Results of Orient 15 indicated the health benefits to patients with local advanced or metastatic oesophageal squamous cell carcinoma (OSCC). This study aimed to evaluate the cost-effectiveness of sintilimab plus chemotherapy in treating OSCC from the perspective of Chinese healthcare system.A partitioned survival model was constructed to evaluate the cost-effectiveness of sintilimab plus chemotherapy vs. chemotherapy in treating OSCC. Baseline characteristics of patients and key clinical data were extracted from Orient 15. Costs and utilities were collected from published studies and open-access databases. Costs, quality-adjusted life-years (QALYs), life-years gained, and incremental cost-effectiveness ratios (ICER) were chosen as economic outcome indicators. We also performed sensitivity analyses and subgroup analyses to verify the stability of results.Combination therapy provided additional 0.84 QALYs and 1.46 life-years with an incremental cost of $25,565.48 than chemotherapy, which had an ICER of $30,409.44 per QALY. The probabilistic sensitivity analysis indicated that combination therapy had a 98.8% probability of cost-effectiveness at the willingness-to-pay threshold (WTP) of $38,184 per QALY. Deterministic sensitivity analysis showed that model outcomes were sensitive to the utilities of progression-free survival and progression disease. The subgroup analysis revealed that combination therapy was cost-effective in patients with high expression of PD-L1 and several specific subgroups.In this economic evaluation, sintilimab plus chemotherapy was likely to be cost-effective compared with chemotherapy in the first-line therapy of advanced OSCC from the perspective of Chinese healthcare system. Our findings may provide evidence for clinicians to make optimal decisions in clinical practice and for decision-makers to evaluate the cost-effectiveness of sintilimab. Oesophageal cancer ranks seventh in terms of incidence and sixth in mortality worldwide in 2020 . OesophaThe emergence of immune checkpoint inhibitors has drastically altered the landscape of cancer treatment. Sintilimab, a fully recombinant human IgG4 anti-PD-1 monoclonal antibody, showed clinical efficacy in many cancers. Sintilimab was also approved by the National Medical Products Administration of China in the treatment of classical Hodgkin’s lymphoma, non-small cell lung cancer, and hepatocellular carcinoma –12.Recently, the randomized, double-blinded, phase III trial Orient 15 reported the efficacy and safety of sintilimab versus placebo in combination with chemotherapy (cisplatin plus paclitaxel or cisplatin plus 5-fluorouracil) as the first-line treatment of unresectable locally advanced, recurrent, or metastatic OSCC . The resDespite these encouraging clinical results, evidence of cost-effectiveness should not be ignored since the combination therapy had a relatively higher cost than chemotherapy alone. Therefore, this study aimed to investigate the cost-effectiveness of sintilimab plus chemotherapy as the first-line treatment for unresectable locally advanced, recurrent, or metastatic OSCC from the perspective of Chinese healthcare system. The evidence may provide information for clinicians and decision-makers to allocate resources reasonably in health decisions.We reported this study followed the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) reporting guideline . Targete2 every cycle) plus paclitaxel (175 mg/m2 every cycle) or 5-fluorouracil (800 mg/m2 continuous administration for five days in each cycle). A maximum of six cycles was recommended for chemotherapy. When disease progressed, or intolerable toxicity occurred, patients could alter from the first-line treatments to the second-line treatments. In Orient 15, 41% (134/327) in sintilimab plus chemotherapy group and 54% (179/332) in placebo plus chemotherapy group received subsequent therapies (Included patients received sintilimab or placebo intravenously at a dose of 3 mg/kg in patients weighing <60 kg or 200 mg in patients weighing ≥60 kg every three weeks (one cycle). Chemotherapy included cisplatin  and Microsoft Excel 2019 .We constructed a partitioned survival model with three health states in our economic evaluation . The thrhttp://getdata-graph-digitizer.com). We precisely reproduced the digitized KM curves since the reconstructed patient-level data whose event and censor times were equal in number to the initial number at risk. Parametric models were applied to extrapolate the survival data to the long term. Following parametric survival models were considered: exponential, Weibull, Gompertz, gamma, log-logistic, log-normal, generalized gamma, genf, fractional polynomial (FP), restricted cubic spline models (RCS) and Royston-Parmar models (RP)  was unavailable, we extracted the PFS and OS data from the Kaplan-Meier (KM) curves of the Orient 15 trial. Then, we used the method of Guyot et.al . to recoels (RP) –23. The els (RP) , 24. TheIn our study, only direct medical costs were considered, including costs of acquiring drugs, costs attributed to the patient’s diagnosis and hospitalization, costs for the management of adverse events (AEs), and costs for end-of-life care (Eol) were analyzed. Drug prices were obtained from public databases and were up to date in 2021 , 26. We The utilities of PFS and PD states associated with advanced OSCC were 0.75 and 0.67 respectively, which were derived from a cost-effectiveness analysis based on the E-DIS trial . The disSensitivity analyses were conducted to test the robustness of the model. In deterministic sensitivity analysis (DSA), all parameters were adjusted within the reported 95% confidence intervals (CI), which were derived from the previous studies or assuming reasonable ranges of the base case values ( ± 20%). We conducted the probabilistic sensitivity analysis (PSA) through a Monte Carlo simulation of 10,000 iterations. We selected a gamma distribution for cost and a beta distribution for probability, proportion, and utility. We used the scatter plot and cost-effectiveness acceptability curves (CEAC) to analyze the cost-effectiveness for each treatment with different WTP thresholds.In subgroup analysis, we first analyzed the results for patients with combined positive scores (CPS) of ≥10 for expression of PD-L1 through the methods of base case analysis since the Orient 15 reported the independent KM curves for these patients. For other subgroups, the ICERs were calculated using the subgroup-specific HRs for OS and PFS obtained from Orient 15. We considered the subgroup of patients with different sex, age, baseline weight, country or region, Eastern Cooperative Oncology Group (ECOG) performance status score, disease type, Hepatic metastasis, and chemotherapy. Data for all subgroups except for the HRs for OS and PFS was assumed to be the same since the lack of sufficient data. Therefore, proportional hazards were assumed here. The composition of costs would be calculated based on specific subgroups.Results of cost, utilities, and life-years gained are shown in Results of DSA are shown in Results of PSA are shown in In the first subgroup, we considered comparing the cost-effectiveness of two therapies among patients whose CPS ≥10. Results of the scenario analysis were similar to the results of base case analysis, whose ICER was $32,376.78/QALY Table 2.Assumed HR to be constant, we conducted the subgroup analysis for other subgroups like sex, age, and weight for both OS and PFS. ICERs changed with HR were selected as the outcome indicator, and the probability of being cost-effective for sintilimab plus chemotherapy was also calculated. Summary results of subgroup analysis are concluded in Our study is the first to conduct the economic evaluation of sintilimab plus chemotherapy versus chemotherapy alone in patients with unresectable locally advanced, recurrent, or metastatic OSCC. Based on the results of Orient 15, our base-case analysis showed that sintilimab plus chemotherapy had additional life-years gained and increased QALYs than chemotherapy. Combination therapy was also more cost-effective for OSCC under the WTP threshold of $38,184 per QALY. Base-case analysis results were robust according to the results of DSA and PSA. According to our DSA results, utilities of PFS and PD, cost of salvage therapy, sintilimub, laboratory tests and radiological examinations were significantly associated with the model outcomes. In the subgroup of patients with CPS ≥10, sintilimab plus chemotherapy also exhibited cost-effectiveness under the given WTP threshold. 12 subgroups, including sex (men or women), age ≥ 65 years, baseline weight ≥60kg, region China, disease type of metastatic, ECOG PS score, hepatic metastasis, the chemotherapy of cisplatin plus 5-fluorouracil and PD-L1 expression TPS showed that sintilimab plus chemotherapy had a higher than 50% probability of cost-effectiveness compared with chemotherapy.According to DSA, utilities of PFS and PD were two factors with the most significant influences. Utilities used in this study were extracted from a published economic evaluation study that targeted on the cost-effectiveness of continuation versus discontinuation of first-line chemotherapy in patients with metastatic OSCC. Some biases might occur since patients included in this study were not Chinese. However, our DSA results showed that with utilities fluctuating, ICERs were still below the given WTP threshold, which indicated that the fluctuation of these two factors did not affect the conclusion.Patients with CPS ≥10 were a key subgroup in Orient 15. Significant improved clinical benefits were observed in this subgroup for both PFS and OS. Similar results were found in other clinical trials: Keynote-590, Checkmate-648, and ESCORT-1st, which indicated that patients with higher expression of PD-L1 derive more clinical benefit –44. SubgThe ability of sintilimab plus chemotherapy to improve the PFS and OS benefits over chemotherapy was associated with economic outcomes. Our base-case analysis found that combination therapy provided patients with more clinical benefits in both PFS and OS. By assuming HR to be constant, the cost-effectiveness of each subgroup could be calculated. We found that for PFS, the fluctuation of HRs did not have significant impacts on conclusions. However, for OS, the fluctuation of HRs in some groups could affect the results. This indicated that for specific subgroups, sintilimab plus chemotherapy was still cost-effective. This finding might provide evidence for clinicians to make optimal decisions with limited healthcare resources.Currently, few studies are targeted on the economic evaluations of immunotherapies for advanced OSCC. After a systematic search on PubMed, nine CEA studies were found to assess different immunotherapies for advanced OSCC. Seven of them focused on second-line therapies. One of the remaining two focused on the cost-effectiveness of first-line camrelizumab plus chemotherapy for advanced or metastatic OSCC in China from the perspective of the Chinese healthcare system . CamreliThere are several limitations in this study. First, owing to the lack of head-to-head study, we did not include other first-line immunotherapies, such as pembrolizumab, nivolumab, camrelizumab and toripalimab, which have shown favorable health benefits for patients with advanced OSCC , 47–50. The findings of this cost-effectiveness analysis suggested that from the perspective of Chinese healthcare system, sintilimab plus chemotherapy is likely to be cost-effective at WTP thresholds of $38,184 per QALY compared with chemotherapy for patients with advanced OSCC. Patients with high expression of PD-L1 and specific subgroups could achieve more clinical benefits and favorable cost-effectiveness from this new combination therapy. Future long-term data are needed to validate and improve this study.The original contributions presented in the study are included in the article/All authors developed the concept and design of the study. TS and MZ performed the data collection, analyzed the data, and prepared the manuscript, and WT performed critical revision of the manuscript. All authors contributed to the article and approved the submitted version.


Platelet-related parameters and HDL-C have been regarded as reliable and alternative markers of coronary heart disease (CHD) and the independent predictors of cardiovascular outcomes. PDW is a simple platelet index, which increases during platelet activation. Whether the PDW/HDL-C ratio predicts major adverse cardiovascular and cerebrovascular events (MACCEs) in patients who complained of chest pain and confirmed coronary artery calcification remains to be investigated. This study aimed to investigate the prognostic value of the PDW/HDL-C ratio in patients with chest pain symptoms and coronary artery calcification.A total of 5,647 patients with chest pain who underwent coronary computer tomography angiography (CTA) were enrolled in this study. Patients were divided into two groups according to their PDW/HDL-C ratio or whether the MACCE occurs. The primary outcomes were new-onset MACCEs, defined as the composite of all-cause death, non-fatal MI, non-fatal stroke, revascularization, malignant arrhythmia, and severe heart failure.P < 0.001). Multivariate Cox hazard regression analysis reveals that the PDW/HDL ratio was an independent predictor of MACCEs . Cox regression analysis showed that participants with a lower PDW/HDL-C ratio had a higher risk of MACCEs than those in the higher ratio group. The incidence of MACCEs was also more common in the PDW/HDL-C ratio > 13.33 group among different severities of coronary artery calcification. Furthermore, adding the PDW/HDL-C ratio to the traditional prognostic model for MACCEs improved C-statistic (P < 0.001), the NRI value , and the IDI value .All patients had varying degrees of coronary calcification, with a mean CACS of 97.60 , and the level of CACS in the MACCEs group was significantly higher than that in non-MACCE (P<0.001). During the 89-month follow-up, 304 (5.38%) MACCEs were recorded. The incidence of MACCEs was significantly higher in patients with the PDW/HDL-C ratio > 13.33. The K–M survival curves showed that patients in the high PDW/HDL-C ratio group had significantly lower survival rates than patients in the low PDW/HDL-C ratio group (log-rank test: The higher PDW/HDL-C ratio was independently associated with the increasing risk of MACCEs in patients with chest pain symptoms and coronary artery calcification. In patients with moderate calcification, mild coronary artery stenosis, and CAD verified by CTA, the incidence of MACCEs increased significantly in the PDW/HDL-C ratio > 13.33 group. Adding the PDW/HDL-C ratio to the traditional model provided had an incremental prognostic value for MACCEs. The growing global burden of coronary heart disease (CHD) has long been at the forefront of the global health agenda , 2. ChesAtherosclerosis is considered the predominant pathological basis of CHD, which is characterized by the development of lipid-rich plaques, in the artery wall. Platelets contribute to the early steps of plaque formation and participate in thrombus formation on ruptured plaque obviously , so platExploring new reference indicators appears to have a significant value not only for atherosclerosis itself, but also for adverse high-risk plaque characteristics and prognosis of CHD. Whether the combined index of platelet and HDL-C can be a new reference indicator needs further research and analysis. To address the knowledge gap, this study aimed to specifically investigate whether the PDW/HDL ratio has a prognostic value for major adverse cardiovascular and cerebrovascular events (MACCE) in patients with chest pain and coronary artery calcification.n = 277) and myocardial infarction and underwent PCI or CABG surgery (n = 589), coagulation disorders (n = 41), chronic hepatic and renal insufficiency or malignant tumor (n = 520), congenital heart disease or severe heart valve diseases (n = 404), other severe medical illnesses (n = 606), and insufficient clinical data . A total of 6,509 patients were followed up from January 2014 to April 2021 by telephone or outpatient clinical visit each year, and 5,647 (86.8%) patients completed the follow-up. Finally, a total of 5,647 participants were included in the study. The detailed recruitment process is depicted in We performed a retrospective cohort study in the First Affiliated Hospital of Xinjiang Medical University, from January 2014 to October 2020. This study was approved by the local research ethics committee. Given the retrospective nature of this research, no informed consent was required. All these patients complained of chest pain and underwent CTA detection which diagnosed them with coronary artery calcification (CACS > 0). The study excluded people who had non-cardiac chest pain , heart rate (HR), and coronary artery calcification score (CACS), and medication at discharge. Peripheral venous blood samples were collected early in the morning after an overnight fast on admission and analyzed shortly after sampling. Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), fasting blood glucose (FBG), platelet count (PC), mean platelet volume (MPV), PDW, and platelet crit (PCT) levels were analyzed. All the patients underwent the first CTA during this hospitalization.Type 2 diabetes mellitus (T2DM) was defined as a history of diabetes or intake of antidiabetic medication. Hypertension was defined as the current use of anti-hypertensive medications or a previous diagnosis. Smoking was defined as either active smoking or having a previous smoking history. Family history of CHD was defined as having more than 3 family members in the immediate or collateral family with a history of CHD.Those CTA were uniformly acquired by using multi-detector row CT scanners consisting of 64-rows or greater. CACS was measured by using the scoring system developed by Agatston. For this investigation, we evaluated the severity of coronary artery calcification from three different perspectives as follows: severity of coronary calcification , extent of coronary stenosis, and obstructive disease classification. The extent of coronary stenosis was categorized as “mild stenosis” , “moderate stenosis” , and “severe stenosis” . The obstructive disease was categorized as “no CAD” (absence of any plaque), “non-obstructive CAD” , or “obstructive CAD” .The primary endpoint was new-onset MACCEs, defined as the composite of all-cause death, non-fatal MI, non-fatal stroke, revascularization, malignant arrhythmia, and severe heart failure. All-cause death referred to the death attributed to cardiovascular or non-cardiovascular causes.t-test, Mann–Whitney test, and chi-square test were used to investigate for any statistically significant difference between the baseline characteristics. Survival analysis was performed by using the Kaplan Meier’s survival curves in the followed up patients from the time of enrollment until the time of their death for the deceased, and until the time of follow-up for the survivors. Univariate and multivariate stepwise Cox proportional hazard regression analyses were constructed to identify the independent predictors of MACCEs. Cox regression analysis was also used for predicting MACCEs after adjusting conventional cardiovascular risk factors such as age and T2DM, and we stratified the analyses by (1) severity of coronary artery calcification, (2) extent of coronary stenosis, and (3) obstructive disease classification. Multivariate analysis of variance was used to compare the incidence of MACCEs in the high and low ratio groups with different degrees of coronary artery calcification. Finally, the ability of the PDW/HDL-C ratio to improve MACCE prediction beyond using traditional risk factors was assessed by using the indexes of discrimination as measured by the c-statistic and integrated discrimination improvement (IDI), and reclassification as measured by the category-less net reclassification index (NRI).Data analysis was carried out using SPSS 19.0 and R version 4.1.1. Continuous variables were reported as mean ± standard deviation when normally distributed. The CACS, TG, FBG, and PDW were not normally distributed; therefore, those variables were expressed as medians with interquartile ranges. Categorical variables were expressed as frequencies. Two-samples independent P < 0.05).The study patients had an average age of 62.55 ± 10.39 years and 3,740 (66.2%) patients were male. During the 89-month follow-up, 304 (5.38%) MACCEs were recorded. According to this, we stratified our population by whether MACCEs occur and found that the age was markedly higher in the MACCEs group. Baseline clinical characteristics and clinical event data of the two groups are listed in P < 0.001). Accordingly, participants were divided into the higher ratio group (PDW/HDL-C ratio > 13.33) and the lower ratio group (PDW/HDL-C ratio < 13.33). Baseline clinical characteristics and clinical event data of the two groups are listed in The optimal PDW/HDL-C ratio cutoff value for predicting MACCEs was 13.33, which was calculated from sensitivity, specificity, and area under the curve (AUC) values determined by receiver operating characteristic analysis   .P < 0.001) and CACS  independently predicted the occurrence of MACCEs in patients.Univariate and multivariate Cox proportional hazard regression analysis and predictors for MACCEs are presented in P = 0.007) and MACCEs .P = 0.019)  , especia= 0.019) .P < 0.001). It was especially obvious in the group of mild stenosis that the people with a high PDW/HDL-C ratio had a higher risk of developing MACCEs  (A similar phenomenon has been observed in patients stratified by the extent of coronary stenosis. As shown in = 0.033) .P < 0.001) (P = 0.022) and obstructive CAD . Nevertheless, this result was not obvious in patients diagnosed with non-CAD by CTA  . CompareD by CTA .P < 0.001). Moreover, the addition of the PDW/HDL-C ratio had an incremental prognostic value for predicting MACCEs in terms of NRI  and IDI , especially when compared with established risk factors and models adding single indicators.The incremental predictive value of the PDW/HDL-C ratio for MACCEs is shown in This study investigated the association between the PDW/HDL-C ratio and MACCEs in patients with chest pain symptoms and coronary artery calcification. The results showed that the higher PDW/HDL-C ratio was positively associated with increased MACCEs. The PDW/HDL-C ratio was an independent predictor of MACCEs after adjusting for confounding factors. Furthermore, our results showed that adding the PDW/HDL-C ratio to the predictive model may improve the discrimination of risk prediction for MACCEs in patients complaining of chest pain and who underwent CTA detection which diagnosed them with coronary artery calcification. These findings reveal the prognostic value of the PDW/HDL-C ratio for MACCEs in patients with chest pain and coronary artery calcification. To the best of our knowledge, this study demonstrated, for the first time, that the PDW/HDL-C ratio is a potential predictor for MACCEs. Most importantly, this study suggests that a simple, convenient, and cost-effective method may optimize the prediction of recurrent cardiovascular risk in patients with chest pain and coronary artery calcification.Platelets play a pivotal role in coronary artery calcification, which is the main pathological change and primary cause of CHD. Variations and heterogeneity of blood cell shape are expressed by PDW for platelets . Lower lTo the best of our knowledge, the population in this study represents a relatively large cohort of patients with chest pain symptoms and coronary artery calcification in which the association between the PDW/HDL-C ratio and long-term MACCEs has been investigated. Moreover, our study is the first to report all-cause death, non–fatal MI, non-fatal stroke, revascularization, malignant arrhythmia, and severe heart failure as composite endpoint events. Additionally, patients in our study had higher percentages of non-fatal stroke and revascularization, thus denoting higher risk patients of cardiovascular and cerebrovascular diseases. Our study demonstrated that a higher PDW/HDL-C ratio was significantly associated with a higher risk of MACCEs. The higher risk of MACCEs persisted after adjusting for traditional cardiovascular risk factors, the burden of comorbidities, disease severity, and medications. We also found that the PDW/HDL-C ratio remained independently predictive of adverse cardiovascular and cerebrovascular outcomes after adjusting for important variables and treatment strategies. Although the PDW/HDL-C ratio independently predicted cardiovascular events in patients with chest pain and coronary artery calcification, the association between the PDW/HDL-C ratio and these individual events has not been well demonstrated. The possible reasons for these results are as follows: (1) after coronary artery calcification is diagnosed by CTA, the PDW/HDL-C ratio may not predict the risk of future myocardial infarction, non-fatal stroke, malignant arrhythmia, or severe heart failure in patients with suspected CHD. (2) In this study, the baseline PDW/HDL-C ratio was used to predict MACCEs, and changes in secondary preventive measures including oral drug therapy, diet, lifestyle, etc., in patients after discharge may affect the prediction of MACCEs by the PDW/HDL-C ratio. (3) During the study period, we experienced COVID-19, and some patients went to the local hospital when their disease progressed due to epidemic prevention and control. Therefore, some patients with MACCEs were not recorded by us. This may result in the relatively small number of events observed in our study subjects, so it is difficult to determine the exact relationship between the PDW/HDL-C ratio and these individual events.Identifying and diagnosing coronary CHD in patients with chest pain is one of the great challenges faced by clinicians. Although coronary angiography is the gold standard for evaluating CHD, more interest is currently focused on the use of non-invasive imaging methods, such as CTA. CTA has been proved to be an effective tool for the rapid and accurate diagnosis of CHD in patients with low to moderate cardiovascular risk. In addition, CTA has a unique ability to non-invasively depict the anatomical structure of the coronary artery, allowing not only the visualization of the arterial lumen to detect severe stenosis or occlusion that causes myocardial ischemia but also to evaluate the coronary artery wall by showing the presence or absence of CAD –23. ReceIn this study, we identified the optimal PDW/HDL-C ratio cutoff value for predicting MACCEs. We found that the AUC of the optimal cutoff value of 13.33 was relatively poor, suggesting that it is difficult to predict hard endpoint events based on the PDW/HDL-C ratio alone. However, by adding the PDW/HDL-C ratio to the established risk factors of MACCEs, we found a significant improvement in risk prediction in terms of the C-statistic value, NRI, and IDI. Although previous studies have shown that a higher PDW level or a lower HDL-C level is associated with worse cardiovascular outcomes, none have discriminated against the incremental prognostic value of the PDW/HDL-C ratio in terms of hard clinical endpoints. Our results implied that the use of the PDW/HDL-C ratio may refine the risk stratification of cardiovascular risk. Routinely introducing the PDW/HDL-C ratio into clinical diagnostic models could more accurately identify patients with higher cardiovascular risk, thus enabling a more targeted treatment or prevention.An increasing number of studies have shown that PDW and HDL-C play an indispensable role in the occurrence and development of CHD, and there is a close relationship between PDW and HDL , 18, 28.This study has several potential limitations. First, platelet parameters and lipid levels were measured only at baseline. The PDW/HDL-C ratio may change with follow-up. Therefore, it is not clear whether the changes in PDW/HDL-C ratio predict cardiovascular outcomes. Second, data on other non-statin lipid-lowering drugs and antiplatelet aggregation drugs have not been recorded, which may have potential effects on the PDW/HDL-C ratio levels and cardiovascular outcomes. Third, all patients were patients with chest pain and coronary artery calcification verified by CTA, so the applicability of the conclusions of this study needs further study to examine those patients with no clinical symptoms and no coronary artery calcification. Finally, the subjects of the study were Chinese patients, so these results need to be repeated in other ethnic groups. Despite these limitations, this study has important clinical implications because it is the first to investigate the association between the PDW/HDL-C ratio and MACCEs in patients with chest pain symptoms and coronary artery calcification.The higher PDW/HDL-C ratio was independently associated with the increasing risk of MACCEs in patients with chest pain symptoms and coronary artery calcification. Identically, from different perspectives of the severity of coronary artery calcification, the same result was confirmed particularly in patients with moderate calcification, mild coronary artery stenosis, and CHD verified by CTA. Finally, adding the PDW/HDL-C ratio to the traditional model had an incremental prognostic value for MACCEs. These findings suggested that the PDW/HDL-C ratio is likely to be a useful marker for prognosis in patients with chest pain symptoms and coronary artery calcification.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.X-ML and Y-NY: funding and conception and design of the study. Y-JQ and J-YL: literature search, data collection, and analysis of the manuscript. FL, X-XT, LZ, and Z-RZ: literature search, data collection, and processing. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In the last decade, injectable hydrogels have attracted a lot of attention due to their excellent functional properties in the field of drug delivery for precise, non-invasive and focused tissue locations. Therefore, designing drug delivery systems (DDS) responsive to hydrogel stimuli to release a drug to an external stimulus with various advantages, can be very challenging. Due to their biocompatibility, mucosal adhesion, and hemostatic activity, chitosan (Chitosan)-based hydrogels offer a lot of potential for tissue engineering and drug delivery. It can be difficult to manage the structure of these stimuli-responsive CS hydrogels or they may require additional crosslinking agents, such as hydrogels with dual pH and thermo-responsiveness. Therefore, it is crucial to create these hydrogels for medicinal applications. Potential delivery methods for controlled release of bioactive substances include injectable polymer hydrogels. Numerous stimuli, including pH, temperature, light, enzymes, and magnetic fields, can cause these polymers’ sol-to-gel transitions in aqueous solutions to change. At room temperature, the low-viscous polymer solution may be conveniently combined with therapeutic agents, such as chemotherapeutics, protein medicines, or cells. Through the use of a syringe or catheter, therapeutic agent-containing solutions may be easily injected into the desired locations .Researchers have focused on developing the optimum drug delivery system (DDS) that can adapt to various physiological needs, such as liposomes, microspheres, nanoparticles, polymeric micelles, and hydrogels. The most prevalent form of responsive nano-system for cancer medication delivery is one that responds to pH variations, owing to the fact that tumors have extracellular pH values that are lower than those of healthy tissues and the circulation (pH = 7.4) . More crChitosan (CS) is a polysaccharide identified as suitable for drug/gene delivery depots . Due to In vitro, the obtained CMC-OCS/MPs/KGN showed slower weight loss and a shorter gelation time than control hydrogels. The hydrogel/MPs’ compressive elastic modulus was greatly enhanced, which is good for cartilage healing. While waiting, the PLGA MPs/KGN might release KGN in regulated bursts in response to ultrasound. In vitro rabbit bone marrow mesenchymal stem cells (rBMMSCs) sown in the scaffolds demonstrated noticeably enhanced vitality throughout a 7–10-day culture (An injectable carboxymethyl chitosan-oxidized chondroitin sulfate hydrogel (CMC-OCS) hydrogel containing microspheres (MPs) was made using the Schiff base crosslinking procedure. The kartogenin (KGN) loaded poly (lactide-co-glycolic acid) MPs (MPs/KGN) were made using the PME method.  culture .in vitro cell culture period. One of the vital components of cartilage, HA, may be the result of this biocompatibility. In fact, when seeded in the composite CS/HA/SiHPMC hydrogels, the in vitro chondrocyte survival up to 21 days of culture is much higher. These results demonstrate that the CS/HA/Si-HPMC hydrogels, especially the one containing 3% (w/v) Si-HPMC, exhibited the best overall properties for use in bone regeneration. When coupled with chondrocytes in subcutaneous or nude mice, CS/HA/Si-HPMC hydrogels have been shown to be useful for cartilage tissue engineering. As a result, the injectable CS/HA hydrogel loaded with Si-HPMC is a promising tissue engineering approach with a lot of clinical cartilage tissue healing potential (A novel injectable hydrogel composite made of water-soluble CS/HA and silanized hydroxypropyl methylcellulose (Si-HPMC) was created for cartilage tissue creation . Si-HPMCotential .According to Samiei et al., the hydrogels that incorporate more gelatin have a somewhat looser network than the others. The hydrogel with less gelatin has a slightly higher value of G′, which denotes more elasticity as a result of more CS amine groups being crosslinked through a covalent connection with genipin. Since there were very few dead cells in any of the hydrogels, this demonstrated the remarkable biocompatibility of the made hydrogels for human dental pulp stem cells (hDPSCs). After 21 days, hDPSCs cultivated on produced hydrogels showed a considerable increase in calcium deposition, as shown by the quantitative findings of alizarin red staining. Furthermore, the greatest Alkaline phosphatase (ALP) activity was shown by hDPSCs cultivated on hydrogel that included more gelatin. After 21 days, the hydrogel with more gelatin expressed late osteogenic genes such OCN and BMP-2 6 and 4 times, respectively, more than the control group. The Poly (N-isopropylacrylamide) (PNIPAAm-g-CS) copolymer/gelatin hybrid hydrogel that was created had excellent properties and produced the osteogenic differentiation required for dental tissue engineering .Focusing on the positive benefits of Fmoc-FF self-assembly and its electrostatic interaction with glycol chitosan (GCS), a straightforward method for producing injectable self-healing hydrogels for Doxorubicin (DOX) administration has been reported. The final product, a hydrogel-based technology, showed a novel Ph-responsive DOX release, with the drug release mechanism being improved in moderately acidic environments. This finding may pave the way for controlled drug release in a typical, moderately acidic tumor microenvironment. The new hydrogel was undoubtedly a successful method for the controlled release of DOX, given the slow rate of release of DOX under physiologically normal settings. Notably, the DOX-loaded hydrogel was efficiently cytotoxic to the human tumor cell line A549 in a test tube .® assays were conducted. Dexamethasone (DMT) was released in vitro from the CS-PF and CS-PF-TPP gels over a longer period of time than the PF hydrogel. Studies conducted in vivo revealed that hydrogels kept the fluorescent component in the joint longer than when it was given in Phosphate-buffered saline (PBS) alone. These findings imply that the DMT-loaded CS-PF and CS-PF-TPP hydrogels may provide an effective drug delivery system for the osteoarthritis treatment  produced the hydrogels. Tripolyphosphate (TPP) was further utilized as a crosslinking agent. In comparison to pluronic gel alone, the gel duration was lengthened by the addition of CS. The morphology of the hydrogels generated was altered by the addition of TPP to the substance. To test whether hydrogels were harmful to human chondrocytes, MTS and Live/Deadreatment .in vitro drug release study. Graft copolymer is discovered to have a significantly higher rate of cellular absorption than pure medication, making it a more effective delivery method. The possibility of using the graft copolymers as an injectable hydrogel has been suggested by an in vivo gelation investigation in a rat model  hydrogel, a thermosensitive, injectable halloysite nanotubes (mHNTs)/CS/GP NC hydrogel was developed. The objective was to develop an injectable nanocomposite (NC) CS hydrogel containing modified mHNTs. This research enhances the mechanical robustness of the resultant scaffold mHNTs as well as the proliferation of human adipose tissue-derived stem cells (hASCs) within it. Overall, the results for bone differentiation showed that IC/mHNTs improved the mechanical strength of CS hydrogel and enhanced the difference of encapsulated hASCs into bone tissue because of their stiffness, tubular structure, and ability to dynamically deliver IC as an osteogenic inducer agent .For tissue engineering applications, the optimal chitosan/oxidized-modified quince seed gum/curcumin-loaded (CS/OX-QSG) hydrogel (ratio of 25:75) with varied curcumin-loaded in halloysite nanotubes (CUR-HNT) levels was effectively synthesized. The hydrogel with the highest OX-QSG concentration (CS/OX-QSG 25:75) exhibited a spongy structure with bigger and smaller holes as well as a high crosslinking density. Because of their good mechanical and anti-bacterial capabilities, the results demonstrated that CS/OX-QSG hydrogels with a 25:75 ratio and 30% CUR-HNTs may provide a viable scaffold for tissue engineering applications .in situ forming CS/poly (ethylene glycol) (PEG) hydrogels with better mechanical characteristics. By altering the quantity (0%–100%), molecular weight , and of the PEG derivatives, the impact of PEG on the physico-chemical characteristics of hybrid hydrogels was extensively clarified. The resultant hydrogels, which are equivalent to commercially available fibrin glue, showed outstanding hemostatic activity and are extremely biocompatible in vivo  on the surface, all of the hydrogels were demonstrated to be bioactive after only 1 day of incubation in simulated body fluid (SBF). Incorporating carboxylated single wall carbon nanotubes (COOH-SWCNTs) with CS and collagen (Coll) by the application of -GP increased cross-linking, created the greatest possible thermos-responsive and injectability characteristics, and significantly enhanced mechanical properties. Additionally, the hydrogels with COOH-SWCNTs incorporated significantly increase cell proliferation and support osteogenic differentiation as compared to pure CS/Coll .in vitro cell culture examination, the DIP-PCL NPs- CS2/SF0.5 hydrogel shown the greatest capacity to encourage the proliferation and activity of MG-63 cells, as demonstrated by a significant increase in cell viability, ALP activity, and calcium deposition  thermosensitive hydrogel containing dipyridamole loaded polycaprolactone nanoparticles (DIP-PCLNPs) as a novel and non-invasive local drug delivery approach for bone tissue engineering. DIPPCL NPs were produced using the solvent-emulsification evaporation method, and the formulation factors were optimized using an irregular factorial design. The optimal conditions for producing DIP-PCL NPs were 7 mg DIP, 1.5% PVA, a W/O volume ratio of 4, and a 4-min sonication period. This perfect formulation was put into the CS2/SF0.5 thermosensitive hydrogel. Since the results demonstrated that the amount of the inserted SF altered the network structure of the CS hydrogel, the gel scaffolds containing 0.5% SF had a shorter gelation time and stronger compressive strength when compared to other composite hydrogels. In an position .In order to increase cardiac tissue’s functioning, cardiac tissue engineering supports, replaces, or repairs it. Application of the polysaccharides—of which chitosan is a key component—addresses the main problem with the vitality of the implanted cells. Chitosan aids in the provision of mechanical support, prevents the spread of pro-inflammatory chemicals, and encloses bioactive elements beneficial for the regeneration of heart tissue. Chitosan’s positive charge and hydrophilicity, among other properties, enable the development of a soft tissue milieu, particularly when combined with biomolecules. Scaffolds made of chitosan support stem cell proliferation and differentiation by providing mechanical strength .via pH-sensitive, biodegradable, and dynamic Schiff base connections. These hydrogels were ideal for injectable drug administration because of their exceptional viscoelastic, thixotropic, and self-healing properties. After Dox loading for 5 days, these hydrogels demonstrated a pH-responsive release, with a higher release at tumoral pH than at physiological pH. MTT and hemolytic assays demonstrated the non-toxicity of these hydrogels. The Dox-loaded hydrogel significantly reduced MCF-7 cell viability, with 72% of the cells dying  with numerous aldehyde groups and N, O-carboxymethyl chitosan  ls dying .in vivo tumor resection and recurrence inhibition experiments, the gel may stop lung metastasis, lessen tumor recurrence, improve survival rates, and mend surgically injured tissues. The in-situ injectable DOX@CSSH/HNTs-SH Gel may be developed as a novel drug delivery system to inhibit tumor recurrence and heal surgical wound tissues after tumor removal  were first loaded with DOX before being added to the gel precursor. This gel loaded and gradually released water-soluble DOX to lessen the cytotoxicity of thiolated chitosan (CSSH) Gel while also enhancing its mechanical characteristics. The results showed that the HNTs-SH could be evenly distributed throughout the gel matrix to boost the gel’s compressive strength and that the gel was pH sensitive to release DOX quickly in the tumor’s acidic microenvironment, where MCF-7 cells could take it up and effectively inhibit MCF-7 cell growth. According to  removal .Wang et al. created a CS/CuNPs/black phosphate nanosheets (BPNSs) solution that is sustained-release, tumor microenvironment (TME) sensitive, and has superior photothermal and redox potential in the acidic TME . Under bIn order to create pH-degradable hydrogels, Gao et al. crosslinked carboxymethyl chitosan (CMCS) with an acid-labile ortho ester compound (OEDe). CMCS was subsequently cross-linked with a mixed cross-linker comprising OEDe and ethylene glycol diglycidyl ether (EGDE) at three different molar ratios in order to further adjust the pH-sensitivity and mechanical qualities. Then, hydrogels were wrapped around DOX-loaded gelatin nanoparticles with an average diameter of 50 nm to create a hybrid system that could be inserted into a tumor location in any shape for local chemotherapy. Intravenous injections of free DOX and DOX-loaded nanoparticles were tested for their ability to prevent tumor growth in mice harboring murine hepatoma tumors. The outcomes demonstrated that the cross-linked CMCS hydrogels could greatly extend the DOX release period as well as improve medication absorption into the tumor site. The hybrid hydrogels with the most pronounced anticancer activity were those with released DOX-loaded nanoparticles because they could further enhance the retention and permeation of DOX in tumor site. Therefore, the local therapy of solid tumors with pH-degradable hydrogels has significant potential .Here in in-situ gelling regime. Although a sol-gel transition under moderate circumstances and well-controlled kinetics is required to provide a medical injection, the administration would also have minimal invasion and enable for filling irregularly shaped gaps. However, there are still a few fundamental issues to be concerned about to fully utilize injectable hydrogels in the biomedical area, aside from focusing on developing novel characteristics and sophisticated functionalities. First, while designating “new” materials for prospective clinical trials, it is important to consider the greater risk involved. The second topic is how a hydrogel’s properties could be impacted by the production process. Injectable gels must go through a sol-gel transition close to or at the desired insertion location, unlike in situ forming gels and 3D printing. As a result, the gelation shouldn't be too slow if the pregel isn't excessively viscous. Controlling the dosing of a hydrogel formulation could be challenging. Finding a solution with the right viscosity for injection, the necessary gelation kinetics, and the reliable mechanical characteristics of the harvested gel might not always be possible. The mechanical characteristics of the injected hydrogels are often poorer than those of the in situ generated ones, specifically for hydrogels with higher modulus or strength. Even though some clever approaches, such as the recently developed conjoined-network strategy, have been established for making high-performance hydrogels, a multistep synthesis procedure may reject the attempt to harvest such hydrogels by injection processing. However, given our focus on clinical users, we may anticipate more advancements in the creation of injectable hydrogels with improved and possibly “smart” features.Injectable hydrogels use their carrier capability and processing flexibility as an


The etiology of Parkinson's disease (PD) is unknown, but evidence is increasing that there is a prominent inflammatory component to the illness. Epidemiological, genetic, and preclinical evidence support a role for gut-derived sterile inflammation. Pro-inflammatory bacteria are over-represented in the PD gut microbiota. There is evidence for decreased gut barrier function and leak of bacterial antigen across the gut epithelium with sub-mucosal inflammation and systemic exposure to the bacterial endotoxin lipopolysaccharide. Preclinical evidence supports these clinical findings and suggests that systemic inflammation can affect the CNS through vagal pathways or the systemic circulation. We will review recent preclinical and clinical evidence to support this mechanism and suggest possible treatments directed at the gut-brain axis. The central nervous system (CNS) trail of Parkinson's disease (PD) is defined by α-synuclein (AS) aggregates in cell bodies and neuritis . The etiThe gut-brain axis is a bidirectional system that comprises the enteric nervous system (ENS), the autonomic nervous system, the central nervous system and hormones of the hypothalamic-pituitary-adrenal axis . The ENSSterile inflammation is defined as inflammation in the absence of a pathogen. A number of stimuli, including those caused by trauma, ischemia, or environmental instigators produce damage-associated molecular patterns (DAMPs). These act at innate immune receptors (toll-like receptors (TLR), RIG-1-like receptors (RLS) and Nod-like receptor family, and pyrin domain containing 3  and damage-associated molecular patterns (DAMPs). TLRs activate myeloid-differentiation factor-88—dependent and—independent signaling pathways that unleash the production of inflammatory cytokines. These include tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, IL-6, IL-8 and IL-12). DAMPs also activate the NLRP3 inflammasome, activating caspase-1, resulting in the production of IL-1β and IL-18. Increased levels of inflammatory cytokines have been documented in the brain and cerebrospinal fluid in PD –13, but 2, is the largest body surface exposed to the environment. The intestines form a critical barrier to penetration of pathogens and their antigens, and make up the majority of the surface related innate immune system. In close proximity to the intestinal epithelium are millions of neurons in direct contact through the vagus nerve with the brainstem and through spinal nerves to the spinal cord. Commensal bacteria in the gut, the gut microbiota, include some 100 trillion micro-organisms  were over-represented  identified PD cases with 47.2% sensitivity and 90.3% specificity , particularly butyrate. SCFAs have anti-inflammatory and anti-oxidant activities and are important in the regulation of intestinal and blood brain barrier permeability through effects on tight junction proteins  using high throughput rRNA gene amplicon sequencing of the V4 variable region of the 16S ribosomal RNA gene. PD subjects had greater α-diversity. At the phylum level, Bacteroidetes, Proteobacteria, and Verrucomicrobia were higher, and Firmicutes were lower in PD samples. At the genus level, putative anti-inflammatory bacteria from the genera resented . There hresented –49. In ocificity . A recencificity . In 10 ocificity , 51–53. proteins . Standarproteins . In addiproteins , 55–57. proteins .The potential importance of fecal microbiota changes in the genesis of PD is supported by preclinical studies. In preclinical models, behavioral, and pathologic markers of parkinsonism in mice engineered to over-express AS are significantly reduced in germ free mice, which lack intestinal microbes , 59 and The intestinal barrier is a single epithelial layer lining the GI tract. The barrier regulates the transport of luminal contents across the intestinal wall by paracellular and transcellular transport . ParacelE. coli antigen and inflammation in mucosal biopsy specimens  is an endotoxin specific to gram-negative bacteria . LPS binIntraperitoneal injections of LPS in gravid rats leads to a reduced number of dopamine neurons in the substantia nigra of their offspring, and enhanced sensitivity to 6-hydroxydopamine induced parkinsonism compared to offspring of control animals . LPS hasIt would be premature to say that the mechanisms by which gut-derived sterile inflammation influences the genesis of PD are completely understood. In the gut, it seems well-established in PD that gut dysbiosis is present, is associated with intestinal leak and penetration of bacterial antigens including LPS into the intestinal wall and production of local inflammatory mediators, induced by action at TLRs. This inflammatory trigger may produce a systemic inflammatory process with contributions from both the innate and adaptive immune systems . SystemiIn the same way that changes in intestinal function are associated with non-neurological disorders that share systemic inflammatory drivers, these changes are not specific among neurodegenerative diseases for PD. For example, studies in another synucleinopathy, multiple system atrophy also show evidence for an inflammatory dysbiosis . ChangesDespite these limitations, attention is turning toward potential therapies. Dietary changes targeting the gut-brain axis are a tempting prospect. Diets rich in fish and healthy snacks reduced LPS activity in serum in a non-PD population , and redLiving bacteria, in the form of probiotics, can be delivered orally via dietary supplements, and these can have a salutary effect on inflammatory bowel disease, such as ulcerative colitis. A pilot placebo-controlled study of probiotics in 50 PD patients showed decreased expression of genes associated with inflammation  , and anoModerate-intensity exercise reduced systemic inflammation and decreased TNFα in PD subjects . DespiteFecal microbial transplant has the potential to restore the gut microbiota to normal . Althougclinicaltrials.gov listed 378 interventional clinical trials in PD. Among these, only 8 targeted the gut, including studies or prebiotic fiber supplements, probiotics, anti-inflammatory diet, nutritional supplement and fecal transplant .At the time of this writing, This article was written solely by KS, but reflects collaborative work with a number of investigators, principally among them from Rush University Medical Center , University of Chicago , California Institute of Technology  University of Gainsville, Florida (MT), and Emory University (MH).The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cancer is one of the most difficult diseases to be treated in the world. Immunotherapy has made great strides in cancer treatment in recent years, and several tumor immunotherapy drugs have been approved by the U.S. Food and Drug Administration. Currently, immunotherapy faces many challenges, such as lacking specificity, cytotoxicity, drug resistance, etc. Nanoparticles have the characteristics of small particle size and stable surface function, playing a miraculous effect in anti-tumor treatment. Nanocarriers such as polymeric micelles, liposomes, nanoemulsions, dendrimers, and inorganic nanoparticles have been widely used to overcome deficits in cancer treatments including toxicity, insufficient specificity, and low bioavailability. Although nanomedicine research is extensive, only a few nanomedicines are approved to be used. Either Bottlenecks or solutions of nanomedicine in immunotherapy need to be further explored to cope with challenges. In this review, a brief overview of several types of cancer immunotherapy approaches and their advantages and disadvantages will be provided. Then, the types of nanomedicines, drug delivery strategies, and the progress of applications are introduced. Finally, the application and prospect of nanomedicines in immunotherapy and Chimeric antigen receptor T-cell therapy (CAR-T) are highlighted and summarized to address the problems of immunotherapy the overall goal of this article is to provide insights into the potential use of nanomedicines and to improve the efficacy and safety of immunotherapy. Cancer has become the second fatal disease in the world after cardiovascular diseases, and the number of patients is increasing year by year . CurrentNano drug is a new way cancer immunotherapy with a safer and more controlled manner , allowinin vitro amplification, cells are injected back into the patient, and then continue to proliferate and find target cells in vivo  has become the focus of clinical immuno-oncology because of its MHC-independence, broad-spectrum replicability and immune memory function . This th in vivo . To simp in vivo , the nant effect . In addit effect  found thTumor immunotherapy is a sort of cancer treatments by activating the immune system , which hImmune checkpoints regulate the immune syste, where stimulatory immune checkpoint molecules promote the activation of T-cells and activate the immune response suppress the immune response of the body and prevent the development of autoimmunity . Up to nImmune checkpoint molecules include CTLA-4, PD-1, PD-L1, which are the most widely researched, and other potentially novel immune checkpoint molecules that has not been used in the clinic, such as lymphocyte activationgene3 (LAG-3), T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) structural domain proteins, T-cell immunoglobulin mucin 3 (TIM-3), and T-cell activation inhibitor immunoglobulin variable region structural domain ISTA) .+ and CD8+ T cells and binds to the ligands CD80 (B7-1) and CD86 (B7-2). It is also a T-cell surface receptor that acts as an immunosuppressive molecule and can participate in immunosuppressive signaling , immune adjuvants, and short peptides to enhance the body’s immune response. Substances used to regulate immunity are often referred as immune system modulators. The initial cytokine drugs approved by the FDA were IFNα-2a (Roferon-A) and IFNα-2b (Intron-A). Subsequently, high doses of IL-2 (Proleukin) were approved for the clinical treatment of metastatic melanoma and renal cancer . In addiazotemia . It was azotemia . IL-2 thazotemia . Thus, aazotemia .Tumor immunotherapy mainly relies on regulating or activating the host’s immune system to inhibit or kill tumors, which has the advantages of low toxicity and high efficiency. However, challenges in clinical tumor immunotherapy are still need to be solved. . First, Nanomedicines, as an emerging technology, have great efficiency in tumors, cellular oxidative damage, and many other diseases because of features including small particle size and stable surface function . One of Nanomedicines include polymeric micelles, liposomes, nanoemulsions, dendritic macromolecules and inorganic nanoparticles. Their main characteristics are shown in d-α-tocopherol polyethylene glycol 1000 succinate (TPGS)-modified carboxymethyl chitosan rhodopsin (TCR) polymeric micelles (PMs) for the oral administration of paclitaxel (PTX)  . The resel (PTX) , throughel (PTX) , dendritel (PTX) , inorganel (PTX) , cisplatel (PTX)  and noveBased on the properties of each material, it is known that polymeric micelles can be used for oncology treatments, drugs with high targeting requirements, etc.; nanoliposomes are recommended for transporting small polar drugs such as paclitaxel and artemisinin; nanoemulsions can be used for drugs with poor solubility and encapsulating capacity, like ophthalmic drugs, oral agents and injectables; dendritic macromolecules can be used when there is a need for large volumes and rapid detection; apheresis nanoparticles can be used when metal trace elements are shortening and biological barriers cannot be overcome.Drug delivery of nanoparticles mainly contains three ways: cellular uptake, cytosolic delivery by endosome escape and direct intracellular delivery, as detailed in Nanoparticles (NPs) can enter into cells by endocytosis. In thisprocess, cell membrane engulfs NPs and forms a separate vesicle in the cell. The mechanisms of endocytosis include phagocytosis, latticin- and niche protein-mediated endocytosis, and latticin- and niche protein-independent pathways . PhagocyNanoparticles with enhanced endosomal escape ability are the best choice for bio-delivery. Commonly used nanoparticle that are commonly used for endocytosis and biological agents’ delivery includes liposomal and lipid-based NPs, polymer micelles and polymer NPs, lipid-polymer hybrid NPs, nanocrystals, and inorganic NPs such as gold NPs, magnetic NPs, graphene, quantum dots, and silicon NPs. The mechanism of intranuclear body escape involves instability of the intranuclear body membrane, which can be solved by increasing the interaction between the intranuclear body membrane and intranuclear solvents such as pH-sensitive solvation compounds or polymers . CurrentVarious methods have been developed to enhance intranuclear body escape, including delivering cytoplasmic directly bypassing intranuclear body wrapping and delivering organisms to the cytoplasm directly. The solutions contain fossa-mediated pathways, contact release, and intermembrane transfer, membrane fusion, direct intracellular translocation, membrane rupture, and microinjection .Although nanocarriers is developing rapidly, many challenges are still to be addressed. The intracellular drug delivery and efficacy of nanocarriers are mainly controlled by the mode of internalization and intracellular transport. Endocytosis of NPs is a major pathway, but intranuclear endosomal encapsulation leads to a lower possibility of cytoplasmic delivery. Materials for inducing intranuclear endosome escape can be limited by toxicity . To solvNanoparticles and coupling compounds are widely used in the following six areas.Immune cells play a crucial role in the immune response process. However, the continuous activation of the immune system may disrupt immune homeostasis and lead to inflammatory diseases. Targeted immunomodulators may be useful in the treatment of such diseases and their complications. However, the disadvantages of immunomodulators include high immune-mediated toxicity, low solubility, and) loss of biological activity after long-term circulation . At thisin vivo of specific T cells  to the surface of lipid nanoparticles, and the principle of achieving specific hepatocyte targeting. Studies have shown that lipid nanoparticle particles have better stability, biodistribution, and targeting capacity, making this technique suitable to be developed, having the potential of an extrahepatic targeting to develop a novel gene therapy . These b-mRNAs are structurally and functionally similar to conventional mRNAs and contain barcodes that allow them to be quantified by deep sequencing  are shown below , also affect the number of regulatory T-cells. Irvine’s group investigated the immunosuppressive effects of small molecule inhibitor TGF-β by T-cell-targeted delivery a (SB525334) . Fahmy and others co-loaded the nanoparticles with TGF-β inhibitors and interleukin 2, attenuating immunosuppression and enhancing T-cell proliferation. Huang et al.  developein vitro, they are transfused back to patients specifically binding of CAR-T cells to tumor-associated antigens in vivo, activating T-cells through signal transduction, promoting T-cell proliferation and releasing cytokines to exert anti-tumor effects  has shown greater clinical advantages and potential. CAR-T therapy is worked by collecting peripheral blood from patients and isolating lymphocytes, introducing CAR genes into T-cells through genetic engineering techniques to express specific receptors to bind specific tumor-associated antigens. After being a amplified  effects . Typical effects . After b effects .CARs are classified into five generations based on intracellular structural domains: The first generation has a single activating structural domain, mainly CD3ζ, and some research studied on the gamma chain of the Fc receptor. The second generation added a co-stimulatory structural domain obtaining from co-stimulatory molecules such as 4-1BB or CD28 attaching to activate structural domain (CD3ζ/γ chain of the Fc receptor) to enhance the cell proliferation and cytotoxic capacity of CAR-T cells . The thiTraditional cancer treatments often cause damage to normal cells in the body, or residual malignant tumor cells flow into the bloodstream through the wounded area, making poor prognosis. Moreover, single immunotherapy modalities have limitations in their efficacy when treating solid tumors. Based on this, the use of combined nanoparticles for cancer immunotherapy can achieve synergistic effects. The current limitations of CAR-T immunotherapy in clinical tumor immunotherapy include the following: stimulating T-cell expansion and persistence; improving T-cell trafficking; stimulating intrinsic T-cell activity; resetting immunosuppressive cells and vascular microenvironment; methods to monitor the therapeutic efficacy of CAR-T cell therapies, etc.The complex preparation procedures and severe toxicities are two major obstacles facing the wide use of chimeric antigen receptor-modified T (CAR-T) cells in clinical cancer immunotherapy. The nanotechnology-based T-cell temporary CAR modification may be a potential approach to solve these problems and make the CAR-T cell-based tumor therapy feasible and broadly applicable. To solve this problem, Judith Guasch  propose in vitro correlates positively with performance of CAR-modified immune cells in vivo. Therefore, they propose that the quality of the IS predicts the effectiveness of CAR-modified immune cells, which provides a novel strategy to guide CAR therapy. Yingmei Luo et al. -modified T-cell therapy has the potential to improve the overall survival of patients with malignancies by enhancing the effectiveness of CAR T-cells. Precisely predicting the effectiveness of various CAR T-cells represents one of today’s key unsolved problems in immunotherapy. Qin Liu  encapsulo et al.  develop via bio-orthogonal click chemistry before adoptive transfer. The spatial-temporal co-existence of aPD1 with ACT T-cells and the responsive drug release significantly improved the treatment outcome of ACT in murine solid tumor model. The average tumor weight of the group treated by cell-surface anchored aPD1 was only 18% of the group treated by equivalent dose of free aPD1 and T-cells. The result of this research indicates that technology can be broadly applicable in ACTs employing natural or Chimeric Antigen Receptor (CAR) T-cells. In addition, the exploitation of embedded protein-targeted nanoliposomes (The efficacy of Adoptive Cell Therapy (ACT) for solid tumor is still mediocre. This is mainly because tumor cells can hijack ACT T-cells’ immune checkpoint pathways to exert immunosuppression in the tumor microenvironment. Immune Checkpoint Inhibitors such as anti-PD-1 (aPD1) can counter the immunosuppression, but the synergizing effects of aPD1 to ACT was still not satisfactory.  demonstriposomes  and γδ-Tiposomes  would bevia reduction-sensitive coupling. The activation state of the T-cell and liposome composition was shown to strongly influence the up-sampling efficiency. Loading cells with liposomes did not impair the activation status of the T-cells and the liposome composition are shown to strongly influence the loading efficiency. Loading the cells with liposomes does not compromise T-cell functionalities like proliferation and cytolytic function.Laura Wayteck et al.  concludein vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T-cell proliferation in vitro and lead to effective T-cell stimulation and inhibition of tumor growth in vivo. This team of investigators have manufactured two types of nanoscale particle platform-based aAPCs and demonstrates that both iron-dextran particles and quantum dot nanocrystals enhance tumor rejection in a melanoma model, providing the first description of nanoscale aAPCs that lead to effective T-cell stimulation and inhibition of tumor growth.Karlo Perica’s team  manufactAdoptive cell transfer of targeted chimeric antigen receptor (CAR) T-cells has emerged as a highly promising cancer therapy. The pharmacodynamic action or CAR T-cells is closely related to their pharmacokinetic profile; because of this as well as the risk of non-specific action, it is important to monitor their biodistribution and fate following infusion. Stefan Harmsen  developeCombination with nanotechnology, CAR-T cells can construct a lentiviral expression vector and complete the culture of a complete CAR-T cell expansion system. Clinical studies  have shoIn recent years, the rapid development of nanomedicine provides new insights to cancer immunotherapy. The most obvious advantage of nanoparticles is tunability. They can be designed in various sizes and shapes. Being modified with targeting molecules or loading various drugs shows the ability of targeted delivery and therapeutic delivery simultaneously. Nanomedicines can also be targeted for immunotherapy applications based on different needs because of different materials properties, such as high targeting requirements, transporting small polarities, poor solubility, not easily encapsulated, and unable to overcome biological barrier situations. Finally, compared to low molecular weight immunomodulators, nanoscale immunomodulators control pharmacokinetic behavior and have the potential to enhance immune activation through synergistic effects due to their unique size effect and co-loading capacity. Indeed, the presence of multiple functional domainsovercome barriers to effective immunotherapy in solid tumors. This paper introduces the characteristics and advantages of immunotherapy, nanomedicines and their current research status in immunotherapeutic approaches, focusing on reviewing the application and prospects of various types of nanoparticles in CAR-T therapy for tumor treatment, learning about the advantages of nanomedicines in immunotherapy and preparing for the development of more nanomedicines in CAR-T therapies for oncology.Currently, CAR-T therapies mainly work in hematologic tumors and lymphomas. It has much less effect in solid tumors with toxicity. This variation is caused by various factors, For example, multiple cells and molecules with different immunosuppressive effects are appeared in solid tumors; the level of antigen expression in solid tumor cells varies between tumor types and different stages; and antigens in solid tumors are generally expressed in small amounts in other sites, such as heart, lung, and liver, accompanying by off-target effects. Therefore, nanoparticle immunotherapy can be used in combination therapies to address the limitations solid tumors treatments, including chemotherapy, radiotherapy, and photodynamic therapy. For example, compared to immunotherapy, radiotherapy combing with immunotherapy significantly improves the efficiency of patient treatment in clinical trials, which overturning the traditional view that radiotherapy kills immune cells and has an immunosuppressive effect. This suggests a synergistic effect of radiotherapy and immunotherapy .Partial radiotherapy activates the immune system, inducing immune cells to attack tumor cells outside the radiotherapy area. It is rare for clinical radiotherapy but immunotherapy can enhance the immune-inducing effects of radiotherapy and increase the incidence of distant effects . Althoug


Recently, ubiquitination has been reported to be closely involved in the progression of tumore. The purpose of this study was to identify the ubiquitination-associated genes and co-expressed lncRNAs on the prognosis of clear cell renal cell carcinoma (ccRCC) patients.We downloaded 530 cases and the corresponding transcriptome profiling from The Cancer Genome Atlas (TCGA) database. We distinguished mRNA and lncRNA expression data from the transcriptome profiling and then extracted the expression of mRNAs that regulate protein ubiquitination. We obtained lncRNAs associated with protein ubiquitination regulation from the lncRNA data by gene co-expression analysis. Cox regression analysis of survival time, survival status, and lncRNA expression level was carried out, and a prognostic index (PI) was constructed.The PI was established based on 8 prognostic lncRNAs that regulate protein ubiquitination and distinguish the high-risk group patients from all patients. Multivariate analysis indicated that this PI was an individualized clinical prognostic factor for patients with ccRCC. Regarding clinical characteristics, a ubiquitination-associated clinical-prognostic index (UCPI), containing 8 ubiquitination-related lncRNAs and age, was established and tested with AUC of 0.80.We established a UCPI containing 8 lncRNAs related to protein ubiquitination. This UCPI may become an appropriate model to predict the prognosis in ccRCC patients and guide clinicians to adjust the follow-up regimen.The online version contains supplementary material available at 10.1186/s12957-022-02618-x. Renal cell carcinoma (RCC) was the sixth deadliest cancer worldwide in 2018, accounting for approximately 4% of adult malignancies . ConsideLong noncoding RNAs (lncRNAs) are defined as transcripts with a length of less than 200 nucleotides and no protein-coding function . SeveralVHL loss, either through mutation, hypermethylation, or chromosomal loss, has been widely reported in most ccRCC patients, indicating the pivotal role of VHL in the genetic pathogenesis of ccRCC  transformation. The R survival package was used to perform a univariate Cox regression analysis of survival status and time using lncRNAs related to protein ubiquitination. A multivariate Cox regression analysis was applied to eliminate genes that failed to become independent indicators, and the remaining lncRNAs were used to establish a clinical-prognostic index with the best Akaike information criterion (AIC) value. The prognostic index (PI) formula was based on a linear combination of the relative expression level of lncRNAs multiplied regression coefficients (coef), which represented the relative weight of lncRNAs in the multivariate Cox regression analysis. This formula distinguished all ccRCC patients into a low-risk group and a high-risk group.Ubiquitination-related lncRNAs expression profiles were normalized by [logThe Survminer package in the R was used to plot the survival curve, while the R pheatmap package was applied to draw the risk score scatter diagram, survival state plot, and risk heatmap, which indicated the diagnostic efficacy of this clinical-prognostic index. To determine if the PI was an individualized clinical-prognostic model, we perform a multivariate Cox regression analysis of the patients’ risk score, and their age, gender, pathological stage, historical grade, and TNM status to validate the independence of this PI. The survival ROC package was applied to calculate the Area Under Curve (AUC) of this PI and visualize the Receiver Operating Characteristic (ROC) curve. Clinical correlation analysis uses the ggpubr package for visual processing.http://www.r-project.org/) and strawberry Perl language , PCA analysis was performed in the LIMMA package, and PCA visualization was performed using the scatterplot3d package. A p-value < 0.05 was considered in statistical analysis.All statistical analyses and visualizations of results were conducted using R software , stage , TNM stage  and RiskScore , whereas only age and RiskScore were identified as independent risk factors for ccRCC patients OS by multivariate Cox regression analysis  prognosis reported a seven immune-related lncRNA model containing AP000695.1 . The lncUbiquitination and its biological processes were significantly correlated with the VHL-hypoxia-inducible factors (HIFs) axis — well-characterized and implicated as connections to the pathogenesis of ccRCC . The ubiIn this study, we constructed one UCPI based on ubiquitination function and clinical variables, which performed well in survival outcome prediction of ccRCC patients. Previous studies have been performed to explore biomarkers for ccRCC prognosis. Recently, similar methods have been used, and a brand new autophagy-related seven-gene prognostic risk signature was reported . HoweverThere were some limitations in our study. First, this UCPI was established from the TCGA database instead of ccRCC tumor specimens, which should be validated in a prospective and long-term cohort in clinical practice. Then, we considered the confounding factors of clinical variables on ccRCC prognosis and constructed the nomogram containing the significant clinical factors. The inherent bias and unknown confounders may contribute to the limitations. Last but not least, only ccRCC tumor issues were included for the dectection and validation of ubiquitination-related RNAs, while the serological samples were ignored. We belived that biomarkers derived from serological samples should contribute to the construction of UCPI, and further exploration should be carried out.Finally, we developed and validated an eight-lncRNA UCPI for ccRCC patients’ prognosis. This UCPI performed well in the prediction of ccRCC patients’ survival status with an AUC of 0.800, providing a novel insight for ccRCC patients’ prognosis and a guide for further individualized therapy. Further extensive study should be designed to prospectively validate the prognostic UCPI model in a large-scale cohort. Moreover, our ubiquitination-related model strongly suggested the crucial effect of ubiquitination on the progression of renal carcinoma.Additional file 1: Supplementary Fig. 1. Coexpression analysis of 9 ubiquitination-related mRNAs and 29 lncRNAs.Additional file 2: Supplementary Fig. 2. Univariate Cox regression analysis of 29 ubiquitination-related lncRNAs in 507 tumor specimens identified 14 lncRNAs to be significantly associated with the prognostic status of ccRCC.Additional file 3: Supplementary Fig. 3. Expression analysis of 8 ubiquitination-related lncRNAs on clinical characteristics, including age (A), gender (B), tumor grade (C), tumor stage (D), tumor TNM stage (E-G).


Suicide is a major public health concern. Suicide rates have increased steadily in the U.S. and are highest among middle-aged (ages 45-64) and older (age 75+) adults. Help-seeking represents an important coping behavior that can mitigate suicide risk. However, research on this topic is sparse, especially among older adults. To address this gap, a systematic review of the existing literature describing help-seeking for suicide risk among middle- to old-age adults was conducted. Using PRISMA guidelines, we searched electronic databases  and key journals with suicide and/or gerontology focuses for peer-reviewed publications in English between 2010-2020. The search yielded 4,732 unduplicated publications. After screening articles for relevance based on titles and abstracts, 52 articles were reviewed in full text. A total of 24 articles met inclusion criteria and were included in this review. The articles reviewed included a range of topics, including prevalence of service utilization, service use prior to suicide-related behaviors, and correlates of help-seeking. Overall, prevalence of service utilization was generally low and varied by suicidal history . The systematic review also identified key service use facilitators  and barriers . Results of this systematic review highlight the need for future research and tailored services to improve suicide prevention and intervention strategies for middle-aged and older adults.


Sex-related differences in psychopathology are known phenomena, with externalizing and internalizing symptoms typically more common in boys and girls, respectively. However, the neural correlates of these sex-by-psychopathology interactions are underinvestigated, particularly in adolescence.N = 1526) community-based sample. To test for sex-by-psychopathology interactions in structural grey matter volume (GMV), we used whole-brain, voxel-wise neuroimaging analyses based on robust non-parametric methods. Psychopathological symptom data were derived from the Strengths and Difficulties Questionnaire (SDQ).Participants were 14 years of age and part of the IMAGEN study, a large , bilateral anterior and mid-cingulum , right cerebellum and fusiform  and left frontal superior and middle gyri . Higher symptoms of hyperactivity/inattention were associated with lower GMV in all four brain clusters in boys, and with higher GMV in the temporoparietal-opercular and cerebellar-fusiform clusters in girls.We found a sex-by-hyperactivity/inattention interaction in four brain clusters: right temporoparietal-opercular region (Using a large, sex-balanced and community-based sample, our study lends support to the idea that externalizing symptoms of hyperactivity/inattention may be associated with different neural structures in male and female adolescents. The brain regions we report have been associated with a myriad of important cognitive functions, in particular, attention, cognitive and motor control, and timing, that are potentially relevant to understand the behavioural manifestations of hyperactive and inattentive symptoms. This study highlights the importance of considering sex in our efforts to uncover mechanisms underlying psychopathology during adolescence. Heterogeneity in mental disorders is a significant and largely unresolved problem that stands in the way of the development of predictors and targeted therapies. A clear example of this heterogeneity is sex-related, which is a ubiquitous phenomenon in psychopathology – many disorders show uneven sex distributions of prevalence, symptoms, age of onset and treatment response Martel, . For exaa priori selection of ROIs  in limbic regions in females . For the behavioural analyses alone, a larger subset of the data (N = 2046) was available . The socioeconomic and housing subscale of the Family Stresses questionnaire  reduce genetic diversity by maximizing European descent homogeneity, and (ii) maximise socioeconomic status (SES), emotional and cognitive development diversity by sampling equally from private, state-funded and special units. Details on recruitment and assessment can be found elsewhere  Goodman, , a briefhttp://adni.loni.usc.edu/methods/documents/mri-protocols/) to give good agreement in the final images despite scanner differences. T1-weighted images were spatially normalised and segmented into GM using a voxel-wise-based morphometry (VBM) pipeline involving unified segmentation  and smoothing to 8 mm .The MRI scanners used across the eight sites were 3-Tesla and of various manufacturers . To ensure comparability across sites, scan parameters were standardised to the highest degree possible and were optimised based on the ADNI1 protocol (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/) randomise function  and hence used two-tailed statistical significance values. In addition, we corrected for multiple comparisons experiment-wise  using Bonferroni correction by multiplying the p value by four (i.e. number of tests conducted).To address the main research question of whether sex-by-psychopathology interactions are detected for GM outcomes, we ran four general linear models (GLM), one for each of the four psychopathological symptom subscale scores  with GMV at the voxel-level as the dependent variable. The sex-by-psychopathology interaction term was calculated by multiplying sex to the relevant subscale score and used as the covariate of interest. Sex, the relevant subscale score, site, handedness, age, puberty, SES and total intracranial volume (TIV) were added as covariates  and 2. ATo test the direction of any resulting interaction, a post-hoc test was run by splitting the sample by sex and running a GLM for each significant cluster, with GMV in each cluster as the dependent variable, the relevant psychopathology symptom subscale (hyperactivity/inattention) as the covariate of interest and site, handedness, age, puberty, SES, TIV and the remaining three psychopathology symptom subscales as covariates. To test if an interaction effect was also global, not just regional, a second post-hoc test used an analogous GLM on the whole sample using whole-brain GMV as the dependent variable and sex-by-hyperactivity/inattention as the covariate of interest. A third post-hoc test investigated whether the sex-by-hyperactivity/inattention GMV interaction within the significant clusters remained significant even when covarying for an additional measure of brain volume, normalised brain volume, corresponding to the sum of the volumes of grey and white matter, divided by TIV. See Supplementary materials for post-hoc test details.p value-oriented) approach. The former uses observed data to calculate probability estimates for different hypotheses, reflecting which of these has higher or lower credibility  and internalizing symptoms subscale scores  were higher in boys and girls, respectively. This was achieved using four, Bonferroni-corrected, GLMs with the relevant subscale score as the dependent variable, sex as the covariate of interest and site, handedness, age, puberty, SES, TIV and the remaining three subscale scores as covariates. GLMs that yielded null findings were supplemented with Bayesian linear regressions to quantify the probability of the null finding. A Bayesian framework is different from a frequentist (We report a sex-by-psychopathology interaction in GMV with hyperactivity/inattention problems in four brain clusters: (i) a right cluster including the superior temporal gyrus and extending to the supramarginal, inferior parietal Heschl gyrus, and rolandic operculum; (ii) a frontal medial cluster including the bilateral cingulum anterior and extending to the mid-cingulum; (iii) a right cluster involving the cerebellum, fusiform and the inferior temporal gyrus; (iv) a left frontal cluster including the superior and middle frontal gyri and extending to the precentral gyrus . Excludit-statistics with a Cohen's d ranging from d = −0.20 to d = −0.27), positive for girls  and the directions of these associations were significant for all clusters (p < 0.05) except for two clusters for girls, the fusiform-cerebellar and cingulum, which were at borderline significance . The third post-hoc test showed that sex-by-hyperactivity/inattention interaction in GMV within the significant clusters remained significant even when covarying for normalised brain volume . The latter result suggests that the main findings at the voxel-level were sufficiently robust, surviving additional brain volume correction.Post-hoc tests indicated that the associations between hyperactivity/inattentive symptoms and GMV were negative for boys  and peer problems were significantly higher in boys  compared to girls, whilst emotional problems were higher in girls  compared to boys  have been previously reported in both structural neuroimaging studies on ADHD showing lower GMV   but comparable and nominally larger than mega-studies on structural brain differences in ADHD  were higher in boys and emotional difficulties  higher in girls. These results are congruent with the existing literature indicating that externalizing disorders are more frequent in boys and internalizing disorders are more frequent in girls (Becker et al., v. controls, Marquand et al., Clinically, our findings have two impacts. First, they inform the clinician that there are biological differences across the sexes in terms of hyperactivity/inattention symptoms. Second, they provide a lead to translational research. This includes research aimed at intervention practices, for example, pharmacological treatment as well as research on biomarkers of hyperactivity/inattention specific to boys and girls. A biomarker would aid the stratification of inattentive and hyperactive type of disorders by including a more objective biological-based approach instead of one only based on behavioural symptoms (Abi-Dargham & Horga, Sex information was limited to biological sex and did not include any gender information, potentially limiting the interpretability of the results Krieger, . Also, oThe brain regions we have identified via a sex-by-hyperactivity/inattention interaction effect are the first to be reported from a large, sex-balanced and community-based sample, using an exploratory whole-brain VBM approach. These study characteristics make our study not only powered to confirm previous findings but also to explore new ones and, within a sample that is more representative of the distribution of psychopathological symptoms in the general population. By addressing these common issues of previous studies, our results are valuable as they offer a reliable reference on sex-by-psychopathology interactions in GMV. Overall, these findings provide important leads for future research on mechanistic accounts of psychopathology and their sex-biases that are crucial to the advancement of improved targeted early interventions.


Flipped classroom models encourage student autonomy and reverse the order of traditional classroom content such as lectures and assignments. Virtual learning environments are ideal for executing flipped classroom models to improve critical thinking skills. This paper provides health professions faculty with guidance on developing a virtual flipped classroom in online graduate nutrition courses between September 2021 and January 2022 at the School of Health Professions, Rutgers The State University of New Jersey. Examples of pre-class, live virtual face-to-face, and post-class activities are provided. Active learning, immediate feedback, and enhanced student engagement in a flipped classroom may result in a more thorough synthesis of information, resulting in increased critical thinking skills. This article describes how a flipped classroom model design in graduate online courses that incorporate virtual face-to-face class sessions in a virtual learning environment can be utilized to promote critical thinking skills. Health professions faculty who teach online can apply the examples discussed to their online courses. In traditional classrooms, students listen to lectures and accomplish most assignments outside of the classroom. Flipped classrooms reverse traditional course components such as lectures, projects, and assignments. This pedagogy highlights pre-recorded lectures as the primary mechanism for teaching, which is often complemented with additional videos, podcasts, and readings housed in a virtual learning environment. During face-to-face class time, students have the opportunity to ask questions and apply their knowledge through active learning techniques, including but not limited to discussion of case studies, role-playing, or problem-based learning. Autonomy is encouraged, as students are active participants in their learning. Unlike teaching methods in traditional classrooms, which are more passive, the expectation is that students come to the flipped classrooms prepared; therefore, a greater responsibility lies with the students to understand the material. Course faculty facilitate face-to-face class activities, provide feedback in real-time, and engage students while assisting in their critical thinking skills development. Flipped classrooms may improve students’ ability to synthesize complex information and increase their critical thinking skills .A meta-analysis by Hew and Lo  examinedThis study aims to provide guidance to health professions faculty with examples of how to use flipped classrooms that incorporate virtual face-to-face class sessions in a virtual learning environment to promote critical thinking skills. A discussion follows on the virtual flipped classroom model design and its use in an online graduate nutrition curriculum. This guidance was developed between September 2021 and January 2022 at the School of Health Professions, Rutgers The State University of New Jersey.There are key components to ensure that flipped classrooms are established to promote critical thinking skills. Virtual face-to-face learning activities should incorporate problem-solving and reflective practice via weekly synchronous virtual face-to-face class sessions using a web conferencing program such as Zoom. In addition, course learning objectives should address higher-level thinking and communication skills, critical thinking skills, and teamwork. Faculty must educate students on the objectives of the flipped classroom model before initiation of their courses to provide them with a clear understanding of faculty expectations and ensure accountability. It is necessary to consult the institution’s instructional designers for assistance in the initial design of each course to establish a standardized course structure and accessibility. Furthermore, consideration should be given to providing a designated area for pre-class, face-to-face, and post-class activities in each weekly course module  to promoFaculty will provide pre-class material or foundational knowledge content to students through pre-recorded lectures that students can access through their virtual learning environment. This content should be available to students before their virtual face-to-face class session; adequate pre-class time is necessary to complete these learning activities. Dividing video lecture content into concise material is more manageable, with a maximum time of 15 minutes per recording. Students view shorter videos more often and are more likely to watch their entirety, resulting in more excellent material retention than longer videos . It is eExample: In an online medical nutrition therapy course, students take a 5-question ungraded quiz before class, which unlocks weekly module content . The quiThe virtual face-to-face class sessions create a bridge between the pre-class lecture material and practical applications that resemble simulated clinical scenarios and can assist in developing critical thinking skills. This time reinforces foundational knowledge and key concepts through the application of active learning and problem-solving. These activities align with the objectives communicated in the pre-class content. Active learning in the classroom includes simulations , role-plExample 1: Students are divided into pairs in breakout rooms during the virtual face-to-face class session during an online nutrition assessment and physical examination course and practice food and nutrition-related history-taking techniques. Once back in the large group, students share their reflections on the experience and discuss how to improve their techniques in preparation for supervised practice. Students reflect or debrief and faculty guide them through their experience to understand, analyze, and synthesize what they learned .Example 2: In an online ethics, professionalism, and leadership course, virtual face-to-face class activities include large- and small-group discussions that apply concepts from pre-class readings, debates on ethical issues in the clinical setting and student presentations related to current nutrition controversies. Debates allow students to argue about controversial problems using higher-order thinking skills. Both the actual debate and the preparation for the debate are considered active learning tools and can assist students in critical thinking skills development .Example 3: An online course on ethically culturally sensitive interviewing and counseling integrates various role-playing encounters in the virtual face-to-face class session. The course instructors visit each breakout room to provide immediate feedback and redirection if necessary. Role-playing scenarios that involve a patient and health practitioner increase critical thinking skills .Example 4: Students develop skills in formulating research questions in an online evidence-informed practice and scientific inquiry course. Students are placed in breakout rooms in pairs during the virtual face-to-face class sessions, receive a short clinical scenario, and work as a team to develop a clinical research question. Upon return to the main room, critical thinking skills are exhibited as student pairs present their case, research question, rationale, and classmates critique each other’s research question. Incorporating a flipped classroom model with pre-recorded evidence-based practice lectures and small group discussions as an active learning strategy during classroom sessions may promote critical thinking skills .Assessment of learning and critical thinking skills can be achieved with formative and summative assessments using active learning techniques, quizzes, exams, self-assessment, case study presentations, and assignments that emphasize analysis, synthesis, and evaluation of student performance. Formative assessments are designed to provide the learner with quick and informative feedback, while summative assessments are utilized to evaluate knowledge and comprehension. Assessment of student performance occurs in virtual face-to-face class sessions and post-class activities. Active learning techniques such as simulation, role-playing, live presentations, and self-assessment of physical examination all occur during virtual face-to-face class sessions. It allows the faculty to provide the student with quick and informative feedback in real-time, fostering effective learning. After the virtual face-to-face class sessions, students complete post-class assignments to enhance critical thinking skills, allowing faculty to assess student performance further. Post-class formative and summative assessment strategies in flipped classrooms include short post-quizzes where students obtain immediate feedback and the rationale for the answers. Additional examples that can be assigned in many health professions programs include case studies that incorporate documentation of patient care plans, formulating research questions, and recording physical examinations performed on a volunteer. Each assignment is graded and returned to the student with extensive narrative feedback, which allows the student to improve foundational knowledge and critical thinking skills.The flipped classroom model has been found to promote critical thinking skills in health professions students ,5,6. Hea