The global health emergency caused by COVID-19 concluded in May 2023, marking the beginning of an endemic phase. This study aimed to evaluate the association between vaccination status and other patient characteristics and the risk of severe disease during this new endemic period. A nationwide cohort study was conducted in Mexico, where we analyzed data from 646 adults who had received positive confirmation of COVID-19 through PCR testing from May to August 2023. The overall risk of severe symptoms in the study sample was 5.3%. The average time elapsed from the last vaccine shot to symptom onset was over six months in all the immunized groups . Compared to unvaccinated patients, those with three vaccine doses showed an elevated risk of severe symptoms. Advancing age and various chronic comorbidities  were associated with a heightened risk of severe COVID-19 manifestations. These findings underscore the ongoing seriousness of COVID-19, even in an endemic phase, underscoring the urgent need for tailored interventions aimed at high-risk patients. The emergence of the COVID-19 pandemic, resulting from the infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had high social and economic implications .Mexico’s COVID-19 vaccination plan was characterized by a tiered approach prioritising high-risk populations and essential workers . The iniIn Mexico, a total of eight COVID-19 vaccines were authorized and administered. These vaccines include BNT162b2 , AZD1222 , Ad26.COV2-S , CX-024414 , BBIBP-CorV , CoronaVac , BBV152 , and Ad5-nCoV  .The post-global emergency phase resulting from COVID-19 is marked by the prevailing presence of the XBB.1.5 subvariant of Omicron SARS-CoV-2, recognized for its heightened transmissibility .In addition, the identification of predictors can aid in refining risk stratification models, enabling healthcare systems to allocate medical interventions better and prioritize vulnerable individuals . FinallyHence, this study aimed to assess the effect of COVID-19 vaccination status on the risk of severe laboratory-confirmed illness in adults during the endemic phase of the disease. Furthermore, the study evaluated the association between different patient characteristics and the risk of severe manifestations. Our hypothesis posited that vaccinated adults, compared to their unvaccinated counterparts, might experience a decreased risk of severe symptoms during the endemic phase of COVID-19.We conducted a nationwide and retrospective cohort study in Mexico during August 2023. Potentially eligible subjects  were sourced from nominal records within an epidemiological surveillance system for viral respiratory diseases, targeting SARS-CoV-2, influenza virus, and other pathogens of public health concern. This system, known as SINOLAVE, primarily sourced data from patients’ medical records and, when applicable, death certificates. SINOLAVE is managed by the Mexican Institute of Social Security (IMSS), which provides healthcare services to approximately 60% (72 million people) of Mexico’s total population .Adult patients aged 18 years and above, who exhibited respiratory symptoms suggestive of COVID-19 between 5 May and 10 August 2023, were eligible. Patients with a negative RT-PCR test result and those lacking complete clinical and epidemiological data of interest were excluded. A total of 172 patients under 18 years of age and 21 patients with insufficient data of interest were excluded.In accordance with normative guidelines , clinicaDemographic and clinical variables were extracted from the audited surveillance system. Vaccination status was assessed based on the number of COVID-19 vaccine doses received . When applicable, the dates of vaccine shots were utilized to calculate the number of days elapsed between the last shot and the onset of symptoms. The analyzed comorbidities were determined by physician-diagnosed history (no/yes). The following conditions were assessed: current tobacco use, obesity (defined by a body mass index [BMI] of 30 or above), asthma, chronic obstructive pulmonary disease (COPD), type 2 diabetes mellitus, cardiovascular disease, chronic kidney disease (CKD), and immunosuppression (due to any cause).Severe COVID-19, our primary binary outcome (no/yes), was defined by the occurrence of pneumonia. The latter was characterized by the presence of respiratory clinical symptoms , coupled with radiographic evidence of pneumonia  necessitating hospitalization .Summary statistics were calculated. We utilized risk ratios (RR) and 95% confidence intervals to evaluate the association between vaccination status and other exposures of interest with the risk of severe COVID-19 manifestations. These estimations were derived using generalized linear regression models. We constructed 11 models, one for each independent variable and a final multiple model that incorporated all explanatory variables. In the construction of the multiple regression model, we adhered to the Hosmer and Lemeshow approach. Thus, variables that exhibited a  p-value .In the generalized linear regression analysis context, the unvaccinated group was designated as the reference category for calculating the relevant estimates. This choice of reference category was made to establish a baseline for comparing vaccination effects , as prevThis study obtained approval from the Local Committee of Ethics in Health Research (601) of the IMSS . None of the participants were physically present or interviewed during any phase of this study, and all researchers adhered to stringent ethical guidelines.Data from 646 patients with laboratory-confirmed COVID-19 were analyzed. As presented in The average time interval between the date of the final vaccine administration and the onset of symptoms was 304.3 ± 111.4 days for individuals who had received one dose, 218.7 ± 63.3 days for those who had received two doses, and 202.1 ± 49.7 days for individuals who had received a vaccine booster (third dose). Participants who experienced severe COVID-19, when compared to those with non-severe disease, were found to have a higher likelihood of being vaccinated  did not show an elevated risk for severe disease compared to unvaccinated adults, this finding should be interpreted cautiously. To gain a more comprehensive understanding of the vaccine’s effects in the endemic phase of COVID-19, further research with a larger sample size is imperative.The presented results underscore that specific patient-related factors, especially age and the presence of pre-existing chronic conditions, significantly contribute to an elevated risk of encountering severe cases of COVID-19 during the endemic phase of the illness. The association between advancing age and the presence of chronic diseases with an escalated risk of severe disease has been widely documented throughout the entire course of the pandemic’s progression ,25.These insights highlight the need for tailored and focused public health strategies that prioritize managing and caring for individuals burdened with chronic health conditions . Such anWithin our study sample, individuals who had received a vaccine booster were at increased risk for severe disease compared to those who remained unvaccinated. It is worth noting, however, that the mean interval between the vaccination date and the onset of symptoms extended beyond six months for participants who had received a booster shot and those who had been administered one or two vaccine doses. Consequently, it was anticipated that the antibody titers would fall below protective levels .p = 0.047) than that of unvaccinated individuals  (data not presented). These factors may collectively contribute to the observed scenario, and the documented association must be interpreted cautiously.Additionally, it is important to highlight that the median age of patients who had received three vaccine doses  was higher  of the Mexican population had received at least one dose of the COVID-19 vaccine 7.5% of t, the facThis latter hypothesis is based on the concept that individuals who have recuperated from previous infections might perceive a unique risk-benefit equilibrium in relation to vaccination ,33. SuchOur study did not observe a significant association between obesity, defined by a BMI of 30 or higher, and the risk of severe symptoms . We hypoA personal history of asthma was associated with the risk of severe symptoms in the bivariate analysis but did not maintain significance in the multiple models. This observation aligns with findings reported in prior published studies ,37,38.The incorporation of cases verified through RT-PCR testing is a major strength of this study. Nevertheless, it is essential to acknowledge and address potential limitations inherent in our research methodology. First, our research did not encompass an evaluation of the antibody titers within vaccinated adults. Consequently, we could not evaluate a correlation between these antibody levels and the risk of experiencing severe manifestations of COVID-19. This aspect represents a gap in our study that warrants consideration in future research endeavors.Second, the personal history of COPD was associated with a 23% escalation in the risk of severe COVID-19 symptoms  subvariant of the Omicron lineage was notably prevalent throughout the study period .Fifth, information regarding concurrent infections  is not systematically collected by the analyzed system, so we cannot assess its impact on the risk of severe manifestations in the analyzed cohort.As the world transitions into an era wherein COVID-19 evolves into a persistent public health challenge, prioritizing tailored approaches aimed at safeguarding and aiding these susceptible populations should continue to stand as a paramount objective for healthcare systems and policymakers. Our study may enrich our comprehension of the evolving dynamics characterizing COVID-19 within an endemic phase. The heightened susceptibility to severe manifestations among specific groups accentuates the significance of upholding vigilant surveillance protocols, bolstering vaccination initiatives, and devising healthcare strategies that effectively attend to the distinct requirements of individuals at an elevated risk.


Hot flashes are thought to be due to changes in hormones involved in body temperature regulation, such as estrogen and neurokinin B.8 At the time of this publication, a regulatory decision was still pending.Fezolinetant (Astellas Pharma Inc.) is a once-daily oral nonhormonal therapy being investigated for the treatment of moderate-to-severe VMS associated with menopause. It acts by regulating the neurokinin-3 receptor in the hypothalamus, thereby affecting temperature regulation. If approved, it would be the first selective neurokinin-3 receptor antagonist available in the United States. Astellas has submitted a New Drug Application for fezolinetant 45 mg to the US Food and Drug Administration.https://icer.org/assessment/vasomotor-symptoms-menopause-2022/.The Institute for Clinical and Economic Review (ICER) conducted a systematic literature review and cost-effectiveness analysis to evaluate the health and economic outcomes of fezolinetant, MHT, antidepressants (selective serotonin reuptake inhibitors [SSRIs]/serotonin norepinephrine reuptake inhibitors [SNRIs]), and gabapentinoids  in women with moderate-to-severe VMS associated with menopause. We present the summary of our findings and highlights of the policy discussion with key stakeholders held at a public meeting of the Midwest Comparative Effectiveness Public Advisory Council (CEPAC) on December 16, 2022. The detailed report is available on ICER’s website at https://icer.org/wp-content/uploads/2022/06/ICER_Menopause_FinalReport_01232023.pdf. A network meta-analysis was not performed because of significant heterogeneity in the assessment of study endpoints .Below we review studies for fezolinetant, MHT, SSRIs/SNRIs, and gabapentin that met our inclusion criteria. There were no studies of pregabalin that met our inclusion criteria. For key results across all trials, see Table 3.2 in the ICER report found at 11 The evidence base also includes two 52-week trials examining safety and tolerability (Skylight 4 and Moonlight 3).14 An additional RCT examined the efficacy of a lower 30 mg dose only in an Asian population (Moonlight 1). Participants in all fezolinetant trials were women aged 40-65 years who had a body mass index no higher than 38kg/m2 with a minimum average of 7-8 moderate-to-severe hot flashes per day or 50-60 per week.The pivotal trials of fezolinetant 45 mg are two 12-week randomized controlled trials (RCTs) with an extension phase lasting an additional 40 weeks (Skylight 1 and Skylight 2).16 although the difference did not meet the minimum clinically important difference (MCID) defined in the literature (> 3.57 hot flashes daily).17 Participants treated with fezolinetant also had a significantly greater reduction in VMS severity at 12 weeks, exceeding the MCID for reduction in severity (> 0.225 hot flashes daily)18 in the Skylight 2 trial, but not in Skylight 1.16 Pooled data from the Skylight trials reported that fezolinetant significantly improved scores on the Menopause Quality of Life Questionnaire (MENQoL) compared with placebo,14 but the difference did not meet the MCID (improvement > 1 on any MENQoL domain).19In the Skylight 1 and 2 trials, participants in the fezolinetant group had a significantly greater reduction in VMS frequency at 12 weeks,13 The safety trials reported no additional concerns.14Fezolinetant was generally well tolerated, with headache as the most common adverse event, and with 2%-3% experiencing elevated liver enzymes. Elevations in liver enzymes were generally asymptomatic and resolved after cessation of the study intervention; there were no cases of drug-induced hepatocellular injury with jaundice.28 Overall, trials reported significantly greater reductions in VMS frequency and severity and significant improvements in MENQoL in the MHT group compared with the placebo group,31 with the majority meeting the MCID. Short-term adverse events were mostly mild to moderate in severity. There are additional benefits and harms to consider with long-term use. For women age 50-59 years, who represent the majority of women making menopause treatment decisions, significant hazard ratios were greater than 1 for breast cancer (P > 0.05), stroke (P > 0.05), and pulmonary embolism (P < 0.05) and were less than 1 for colorectal cancer (P < 0.05), hip fracture (P < 0.05), death (P > 0.05), and coronary heart disease (P > 0.05).33Ten RCTs in postmenopausal women evaluated the efficacy of combined estrogen and progesterone or estrogen only in postmenopausal women (either a standard dose [1 mg] or low dose [0.5 mg]) for the treatment of VMS.41 The results were inconsistent across trials. Although some trials reported statistically significant improvements in VMS, with desvenlafaxine appearing to have the most consistent treatment effect , none of the antidepressants reviewed achieved the MCID in VMS frequency or MENQoL when compared with the placebo group. Adverse events in the SSRI and venlafaxine trials were mostly mild or moderate in severity, with the most common events being fatigue, dry mouth, nausea, and decreased appetite.36 Desvenlafaxine trials reported more adverse events in the treatment group than the placebo group, and differences in discontinuation were more apparent at higher doses.38The efficacy of SSRIs/SNRIs for the treatment of VMS has been evaluated in 10 RCTs. 44 Although demonstrating statistical significance, all trials failed to show clinically meaningful differences in VMS frequency or severity. The MENQoL was not assessed in any of the trials. There were more adverse events in the gabapentin groups compared with the placebo groups, but these were mostly mild to moderate, with the most common events being dizziness, headache, and somnolence.44The efficacy of gabapentin for the treatment of VMS has been evaluated in 3 RCTs.Although the Skylight 1 and 2 trials reported statistically significant improvements in VMS frequency and severity with fezolinetant, the improvement in VMS frequency did not meet the MCID, nor did improvements in MENQoL reach the MCID. Only 1 of the Skylight trials met the MCID for improvement in VMS severity. Adding to our uncertainty about the relative benefits of treatment with fezolinetant is a lack of published data  and a lack of evidence on long-term efficacy and safety. As the median total duration of moderate-to-severe VMS among women in the United States is 9.4 years and this is a first-in-class medication for which we cannot rely on safety data from medications in the same therapeutic class, more long-term data for fezolinetant are needed.We developed a de novo decision analytic model to evaluate fezolinetant for the treatment of VMS compared with no pharmacologic treatment. We performed separate analyses looking at outcomes and costs within the health care sector and inclusive of broader societal effects, including productivity. The health outcome of each intervention was evaluated in terms of symptom improvements , life-years gained, equal value of life-years (evLYs) gained, and quality-adjusted life-years (QALYs) gained. Outcomes were estimated over a lifetime time horizon to capture short-term and ongoing morbidity and mortality. Costs and outcomes were discounted at 3% per year.45 Health state occupancy  was derived using survival extrapolation methods of the proportion of women with and without VMS during the menopause transition using Kaplan-Meier curves. Full details on ICER’s cost-effectiveness analysis and model are available at https://icer.org/news-insights/press-releases/icer-publishes-final-evidence-report-on-fezolinetant-for-vasomotor-symptoms-associated-with-menopause/.The model was focused on an intention-to-treat analysis with a hypothetical cohort of women with VMS being treated with fezolinetant. The first model cycle included treatment costs for all patients until discontinuation, which was based on the typical duration of VMS.Results of the base case analysis of fezolinetant vs no pharmacologic treatment are shown in 46The price of fezolinetant is currently a placeholder price based on market projections for similar drugs, and thus cost-effectiveness estimates must be interpreted with caution. Because of inconsistency in the trial endpoints, we were unable to compare the cost-effectiveness of fezolinetant vs other comparators . Health-related quality of life was derived using a mapping algorithm between the MENQoL  and the EuroQol-5D. The changes in utility scores are a function of the total changes in MENQoL to allow for health-related quality of life to be associated with VMS and other symptoms correlated with VMS.The Midwest CEPAC is one of the independent appraisal committees convened by ICER to engage in the public deliberation of the evidence on clinical and cost-effectiveness of health care interventions. The CEPAC is composed of medical evidence experts, including clinicians, methodologists, and patient advocates. The ICER report on fezolinetant for moderate-to-severe VMS associated with menopause was the subject of a CEPAC meeting on December 16, 2022. Following the discussion, CEPAC panel members deliberated on key questions raised by ICER’s report.A majority of the panel (11 to 1) voted that the current evidence is inadequate to demonstrate that the net health benefit of fezolinetant for VMS is superior to that provided by no pharmacologic treatment. The panel voted unanimously that the currently available evidence was inadequate to distinguish the net health benefit between fezolinetant and MHT for VMS .The Midwest CEPAC also voted on important “potential other benefits” and “contextual considerations”  and 4 thhttps://icer.org/news-insights/press-releases/icer-publishes-final-evidence-report-on-fezolinetant-for-vasomotor-symptoms-associated-with-menopause/). Select key policy recommendations relevant to the potential introduction of fezolinetant into practice are as follows:Following the discussion of the evidence, a policy roundtable was convened to deliberate on how best to apply the evidence on fezolinetant to treatment for VMS associated with menopause. The full set of policy recommendations can be found in the Final Evidence Report (1.All stakeholders have a responsibility and an important role to play in ensuring that women have access to effective new treatment options and that new treatments are introduced in a way that encourages shared medical decision making and equitable access.2.Given the uncertainty in long-term outcomes and the fact that this is a first-in-class therapeutic, it is not unreasonable for payers to consider whether to use the specific trial eligibility criteria to define a threshold for the frequency/severity of VMS that merits coverage. However, clinical experts did not believe that it was appropriate to use strict thresholds based on the frequency of VMS given that patients may have fewer very severe episodes that still have a substantial impact on quality of life.3.Given that many patients may benefit from readily available, effective, and low-cost MHT, clinical experts agreed that it would be reasonable for payers to require prescriber attestation that patients are not appropriate candidates for MHT prior to prescribing fezolinetant.4.Manufacturers should set prices that will foster affordability and access for all patients by aligning prices with the therapeutic value, moderated by the substantial uncertainty about longer-term safety and effectiveness.


Immunotherapy as a rapidly developing therapeutic approach has revolutionized cancer treatment and revitalized the field of tumor immunology research. 3D in vitro models are emerging as powerful tools considering their feature to maintain tumor cells in a near-native state and have been widely applied in oncology research. The novel 3D culture methods including the co-culture of organoids and immune cells, ALI culture, 3D-microfluidic culture and 3D-bioprinting offer new approaches for tumor immunology study and can be applied in many fields such as personalized treatment, immunotherapy optimizing and adoptive cell therapy. In this review, we introduce commonly used 3D in vitro models and summarize their applications in different aspects of tumor immunology research. We also provide a preliminary analysis of the current shortcomings of these models and the outlook of future development. Tumor microenvironment (TME) is a complex and continuously evolving entity . It inclFor traditional tumor therapeutic approaches, tumor cells are the direct targets of therapy. However, as the role of immunity in tumor development has been constantly illustrated, such as the generation and progression of tumors in immunodeficiency or inflammatory response states, other components of the TME are also emerging as new targets for tumor therapy . These i-/- mice, in which CD4 + T cells were found to promote early tumor development through a DC-dependent pattern and the process could be partially inhibited by antagonists of IL-13 [Co-culture of 2D tumor cells with exogenous immune cells are the commonly used tools for basic research on tumor immunology and preclinical trials of relevant immunotherapeutic drugs. It is not only easy and cost effective, but also allows easy gene editing and drug intervention. Besides, the model has a wide range of assays and well-established evaluation techniques, making the experimental process very convenient and reproducible. However, tumor cell linages do not reflect the growth pattern of tumor cells in vivo and the heterogeneity, diversity and individuality of patient-derived tumors. The additional immune cells cannot restore the spatial and temporal characteristics and interaction patterns of the TME, and genetic mutations and phenotypic variations may occur during long-term passaging cultures . As for of IL-13 . These sof IL-13 . Therefo3D in vitro models are newly developed techniques that can simulate the 3D structure in vivo, thus reproducing the physiology and physiopathological characteristics of the original tissues . These mAn organoid initially refers to a 3D structure grown from stem and progenitor cells. It consists of variant organ-specific cell types and can self-organize via cell differentiation and spatially restricted lineage commitment –17. OrgaOrganoid construction methods can be divided into two types: scaffold-dependent methods and scaffold-free methods. In the scaffold-dependent system, scaffolds provide physical support for cell growth in which cells aggregate and self-assemble to form 3D structures. Depending on the different sources and components, extracellular scaffold can be further divided into matrix derived from decellularized tissue , natural polymer-based hydrogel  and synthetic hydrogel  . As for In the above culture models, tumor stem cells can proliferate and differentiate to form tumor-like structures, but the stromal component of the original tumor tissue cannot be preserved. To apply organoid to tumor immunology research, exogenous immune cells need to be added to interact with tumor organoids. Here we introduce the commonly used methods for co-culturing tumor organoids and immune cells Fig. A.+ T cells isolated from peripheral blood mononuclear cells (PBMCs) were added to the medium of pancreatic cancer organoids. After co-cultured for 72 h, it was found that T cells were viable and Ki67 positive in organoid growth medium but the cell activity was weaker than in RPMI medium. When tumor organoids were present in the matrigel, T cells migrated and infiltrated into matrigel in response to pancreatic cancer cells. Otherwise, they mostly gathered at the edge to form clear boundaries [The easiest way to co-culture organoid with immune cells is by directly adding exogenous immune cells into the culture medium Fig. A. In thiundaries . T cell undaries , 24. To undaries  diluted However, the above methods are more suitable for studying the indirect interaction between immune cells and tumor cells. The direct contact is still not sufficient, even if the immune cells have a strong migration and infiltration ability and the matrigel is diluted. Moreover, the components in matrigel may affect the function of immune cells, leading to non-specific activation. Therefore, when considering direct intercellular interactions, mature organoids can also be isolated from the matrigel and cultured in suspension with immune cells Fig. B.Based on this method, the researchers co-cultured patient-derived MSI-H CRC organoids and NSCLC organoids with homologous PBMCs. The tumoriods were first separated from matrigel and digested into single cell suspensions, which were treated with IFN? to increase the expression of MHC-I molecules on the surface of tumor cells and thus promote antigen presentation. PBMCs isolated from peripheral blood were simultaneously incubated with anti-CD28 and anti-PD-1 antibodies to provide a co-stimulatory signal and counteract the upregulation of PD-L1 expression caused by IFN? treatment. The treated tumoroid single cells were mixed with PBMCs at a certain ratio and co-cultured in T-cell medium containing IL-2 for 7 days. After 2 rounds of co-culture, the T cells were activated and can specifically kill the corresponding tumoroids. In this way, tumor-reactive T cells could be enriched in peripheral blood and their tumor-killing efficiency could be measured at the individual level, providing a good model for cell therapy optimization . Qingda +CD11b+ tumor associated macrophages (TAMs) from ovarian cancer-bearing mice with ID8 tumor cells at a ratio of 1:10 in medium containing 2% matrigel. The mixture was planted in the pre-coated plates to investigate the role and mechanism of TAMs in the growth and metastasis of ovarian cancer. This method allowed direct contact between tumor cells and immune cells, and also prevented the adherence of organoids, making the co-culture system more stable and suitable for long-term studies  or other physical supports , 32. DueIn addition to the two co-culture methods mentioned above, it is also possible to co-culture tumor cells and immune cells by mixing them directly in matrix Fig. C. Follow+ hematopoietic cells were still present after 8 days of culture but the number of CD3+ T cells decreased significantly, suggesting that the culture environment with organoid medium might not be the most suitable for T cell survival and further addition of T cell-promoting growth factors was needed to adjust the culture conditions [The ALI culture is a special organoid culture model. In this model, tumor tissues are mechanically minced into pieces of about 1 mm in diameter without enzymatic digestion, mixed with collagen gel and layered on top of pre-solidified collagen gel within a permeable, membranous inner transwell insert. The insert is placed in a cell culture dish, and culture medium is added between the dish and the insert to form the double dish culture system. This way, the upper part of mixture of tumor tissues and collagen is directly exposed to air, and the culture medium infiltrates the bottom of the collagen through the micropores to create an air-liquid interaction model  in microscopic channels (10- 4 to 10- 5 m in size) [Microfluidics refers to a technique for controlling the flow of micro fluids 10in size) . In thisin size) . Combiniin size)   and biological materials according to the requirements of bionic morphology and organism function using 3D printing techniques . ConventIn 2020 Kunyoo Shin’s team  introducThe above 3D in vitro models have been widely used in simulating TME and tumor immune-related studies. They all have their own advantages and disadvantages and are suitable for different application scenarios. The following table summarizes and compares the characteristics of different models Table . HoweverZhiyi Gong et al.  designedTumor cells and their microenvironment are a functional entity. The microenvironment interacts and co-evolves with tumor cells and play important roles in multiple processes of tumor development. Therefore, studying the interaction between tumor and microenvironment can help us understand the biological behavior of tumor and lay the theoretical foundation for finding new therapeutic targets and exploring new methods of tumor immunotherapy.+ T cells. DKK1 inhibitors combined with PD-1 monoclonal antibodies could achieve better tumor control [3D in vitro models are good models for studying the TME because of their simplicity, convenience, flexibility and high similarity to original tumor tissues Fig. E. For ex control . Similar control . By dete control . In addi control . Using m control . Using t control . These sWith the increasing research on the immune microenvironment, immunotherapy has been more and more widely used in the treatment of tumors, among which the most frequently applied is immune checkpoint inhibitor therapy. ICI therapy targeting PD1/PD-L1 and CTLA-4 has been clinically employed in progressive melanoma , squamouFor precise patient selection, a number of biomarkers have been demonstrated to predict the efficacy of ICI therapy, such as MSI status , TMB 6363, neoanPaula Voabil et al.  culturedDespite the increasing application of immunotherapy, its effectiveness is still limited. Only a minority of patients can benefit from immunotherapy, and even in patients who are sensitive, problems of acquired drug resistance may occur. Therefore, new strategies are needed to optimize the effectiveness of immunotherapy. 3D in vitro models have been widely used in the evaluation of different treatment options and in vitro drug screening because of their ability to better reflect tumor characteristics and manipulability Fig. G. For ex+ T cells, increased the expression of cytokines such as IFN?, IL12, IL6 and TNFa, and prevented T cell depletion by downregulating PD-L1, CTLA-4, TIM-3 and LAG-3. These results showed that MEK inhibitor had PD-L1 monoclonal sensitizing effects. Small molecule drugs were screened using a 3D microfluidic organoid culture system, and CDK4/6 inhibitors were found to significantly increase T cell activation and infiltration. Simultaneous application of PD-1 monoclonal antibodies and CDK4/6 inhibitors also showed enhanced T cell killing of tumor cells [+ T cells [+ T cell for the treatment of pancreatic cancer. Marcel Alexander Heinrich et al. [PD-1/PD-L1 inhibitors are commonly used immunotherapies in clinical practice, so enhancing the efficacy of PD-1/PD-L1 therapy through different pathways is at the forefront of research. Carminia Maria Della Corte et al.  used a cor cells . Meanwhi T cells , 75. In  T cells  co-cultuh et al.  applied In the field of tumor immunotherapy, apart from immune checkpoint inhibitors, adoptive cell transfer therapy (ACT) is also developing rapidly. This treatment involves isolating immunologically active cells from tumor patients, expanding and functionally characterizing them in vitro, and then re-infusing them into patients for the purpose of killing the tumor directly or stimulating immune response to eliminate tumor cells. According to the development of ACT, the adopted cells include lymphokine-activated killer cell (LAK), cytokine-induced killer cell (CIK), tumor infiltrating lymphocyte (TIL), natural killer cell (NK), cytotoxic T lymphocyte (CTL), chimeric antigen receptor T cell (CAR-T) and T cell engineered with T-cell receptor (TCR-T) , 77. AltFirstly, 3D in vitro models can be used as a source of tumor antigens for the preparation of tumor-specific T cells because of their high consistency with the original tumor. Krijn K. Dijkstra et al.  co-cultu+ tumor cells but not negative cells. This indicated that CAR-T cells were able to specifically kill target cells instead of complete elimination of all tumor cells. Such co-culture models provide a feasible way to test the efficacy of CAR-T therapy. Similarly, co-culture of human bladder cancer organoids with specific CAR-T cells allows detecting the killing ability and specificity of CAR-T cells [Secondly, 3D in vitro models can also be used to assess the efficacy of adoptive cell therapy. By co-culturing the specific immune cells used in the adoptive cell therapy with organoids or other 3D in vitro cultures, the activation of immune cells and the apoptosis of tumors can be assessed. Fadi Jacob et al.  co-cultu-T cells . CAR-NK9-T cells . EvaluatAlthough 3D in vitro models have an increasingly wide range of applications in various research fields, there are still some problems in the process of model construction. First, there is no uniform standard for the culture of in vitro models. For example, the culture protocols of the same tumor-derived organoids in different laboratories often differ, such as different culture media components. Some niche factors commonly used in organoid culture are produced by different cell lines and often have batch effects. These unstable factors can have an impact on the experimental results . In addiAnother important difficulty faced when using 3D models for research is the frequent lack of validated evaluation tools. Evaluation methods commonly used in 2D cultures may not be applicable to 3D culture systems due to the unique construction methods and growth characteristics of 3D cell clusters. In various Label-dependent and reader-based assays for cell viability and cytotoxicity, such as MTT assay, LDH activity assay, CellTiter-Glo®3D Cell Viability and fluorescence imaging, the presence of extracellular matrix and the tight intercellular junctions in 3D aggregated cell clusters lead to difficulties in cell lysis and poor penetration of dyes and reagents, resulting in reduced sensitivity of the assay and biased results . In addiIn addition to the problems in the construction of 3D in vitro models, there are also some challenges in using these models to reconstruct the in vitro tumor immune microenvironment. The tumor immune microenvironment is a complex system composed of multiple immune cells and stromal cells, which is highly heterogeneous and dynamic. How to simulate the real state of the in vivo tumor microenvironment as much as possible in vitro needs to be further optimized and refined.As in the organoid/immune cell co-culture model, the type of immune cells added externally is more limited, and the status of the additional immune cells is not consistent with the original cell in the immune microenvironment, and therefore differs significantly from the original microenvironment of the tumor. Moreover, the organoid culture medium cannot maintain the function of immune cells in an optimal condition, so the cellular activity and survival time of immune cells may be affected .In contrast, in ALI or microfluidic culture models a portion of the original tumor infiltrating immune cells are retained in the organoid. These immune cell fractions are maintained for only a limited period of time and are subject to greater loss during passaging or freezing and thawing. Therefore these methods are only suitable for short-term studies . In addiIn summary, different in vitro culture models have their unique advantages and corresponding shortcomings, and therefore the most suitable model needs to be selected according to the research objectives. Combining multiple models, integrating their advantages, and further optimizing the culture conditions are ways to refine in vitro 3D culture models for better application in tumor immunology research in the future.The emerging 3D in vitro models have greatly facilitated tumor immunology research. However these models can be further optimized to improve the clinical translation capability and to expand the application. More integrated 3D in vitro models are expected in the future.First is the integration of multiple models. The advantages of multiple models should be utilized to establish a more complete immune system in vitro. For example, in the ALI model, the original immune cells in the TME can be preserved. In contrast, the infiltration of immune cells into tumor organoids in the co-culture model better mimics the process of circulating immune cells trafficking into the tumor and undergoing phenotypic changes. If the two models are combined together, a more comprehensive simulation of the immune response process can be achieved.Second is the functional integration of 3D in vitro models. Simulating the immune microenvironment in vitro does not simply mean putting tumor cells and immune cells in the same spatial space, but more critically, allowing them to interact more functionally. In order to achieve this, it is necessary to restore the in vivo physiological state as much as possible. For example, cells can be exposed to mechanical stress, substrate stiffness and physiologic shear flow. It is also necessary to control the shape and size of the model as well as the ratio of each cell according to the characteristics of different tumor tissues. In these areas, 3D-microfluidic culture systems may have a strong potential.Finally is the integration of other components of the TME. In the TME, there are various components besides immune cells that play important roles, such as tumor-associated fibroblasts, blood vessels and neurons. The microbiota also has close interactions with the TME. Therefore, if multiple microenvironment components can be integrated into the same system in future studies, 3D in vitro models will have more in-depth applications in oncology research.In a few years of recent, researchers have taken great efforts to reconstruct tumor immune microenvironment using various 3D models in vitro. The newly developed models including Organoid/immune cell co-culture models, ALI culture model, 3D-Microfluidic based culture method and 3D-bioprinting model have been used in different fields of tumor immunology research and greatly helped the selection of patients benefit from immunotherapy, the development of novel treatment approach and the research of resistance to immunotherapy. In the future, 3D in vitro models can be further enhanced to simulate the tumor microenvironment in a more integrated way both structurally and functionally, to better facilitate tumor immunology research.


Accurate phylogenies are fundamental to our understanding of the pattern and process of evolution. Yet, phylogenies at deep evolutionary timescales, with correspondingly long branches, have been fraught with controversy resulting from conflicting estimates from models with varying complexity and goodness of fit. Analyses of historical as well as current empirical datasets, such as alignments including Microsporidia, Nematoda, or Platyhelminthes, have demonstrated that inadequate modeling of across-site compositional heterogeneity, which is the result of biochemical constraints that lead to varying patterns of accepted amino acids along sequences, can lead to erroneous topologies that are strongly supported. Unfortunately, models that adequately account for across-site compositional heterogeneity remain computationally challenging or intractable for an increasing fraction of contemporary datasets. Here, we introduce “compositional constraint analysis,” a method to investigate the effect of site-specific constraints on amino acid composition on phylogenetic inference. We show that more constrained sites with lower diversity and less constrained sites with higher diversity exhibit ostensibly conflicting signals under models ignoring across-site compositional heterogeneity that lead to long-branch attraction artifacts and demonstrate that more complex models accounting for across-site compositional heterogeneity can ameliorate this bias. We present CAT-posterior mean site frequencies (PMSF), a pipeline for diagnosing and resolving phylogenetic bias resulting from inadequate modeling of across-site compositional heterogeneity based on the CAT model. CAT-PMSF is robust against long-branch attraction in all alignments we have examined. We suggest using CAT-PMSF when convergence of the CAT model cannot be assured. We find evidence that compositionally constrained sites are driving long-branch attraction in two metazoan datasets and recover evidence for Porifera as the sister group to all other animals.  Understanding the biological foundations of contemporary life on Earth requires detailed knowledge of evolutionary history. The history of speciation events informs us about the appearance of advantageous innovations and the loss of dispensable traits in a continuously changing environment. Consequently, the development of phylogenetic models inferring the history of speciation events has continued at an impressive pace during the past decades.Models of sequence evolution are inevitably simplifications of the complex processes that generate real-life biological sequences. Unfortunately, overly simplistic models can lead to model misspecification and long-branch attraction  symmetric exchangeabilities, describing differences in rates of exchange between pairs of states, and (ii) stationary frequencies of the target states e.g., . FiguratWe can distinguish between general distribution mixture models estimated from curated training databases, and distribution mixture models directly estimated from the datasets at hand. For example, Finite mixtures can be used in the context of maximum likelihood (ML) phylogenetic inference. However, statistical analyses of model fit and investigation of known cases of LBA indicate that a large number of components are necessary for robustness against LBA  which isBayesian approaches can more easily accommodate richer mixtures. Nonparametric Bayesian methods do not require explicit specification of the number of mixture components nor their stationary distributions. In particular, the CAT model  uses a DIn all cases, mixture modeling approaches accounting for across-site compositional heterogeneity are complex and require considerable computational resources e.g., . In ordeIn this work, we follow a multistep procedure similar to the PMSF model and address two points: First, on the computational side, we extend the PMSF approach by using the CAT model instead of an empirical profile mixture model as the reference model for computing the profiles. Importantly, the CAT model is used under a fixed guide topology. Thus, the problem of simultaneous inference of both the site-specific stationary distributions and the tree is reduced to a search of site-specific stationary distributions, and tree branch lengths only. We termed our approach CAT-PMSF.Second, we investigate the contribution of sites with different degrees of compositional constraints to LBA. In particular, we test to what extent more severely constrained sites exhibit bias toward LBA trees under models that do not adequately account for across-site compositional heterogeneity. We employ “compositional constraint analysis,” which examines phylogenetic signal for alternative topologies as a function of per-site compositional diversity measured by the effective number of amino acids .Examining simulated alignments as well as classic empirical datasets including Microsporidia , Nematodsite-homogeneous and site-heterogeneous when referring to models ignoring and accounting for across-site compositional heterogeneity, respectively. Furthermore, we use the term tree to denote a directed acyclic graph with node labels and branch lengths, in which exactly one branch connects any two nodes. We use the term topology to denote a tree without information about branch lengths but with node labels. We specify an evolutionary model with exchangeabilities, EX, and across-site compositional heterogeneity model, ASCH as EX + ASCH. All discussed evolutionary models used for simulations as well as inferences implicitly use discrete gamma rate heterogeneity with four components. We add a flag +PMSF to denote usage of the posterior mean site frequency model . The scripts and the simulated data are available in the Dryad Digital Repository: https://doi.org/10.5061/dryad.g79cnp5rh and at https://github.com/drenal/cat-pmsf-paper.We used Felsenstein-type trees with four leaves , let us denote them topology A positive value of Supplementary Figs. S13–S15 available in the Supplementary material which can be found in the Dryad Digital Repository: https://doi.org/10.5061/dryad.g79cnp5rh). For the simulation study, we used the actual http://phylopic.org/), the silhouette for Microsporidia is based on We ordered and binned the sites according to their Supplementary Figs. S1 and S2 show simplified and complete species trees, respectively.Supplementary Fig. S3 show simplified and complete species trees, respectively.The dataset provided by Supplementary Fig. S4 show simplified and complete species trees, respectively.The placement of Ctenophora on the tree of Metazoa is still a matter of debate. We apply CAT-PMSF to two datasets. First, the alignment provided by https://github.com/lsjermiin/Homo.v2.1) performs this test for all pairs of sequences in an alignment. We applied Homo v2.1 to the simulated as well as empirical alignments.CAT-PMSF assumes homogeneity of the evolutionary process across the branches of the tree. The matched-pairs test of symmetry  tests foIn brief, CAT-PMSF comprises three steps: (i) estimate a guide topology using a site-homogeneous model, (ii) estimate site-specific stationary distributions with the CAT model in PhyloBayes  using thWe assessed and compared the accuracy of CAT-PMSF with other site-homogeneous and site-heterogeneous models. To this end, we simulated amino acid sequence alignments with a length of 10,000 sites along Felsenstein-type quartet trees . Supplementary Figs. S7 and S8 show results for other values of The true topology was not recovered with site-homogeneous models when Supplementary Figs. S7 and S8). For site-homogeneous evolutionary models, more constrained sites with a lower value of At odds with this expectation, site-homogeneous evolutionary models exhibit conflicting phylogenetic signal between sites with lower and higher Supplementary Tables S3, S5, and S7). For the Pearson’s correlation coefficient, site-homogeneous models exhibit large and significant correlation for To ascertain the statistical significance of the compositional constraint analysis we calculated Pearson’s correlation coefficients, as well as Spearman’s and Kendall’s rank correlation coefficients and associated p-values between the log-likelihood differences and the site-specific Supplementary Table S9). AU tests of the Poisson + CAT-PMSF model show similar results in that the topology exhibiting LBA is rejected for Supplementary Table S10). The LG + CAT-PMSF model only rejects the topology exhibiting LBA for Supplementary Table S10).Approximately unbiased (AU) tests  of ML trSupplementary Figs. S7 and S8). In all cases excluding the LG + CAT-PMSF and GTR + CAT-PMSF models for large Supplementary Figs. S7 and S8).Finally, we note that site-heterogeneous models with GTR exchangeabilities perform well if Supplementary Figures S9 and S11 show the cumulative site-specific log-likelihood differences and Supplementary Figures S13–S15 provide results for alternative bin sizes.Similar to the simulation study above, for empirical alignments the site-specific stationary distributions obtained in Step 2 of the CAT-PMSF pipeline can be used to quantify the strength of compositional constraint measured by For site-homogeneous models, the site-specific log-likelihood differences between the ML trees constrained to the two competing topologies . For Platyhelminthes, the bias is strong enough that the total likelihood across all sites is higher for the LBA topology, while for the datasets involving Nematoda and Microsporidia, the LG + C10 + PMSF model recovers the correct topology, albeit with reduced support. In general, the results of the LG + C10 + PMSF and LG + C60 + PMSF models are consistent with the observation .The site-heterogeneous LG + C60 + PMSF model shows reduced, but still apparent conflict compared to site-homogeneous models and the LG + C10 + PMSF model (ervation  that incSupplementary Table S4). The ML trees inferred by CAT-PMSF are consistent with the accepted phylogenetic relationships and AU tests confirm the rejection of trees with LBA topologies (Supplementary Tables S12–S14).In contrast, CAT-PMSF exhibits consistent signal toward the assumed-to-be-correct topologies across all sites and datasets with no significant correlation between log-likelihood difference and site-specific Finally, we used CAT-PMSF on two metazoan datasets  to invesSupplementary Figs. S10 and S12) for both the alignments from Supplementary Table S4).Compositional constraint analysis under site-homogeneous models, as well as combinations of PMSF and site-heterogeneous mixture models with 20 and 60 components exhibit patterns of conflicting phylogenetic signal for sites with different degrees of compositional constraints . Sites with higher Supplementary Table S3) favoring a Porifera-sister topology and rejecting the Ctenophora-sister topology  with the closest outgroup, Choanoflagellatea . For the alignment published by Under site-homogeneous models, sites with We introduce CAT-PMSF, a method for phylogenetic inference from alignments exhibiting across-site compositional heterogeneity. The CAT-PMSF pipeline uses the site-specific amino acid preferences estimated by a nonparametric Bayesian approach in the context of a downstream ML analysis. Doing so combines the benefits of both approaches: a more accurate inference of the patterns across sites with a computationally more efficient and more reproducible inference of the tree topology. In addition to phylogenetic inference, the CAT-PMSF pipeline can also be used to investigate the consistency of phylogenetic signal for sites under different degrees of compositional constraints. Compositional constraint analyses on both simulated and empirical datasets exhibiting across-site compositional heterogeneity show that site-homogeneous and some site-heterogeneous mixture models indeed have inconsistent signal which contributes to topological bias and LBA artifacts.In the simulation study , site-hoThe threshold Supplementary Figs. S7 and S8).In our simulations, support of site-homogeneous models shifted from the true topology toward the incorrect topology when increasing CAT-PMSF . AlthougWe discovered bias toward one of the topologies in simulation studies because we know the true parameters and trees. Bias is harder to detect in analyses of empirical data. Compositional constraint analysis detects conflicting signal between more and less constrained sites. Detection of such inconsistencies is a strong indicator for bias: Knowing the stationary distribution of a site alone should not provide us with information about the favored evolutionary history. In mathematical terms, the log-likelihood difference of a site between two hypotheses should be conditionally independent given the stationary distribution of that site. In contrast, we expect the signal obtained from more and less constrained sites to be consistent up to the random statistical error.P-values confirm this observation across a wide range of simulated and empirical datasets (Supplementary Tables S3 and S4).In our analyses of empirical data, we observed strong inconsistencies between more and less constrained sites for site-homogeneous models and hardly any inconsistencies when using CAT-PMSF . PearsonSupplementary Table S15). If we add more distant outgroups, the results are less conclusive (Supplementary Table S15). LBA provides an explanation for this observation: outgroups more distant to the ingroup  attract outgroups closer to the ingroup .Compositional constraint analysis seeks to decompose the log-likelihood contributions of more and less constrained sites. We decided to measure how constrained sites are by computing the effective number of amino acids per site based on the concept of homoplasy, because it is inherently related to LBA: A model that underestimates the probability of homoplasy will incorrectly attribute sequence similarities due to homoplasy to a putative close evolutionary distance. Other measures of how constrained sites are or any monotonic transformation of the function computing eristics . To reiteristics –4. More We also note that CAT-PMSF assumes homogeneity of the evolutionary process across branches of the tree. The simulation study conforms to this assumption. Tests for across-branch homogeneity were less conclusive for the empirical datasets  than for the simulation study. If the evolutionary process is stationary and homogeneous, the CAT model should perform well in estimating site-specific amino acid compositions. Even if the evolutionary process is non-stationary or heterogeneous, the site-specific amino acid compositions inferred by the CAT model will capture the spectrum of compositions attained at least somewhere on the tree. In this case, the inferred compositions will not be stationary in the mathematical sense, but still should have a positive impact on the detection of genuine convergent evolution. Ideally, we should model across-site and across-branch compositional heterogeneity for amino acid sequences in a combined way. For example, there has been work in this direction on nucleotide sequences e.g., . In the Supplementary Table S1), and trees that we are convinced to be free from LBA artifacts in the analyses comprising empirical datasets . This observation justifies the usage of site-homogeneous models in Step 1 of the CAT-PMSF pipeline.The results of CAT-PMSF are conservative because the CAT model estimates the site-specific stationary distributions using guide topologies prone to LBA artifacts. That is, the guide topologies are obtained with site-homogeneous models. Even so, CAT-PMSF correctly infers the true trees in the simulation study . In practice, many sites may have evolved under different conditions, so we cannot expect all sites to share a universal stationary distribution. In fact, we do not even expect stationarity. In our opinion, we should analyze data using complex models and decide about which parameters are necessary to grasp the complexities of evolution. With CAT-PMSF we further explored this path. The CAT-PMSF method uses site-specific stationary distributions and therefore is a parameter-rich model.In comparison, the site-specific posterior mean stationary distributions of the classical PMSF approach are a superposition of a finite set of stationary distributions of the underlying mixture model. Consequently, the Finally, the speed benefit of CAT-PMSF originates from fixing the topology during the Bayesian analysis with the CAT model. Of course, estimating the site-specific stationary distributions is still by far the most time-consuming step. In the future, we aim to design improved methods for estimating site-specific stationary distributions. Specifically, we are thinking about methods based on machine learning such as AlphaFold .In conclusion, compositional constraint analyses show evidence for a potential LBA caused by model misspecification and an argument that careful model choice as well as validation is important in phylogenetic inference. We also propose a method, CAT-PMSF, with the potential to produce more accurate phylogenetic estimates using a site heterogeneous, but branch homogeneous, substitution process.https://doi.org/10.5061/dryad.g79cnp5rh.Supplementary material, including data files and/or online-only appendices, can be found in the Dryad Digital Repository:


The 10-day coughing/choking showed significant decreases at the 5-month and 19-month in the exercise-based group and at the 5-month in the non-exercise-based group (p < 0.05). Our findings suggested that regular physical exercise-based swallowing intervention effectively alleviated coughing/choking problems of older adults with dementia and its effectiveness was long-lasting. For those who could not comply with exercise programs, noticeably with more advanced age and dementia, the effective swallowing intervention period was short-term.Swallowing safety is one of the top health concerns of dementia. Coughing and choking (coughing/choking) are signs of impaired swallowing safety. This study aimed to investigate the effectiveness of regular physical exercise-based swallowing intervention for reducing coughing-choking at the dementia day-care center. This was a retrospective analysis with data from medical records, including age, the clinical dementia rating (CDR), and the frequencies of coughing/choking in ten days (10-day coughing/choking). Those who complied with the exercise programs were assigned to the exercise-based group (n = 22), and those who could not comply were assigned to the non-exercised-based group (n = 7). The non-exercised-based group showed more advanced age and higher CDR than the exercise-based group ( Dementia is a syndrome that leads to deterioration in cognitive functions beyond what might be expected from normal aging. Currently, dementia is the seventh leading cause of death and one of the major causes of disability and dependency among older adults worldwide . The preThe interventions for swallowing safety in dementia are usually cataloged into compensatory and therapeutic approaches ,6,7. ComTo implement therapeutic interventions for dementia, an interdisciplinary model is highly recommended because this model relies less on pharmacological treatment-based medical practice and more on integrated interventions from multi-disciplinary specialists and caregivers to deal with the complex range of cognitive, physical, socio-emotional, and self-care deteriorations associated with dementia ,13. The After a reformed long-term care (LTC) policy, called LTC 2.0, implemented in 2017, and a reformed dementia prevention and care policy, called Dementia Plan 2.0, established in June 2018, Taiwan’s long-term care system extended services to include hospital-affiliated dementia day-care units to provide integrated interventions for people living with dementia aged 50 years and more ,15. A phThis was a retrospective study with study cases obtained from medical records of the geriatric psychiatric day-care center (the dementia day-care center) at Hualien Tzu Chi medical center in eastern Taiwan. Information on the physical exercise-based swallowing intervention and data, including gender, age, and clinical data related to safe swallowing, were accessed on 34 individuals admitted to the dementia day-care center from October 2018 to May 2020. Five individuals whose data needed to be clarified were excluded. Twenty-nine individuals were included in the analysis.The participation records of the physical exercise-based swallowing intervention were reviewed, showing that those who could comply with the instruction to perform physical exercises in the first month would extend their participation throughout 19 months, and those who could not cooperate to perform physical exercises in the first month would not comply with the physical exercise programs after. Of the 29 individuals, 22 who received the reactive swallowing interventions and complied with physical exercises were assigned to the exercise-based group. Seven who received the compensatory swallowing interventions and could not comply with the physical exercises were assigned to the non-exercise-based group.The Research Ethics Committee at Tzu Chi General Hospital, Buddhist Tzu Chi Medical Foundation, approved this study (IRB110-137-B).The physical exercise-based swallowing interventions consisting of swallowing-related exercises and compensatory measures were carried out before and during lunchtime every weekday from 11 October 2018 to 11 May 2020 . The proAn SLP and the nurses in charge at the day-care center instructed the exercise-based group to engage in swallowing-related exercises for 20 min before lunchtime every weekday. The instructor-to-participant ratio was 1:3. The exercise programs, modified from Taiwan-SLPU swallowing exercises , includeA comprehensive compensatory intervention of six measures was checked on both groups during lunchtime every weekday: 1. keep the surrounding environment quiet and stable to minimize distraction while eating; 2. keep the oral environment intact to avoid loose dental braces or any obstacle, such as dry mouth; 3. keep sitting upright to avoid bolus pathway blockage; 4. use a small tablespoon (5 mL) for eating to avoid a bolus too large for each swallowing; 5. use a wide-open bowl but not a narrow-open cup for liquid food to avoid tipping the head back while drinking; 6. do not provide solid and liquid food simultaneously to avoid the confusing control of bolus flow. The mealtime compensatory measures were supervised by the nurses in charge, who prepared in advance and stayed with the participants during lunchtime to identify and fix any problems. The nurse-to-participant ratio was 1:3.At home, the attending caregivers were given a health education leaflet on the six compensatory measures and were asked to check every action at mealtime.The clinical dementia rating scale (CDR)  and the The frequency of coughing/choking in 10 days (10-day coughing/choking) was evaluated by nurse practitioners using the cumulative number at the day-care center. The occurrences of coughing/choking were tallied within a timeframe that included the duration of and 5 min following meals. Throughout this period, the participants were under the continuous supervision of attending nurses, with a nurse-to-participant ratio of 1:3.As physical exercises of sustained phonation were expected to be beneficial for improving voluntary cough, maximum phonation time (MPT)  and cougThe 10-day coughing-choking was evaluated ten days before the beginning of the physical exercise-based interventions, and 5 months, 8 months, and 19 months after; the MPT and coughing strength were evaluated at the beginning of the physical exercise-based interventions and 5 months after; the CDR and FOIS were assessed at the beginning of the physical exercise-based interventions .t-test or Wilcoxon rank-sum test and within groups by paired t-test or Wilcoxon signed-rank test, depending on whether they followed a normal distribution or not. The chi-square test or Fisher’s exact test was used for the categorical variables to evaluate the group differences, expressed as frequency with proportion. The Wilcoxon signed-rank test was used to compare follow-up changes for each group. Statistically significant differences were defined as p < 0.05. All statistical analyses were performed using SPSS Statistics for Windows, version 17.0 .The Shapiro–Wilk test was adopted to test the normality of continuous variables. Continuous variables were expressed as mean ± SD and the mean differences between groups were evaluated by independent The main findings of this study were that the regular physical exercise-based swallowing interventions at the dementia day-care center reduced the 10-day coughing/choking among those who complied with exercise programs at the 5-month and the 19-month follow-ups. Among those who could not comply with exercise programs, noticeably those with more advanced age and severe CDR, FOIS, MPT, and coughing strength, the compensatory intervention’s effectiveness for reducing the 10-day coughing/choking was marked at the 5-month post but declined after.As nursing homes report a higher proportion of older adults with dysphagia than other settings, the evidence on interventions for dealing with swallowing safety in the demented elderly has mainly been based on the findings from these settings. According to a recent review, the most commonly implemented interventions to facilitate safe swallowing in nursing homes were “compensatory” in nature, such as modification of diet, ensuring an appropriate environment for swallowing, appropriate feeding assistance, and appropriate posture or maneuver for swallowing . These cAny type of physical activity is more beneficial than being physically inactive, and physical exercise confers greater benefits for physical functions ,22. In tDementia is a syndrome that causes cognitive and other related functions to become worse gradually, leading to confused behavior. It has been suggested that older adults with dementia may have difficulties participating in or performing physical exercise. A meta-analysis by Hong et al. reported that the average attendance rate of the exercise group among sedentary healthy older adults in 47 exercise RCTs was 86% . Among oAmong multiple dementia LTC service models, day-care centers are designed primarily to meet the daily living and social needs of older adults with dementia during the day in a professional care environment to provide respite and support services for family caregivers . PreviouThere were several limitations of this study. First, some areas for improvement of a retrospective study existed in this study, including missing data in charts, loss of follow-up, and differences in baseline characteristics of the groups. Five cases were excluded due to largely missing data. Seven cases in the exercise-based group and one in the non-exercise-based group were lost at 19-month follow-up due to death or the COVID-19 pandemic. The group assignment in this study depended on compliance with the physical exercise programs in a day-care center. The results showed significant age, CDR, and FOIS differences between the exercise- and non-exercise-based groups. We recognized that the association of the physical exercise-based intervention and the 19-month mitigation in coughing-choking were not statistically significant after adjusting for confounding factors of age, CDR, and FOIS, meaning that it was impossible to determine whether the ease of coughing/choking was a result of physical exercise programs. Furthermore, the retrospective nature of this study may have some limitations with respect to participant recruitment and outcome assessment. The absence of a randomized control design and difficulties in recruiting non-exercising individuals within a clinical setting may have resulted in selection bias. Additionally, the subjective rating of cough strength/sound used in this study can be unreliable and biased, thereby affecting the study’s validity. The absence of FOIS and other outcome measures at various time points in this study further restricts the ability to assess the intervention’s overall long-term effects. Secondly, this study needed more sample sizes. A small sample size may decrease statistical power. This also limited further analysis by subgroups.Impaired swallowing safety can be indicated by symptoms such as coughing and choking. An interdisciplinary intervention for swallowing, which combines compensatory swallowing measures and swallowing-related exercises, integrated into the daily treatment routine of a dementia day-care center, can effectively alleviate coughing and choking problems. The effects of this intervention may last for a significant duration. However, for older adults with severe dementia symptoms and advanced age who may struggle to adhere to exercise programs, compensatory swallowing measures may only provide short-term relief. It should be noted that our retrospective study could not establish a direct causal link between swallowing-related exercises and alleviating coughing/choking problems. Further randomized controlled studies are needed to determine the individual effectiveness of swallowing-related exercises in improving swallowing safety for older adults with dementia.


Circular RNA (circRNA) is a special class of noncoding RNA molecules and the latest research hotspot in the field of RNA. CircRNA molecules have a closed loop structure, which is not affected by RNA exonuclease and has the characteristics of more stable expression. Previous studies have shown that circRNA molecules are rich in microRNA (miRNA) binding sites and act as miRNA sponges in cells. By interacting with miRNAs associated with tumors and other diseases, circRNAs play an important regulatory role. However, circRNAs have recently been found to have small open reading frames that enable them to encode peptides/proteins. These proteins have been reported to play an important role in the mechanism of regulation of a variety of diseases and have great potential in the diagnosis and treatment of diseases. In this review, we summarize the mechanism of action of the newly discovered circRNA-coding proteins since 2022 and briefly describe their research process. In addition, we also discuss the prediction model of the functional sites and encoded proteins of circRNAs, which provides a potential idea for future research on circRNAs. Scientists first discovered circular RNA (circRNA) in plant viruses, yeast mitochondrial RNA and hepatitis B virus . DiffereCircRNAs have many functions in cells : 1). As Previously, the view was usually that circRNA was an endogenous noncoding RNA . HoweverThe results of the current studies show that cap-dependent translation of circRNAs encoding proteins is typically initiated by internal ribosome entry sites (IRES) or N6 methyladenosine (m6A)-induced ribosome engagement sites (MIRES). Lacking a 5′cap structure, circRNA follows a cap-independent translation mechanism and is dependent on IRES or MIRES to bind to the initiation factor eIF4G2 complex and anchor the 43S complex for protein translation . IRES arIn vivo, the tumor volume was smaller in the circZKSaa overexpression group. Ultimately, they found that overexpression of circZKSaa promoted the interaction of FBXW7 with mammalian target of rapamycin (mTOR) and promoted the ubiquitination of mTOR, thereby inhibiting the PI3K/AKT/mTOR pathway.in vivo and prolonged the survival time of nude mice.in vitro. Rescue experiments confirmed that the circular RNA MTHFD2L played a role in inhibiting gastric cancer by coding the CM-248aa protein. Mechanistically, they found that CM-248aa competitively targeted the SET nuclear oncogene (SET) and promoted the dephosphorylation of AKT, extracellular signal-regulated kinase, and P65.CircFBXW7 (hsa_circ_0001451) has been reported to play a tumor suppressor role in gliomas by encoding a novel protein . In the in vivo models further confirm the inhibitory effect of circASK1 and its encoded protein on gefitinib resistance, providing a novel therapeutic target for overcoming gefitinib resistance in LUAD patients.In a study by in vivo and in vitro experiments, circMAP3K4-455aa was shown to promote the development of liver cancer and prevent the apoptosis of liver cancer cells. Ultimately, their study showed that circMAP3K4-455aa could organize apoptosis in HCC cells by inhibiting the cleavage and nuclear distribution of AIF.in situ hybridization (FISH) analysis showed that circINSIG1 was enriched in the cytoplasm of CRC cells. They next found that the sequence of circINSIG1 contains an IRES of ORFcircINSIG1-121 with the potential to encode 121 amino acids. The activity that mediates translation of the IRES was confirmed by dual-luciferase assay. The circINSIG1-flag vector was constructed, and the expression of circIN-SIG1-121 was detected. In addition, the circIN-SIG1-121 antibody confirmed the endogenous presence of circINSIG1-121. The specific peptide fragment of the circINSIG1-121 protein was further detected by mass spectrometry and SDS‒PAGE. Finally, they found that circINSIG1-121 promoted the ubiquitination of INSIG1, a regulator of cholesterol metabolism, by recruiting the CUL5-ASB6 complex, thereby inducing cholesterol biosynthesis, which promoted CRC proliferation and metastasis.in vivo and in vitro studies indicate that the effect of circATG4B is attributed to its potential to encode a novel protein, circATG4B-222aa. One IRES in circATG4B was identified, and its activity that mediates translation was confirmed by dual-luciferase assay. CircATG4B-222aa competitively interacts with TMED10 and prevents TMED10 from binding to ATG4B, which leads to increased autophagy and the subsequent induction of chemoresistance. This study suggests that exosomal circATG4B is involved in reducing the chemosensitivity of CRC cells and provides a new basis for a potential therapeutic target for oxaliplatin resistance in CRC.In addition, the proteins encoded by circRNAs can also contribute to cancer progression by enhancing resistance to anticancer drugs. Oxaliplatin is commonly used in chemotherapy for colorectal cancer (CRC) after surgical resection. in vitro and tumor growth in vivo. In addition, they detected a peptide encoded by circMRPS35 (circMRPS35-168aa) that was significantly induced by chemotherapy drugs and promoted cisplatin resistance in HCC. In their study, two internal ribosome entry sites (IRESs) were identified, and luciferase assay results showed that IRES-1 has significantly higher activity than IRES-2. These results suggest that circMRPS35 may be a novel mediator in HCC progression, and they raise the potential of novel biomarkers for HCC diagnosis and prognosis, as well as novel therapeutic targets for HCC patients.The coding information of tumor-associated circRNAs is summarized in Bombyx mori cypovirus (BmCPV) encodes the viral peptide vSP27 against viral infection. First, they identified a novel polypeptide, vSP27, from the BmCPV minus strand, identified a circRNA encoded by the BmCPV genome and verified a head and tail sequence from Sanger sequencing. The pIZT-LcR-circRNA-vSP27 vector was then prepared, and the head and tail sequences of circRNA-vSP27 could be detected in the transfected cells. The translational activity of circRNA-vSP27 was confirmed by Western blotting in pIZT-lcr-circRNA-vSP27-transfected cells. However, the expression level of vSP27 was affected by circ-siRNA but not by linear siRNA treatment, indicating that vSP27 was mainly translated by circRNA-vSP27. They then found that the expression of viral structural protein VP7 and viral structural protein genes (vp1 and vp2) was decreased in BmCPV-infected cells overexpressing circRNA-vSP27. Finally, their study showed that ROS production induced by vSP27 activated the NF-κB signaling pathway and induced the expression of antimicrobial peptides, thereby inhibiting BmCPV infection.Bombyx mori Nucleopolyhedrovirus (BmNPV) and increased with viral infection. First, they found that circRNA-000010 has a small open reading frame (sORF) and an IRES-like element. The expression of VSP39 protein was then detected by Western blotting in BmnPV-infected midgut and BmN cells and in cells transfected with the circ_000010 plasmid. In BmN cells pretransfected with circ_000010, Western blot results showed that knocking down the linear transcript did not change the expression of VSP39, while knocking down circRNA-000010 significantly reduced the expression of VSP39. The translation of VSP39 from circ_000010 was demonstrated. Finally, the VSP39 vector was prepared using the ORF of VSP39 and transfected into BmN-infected cells. The expression of the Bm59 gene and GFP and virus titer increased with increasing vector transfection dose of VSP39. This proves that VSP39 can promote viral replication.However, in the study by in vivo.In a study by For diseases caused by viruses, circRNA-encoded proteins can also play a regulatory role. Transmissible gastroenteritis virus (TGEV), a member of the coronavirus family, is the pathogen that causes infectious gastroenteritis and can cause mitochondrial dysfunction in host cells. Transmissible gastroenteritis virus (TGEV), a member of the coronavirus family, is the pathogen that causes infectious gastroenteritis and can cause mitochondrial dysfunction in host cells. In addition, the mitochondrial permeability transition is caused by the abnormal opening of the mitochondrial permeability transition pore (mptp) regulated by the voltage-dependent anion-selective channel protein 1 (VDAC)-cyclophilin D (CypD) complex, which is a critical step in the process of mitochondrial dysfunction. According to previous reports, circRNAs are involved in the regulation of a variety of pathophysiological processes in the human body and play roles in cardiovascular system diseases, myoblast proliferation, and nervous system diseases. In the latest study, the authors found that circRNAs also play an important role in genitourinary system-related physiological processes and diseases.in vitro rescue experiments showed that circRsrc1 regulates mitochondrial function through its encoded protein Rsrc1-161aa. Further investigation of the mechanism revealed that Rsrc1-161aa interacts with the mitochondrial protein C1qbp directly, enhancing its binding activity to mitochondrial mRNA, thereby regulating mitochondrial ribosome assembly and affecting the translation of OXPHOS proteins and mitochondrial energy metabolism. Therefore, the authors concluded that the circRsrc1-encoded Rsrc1-161aa protein regulates mitochondrial ribosome assembly and translation during spermatogenesis, thereby affecting male fertility.CircRNA has been shown to be abundant in the mammalian testis  and was in vitro and in AKI kidneys in vivo. Blockade of the AKT and mTOR signaling pathways by pharmacological inhibitors reversed the impaired autophagic flow and apoptosis induced by znf609-250a in HK-2 cells. This study demonstrated that the highly expressed circ-ZNF609-encoded ZNF609-250aa induced apoptosis and AKI by impairing autophagic flow through an AKT/MTOR-dependent mechanism. Targeting circ-ZNF609 may be a novel treatment for ischemic AKI.Nontumour-associated circRNA coding information is presented in in vitro differentiation of murine and human myoblasts. They identified conserved circRNAs regulated during murine and human muscle differentiation and that their expression was altered in myoblasts with Duchenne muscular dystrophy (DMD). Using a dedicated knockdown strategy and subsequent high-content functional genomic screening, they identified circRNAs involved in the regulation of myogenesis. Among them, circ-ZNF609 could specifically control the proliferation of myoblasts. Circ-ZNF609 contains an open reading frame that, similar to the linear transcript, begins at the start codon and terminates at an in-frame stop codon created upon circularization. Circ-ZNF609 associates with heavy polyribosomes and is translated into proteins in both a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes.CircRNAs play an important role in the proliferation of myoblasts. in vitro and in vivo, and knockdown of circTmeff1 partially rescued the muscle mass of mice in the established atrophic environment. Mechanistically, circTmeff1 directly interacts with TAR DNA-binding protein 43 (TDP-43) and promotes TDP-43 accumulation in mitochondria, thereby triggering mitochondrial DNA (mtDNA) release into the cytoplasm. It also activates the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. Unexpectedly, TMEFF1-339aa was identified as a novel protein encoded by circTmeff1 that mediates its proatrophic effect, and one functional IRES was identified by dual-luciferase assay.Skeletal muscle atrophy is a common clinical feature of many acute and chronic diseases. In the nervous system, an increasing number of studies have shown that circRNAs are involved in the regulation of related proteins in the pathogenesis of Alzheimer’s disease . AmyloidThe C9ORF72 hexanucleotide GGGGCC repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, it is not clear how these intronic repeats are exported and translated in the cytoplasm. Using a single-molecule imaging approach to examine the molecular identity and spatiotemporal dynamics of the repeat RNA, in vivo. The translation of microproteins is not restricted to the heart and is prominent in the translatome of the human kidney and liver. They linked these microproteins to different cellular processes and compartments and found that many microproteins localized to mitochondria. Importantly, many microproteins are translated from lncRNAs with well-defined noncoding functions, suggesting previously unrecognized biological functions. In the study by In a human heart translatome study , researcin vivo function of circRNAs generated by the sulfate-free gene (circSfl), which was consistently upregulated in multiple long-lived insulin mutants, especially in brain and muscle. Notably, the lifespan extension of the insulin mutant was circSfl dependent, whereas overexpression of circSfl alone was sufficient to extend lifespan. In addition, circSfl is translated into a protein that shares the N-terminus with the full-length Sfl protein encoded by the host gene and may have some function. This study demonstrates that insulin signaling affects global circRNA accumulation and reveals an important role for circSfl during aging in vivo.Proteins encoded by circRNAs may also play a role in regulating the aging process. Researchers  found thDue to the large number of circular RNAs, functional and structural prediction models are essential to improve the efficiency of research. Therefore, in this section, we mainly discuss the novel structure and function prediction models of circRNAs.https://github.com/Xuezg/JLCRB. . Du et al. and Li et al. proposed multiview-based circRNA binding site prediction models. Duan et al. first used four different feature coding methods to extract the feature information of RNA. Then, a global union of multiple views is generated to construct the intrinsic connection between views, which is used for feature calibration of each view, highlighting important features and suppressing unimportant features. Finally, the binding sites of the circRNAs were detected using the depth features obtained by multiview fusion. In 37 ciRNA-RBP databases, the area under the curve (AUC) of this method was as high as 93.68%. The code and data sets are available from g/JLCRB. . The advp/ASCRB. . CompareIn the study of circRNAs, the association between diseases and circRNAs is also very important. The emergence of prediction models for circRNA-disease associations helps to discover more important circRNAs. A computational framework for predicting potential circRNA-disease associations, RGCNCDA, was proposed in the study by In their study, This article summarizes the latest research results on circRNA-encoding proteins. There is increasing evidence that circRNAs have protein-coding capabilities, produce functional proteins or peptides, and participate in various biological processes. Most previous studies have focused on the role of circRNAs in the occurrence and progression of cancer. CircRNA-encoded peptides can act as either oncogenes or tumor suppressors, residing in the nucleus, cytoplasm, cell membrane, or secreted proteins. Because circRNA-encoded proteins have significant sequence overlap with their cognate mRNAs and use the same reading frame, most circRNA-encoded proteins exert their function by enhancing or attenuating the activity of the original protein encoded by the corresponding mRNA. In recent years, researchers have identified the role of circRNAs in cardiovascular system diseases, myoblast proliferation, and nervous system diseases. Recent studies have also reported that circRNAs play an important role in genitourinary system-related physiological processes and diseases. Although most studies have used Western blot and immunofluorescence assays to analyze the expression level of the target protein, a rescue assay is still needed to determine whether the protein, rather than the circRNA itself, plays a physiological or pathological role.At present, substantial progress has been made in the regulation and function of circRNAs, but there are still many valuable directions for future research. First, most of the activities of circRNA-encoded proteins reported thus far are associated with cancer. Future studies should further explore whether these proteins encoded by circRNAs play important roles in other physiological and pathological processes, such as developmental lung diseases and cell proliferation and differentiation during embryonic development. Second, many circRNAs can either encode proteins or possibly act as sponge molecules to inhibit the action of microRNAs; which of these two functions is the primary activity of these circRNAs needs to be further investigated. Third, the physiological and pathological processes in the human body are the result of the interaction of many factors. Therefore, the correlation between the expression level of the circRNA-encoded protein and physiological or pathological processes should be fully investigated. In summary, the current research achievements on circRNAs are only the tip of the iceberg in this field. Further study of circRNA-related networks in the pathogenesis of cancer, as well as their role in other physiological and pathological processes, will promote our understanding of complex cell signaling pathways and regulatory mechanisms and will be one of the most popular research directions in the future.