contains supplementary material available at 10.1007/s40820-022-00965-8. Wearable electronics have experienced development and progress in the past decades because of their significant contributions to various fields –6. WearaInternet of things (IoT) consists of a series of devices that are interconnected over the internet or other communication networks. It has shown significant potential in activity recognition, intelligent monitoring, and real-time response systems –22. UltrHowever, the remarkable advancement of wearable electronics necessitates the widespread use of mobile power sources, which in many situations are still traditional devices with limited lifetimes –40. ThusIn past few years, TENG-based HMIs have been developed to track a trajectory pattern or identify letters leading to a real-time response and sustainable monitoring –63. For Here, HMI-enabled wearable electronics based on a triboelectric mechanism are constructed to achieve an intelligent, highly accurate, and real-time response writing system and carboxymethyl chitosan-silk fibroin-TENG (CSF-TENG) with a contact-separation mode fabricating the portable electronics, which uses carboxymethyl chitosan-silk fibroin (CSF) film as friction electrification material. CSF film is prepared by crosslinking carboxymethyl chitosan and silk, resulting in biodegradability, flexibility, and softness. Weight loss and UV–visible absorption change are used to investigate the biodegradation behavior of CFS film by trypsin and lysozyme. The impact of silk content on the electrical properties of CSF-TENG and the high electrical output performance of the optimal device is evaluated. Most importantly, the CSF-TENG-based HMI is successfully demonstrated in practicing letters and correcting the writing steps. Finally, 3D virtual reality (VR) applications including letters writing and healthcare based on the CSF-TENG-based HMI are achieved.Sodium Carbonate, lithium bromide, and phosphate buffer  were purchased from Sinopharm Chemical Reagent Co., Ltd., China; EDC hydrochloride was obtained from BOSF Biotechnology Co., Ltd; trypsin (EC 3.4.4.4) and lysozyme (EC 3.2.1.17) were supplied by Shanghai Macklin Biochemical Co., Ltd; deionized water was obtained from a ULUPURE water system; carboxymethyl chitosan was purchased from Aladdin Reagent Company, and raw silks were obtained from the laboratory.2CO3 solution at 98 °C for 30 min and washed thoroughly with deionized (DI) water to remove impurities and wax; the purified products were submerged in a 9.3 M LiBr solution with a mass ratio of 1:20 at 60 °C. Further, the solution was collected and dialyzed (Mw = 3500) to eliminate residues, and a silk fibroin solution was obtained and denoted as SF.Raw silk was boiled in a 0.02 M NaTo achieve a uniform solution, 1 g of carboxymethyl chitosan was dissolved in 25 mL of DI water through stirring. After stirring, the obtained SF was added to the as-formed solution, and 1 mL of EDC hydrochloride (concentration: 30% of the amount of carboxymethyl chitosan) was subsequently added to the solution dropwise after stirring. Further, the mixture was dried in an oven at 60 °C, and the final (CSF) film was obtained. Following the same process, CSF films prepared with a different mass ratio of SF were abbreviated as CSF 0:1, CSF 1:1, and CSF 2:1.2 was selected as one electrode, and the PTFE film of the same size was attached to the CSF film. Further, the conductive sponge was chosen as another electrode to cover the PTFE film; a dielectric layer of PTFE film (5 × 5 cm2) was attached to the surface between the CSF film and conductive sponge.The as-prepared CSF film with a size of 5 × 5 cmThe CSF-TENG writing system was designed as a woven structure to sensitively respond to mechanical inputs during the handwriting process. The writing system was consist of an 8 × 8 pixel overlapping region on the weft and warp side.−1. At fixed intervals, 2 mL of the reaction solution was sampled and measured using UV–vis spectrophotometry. The reaction solution was manually changed daily throughout the degradation process to enhance the degradation efficiency.CSF film was incubated in a 30 mL solution of trypsin and lysozyme in PBS at 38 °C for 11 days. To obtain a uniform solution, 15 mg each of trypsin and lysozyme was dissolved in 30 mL of PBS, and 100 mg of CSF film was added to the resultant solution. The solution was placed on a shaking table, degraded at 38 °C, and shook at 100 r minThe microstructures and morphologies of the samples were obtained using scanning electron microscopy (SEM) and a su1510 microscope. Fourier-transform infrared (FTIR) spectra were obtained on a Nicolet iS10 spectrometer (Thermo Fisher). The chemical states of the samples were scanned by x-ray diffraction  under Cu Kα radiation from 10 to 90 °C. UV–vis spectra of the solution were performed by UV-3600 . An electrometer was used to measure the TENG’s output voltage and current (Keithley 6514).Figure The SEM images in Fig. The crystal phases of all samples were measured using XRD. The XRD pattern of carboxymethyl chitosan shows a broad peak at 20.5° , N–H (amide II), and C–N&N–H (amide III), respectively. The character peak at 3279 cm−1 is assigned to the hydrogen-bonded N–H and O–H stretching vibration. The spectrum of carboxymethyl chitosan shows absorption peaks at 1050 and 1130 cm−1, which are typical peaks of the saccharide structure. The amide I and amide II absorption peaks at 1652 and 1573 cm−1, respectively, indicate that chitosan has a significant deacetylation degree. Further, the CSF 1:1 film exhibits all the characteristic peaks mentioned earlier, indicating that the SF and carboxymethyl chitosan are effectively crosslinked.FTIR measurements were also performed to adequately account for the interaction structure of SF and carboxymethyl chitosan. Figure 2O2, etc.) can cause diverse diseases in living things and pose severe threats to ecosystems. Biodegradation is an effective degrading process in which microorganisms or their active by-products  decompose waste in the natural environment [Chemical reagents  for verification. Figure S2 shows that the entire electrical outputs are increased linearly as the pressure increases from 0.65 to 12.67 N. This phenomenon is attributed to the increased pressure, which can enlarge the contact-separation area, resulting in higher electrical outputs. The peak voltage was measured as different external resistances to obtain additional details on the behavior of the electrical energy output under external load  were fabricated to obtain a high triboelectric patch. The effect of the amount of SF on the electrical output performance of CSF-TNEG was evaluated under mass ratio (CC: SF) ranging from 0:1, 1:1, 1:2, and 1:0 Fig. c. The reely Fig. d–f. Furtoad Fig. g. At a rces Fig. h. By man–,” “/,” and “|”) obtained by the acquisition card. For the stroke “–,” the user briefly touches the first point of the stroke array, and proceeds from electrode 2 to 6, where an output voltage pulse with the regular magnitude is first produced on electrode 2, and then the output voltage of various magnitudes are rapidly generated on electrodes 3, 4, 5, and 6 until the user completes the strokes array without discovering an output voltage . Because of the real-time response-ability, it is obvious that no matter what kind of strokes the user writes, the handwriting signals and tracks exhibit instant response in the time domain. We selected three letters that are composed of strokes “–,” “/,” and “|” in all letters as an exhibition . Leveraging the various output information from CSF-TENG, labview is utilized to analyze the signals to identify and correct letters. Figure |,” “–,” and “–.” The correlation judgment of the letter F is performed based on the standard. A real-time trend can be observed in the voltage on F writing from the output signals  acts as the upper triboelectric layer because of its woven structure. Each point’s output signal is recorded with 2 channels, and varying contact points are connected to 16 channels . Figure Apart from letter F, letters H and K are also explored as representative letters by CSF-TENG-based HMI to make our writing applicable to the normal letters. Figure The flourish advancement of VR and AR technologies provide a creative way for the potential application in social media and personal engagement. Thus, we designed a training program in Unity 3D to verify a VR writing control (Movies S4-S6). Each sensor channel is connected to Arduino for data acquisition. Python processes recieved data in real-time manner and then sends motion command to Unity 3D. Unity 3D receives the command of the predicted steps and then convert it into the movements of virtual pen, as shown in Fig. −2 at a resistance of 1 GΩ is produced. Further, by using CSF-TENG-based HMI for real-time feature response, the writing process and sequence for the three letters can be tracked and reminded in a timely manner, demonstrating the identification and revision abilities of the proposed system for letters with different shapes. The error warning of the writing result can be generated by writing the same letters with different strokes. It only needs to be lightly touched once in the testing experiment because of its numerous advantages like portability, sensitivity, and rapid response properties. Finally, 3D VR controls including writing, healthcare and vehicle monitoring are successfully displayed using the constructed CSF-TENG-based HMI. Looking forward, an intelligent lifestyle can be established via wearable electronics, automatic identification, correction, and VR applications as a prospect under the HMI and AI infrastructure by employing sensory interactive system.In conclusion, a wearable electronic for letters exercise and automatic correction is developed by CSF-TENG-based HMI. It is implemented by combining TENG and advanced HMI-based real-time data processing. A flexible and wearable CSF-TENG with biodegradable CSF triboelectric film is proposed using the simple assembly method. Contrasting experiments to optimize the output capacity of CSF-TENG are performed, followed by a series of performance measurements. A maximum voltage of 165 V and output power density of 72 mW cmSupplementary file1 (MP4 1766 KB)Supplementary file2 (MP4 3060 KB)Supplementary file3 (MP4 2942 KB)Supplementary file4 (MP4 12094 KB)Supplementary file5 (MP4 9402 KB)Supplementary file6 (MP4 14673 KB)Supplementary file7 (MP4 10654 KB)Supplementary file8 (MP4 11969 KB)Supplementary file9 (PDF 1 KB)Below is the link to the electronic supplementary material.


The glymphatic clearance of albumin, globulin, and total protein is preserved in PWH. The analyses of soluble biomarkers in CSF should account for the time of collection.The accumulation of soluble proteins and metabolites during wakefulness and their clearance during sleep via the glymphatic system occurs in healthy adults and is disturbed in some neurological conditions. Such diurnal variations in the cerebrospinal fluid (CSF) proteins produced outside the central nervous system and entering via the blood–brain barrier (BBB) have not been evaluated in people with HIV (PWH). CSF and blood were collected in 165 PWH at six US centers between 2003 and 2007. The time of collection was compared to CSF albumin, globulin, and total protein concentrations using bivariate and multivariate regression. Participants all took antiretroviral therapy (ART) and were mostly middle-aged (median age 44.0 years) men (78.8%), with AIDS (77.0%), plasma HIV RNA ≤ 200 copies/mL (75.8%), and immune recovery (median CD4+ T-cell count 414/µL). CSF was collected at median 1:10 p.m. (range 9:00 a.m. to 5:20 p.m.) and within a median of 15 min of blood collection. A later time of CSF collection was associated with higher total protein ( The glymphatic system is important in the clearance of proteins, metabolites, waste products, and toxins from the central nervous system (CNS). This system operates by promoting the removal of these molecules from the CNS extracellular fluid (ECF) into the lymphatics . For exaAdditionally, the dysfunction of the BBB, seen in several immune-altering conditions, may allow for entry of other cell types and proteins and thus subsequent removal via the glymphatic system . For exaThe diurnal variation of albumin, globulins, and other blood-derived molecules found in the CSF such as inflammatory cytokines has not been studied in people with HIV (PWH). Clearance of these proteins might be influenced by the glymphatic system. We evaluated whether diurnal variations occur in CSF albumin, globulin, and total protein. We predicted that, if glymphatic clearance is at least partially preserved in PWH, then CSF levels of these components would be lower in the morning, following glymphatic clearance during sleep, as compared to later in the day.Participants were 165 PWH who enrolled in the CHARTER study between 2003 and 2007 who had CSF and matched plasma stored. The median age of the participants was 44.0 years , and 130 (78.8%) identified as male. Black participants were 42.4% of the cohort; 13.9% participants were Hispanic and 43.6% were non-Hispanic white. Median (IQR) nadir CD4+ T-cell count was 101/µL  and median (IQR) CD4+ T-cell count was 414/µL  at the time of evaluation. Median estimated duration of HIV infection was 11.1 years  and 127 (77.0%) had a diagnosis of AIDS. All participants had been taking antiretroviral therapy (ART) for at least 1 month at the time of data collection, with the median duration of the current ART regimen being 8.7 months . Most participants had HIV RNA ≤ 200 copies/mL in plasma  and in CSF .p = 0.0077; n = 123, r = 0.167, p = 0.0644). The relationship between serum total protein and CSF collection time was weaker and non-significant . The median (IQR) CSF albumin was 16  mg/dL; 1.8% were above the upper limit of the reference range for males 18–50 years (10–45 mg/dL). A similar relationship was seen for CSF albumin: the later collection time more strongly correlated with higher albumin in CSF . Additionally, the later CSF collection time significantly correlated with higher globulin in CSF ; 12% of values were above the upper limit of the reference range (60 mg/dL). Higher CSF total protein correlated with a later CSF collection time . The median (IQR) CSF-to-serum albumin ratio (CSAR) was 3.95 ; 8.6% of ratios were above the reference range for persons under 45 years (6.8). The CSF collection time did not correlate with the CSAR .Threshold values in the data were explored with recursive partitioning, which identified that mid-afternoon may be important for CSF collection. Participants who had CSF collected after 2:50 pm  had higher concentrations of albumin . Similarly, the blood collection time did not correlate with HIV RNA in plasma among participants with detectable levels . Fifteen participants (9.09%) had CSF pleocytosis (white cells > 5/µL). CSF collection time did not correlate with CSF glucose , or with CSF leukocyte  or erythrocyte  counts. In addition, IL-6 , IP-10 , and MCP-1  were not associated with the time of CSF collection.Among participants with detectable HIV RNA in CSF, the CSF collection time did not correlate with HIV RNA in CSF , ethnicity (p = 0.436), current CD4 (p = 0.946), nadir CD4 (p = 0.999), or viral detectability in blood (p = 0.288) or CSF (p = 0.916). Older participants tended to undergo lumbar punctures later in the day ; however, age was not correlated with CSF levels of total protein , albumin , IL-6 , IP-10  or MCP-1 .Other variables were considered potential confounders of the relationship between CSF collection time and the outcomes of interest  when these variables were significantly related to both CSF collection time and the outcome. CSF collection time was not related to gender  were higher in participants whose lumbar punctures (LPs) were performed later in the day compared to earlier. The effect size for this relationship (Cohen’s d) was medium when comparing those whose LPs were performed later in the afternoon to those carried out earlier in the day. This finding was not explained by changes in blood–CSF barrier function  or by differences in serum albumin concentration, and was robust in consideration of the demographic and other potential confounders. We propose that diurnal variation in glymphatic clearance from CSF accounts for these results. The major source of CSF albumin is its transport from the blood, via binding glycoprotein receptors on epithelial cells in the choroid plexus, and subsequent transfer into the ventricular CSF . CSF is The glymphatic system is responsible for clearing soluble proteins and other molecules from the central nervous system by shunting these molecules, accumulated during the day in the interstitial fluid and CSF, into the lymphatic system. In people without HIV (PWoH), this results in a diurnal pattern in which levels of macromolecules, such as albumin and globulins, increase over the course of the day as they accumulate in the CSF and decrease at night as they are cleared through the glymphatic system. Until now, it was unknown whether this pattern exists in PWH, who are commonly prone to sleep disturbances which could interfere with glymphatic clearance, as occurs in other neurodegenerative conditions, such as Alzheimer’s disease (AD) ,15.Alternative explanations for our findings may be proposed. For example, diurnal changes in albumin/globulin/protein entry, CSF production, blood–brain and blood–CSF barrier permeability, or transport via trafficking cells might occur that could in turn affect albumin, globulin, and total protein. Arguing against some of these hypotheses, however, we did not observe a relationship between time of day and CSF cell counts or CSAR. Recognition of the diurnal pattern is important in the context of CSF collection for clinical or research studies, as timing of collection may be a confounding factor. Therefore, time should be recorded and considered when collecting levels of CSF components, such as albumin.The strengths of this study are the relatively large number of PWH who underwent lumbar puncture and the racial and ethnic diversity of the cohort, enhancing generalizability. We replicated a similar diurnal pattern across several CSF components of relevance to HIV. Several limitations may be noted. CSF was collected only during daytime hours; thus, further data would need to be collected to determine if albumin levels decrease over the course of sleep. We did not collect data on sleep quantity or quality to determine if these might influence the findings. Female participants were underrepresented relative to males, although the proportion (21%) was comparable to the percent of women with HIV in the U.S. (20–25%). The rate of viral suppression was lower than in many modern cohorts; however, the correlation between CSF protein and CSF collection time was similar for those with and without viral suppression. Future directions might include performing studies involving matched PWoH to assess the extent to which diurnal variations are preserved in PWH. Future studies also might determine the impact of various sleep parameters on CSF albumin, globulin, total protein and HIV RNA. The CSF molecules of importance for CNS function, such as metabolic waste products cleared by the glymphatic system, would be of particular interest. We performed a cross-sectional analysis of people with HIV (PWH) from the CNS HIV AntiRetroviral Therapy Effects Research (CHARTER) study, which enrolled research volunteers at six U.S. sites: Johns Hopkins University, Icahn School of Medicine at Mount Sinai, University of Texas Medical Branch Galveston, University of California San Diego, University of Washington, and Washington University in St. Louis. Participants were all ambulatory, community-dwelling PWH. Inclusion criteria were willingness to undergo lumbar puncture (LP) and phlebotomy. Exclusions were active opportunistic disease; inherited or acquired bleeding disorder or other contraindications to LP; active, uncontrolled psychotic disorder; or neurological disorder unrelated to HIV, such as multiple sclerosis or Parkinson’s disease. The protocol was approved by Institutional Review Board at each research site. All participants provided informed consent for the study procedures outlined by the protocol. Visits described here were conducted between 2003 and 2007.n = 36), interferon gamma-induced protein 10  and monocyte chemoattractant protein 1 CSF was collected by lumbar puncture performed by trained clinicians. Current antiretroviral therapy (ART) was gathered through a structured interview. Albumin, total protein, and HIV RNA were measured in CSF and blood, which were collected within 90 min of each other , using standard methods. Unless noted, most samples presented here were measured at local, CLIA-certified reference labs. Serum albumin was measured as part of the comprehensive metabolic panel using a standard colorimetric assay. Serum globulin was calculated by subtracting albumin from total protein. CSF albumin was measured by immunonephelometry . Expected albumin ranges in healthy adults were 0–35 mg/dL (CSF) and 3.5–5.2 g/dL (serum). CSF total protein was measured by spectrophotometry. We calculated the CSF-to-serum albumin ratio as an index of blood–brain barrier permeability. Peripheral blood CD4+ T cell count was measured by flow cytometry. CSF cells were counted by automated methods. HIV RNA was quantified in plasma and CSF by reverse transcriptase-polymerase chain reaction  with a lower limit of quantification of 50 copies/mL. In a subset of participants, CSF levels of cytokines were also available, measured using commercially available immunoassay kits manufactured by Millipore : interleukin-6  as an index of blood–CSF barrier (BCB) permeability. Potential confounders assessed included demographics, current and nadir CD4+ T-cells, viral suppression status (plasma HIV RNA copies ≤ or >200/mL) and CSF leukocyte counts. When potential confounders such as age, demographic and disease variables were significantly related to both the predictor (CSF collection time) and outcomes of interest , we evaluated these further in multivariable regression analyses. We calculated effect sizes as Cohen’s d. Analyses were conducted using JMP Pro version 15.0.0 .


Extensive structural and functional studies have established that a membrane-exposed hydrophilic groove in TMEM16 PLS can serve as a translocation pathway for lipids. However, it is still unclear how the TMEM16 PLS conduct ions. A “protein-delimited pore” model suggests that ions are translocated through a narrow opening of the groove region, which is not sufficiently wide to allow lipid movement, whereas a “proteolipidic pore” model envisions ions and lipids translocating through an open conformation of the groove. We investigated the dynamic path of potassium ion (K+) translocation that occurs when an open groove state of nhTMEM16 is obtained from long atomistic molecular dynamics (MD) simulations, and calculated the free energy profile of the ion movement through the groove with umbrella sampling methodology. The free energy profile identifies effects of specific interactions along the K+ permeation path. The same calculations were performed to investigate ion permeation through a groove closed to lipid permeation in the nhTMEM16 L302A mutant which exhibits a stable conformation of the groove that does not permit lipid scrambling. Our results identify structural and energy parameters that enable K+ permeation, and suggest that the presence of lipids in the nhTMEM16 groove observed in the simulations during scrambling or in/out diffusion, affect the efficiency of K+ permeation to various extents.The TMEM16 family of transmembrane proteins includes Ca Some family members, such as TMEM16A  and TMEMfication . Others,channels . While nchannels . Inferenchannels  have reachannels  was firschannels . Subsequed forms . The str of Ca2+ . In conty is low . Of the y is low  has thusectively .Computational studies using molecular dynamics (MD) simulations based on the structural information available for the various TMEM16 family members  have aim+) translocation through the open groove of nhTMEM16 observed in the long MD simulations, and present the free energy profile of the ion movement through the groove calculated with umbrella sampling methodology . Snapshots representing various stages of the ion translocation during the two events are shown in We have previously reported unbiased 10 BID 4WIS ] in 3:1 IS (+Cl−  (see Mat+ ion permeation occurring at the 5.8 µs time-point in the MD trajectory shown in + traverses the groove in the IC-to-EC direction. The duration of the permeation events was 11 ns for the first and 63 ns for the second one (see panels A and B in + ion forms interactions at two main sites in the groove during both events. These are 1) the EC gate , akin to the ion-binding sites in ion channels that are considered to serve as “stepping stones” for the ions to pass through the conducting pore : System 1—the original system in which the two K+ permeation events were observed, consisting of the scramblase with the POPG lipid in the groove, embedded in a 3:1 POPE/POPG membrane; System 2—the same a System 1 but without the in-groove POPG; System 3—a system consisting of the scramblase with a POPC in the groove (instead of POPG), embedded in a POPC membrane; System 4—the same a System 3 but without the in-groove POPC. The headgroup of the in-groove POPC in System 3 is at the same Z position along the membrane axis as the head group of the in-groove POPG of System 1 . The resulting free energy curves are shown in + ion with the negatively charged headgroup of POPG.The PMF in each of the four Systems was calculated from the umbrella sampling simulations along the reaction coordinate taken as the distance in the Z direction (perpendicular to the membrane) between the K+ ion samples similar (open) groove conformations without lipid present in the middle of the groove.However, the similarity between the free energy barriers for Systems 1 and 2 suggests that the free energy contribution from the favorable electrostatic attraction between the ion and POPG lipid at a longer distance is compensated at shorter interaction distances by unfavorable steric interactions due to the presence of the POPG lipid in the groove. The greater similarity between the PMF profiles of System 2 and 4 is expected as the translocating K+ ion is engaged in >50% of the trajectory frames in that window. Thus, for the umbrella window shown in + permeation path, assigned to regions of the PMF profiles in + ion interacts with E313 coincides with the local minimum at the EC end in the PMF profile, suggesting that E313 serves as an attraction point for the ion at the EC end of the groove. The PMF maximum  for the in-groove POPC construct, System 3, is located in the interval where the K+ ion interacts with the POPC lipid (from Z = −3 Å to Z = 4.75 Å), suggesting that indeed the presence of POPC in the groove results in the free energy barrier that is ∼5 kcal/mol higher than in the other three systems.The nature of the interactions shaping the PMF profiles in +/POPC interactions in System 3 is longer than for the K+/POPG interactions (from Z = −3.25 Å to Z = 1.25 Å) in System 1. This difference could be due to the smaller size of the PG head group compared to PC. Notably, this short K+/POPG interacting range does not include the local minimum at Z = 2.7 Å in the PMF profile for System 1. Rather, this minimum appears to be due to the combined longer range electrostatic interaction as well as a favorable environment generated by the polar residues T381 and S382  which considers only geometric measures of the molecular components . The paths of the translocated ion shown in The “average trajectory” paths for the Kshown in  were obt2+-activated state, the presence of lipids that are being scrambled or are diffusing in and out of the groove as observed in simulations, can reduce potassium ion permeation to various extents. To interpret the mechanistic reasons for the calculated free energy profile for the translocation, we identified the effects of specific residues involved in K+ permeation along the ion path. These include multiple polar residues, such as E313 , T333, e region , as well2+ , because the membrane is negatively charged, reducing the probability of a successful Cl−/protein encounter. Indeed, our unbiased 15 µs long MD simulation of nhTMEM16 in POPC membrane system (− is similar to that of K+ (124 Cl− and 130 K+), we did observe spontaneous Cl− ion penetration into the groove. However, coming from the IC end and moving up only to the middle of the groove, the ion did not complete the translocation in this run, precluding the initiation of a comparative umbrella sampling simulations for Cl− similar to those for K+. In future simulations of much longer times this permeation is expected to be completed and thereby enable full comparative evaluations.In our unbiased 10 µs long MD simulation of the nhTMEM16 wild type in 3:1 POPE/POPG membrane system, we did not observe any instance of chloride passing through the groove in the absence of an applied external voltage across the membrane that is known to modulate ion conductance in TMEM16 scramblases . Notablye system , where t+Cl−, resulting in a total of 388,447 atoms. The POPC membrane containing the wild type scramblase had 679 lipids in a solution box of 90,189 water molecules and 150 mM K+Cl−, for a total of 385,207 atoms. The 3:1 POPE/POPG membrane containing the L302A mutant had 1,505 lipids and 146,737 waters in addition to the 150 mM K+Cl− , resulting in a total of 652,878 atoms. For all simulations we used the latest CHARMM36m force-field for proteins and lipids  were equilibrated for 11.8 and 6.2 ns after the in-groove lipid was removed from Systems 1 and 3 respectively. Because ion permeation is generally much faster than lipid translocation (104 lipids per second ), 1D umb2. Additional windows were added with force constant of 20 kcal/mol/Å2 to improve the sampling for Z = 3.6 Å window in System 1   , and Mon2 and movement velocity of 10 Å/ns. The setup of the umbrella sampling for the L302A system was the same as the one used for the wild type nhTMEM16, except that we used 96 umbrella sampling windows covering Z = −12.25 Å to Z = 11.5 Å range.For the mutant nhTMEM16 L302A system, the initial structural configuration is from a frame at the end of a 2 μs unbiased MD simulation of the system. To obtain starting configurations for all the umbrella sampling windows for this system, a steered MD simulation was performed with force constant of 5 kcal/mol/ÅP = 2. Its TM region was embedded in a membrane slab, which includes a 26-Å-thick hydrophobic core of the bilayer (with Ecore = 2 dielectric constant), surrounded by two 8-Å-thick regions, representing the lipid headgroups (Ehead = 30). The membrane slab contained a cylindrical hole (with the dielectric of EW = 80) with a radius of 18 Å centered around the groove of the protomer. The system was surrounded by solvent (EW = 80) containing 150 mM of monovalent mobile ions. The cylindrical hole was accessible to the ions, but ions were not permitted into the membrane region.The electrostatic potential was calculated by solving the linearized Poisson-Boltzmann equation  in CHARM