Cancer cell lysosomes contain various hydrolases and non-degraded substrates that are corrosive enough to destroy cancer cells. However, many traditional small molecule drugs targeting lysosomes have strong side effects because they cannot effectively differentiate between normal and cancer cells. Most lysosome-based research has focused on inducing mild lysosomal membrane permeabilization (LMP) to release anticancer drugs from lysosomal traps into the cancer cell cytoplasm. In fact, lysosomes are particularly powerful “bombs”. Achieving cancer cell-selective LMP induction may yield high-efficiency anticancer effects and extremely low side effects. Nanodrugs have diverse and combinable properties and can be specifically designed to selectively induce LMP in cancer cells by taking advantage of the differences between cancer cells and normal cells. Although nanodrugs-induced LMP has made great progress recently, related reviews remain rare. Herein, we first comprehensively summarize the advances in nanodrugs-induced LMP. Next, we describe the different nanodrugs-induced LMP strategies, namely nanoparticles aggregation-induced LMP, chemodynamic therapy (CDT)-induced LMP, and magnetic field-induced LMP. Finally, we analyze the prospect of nanodrugs-induced LMP and the challenges to overcome. We believe this review provides a unique perspective and inspiration for designing lysosome-targeting drugs. Cancer burdens global public health; it accounts for nearly 10 million deaths annually worldwide, and this number keeps increasing . The batLysosomes, discovered in 1955, have long been regarded as mere “digesting” machines . These oThese lysosomal bombs have also been called “suicide bags” . BesidesThe tremendous progress in nanodrugs has enabled the discovery of novel effective “detonators” to ignite lysosome bombs in tumor cells. First, most cellular uptake pathways of nanomedicines mediate endocytosis into lysosomes, including phagocytosis, clathrin-mediated endocytosis, and macropinocytosis , making Cancer cells present an altered lysosomal membrane structure making them more susceptible to osmotic swelling . TherefoN,N,N-trimethyl (11-mercaptoundecyl) ammonium chloride (TMA) and the negatively charged 11-mercaptoundecanoic acid (MUA) at different ratios to specifically induce LMP in cancer cells. A decrease in pH protonates the carboxyl group of the MUA and decreases the negative charge, rendering MUA hydrophobic. Interestingly (+/−) NPs with a TMA/MUA ratio of 80:20 only aggregated and triggered LMP in the lysosomes of cancer cells, but not in multilamellar bodies of normal cells. These (+/−) NPs (χTMA:χMUA = 80:20) had a particle size of only 5.3 nm and formed very large aggregates with a diameter >2 μm in cancer lysosomes, ultimately resulting in a powerful and broad-spectrum LMP-induced anticancer effect. Furthermore, in normal cells, the 80:20 (+/−) NPs were only present in multilamellar bodies, and most of them were excreted by exocytosis, indicating the potentially low adverse effects.The nano-aggregation strategies aim to selectively target cancer cells to achieve an excellent anticancer effect with few side effects. Currently, two nano-aggregation-induced LMP strategies exist. The first strategy  is based2PO3)-Gly-Lyso (Py-Yp-Lyso) to treat cervical cancer through nano-aggregation-inducing LMP. Py-Yp-Lyso consisted of four parts: Glu-Tyr (H2PO3) was dephosphorylated by alkaline phosphatases to form Py-Y-Lyso and further self-assembled into nanofibers. The pyrene molecule (Py) served as a monitor due to its concentration-dependent luminescence. Phe-Phe conferred better biostability as a self-assembling structure. Finally, the 4-(2-aminoethyl) morpholine served as a lysosome targeting molecule (Lyso). It is prone to protonation, becomes more hydrophilic into the acidic lysosome, and remains there instead of diffusing to the cytoplasm. Py-Yp-Lyso had a negative charge and was stable in the blood circulation system because it contained a phosphate group. When Py-Yp-Lyso reached the cancer cells, alkaline phosphatases on the surface of the cancer cell plasma membrane dephosphorylated it, reversing its charge. The positively charged surface of the dephosphorylated Py-Y-Lyso promoted endocytosis and protonated the 4-(2-aminoethyl) morpholine moiety, preventing Py-Y-Lyso from diffusing out of the lysosome. In acidic lysosomes, Py-Yp-Lyso aggregation formed large nanofibers which triggered LMP. Py-Yp-Lyso had significantly higher cytotoxicity in Hela cells than in normal cells and inhibited tumor growth to 55.4% of that of control tumors at day 15 following the injection. Similarly, Ji et al.  are highly reactive and, at high concentrations, they can react with unsaturated lipids in lysosomal membranes to trigger LMP . CDT kilreaction . CDT hasl tissue . The CDTl tissue  or cytopl tissue , promptil tissue  to searcl tissue . Moreovel tissue  but do el tissue . Combiniin situ growth of CuS on LDH nanosheets. The group chose CuS nanodots for their photothermal activity and better biosafety and performed near-infrared (NIR) excitation to release thermal effects-inducing copper ions to initiate Fenton-like reactions. This process is called NIR-induced CDT. Moreover, the sheet-like structure of LDH allowed CuS to stay in or adhere to lysosomes for longer periods, as 1D and 2D materials remain longer in endocytosis than spherical nanoparticles do. Therefore, LDH-CuS exhibited strong lysosomal localization ability, while free CuS nanodots are prone to diffuse into the cytoplasm. Finally, NIR-excited LDH-CuS efficiently generated OH in lysosomes, inducing LMP and achieving complete tumor suppression, while free CuS nanodots had poor tumor-suppressive effects, demonstrating the importance of lysosomes in CDT.Copper-based nanoparticles can be used in a wide range of acidic environments  and exhi2O2 supply is essential to maintain CDT and the subsequent LMP process. The exhaustion of endogenous H2O2 in lysosomes of tumor cells hampers CDT efficacy (2+ and H2O2 in the presence of poly (vinylpyrrolidone) (PVP) as a stabilizer. These nanodots simultaneously release Cu2+ and H2O2 in the acidic environment, especially in the lysosomes of tumor cells. CP nanodots significantly inhibited the tumor growth of U87MG tumor-bearing mice via CDT-inducing LMP. To further explore the retention behavior of copper-based nanomaterials in lysosomes, Deng et al.  have been developed, especially those made from iron oxides, since they have favorable biocompatibility, abundant raw materials, and outstanding biological effects . The MNPPucci et al.  also conVery recently, Clerc et al.  construc2O2 concentrations  to overcome the dark toxicity of many photosensitizers . To the In general, therapeutic strategies that utilize the characteristics of lysosomes in tumor cells are efficient. In addition, the lysosomal cell death mechanism is different from that of most antitumor drugs and provides more opportunities for coping with drug resistance. In addition, processes such as development and aging may be related to the instability of lysosomal membranes. Therefore, LMP-inducing strategies can not only be applied to tumors but may also be worth studying in other aspects leading to treatments for other diseases.


Components of metabolic syndrome might be predictors of the therapeutic outcome of psychiatric symptom in schizophrenia, whereas clinical results are inconsistent and an intrinsic therapeutic link between weaker psychiatric symptoms and emergent metabolic syndrome remains unclear. This study aims to reveal the relationship and illustrate potential mechanism by exploring the alteration of cerebellar functional connectivity (FC) in schizophrenia patients with comorbidity metabolic syndrome. Thirty-six schizophrenia patients with comorbidity of metabolic syndrome (SCZ-MetS), 45 schizophrenia patients without metabolic syndrome (SCZ-nMetS) and 39 healthy controls (HC) were recruited in this study. We constructed FC map of cerebello-cortical circuit and used moderation effect analysis to reveal complicated relationship among FC, psychiatric symptom and metabolic disturbance. Components of metabolic syndrome were significantly correlated with positive symptom score and negative symptom score. Importantly, the dysconnectivity between cognitive module of cerebellum and left middle frontal gyrus in SCZ-nMetS was recuperative increased in SCZ-MetS, and was significantly correlated with general symptom score. Finally, we observed significant moderation effect of body mass index on this correlation. The present findings further supported the potential relationship between emergence of metabolic syndrome and weaker psychiatric symptom, and provided neuroimaging evidence. The mechanism of intrinsic therapeutic link involved functional change of cerebello-cortical circuit. Schizophrenia is a common, complex, heterogeneous cognitive syndrome that seems to originate from a disruption of brain development caused by genetic and/or environmental factors . AntipsyHowever, some studies found that components of metabolic syndrome might be predictors of the improvement in schizophrenia psychopathology ,12,13,14Cerebellum is a pivotal hub underlying pathological mechanism of schizophrenia . In morpThe development of research on cerebellum supported functional heterogeneity of different cerebellar modules . The stuIn this study, we aim to evaluate the potential relationship between emergent metabolic syndrome and therapeutic outcome of schizophrenia and explore potential mechanism of therapeutic link from the viewpoint of neuroimaging in a sample including schizophrenia patients with comorbidity of metabolic syndrome (SCZ-MetS), schizophrenia patients without metabolic syndrome (SCZ-nMetS), and healthy controls (HC). We hypothesized that (1) SCZ-MetS group had weaker psychiatric symptom compared with SCZ-nMetS group; (2) The emergence of metabolic symptom correlated with lower psychiatric symptom score; Next, we constructed resting-state FC in cerebello-cortical circuit. We presumed that (3) the abnormal FC in cerebello-cortical circuit of patients compared with HC significantly improved in the SCZ-MetS group, underlying the severity psychiatry symptom. Finally, we explored the potential moderation effect of metabolic components on correlations between FCs and PANSS scores to confirm this relationship.A total of 120 subjects were acquired in this study, including 36 schizophrenia patients with comorbidity of metabolic syndrome (SCZ-MetS) and 45 schizophrenia patients without metabolic syndrome (SCZ-nMetS) and 39 healthy controls (HC). All the chronic schizophrenia patients were recruited from the Clinical Hospital of Chengdu Brain Science Institute. The patients were diagnosed using the structured clinical interview for the DSM-IV axis 1 disorders-clinical version (SCID-I-CV), and all were being treated with medication. The illness duration of the patients was at least 2 years. The severity of psychiatric symptoms was evaluated using PANSS. HC were recruited from the local community through advertisements and word of mouth to participate in this study. HC were excluded based on current or past axis I disorder as verified using the Structure Clinical Interview for DSM-IV, history of neurological illness, traumatic brain injury, substance-related disorders, or first-degree relatives with history of psychosis. Four patients and four HC were excluded due to exceeded head motion. Thirty-four SCZ-MetS (44.42 ± 9.24 years old), 43 SCZ-nMetS (40.65 ± 12.24 years old) and 35 HC (39.49 ± 13.56 years old) were included in the final analysis. The Ethics Committee of the Clinical Hospital of Chengdu Brain Science Institute in accordance with the Helsinki Declaration approved this study. Written informed consent was obtained from each subject and his/her guardian before the participating in the study.2, flip angle [FA] = 90°, matrix = 64 × 64, slice thickness/gap = 4 mm/0.4 mm). The brain fMRI scanning lasted for 510 s, and a total of 255 volumes were collected. The subjects were instructed to keep eyes open and look at the cross/plus on the screen. Subjects were also instructed to keep head motionless without falling asleep during the scanning.All magnetic resonance imaging (MRI) data were acquired on a 3T MRI scanner  in the Center of Information Medicine Research in University of Electronic Science and Technology of China. The head was fixed using foam pads and ears plugs to minimize the motion and scanning noise, respectively, during scanning. Resting-state brain fMRI was collected using gradient-echo EPI sequences  was carried out. All participants whose maximum translation in any of the orthogonal directions larger than 2.5 mm or rotation larger than 2.5° were excluded from subsequent analysis. Then, six motion parameters and their first temporal derivative, white matter signal, cerebrospinal fluid signal were removed through linear regression. The global signal was not regressed out as had been recently suggested in processing the schizophrenia functional data [Functional imaging data were preprocessed using DPABI  and NIT nal data . FinallyTo assess the resting-state FC between cerebellum and cerebral cortex, we segregated cerebellum into motor and cognitive cerebellum modules according to our previous study . The cer2). Systolic blood pressures (SBP) and diastolic blood pressures (DBP) were assessed by a digital blood pressure monitor. A peripheral venous blood sample was collected from patients. FBG level and liver function examination was performed for each individual patient. Lipid profile  was obtained from liver function examination.All the components of metabolic syndrome and metabolic syndrome case history of patients with schizophrenia were collected in their medical histories, including height, weight, fast blood glucose (FBG), blood pressure (BP), triglyceride (TG), high-density lipoprotein (HDL), and treatment of metabolic disease. Given that total cholesterol (TC) and low-density lipoprotein (LDL) had been included in studies verifying associations between metabolic dysregulation and psychiatric symptoms ,13,15,16−2 to 27 kg m−2. Besides that, due to the standpoint that central obesity should be an “optional” rather than “essential” criterion for defining metabolic syndrome in an Asian cohort, whether the patients with schizophrenia were subdivided into SCZ-MetS group was decided according to a slightly altered definition based on diagnostic criteria proposed by International Diabetes Federation (IDF) in 2005 [2); (2) FBG ≥ 5.6 mmol/L or treatment for diabetes mellitus; (3) BP ≥ 130/85 mmHg or on anti-hypertensives; (4) TG ≥1.695 mmol/L or on lipid lowering agent; (5) HDL < 1.036 mmol/L for men, < 1.295 mmol/L for women or treatment for dyslipidemia with lipid-lowering agent.For the definition of metabolic syndrome in this study, firstly, taking Asians’ ethnic specificity of relationship between BMI and body fat percentage into consideration , we lowe in 2005 ,34. The p < 0.05). However, on account of less obvious differences between two groups of patients, we described findings of post hoc analysis between SCZ-MetS and SCZ-nMetS within the uncorrected statistical threshold (p < 0.005).To detect the difference in psychiatric symptoms scores, medication dosage and illness duration between SCZ-MetS and SCZ-nMetS, PANSS scores, chlorpromazine equivalent and illness duration were compared using two-sample t-test. One-way analysis of Covariance analysis (ANCOVA) among SCZ-MetS, SCZ-nMetS and HC was performed with age and years of education as covariates to reveal difference of FC. Then, pairwise post hoc analysis was performed with age and years of education as covariates to assess difference of FC between individual groups. Moreover, in the pairwise post hoc analysis between SCZ-MetS and SCZ-nMetS, we appended medication dosage as a covariate to reduce the influence of drug factors on the results. In the post hoc analysis between two groups of patients and HC, the statistical threshold used was corrected using the false discovery rates (FDR) , respectively. Firstly, FC values of regions of interest (ROIs), as brain FC features, were extracted by creating corresponding spheres (radius: 3 mm) whose center is corresponding to coordinates of significantly different subregions according to pairwise post hoc analysis between SCZ-MetS and SCZ-nMetS. We obtained the following 5 ROIs: 3 for ROIs based on CBCc-cortical circuit ; 2 for ROIs based on CBCm-cortical circuit . Secondly, the partial correlation analysis was performed among FC values, indicators related to metabolic syndrome and PANSS scores in pairs with age, years of education and medication dosage as covariates. Thirdly, moderation effects of metabolic symptoms on the association between brain features and PANSS scores was performed using Hayes’s PROCESS macro (Model 1)  in SPSS p < 0.05, n = 58). No group difference was observed in negative symptoms and general symptoms , fast blood glucose (FBG) level , triglyceride (TG) level  and lower high-density lipoprotein (HDL) level  than SCZ-nMetS group.The clinical characteristics of metabolic syndrome were presented in p = 0.039, n = 27) and in all patients (SCZ-MetS group plus SCZ-nMetS group) . FBG level was reversely correlated with negative symptom  in SCZ-MetS group. Finally, HDL level was positively associated with positive symptom  in all patients (SCZ-MetS group plus SCZ-nMetS group).Crucially, we observed inverse correlation between positive symptom and BMI in SCZ-MetS group  . A summap < 0.05).The one-way analysis of Covariance analysis (ANCOVA) among SCZ-MetS, SCZ-nMetS, and HC on the FC between cortical regions and two cerebellar modules revealed a main effect of the diagnostic group in the extensive cortical region (p < 0.05). Compared to the motor module, the cognitive module exhibited more difference in the cerebral cortex related to the higher cognitive function, including bilateral insula and medial superior frontal gyrus.We found that FC between both modules of cerebellum and cerebral cortex represented the significant difference in primary motor cortex, primary somatosensory cortex, primary visual cortex and primary auditory cortex  based on the Schaefer atlas [p < 0.05). However, there is no significant difference in cerebellar motor cluster (CBCm)-cortical circuits  in SCZ-nMetS. The post hoc analysis between SCZ-MetS and SCZ-nMetS revealed that SCZ-MetS showed increased FC between cognitive module of cerebellum and bilateral posterior precuneus (p < 0.05). Interestingly, the SCZ-MetS showed increased CBCc-MFGL FC (p < 0.05). Additionally, SCZ-MetS also showed increased FC between CBCm and bilateral middle frontal gyrus (p < 0.01).The post hoc analysis showed that SCZ-MetS and SCZ-nMetS had significantly abnormal FC in cerebellar cognitive cluster (CBCc)-cortical circuits compared with HC, including abnormally increased FC within dorsal attention network (DAN), sensorimotor network (SMN), default mode network (DMN), salience ventral attention network (SVAN), visual network and abnormally reduced FC within DMN, DAN . Besides that, in SCZ-nMetS group, FC between CBCm and bilateral middle frontal gyrus (CBCm-MFGL and CBCm-MFGR) was negatively related to PANSS general symptom score , PANSS negative symptom score  and PANSS total symptom score . However, we did not find any significant relationship between FC of CBCc-MFGL, CBCm-MFGL, CBCm-MFGR and indicators of metabolic syndrome. For the FC between CBCc and right posterior precuneus, we observed significantly negative correlation between the FC and metabolic components including TC  and LDL  in SCZ-MetS group, which is not significant in SCZ-nMetS group. Finally, we did not find correlation of FC between CBCc and right posterior precuneus with psychiatric symptoms.Based on the post hoc analysis between SCZ-MetS and SCZ-nMetS, we chose 5 regions of interest (ROIs) a,b for pUsing Hayes’s PROCESS macro (Model 1)  in SPSS This study attempted to explore a therapeutic link between emergent metabolic dysregulation and weaker psychiatric symptom in schizophrenia. Besides that, we endeavored to reveal a possible mechanism of the therapeutic link by investigating FC pattern between cerebellum and cerebral cortex in patients with schizophrenia. We found weaker positive symptom in SCZ-MetS as well as significant correlations between metabolic disturbance, positive and negative symptom score of schizophrenia, which corroborated our hypothesis that the worse metabolic symptoms correlated with better therapeutic outcome of psychiatric symptoms. No significant correlation was found between medication dosage, metabolic components, and positive symptoms, which supported that larger dosages of antipsychotics could not fully explain the lower psychotic symptoms. Compared with SCZ-nMetS, SCZ-MetS showed different cerebello-DMN FC pattern. Importantly, CBCc-MFGL dysconnection of SCZ-nMetS could relieved in the SCZ-MetS. Finally, we observed moderation effect of BMI on association between CBCc-MFGL FC and general symptom score in all patients (SCZ-MetS group plus SCZ-nMetS group), supporting the moderating role of metabolic disturbance on relationship between FC and psychiatric symptom. These results had converged to suggest that cerebello-frontal circuit involved in mechanism of intrinsic therapeutic link between emergence of metabolic syndrome and weaker psychiatric symptom.Whether an association exists between metabolic dysregulation and improvement of psychiatric symptom severity in patients with schizophrenia has not been completely determined, although some results with large cohort and meta-analyses have suggested that the most efficacious of antipsychotics, for example clozapine, might be associated with the most severe metabolic disturbance ,38. In tCerebellum as a pivotal hub extensively connected with cortical regions ,45. AlteOperation of DMN was considered the neural basis of spontaneous human thoughts and feelings . ImpaireFor cerebello-DMN FC, results here have revealed that SCZ-MetS showed increased FC between cognitive module of cerebellum and bilateral posterior precuneus. Albeit there is no significant correlation between the increased FC and psychiatric symptoms, negative correlation with metabolic components was observed in SCZ-MetS group. Previous studies demonstrated that change of functional connectivity strength in precuneus and posterior cingulate was implicated with obesity-inducing and obesity-preventing behavior ,59. BesiSimultaneously, we found that all patients showed reduced CBCc-MFGL FC compared with HC. Importantly, both HC and SCZ-MetS showed increased CBCc-MFGL FC compared with SCZ-nMetS. Additionally, increase of FC in CBCc-MFGL was negatively correlated with reduction in PANSS general symptom scores in all the patients. Cerebello-cortical connectivity has been identified as the pivotal connectivity pattern determining severity of developmental mental disorders including schizophrenia . ConnectFor the relationship between metabolic syndrome and improvement of psychiatric symptoms of schizophrenia, there are some potential explanations. Firstly, Previous studies suggested that it was reasonable to hypothesize the existence of overlapping mechanism between therapeutic action and metabolic side effect of antipsychotics, in which insulin and 5-hydroxyptrytamine receptor type 2 (5-HT2) participate as a cross point ,15. AntiThere are some limitations to the current study. Firstly, we did not collect complete clinical information for all the patients, including components of metabolic syndrome and PANSS scale scores. Soundly, though there is no significant difference in chlorpromazine equivalent between SCZ-MetS and SCZ-nMetS, we did not control the type of antipsychotics patients took. In addition, we did not control the influence of drugs to relieve metabolic syndrome such as lipid-lowering agents, which could conceal actual symptoms of metabolic syndrome. Lastly, the current study was a preliminary study to explore possible mechanism of intrinsic therapeutic link between weaker psychiatric symptom and emergent metabolic dysregulation, and many conclusions are still speculative. In future work, we tend to investigate this relationship in first-episode patients treated with single antipsychotics in a long-term longitudinal study.In summary, we found CBCc-MFGL dysconnectivity of SCZ-nMetS was reconstructed in SCZ-MetS underlying weaker psychiatry symptom. The recuperative increased CBCc-MFGL FC in SCZ-MetS suggested a link between emergence of metabolic syndrome and better relieved psychiatric symptom in schizophrenia patients with comorbidity of metabolic syndrome. These results supported the potential relationship through multimodal neuroimaging and indicated that mechanism of intrinsic therapeutic link involved functional change of cerebello-cortical circuit. The current study offered new insights into clinical relationship between therapeutic benefit and metabolic health in schizophrenia.


Starting in December 2019, the coronavirus SARS-CoV-2 emerged and soon acquired a pandemic dimension. The evidence that 1 in 3 patients presented neuropsychiatric symptoms highlighted SARS-CoV-2 neurotropic properties. The involvement of the Central Nervous System (CNS) seems to be associated with poor prognosis, and it can occur independently of the respiratory system.To assess neuropsychiatric symptoms in SARS-CoV-2 patients and possible mechanisms of CNS invasion; to reflect on what changes should be made in order to avoid short and long-term complications.A non-systematic literature review was performed, including publications between January and August 2020.The most frequent CNS presentations included fatigue (38-75%), headache , nausea or vomiting . Regarding PNS involvement, three kinds of hypoesthesia  were commonly present. Additionally, cases of neurological syndromes associated with SARS-CoV2 were reported, being related to a poor prognosis in cases such as brainstem infiltration. Another major concern regarding CNS involvement is the possibility of permanent neurological disabilities. Importantly there are reports of patients who tested positive for SARS-CoV-2 in CFS, without samples from nasopharyngeal swabs. Different hypothesis are postulated to explain possible mechanisms through which SARS-CoV-2 affects CNS, including: direct invasion through the olfactory nerve, hematogenous route through ACE-2 (angiotensin-converting enzyme) receptor expressed in blood-brain-barrier; or indirect mechanisms.Here we discuss the neuropsychiatric manifestations of SARS-CoV-2 infection and the potential mechanisms by which they occur at an early stage. Awareness, prevention and early treatment of potential neuropsychiatric symptoms of COVID-19 should not be overlooked, especially because they seem to predict a worse prognosis.No significant relationships.


Chimeric antigen receptor (CAR)-T cell therapy represents a landmark advance in personalized cancer treatment. CAR-T strategy generally engineers T cells from a specific patient with a new antigen-specificity, which has achieved considerable success in hematological malignancies, but scarce benefits in solid tumors. Recent studies have demonstrated that tumor immune microenvironment (TIME) cast a profound impact on the immunotherapeutic response. The immunosuppressive landscape of TIME is a critical obstacle to the effector activity of CAR-T cells. Nevertheless, every cloud has a silver lining. The immunosuppressive components also shed new inspiration on reshaping a friendly TIME by targeting them with engineered CARs. Herein, we summarize recent advances in disincentives of TIME and discuss approaches and technologies to enhance CAR-T cell efficacy via addressing current hindrances. Simultaneously, we firmly believe that by parsing the immunosuppressive components of TIME, rationally manipulating the complex interactions of immunosuppressive components, and optimizing CAR-T cell therapy for each patient, the CAR-T cell immunotherapy responsiveness for solid malignancies will be substantially enhanced, and novel therapeutic targets will be revealed. The past decade has witnessed a revolutionary evolution in the application of immunology to oncology treatment. Immunotherapy has made striking advances in malignancies, far outperforming conventional chemotherapy and radiotherapy. Frustratingly, the immunosuppressive properties of the tumor microenvironment (TME) pose the challenge of limited clinical efficacy and severe side effects to tumor immunotherapy in vitro. These T cells are then infused back into the patient to proliferate robustly and elicit potent anti-tumor activity Figure A. These synthetic receptors recognize their corresponding specific antigens using a single-chain variable fragment (scFv) from the variable region in a major histocompatibility complex-independent manner. The majority of scFv possesses binding properties similar to antibodies. CAR-T cell therapy has shown dramatic clinical responses and high rates of complete remission in hematologic malignancies Chimeric antigen receptor (CAR)-T cell therapy is novel immunotherapy through genetically engineered T cells to express CAR targeting molecules. CAR-T cell therapy aims at introducing CARs genes into patient-derived T cells of peripheral blood in a fairly short period, where the biological properties of T cells are redirected and reprogrammed The immune characteristics of the tumor microenvironment (TME) have been categorized as one of the ten tumor characteristics Figure B. As proof: 1) The tumor-associated stroma such as fibroblasts, mesenchymal cells, and various extracellular matrices formed stumbling block against the entry of T cells Figure C.TME contributes to unfavorable immunotherapy efficacy by preventing CAR-T cells from exerting high cytotoxicity against tumor cells Figure .Tumor-associated immune cells possess crucial functions in tumorigenesis, which antagonize and/or promote tumors. Homogeneous immune cells could reshape themselves depending on different tumor ecosystems MDSCs are pathologically activated monocytes and neutrophils with vigorous immunosuppressive behavior and are implicated in the negative modulation of immune responses and poor clinical outcomes de novo generation of regulatory T cells (Tregs) mediated by IL-10 and IFN-γ in vivoin vitroApart from promoting tumor development by forming pre-metastatic niches and angiogenesis Proverbially, macrophages are essential components of innate immunity and leukocyte infiltration present in solid tumors, which regulate cancer-related inflammation and constitute vital regulators of tumor initiation and progression. Circulating monocytes could be recruited into the tumor stroma by multiple chemokines and cytokines such as CCL2, GM-CSF, and VEGF family members TAMs could regulate the immunological activity of T cells to foster tumor progression. Pro-inflammatory cytokines produced by TAMs could trigger the accumulation and expansion of CD4+ Th17 cells to foster angiogenesis and overexpressing CTLA-4, programmed death-1 (PD-1), and glucocorticoid-induced tumor necrosis factor receptor (GITR), thereby promoting tumor development TGF-β in TME upregulated Tim-3 expression on TAMs, which promoted tumorigenesis and tolerance via NF-κB signaling and downstream IL-6 production Commonly referred to as Tregs are CD4+CD25+Foxp3+ T cells, which could chemoattract TME through chemokine gradients such as CCR8-CCL1, CCR4-CCL17/22. Tregs negatively moderate immune responses to maintain autoimmune tolerance and homeostasis. However, excessive suppression of immune responses in the TME promoted tumor progression B cells in TME play a double-edged sword effect either by promoting tumor immunity or enhancing tumorigenesis Tumor-associated neutrophils (TANs) are another type of immune cell that infiltrates various tumors. TANs could be recruited to TME by IL-8 via CXCR1/CXCR2 receptors and abolish the ability of CD8+ T cells through TNF-α production-mediated NO et al. have demonstrated that tumor-associated mast cells (TAMCs), a heterogeneous population, harbor a distinct phenotype compared to MCs present in the non-lesional homologue of lung cancer Mast cells (MCs) are well known to participate in allergy and inflammation. The ability of MCs in TME has been hypothesized to be either pro- or anti-tumor et al. reported that HCC impaired DCs recruitment due to the absence of a tumor-derived chemokine CCL5 via tumor-intrinsic Wnt/β-catenin signaling Dendritic cells (DCs) acting as specialized antigen-presentation cells have long been recognized as a critical factor in T-cell-mediated anti-tumor reactions. DCs uptake and cross-present tumor antigens to naïve T cells accompanied by priming and activating CTLs with the ability to eradicate tumor cells Table 1).Immune cells, endothelial, stromal, and tumor cells within the TME secreted bulk masses of immunosuppressive factors including chemokines, cytokines, and inhibitory molecules to assist and/or restrain each other, which orchestrates a severely immunosuppressive tumor milieu. These factors bind to corresponding receptors via paracrine, autocrine or endocrine means to modulate tumor growth/invasion/metastasis, and immune responses, thereby reshaping TME and mediating intercellular crosstalk. Some of their specific features have been mentioned above. Herein, we dissect the representative participants in the current data , the endogenous factors  could further exacerbate the immunosuppressive landscape of TIME. Identifying tumor-specific antigens as targets is a challenge for CAR-T cell therapy to win the war against solid tumors. The optimal target surface antigen should possess excellent coverage, high expression on solid tumors, and not affect normal cells to avoid on-target and off-tumor cross-reactions With the increasing understanding of TIME, more strategies concentrate on 're-editing' TIME to better arouse tumor-antagonizing immunity, which offers a novel tactic for CAR-T cell therapy.Figure A. It has been reported that MDSCs inhibited the cytotoxicity of different generations of disialoganglioside (GD2)-CAR-T cell. The frequency of circulating MDSCs was inversely correlated with the levels of GD2-CAR-T cell in phase I/II clinical trial, further underlining the importance of targeting immunosuppressive cells Several approaches of targeting immunosuppressive cells to advance the tumor-killing ability of CAR-T cells have been applied in basic studies The combination of CAR-T therapy with immunomodulatory agents was proposed for obtaining satisfactory therapeutic results Engineering CARs to target TAMs is also a pervasive experimental direction. TAMs that expressed appreciable levels of folate receptor beta (FRβ) possessed an immunosuppressive M2-like phenotype, which indicated that targeting FRβ could limit the outgrowth of solid tumors Likewise, depletion of other immunosuppressive cells is an attractive way. Using an anti-CD25 antibody with augmented binding to activating Fc gamma receptor , CD25+ Tregs were depleted. This reinforced cancer-infiltrating CD8+ T cells and improved the eradication of established tumors Figure B. Currently, recombinant cytokine drugs such as IL-2 and IFN-α, have been approved for anti-tumor treatment. This facilitates the generation of “armored” CAR-T cells engineered to secret pro-inflammatory cytokines. To illustrate, CAR-redirected T-cells tailored to release inducible IL-12 could eliminate the antigen-loss cancer cells, recruit macrophages and reinforce the function, and sustain pro-inflammatory responses Induced local dissemination of stimulatory factors could counteract the immunosuppressive dilemma to reinforce CAR-T cell potency More recently, a preclinical study emphasized that CAR-T cells via autocrine IL-23 signaling had the superior anti-tumor capacity and attenuated side effects, with decreased PD-1 expression and increased granzyme B in comparison to those expressing IL-18 in vivo persistence, bolstered cytokine secretion, fought against exhaustion, and induced tumor eradication Another approach to addressing the efficacy conundrum is to modify CAR-T cells by rewiring inhibitory inputs to stimulatory outputs or being refractory to inhibitory cytokines present in TME. Engineering of TGF-β CAR-T cells demonstrated that CARs could express a dominant-negative TGF-βRII (dnTGF-βRII), thereby blocking TGF-β signaling in T cells. DnTGF-βRII increased CAR-T cell proliferation and long-term 'Switch receptor' could also achieve by rewiring negative signals. Mohammed and colleagues Figure Cin vivo and tumor-specific accumulation Given the pivotal role of chemokines and their receptors in recruiting immune cells and trafficking CAR-T cells into tumors, numerous studies have endeavored to integrate chemokines and CAR-T cells to battle tumors in recent years Targeting other chemokines is under active experimentation, some of which have achieved satisfactory therapeutic effects in solid tumors. Some researchers constructed a lentivirus-based CAR gene transfer system targeting CCR4, a chemokine receptor over-expressed in T-cell malignancies and Tregs profiles Undoubtedly, CAR-T cells engineered to acquire responsiveness to cytokines, chemokines, and their receptors exhibit stronger tumor-antagonizing and trafficking functions than the classical second-generation. CAR-T cells against other soluble inhibitors such as PGE2 Figure DImmune checkpoint receptors and ligands, such as PD-1, PD-L1, and CTLA-4, could preclude CAR-T cell cytotoxicity and induce anergy in TME in vitro and animal models Gene-editing technologies that knock out immune checkpoints could also be applied to abrogate the expression of T cell negative regulators. PD-1 knockout through TALEN technology augmented the persistence and tumor clearance capability of intratumoral T cells and established durable anti-tumor memory et al. found that soluble LAG3 promoted tumor cell activation and anti-apoptotic effects et al. modified CAR-T cells to secrete PD-1-blocking scFv Similar to PD-1, LAG-3 and T cell immunoglobulin and mucin-domain containing-3 (Tim-3) known as T cell exhaustion markers functioned as coinhibitory receptors to throttle T cell proliferation and cytokine production In the previous section on targeting immunosuppressive cells and/or factors, we covered part of targeting tumor antigen heterogeneity. Admittedly, as CAR-T cells targeting solid tumors become increasingly effective due to tumor antigen heterogeneity, clinical outcomes will be constrained et al. developed a bicistronic construct to drive CAR expression specific for EGFRvIII and EGFR. Unlike EGFR-specific CAR-T cells, BiTE-CAR efficiently redirected CAR-T cells, recruited untransduced bystander T cells against heterogeneous tumors, and was not toxic to human skin grafts in vivoAnother way to achieve multiple targets is to create bispecific T cell engagers (BiTEs) linking CD3 scFvs and tumor-associated antigens. BiTEs are bispecific antibodies that redirect T cells to target antigen-expressing tumors. Compared with single-target CAR-T cells, BiTE CAR-T cells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive tumors Despite countless researchers working on the clinical translation of CAR-T cells to overcome the unfriendly TIME, there are numerous hard nuts to crack without any clue. Table Figure A.Recently, the rapid advancement of nanomedicine has provided inimitable insights into the security and durability of CAR-T cell therapy. Nanoparticles that achieve targeted delivery and prolong the retention time of carried drugs could better remodel the tumor immunosuppressive environment than traditional drugs in vivo, allowing better infusion timing and dosage optimization to avoid potentially lethal systemic toxicity Figure BRadioactive material is extensively utilized and may have distinct immunomodulatory effects locally and systemically. In addition to boosting local expression of multiple cytokines and promoting vascular normalization, radiotherapy could activate endogenous target antigen-specific immunity to yield complementary benefits, thereby maximizing the effect of CAR-T cells in vivo animal models and in vitro planar cell models, emerging tumor-on-chip platforms integrating microfluidics, tissue engineering, and 3D cell culture have successfully mimicked the key structural and functional properties of TME in vivoet al.Until recently, tumor-on-chip offered a renewed direction for preclinical CAR-T cell research. Compared with conventional CAR-T immunotherapy is revolutionizing the paradigm of cancer therapy. However, unique obstacles posed by solid tumors remain a Gordian knot for CAR-T immunotherapy. The intricate interactions among immune cells, tumor cells, immune molecules, and cytokines form TIME that throttles immune responses and encourages tumor development. By precisely mapping the complex regulatory network of TIME and drawing the optimization strategies, we could provide patients with more effective and tailored CAR-T immunotherapy. To address the hostile TIME and mitigate or even obviate the risk of serious adverse reactions, cutting-edge technologies, such as nanoparticle delivering systems, radionuclide-based molecular imaging, and novel tumor-on-chip platforms are gradually applied for CAR-T immunotherapy. Indeed, the current understanding of TIME is only the tip of an iceberg. Nevertheless, we believe that by progressively regulating the harsh TIME to reshape a friendly microenvironment, CAR-T immunotherapy will produce a more astonishing breakthrough.


The COVID-19 (SARS-CoV-2) pandemic drove acute care surgeons to pivot from long established practice patterns. Early safety concerns regarding increased postoperative complication risk in those with active COVID infection promoted antibiotic-driven non-operative therapy for select conditions ahead of an evidence-base. Our study assesses whether active or recent SARS-CoV-2 positivity increases hospital length of stay (LOS) or postoperative complications following appendectomy.Data were derived from the prospective multi-institutional observational SnapAppy cohort study. This preplanned data analysis assessed consecutive patients aged ≥ 15 years who underwent appendectomy for appendicitis (November 2020–May 2021). Patients were categorized based on SARS-CoV-2 seropositivity: no infection, active infection, and prior infection. Appendectomy method, LOS, and complications were abstracted. The association between SARS-CoV-2 seropositivity and complications was determined using Poisson regression, while the association with LOS was calculated using a quantile regression model.Appendectomy for acute appendicitis was performed in 4047 patients during the second and third European COVID waves. The majority were SARS-CoV-2 uninfected , while 70 (1.7%) were acutely SARS-CoV-2 positive, and 116 (2.8%) reported prior SARS-CoV-2 infection. After confounder adjustment, there was no statistically significant association between SARS-CoV-2 seropositivity and LOS, any complication, or severe complications.During sequential SARS-CoV-2 infection waves, neither active nor prior SARS-CoV-2 infection was associated with prolonged hospital LOS or postoperative complication. Despite early concerns regarding postoperative safety and outcome during active SARS-CoV-2 infection, no such association was noted for those with appendicitis who underwent operative management. Acute appendicitis is one of the most frequent surgical emergencies, and its management is one of the most commonly-performed emergency general surgery procedures –4. WhileThe initial wave of the SARS-CoV-2 (COVID-19) pandemic raised concerns regarding transmission of infection to operating team members during aerosol generating procedures such as airway control, tracheostomy, endoscopy, or laparoscopy. Concomitantly, safety concerns also surfaced during the COVIDSurg study during the initial phase of the pandemic regarding the advisability of undertaking operative management for patients acutely infected with SARS-CoV-2 . Therefo® web application . There were no statistically significant between-cohort differences in body mass index, ASA classification, comorbidities, smoking history, or admission laboratory data. However, the proportion of patients with a respiratory rate above 20 was significantly higher among patients with active and previous SARS-CoV-2 infections compared to patients who had never had SARS-CoV-2  . Relatedly, procedure duration and the crude rate of any or severe complications were also similar across groups. Patients with active SARS-CoV-2 infection demonstrated a longer crude LOS compared to patients without prior SARS-CoV-2 infection  (Table The prevalence of perforation and the time to surgery from admission were similar across groups. Patients with active and prior SARS-CoV-2 infections were significantly more likely to undergo an open surgical procedure (24.3% and 26.7% vs 9.6%, respectively, The treatment of patients with acute surgical emergencies in the context of the COVID-19 pandemic has been challenging throughout different phases of the pandemic—from early knowledge gaps and resource-exhaustion, through concern for excess postoperative morbidity and potential aerosolization of viral particles through laparoscopy, to cancellation of elective surgery and deviation from usual practice patterns, the introduction of population vaccination, and most recently, phased reintroduction of scheduled surgical services –16.The ‘evidence-to-practice gap’ between guideline-based recommendations and widespread adoption and implementation is well chronicled in the surgical literature and a burgeoning focus of implementation science . Under uno untoward consequences for those with active SARS-CoV-2 infection who underwent appendectomy with active infection compared to those without infection, as well as those who recovered from a prior infection [Multinational collaborations rapidly arose to assess outcomes of different therapeutics for those with acute SARS-CoV-2 infection. Some early therapies, such as glucocorticoid administration, remdesivir, monoclonal antibody rescue, and prone position therapy were found to be beneficial when large datasets were interrogated , 29. Othnfection . Since tThe outcomes from this study span 90 days, a sufficient time frame to capture delayed events including hospital readmission for operative domain or pulmonary system failures; these adverse events were not observed. Importantly, our data are different from observations made early in the pandemic. One reason for such differences may be viral evolution, a process that has been repeatedly observed with different variants with some demonstrating enhanced infectivity but less virulence, and vice versa , 35. OthOur prospective time-bound multi-center observational cohort study allowed for a comprehensive defined dataset to be gathered in line with pre-publication, open-access protocols filed with clinical trial repositories. Based on the pre-specified data fields, we did not capture all comorbidities, nor how patients were selected for non-operative versus operative management. Moreover, our data is limited to those with acute appendicitis and may not be applicable to those who required longer or more complex operative procedures, nor those who may require postoperative invasive mechanical ventilation. Similarly, we did not capture SARS-CoV-2 related therapy such as steroids, remdesivir, or the use of non-invasive ventilation or prone position therapy. Relatedly, we did not capture whether patients were ill from their SARS-CoV-2 infection, or asymptomatically infected as existing recommendations addressed such infection in a binary fashion. We were instead interested in assessing outcome across multiple centers for those who underwent operation regardless of how their acute SARS-CoV-2 infection was managed. Indeed, current management leverages some different than what was used during our study period. Additionally, our results present the median effects observed in the study population. We can therefore not eliminate the risk that a particular subgroup, for example frail or elderly patients, might exhibit an increased risk when undergoing surgical management for acute appendicitis with a concomitant SARS-CoV-2 infection. Nevertheless, owing to the age distribution of appendicitis patients, this cohort is relatively small with only 3.5% of patients in this sample being older than 75 and none of them having an active SARS-CoV-2 infection on admission [The current study failed to detect any association between SARS-CoV-2 infection status and post-appendectomy complications or hospital length of stay. This provides evidence that the most common management approach for acute appendicitis—appendectomy—may be safely performed in patients who present with acute or recently recovered SARS-CoV-2 infection.


The public health threat from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to intensify with emerging variants of concern (VOC) aiming to render COVID-19 vaccines/infection-induced antibodies redundant. The SARS-CoV-2 spike protein is responsible for receptor binding and infection of host cells making it a legitimate antibody target. Antibodies mostly target epitopes in the receptor binding domain (RBD). Mutations occurring within epitopes influence antibody specificity and function by altering their 3D architecture. However, the mechanisms by which non-epitope mutations in the RBD influence antibody specificity and function remain a mystery. We used Protein Data Bank (PDB) deposited 3D structures for the original, Beta, Delta, BA.1, and BA.2 RBD proteins in complex with either neutralizing antibodies or Angiotensin-Converting Enzyme 2 (ACE2) to elucidate the structural and mechanistic basis for neutralizing antibody evasion driven by non-epitope amino acid substitutions in the RBD. Since the mechanism behind the extensively reported functional discrepancies between the same antibody when used individually and when used in an antibody cocktail is lacking, we explored the structural basis for this inconsistency. Finally, since SARS-CoV-2 antibodies are viral mutagens, we deciphered determinants for antibody-pressured amino acid substitutions. On the one hand, we show that non-epitope mutations in the RBD domain of SARS-CoV-2 VOC influence the formation of hydrogen bonds in the paratope-epitope interface by repositioning RBD amino-acid sidechains (AASCs). This increases the distance between complementary donor/acceptor atoms on paratope and epitope AASCs leading to weaker or the complete prevention of the formation of hydrogen bonds in the paratope-epitope interface. On the other hand, we show that SARS-CoV-2 VOC employ the same strategy to simultaneously search for complementary donor/acceptor atoms on ACE2 AASCs to form new interactions, potentially favoring increased viral transmission. Additionally, we illustrate that converting the spike protein to an RBD, a deletion mutation, also repositions epitope AASCs and that AASC interactions in the paratope-epitope interface vary when an antibody is used individually versus when utilized as a cocktail with other antibodies. Finally, we show that the process of substituting immunogenic RBD amino acids begins with the repositioning of their AASCs induced by immune/antibody pressure. We show that donor/acceptor atoms from any amino acid can determine cross-reactivity instead, provided they possess and present spatially pairing donor/acceptor atoms. By studying structural alignments for PDB deposited antibody-RBD 3D structures and relating them to published binding and neutralization profiles of the same antibodies, we demonstrate that minor structural alterations such as epitope AASC repositioning have a major impact on antibody effectiveness and, hence, should receive adequate attention given that protein structure dictates protein function.  Protein structures are determined by amino acid sequences and are considered denatured when their biological, chemical, and physical properties are altered due to distortions in their 3D native structures . DistortDonor/acceptor atoms on original RBD and paratope/ACE2 amino-acid sidechains (AASCs) facilitate the formation of hydrogen bonds . HydrogeWe examined several Protein Data Bank (PDB) deposited 3D structures of the original, Beta, Delta, Kappa (a variant of interest), BA1, and BA2 RBD proteins in complex with either antibodies or ACE2. To explore the role of mutation in repositioning RBD AASCs, we obtained 3D structures from the Protein Data Bank (PDB) for the original, Beta, Delta, Kappa, and Omicron (BA1 and BA2) spike or RBD proteins in complex with either SARS-CoV-2 antibodies or ACE2. To assess the generalizability of findings, we obtained PDB 3D structures of viral proteins with functions analogous to the SARS-CoV-2 spike protein which included influenza antibodies in complex with variant hemagglutinin proteins and a Zika/Dengue cross-reactive antibody in complex with either the Zika or Dengue envelope glycoproteins. We also added 3D structures of SARS-CoV-2 antibodies in complex with either the spike or RBD proteins to explore the effect of spike protein truncation on the repositioning of RBD AASCs. Since immunoglobulins are also proteins, we included PDB structures for a Fab in complex with the HIV-gp120 glycoprotein and its parent IgG to explore the generalizability of truncation-induced AASC repositioning. Since induced-fit causes paratope and epitope AASCs to reposition upon binding, we explored the structural implications of induced-fit on the same antibody when used individually or when mixed with other antibodies in antibody cocktails. To undertake this task, we acquired PDB 3D structures for the same RBD in complex with SARS-CoV-2 antibodies either individually or as antibody cocktails. To explore the generalizability of induced-fit on epitope and paratope AASC repositioning, we extended the same analysis to PDB 3D structures for Ebola antibodies in complex with the same glycoprotein protein. http://www.yasara.org/, accessed on 25 October 2022) to display all PDB 3D structures included in the analysis. We analyzed the length of hydrogen bonds because it determines the strength of RBD-antibody/ACE interactions. To analyze the characteristics of hydrogen bonds and highlight the presence of mutation-induced AASC repositioning in the RBD-antibody/ACE2 interface, the original SARS-CoV-2 RBD was structurally aligned with the Beta, Delta, Kappa, BA.1, and BA.2 RBD proteins using MUSTANG [We used YASARA View  /M] × 100%, where M is the largest distance between the same donor/acceptor atoms on paratope AASCs for the same antibody and complementary acceptor/donor atoms on epitope AASCs for the Beta, Delta, Kappa, BA1, and BA2. Analogous to M, N is the donor/acceptor atom distance obtained between the original SARS-CoV-2 variant and the antibody. Antibody effectiveness varies across SARS-CoV-2 VOC ,15,16,17We extended the same analysis to ACE2 in complex with SARS-CoV-2 VOC RBD proteins . We showStructural evidence in support of functional discrepancies between the spike and RBD proteins of the same SARS-CoV-2 variant remains elusive. We structurally aligned spike and RBD proteins belonging to the same SARS-CoV-2 variant in a complex with the same SARS-CoV-2 antibody A–C. InteAntibody-RBD binding can use either the “lock and key” or “handshake” mechanism ,23. The https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614395/pdf/jove-126-56067.pdf, accessed on 25 October 2022) to mimic immune pressure. Although escape SARS-CoV-2 variants are generated when original viruses are exposed to antibody pressure [Epitope characterization normally follows antibody discovery and is essential for vaccine design. Since expensive equipment is required for X-ray 3Dlography or cryo-electron microscopy, one affordable way of characterizing epitopes is through generating escape viruses. This is done by growing viruses in the presence of antibodies 2 exhibited reduced activity, the Fab completely lost activity. These researchers repeated the same experiment but this time using a different antibody and found that the Fab did not lose its binding and neutralization activity relative to the parent immunoglobulin. Therefore, they fairly concluded that antibody valency does not explain the loss of Fab activity [Protein binding and neutralization profiles are highly dependent on their native 3D structures which are determined by amino acid sequences. While it is known that epitope amino acid substitutions influence RBD-antibody/ACE2 interactions, the role of epitope AASC repositioning induced by non-epitope mutations in promoting immune escape and ACE2 binding simultaneously remains unexplored. We show that non-epitope mutations, including truncations, and induced-fit can reposition RBD AASCs leading to the weakening, complete disruption, or establishment of new hydrogen bonds with paratope/ACE2 AASCs. Discordant epitope-paratope/ACE2 interactions facilitated by structural variations might explain inconsistencies in the effectiveness of the same antibodies/ACE2 against SARS-CoV-2 VOC and when antibodies are used in cocktails relative to individual use ,16,25,26activity . Furtheractivity ,32, justactivity , how sevactivity  indicatiVaccines are designed to induce antibodies with spatially positioned acceptor/donor atoms on paratope AASCs to specifically interact with corresponding donor/acceptor atoms on RBD AASCs. Since vaccine-induced antibodies have fixed structures, epitope amino acid substitutions or mutation-induced AASC repositioning in the RBD of VOC could influence the effectiveness of vaccine-elicited antibodies. The spike protein from the original SARS-CoV-2 is still used in current COVID-19 vaccines despite the emergence of VOC with different amino acid sequences and RBD 3D structures. Changes in RDB native 3D structures might explain the consistent reduction in COVID-19 vaccine-induced antibody effectiveness against continuously emerging VOC , underscWaning immunity is believed to cause breakthrough infections while booster COVID-19 vaccine doses have been proposed as one way of increasing the level of waning neutralizing antibodies ,35,36. HFurthermore, AASC repositioning has challenged the notion that amino acid conservation directly translates into maintained binding, and by extension neutralization activity. We have shown that spatially conserved donor/acceptor atoms directly translated into conserved binding and neutralization activity irrespective of the presenting amino acid. Hence, antibodies must be engineered to target conserved RBD donor/acceptor atoms to protect against current and future SARS-CoV-2 VOC. This should be extended to designing universal COVID-19 vaccines. We also showed that AASC repositioning must occur before immune-pressured epitope amino acids are eventually substituted and/or new contacts with ACE2 are established. Identifying immune-pressured AASCs and monitoring trends of donor/acceptor atoms on RBD and ACE2 AASCs will enable modeling of their immune-evasion and transmission capabilities which is essential for pandemic preparedness. Prospective mapping of escape mutations has been explored  concorda


The global pandemic has resulted from the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), causing coronavirus disease 2019 (COVID-19). To control the spread of the pandemic, SARS-CoV-2 vaccines have been developed. Messenger ribonucleic acid (mRNA)-based COVID-19 vaccines have been the most widely used. We present the case of a 65-year-old patient, who was diagnosed with acute disseminated encephalomyelitis, ocular myasthenia gravis, and autoimmune thyroiditis, following his third mRNA COVID-19 vaccination. On admission, the patient showed mild left-sided hemiparesis, contralateral dissociated sensory loss, dizziness, and right-sided deafness. Brain MRI revealed multiple acute inflammatory contrast-enhancing periventricular and brainstem lesions with involvement of vestibulo-cerebellar tract and cochlear nuclei. Despite steroid pulse and intravenous immunoglobulin therapy, clinical symptoms and MRI lesions worsened, and additional signs of ocular myasthenia gravis and elevated but asymptomatic thyroid antibodies developed. After repeated plasma exchange, all clinical symptoms resolved. This is, to the best of our knowledge, the first case report of multiple autoimmune syndromes triggered by COVID-19 vaccination. The rare occurrence of such treatable autoimmune complications should not question the importance of vaccination programs during the COVID-19 pandemic. Besides specific vaccine complications (such as vaccine-induced thrombotic thrombocytopenia after vector-based COVID-19 vaccines), the association between new-onset autoimmune disease and vaccination could not be established yet, most likely due to low incidence. However, cases of vaccine-triggered autoimmune phenomena have been reported, and different mechanisms have been suggested  . Up to tWe report a patient, who developed all of these three autoimmune disorders shortly after being vaccinated for SARS-CoV-2.A 65-year-old male patient was referred for the subacute onset of paresis of the left arm, followed by loss of pain and temperature sensation on the right side of the body, as well as right-sided deafness with vertigo, 3 days after receiving the third dose of the mRNA-based Pfizer-BioNTech COVID-19 vaccine, without any acute allergic reactions.3, respectively), while protein and glucose levels were within reference ranges. Oligoclonal banding was not present in both events. Confirming the diagnosis of ADEM, stereotactic brain biopsy showed typical perivenous inflammatory demyelination. The patient fully recovered under intravenous high-dose corticosteroids both times. A Follow-up MRI of the brain solely showed minor periventricular white matter sequelae; spinal cord lesions were completely resolved.His medical history was relevant for multiphasic ADEM, with two previous clinical episodes, 10 and 11 years prior to this admission. In the first event, the patient manifested mild right-sided sensorimotor hemiparesis and Th11/Th12 paraplegia with urinary incontinence in the second event. At that time, cerebral and spinal cord magnetic resonance imaging (MRI) showed multiple T2-weighted hyperintense lesions involving supratentorial areas and, respectively, the spinal cord, all with T1 contrast enhancement. Cerebrospinal fluid (CSF) showed lymphocytic pleocytosis immune disorders or neurological diseases were denied.3), while protein and glucose levels were normal. Oligoclonal bands were searched in serum and CSF by isoelectric focusing, with negative results (type 1 pattern). Screening for bacterial, viral, and fungal neuro infections was negative. Tests were also negative for antibodies targeting antigens associated with demyelinating disorders of the central nervous system (myelin oligodendrocyte protein and aquaporin-4), as well as onconeural-, and anti-ganglioside antibodies. The CSF cytological analysis excluded circulating malignant cells. Biochemical serum markers for sarcoidosis (angiotensin-converting enzyme and soluble interleukin-2 receptor) were unremarkable, and CD4/CD8 ratio in CSF and bronchoalveolar lavage were not elevated. Interleukin-10 in CSF was normal, and chemokine CXC ligand 13 slightly increased. Complete blood count and markers of systemic autoimmunity  were normal/negative.On the current admission, the patient was alert and oriented and presented mild left-sided hemiparesis (MRC 4/5) with contralateral dissociated sensory loss, and right-sided vestibulocochlear nerve deficit. Brain MRI revealed acute inflammatory gadolinium-enhancing lesions on the right cerebellar peduncle, as well as pons and medulla oblongata. CSF analysis showed lymphocytic pleocytosis (54 cells/mmThe patient was treated with high-dose intravenous methylprednisolone (1 g daily) for five days. Due to non-response, intravenous immunoglobulin therapy with a total dose of 2 g/kg, fractionated in 5 days, was started. Rapid clinical deterioration with the development of severe left-sided hemiparesis (MRC 2/5), hemiataxia, and major difficulties to walk was accompanied by new periventricular and progressive infratentorial and upper cervical spinal cord contrast-enhancing lesions on follow-up MRI .Furthermore, the patient developed fluctuating binocular horizontal diplopia and ptosis of the right eye, with worsened toward the end of the day. Diagnostic pyridostigmine  did not improve ocular symptoms within a 1-h observation period. Immunologic testing showed elevated anti-AChR antibody titers . Autoantibodies against muscle-specific kinase and titin were negative. No thymoma was detected on chest computed tomography (CT). The patient was newly diagnosed with ocular myasthenia gravis and started on oral pyridostigmine 90 mg twice a day.On further laboratory investigations, positive anti-thyroglobulin antibodies , anti-thyroid peroxidase antibodies , and anti-thyroid stimulating hormone (TSH) receptor autoantibodies  were detected. Thyroid function , however, was normal, with no past medical history of thyroid disease. Thyroid ultrasonography was normal. Subacute thyroiditis was first diagnosed based on the patient's laboratory findings.Considering the clinical worsening and the development of multiple autoimmune disorders despite treatment with corticosteroids and intravenous immunoglobulins, plasmapheresis was indicated. Seven plasma exchanges were conducted within 13 days. The hemiparesis improved, and the patient regained walking ability. Follow-up MRI brain and spinal cord scans after the third plasma exchange already revealed reduced lesion size and contrast enhancement. Ocular myasthenic symptoms resolved completely. Anti-thyroid and anti-AChR autoantibodies were no longer detectable. The patient was referred to rehabilitation, where clinical status further improved until 1 month after plasma exchange (three months after onset).Our patient met the diagnostic criteria for ADEM set by the International Pediatric MS Study Group , and altThe rare occurrence and favorable outcomes of vaccination-triggered ADEM, myasthenia gravis, and subacute thyroiditis, as well as the fact that severe (multiple) autoimmune syndromes may also occur after COVID-19 infection –17, do nThe original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.KP conducted the literature search and drafted the first version of the manuscript. SP and UZ made critical revisions. All authors approved the submitted version of the 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.


We thus identified 3619 circQTLs that were also trans-eQTLs and constructed 19,804 circQTL-circRNA-trans-eGene regulatory axes. We conducted two different types of approaches, mediation and partial correlation tests (MPT), to determine the axes with mediation effects of circQTLs on trans-eGene expression through circRNA expression. We showed that the mediation effects of the circQTLs (trans-eQTLs) on circRNA expression were positively correlated with the magnitude of circRNA-trans-eGene correlation of expression profile. The positive correlation became more significant after adjustment for the circQTLs. Of the 19,804 axes, 8103 passed MPT. Meanwhile, we performed causal inference test (CIT) and identified 2070 circQTL-trans-eGene-ASD diagnosis propagation paths. We showed that the CIT-passing genes were significantly enriched for ASD risk genes, genes encoding postsynaptic density proteins, and other ASD-relevant genes, supporting the relevance of the CIT-passing genes to ASD pathophysiology. Integration of MPT- and CIT-passing axes further constructed 352 circQTL-circRNA-trans-eGene-ASD diagnosis propagation paths, wherein the circRNA-trans-eGene axes may act as causal mediators for the circQTL-ASD diagnosis associations. These analyses were also successfully applied to an independent dataset from schizophrenia brains. Collectively, this study provided the first framework for systematically investigating trans-genetic effects of circQTLs and inferring the corresponding causal relations in diseases. The identified circQTL-circRNA-trans-eGene regulatory interactions, particularly the internal modules that were previously implicated in the examined disorders, also provided a helpful dataset for further investigating causative biology and cryptic regulatory mechanisms underlying the neuropsychiatric diseases.Genetic risk variants and transcriptional expression changes in autism spectrum disorder (ASD) were widely investigated, but their causal relationship remains largely unknown. Circular RNAs (circRNAs) are abundant in brain and often serve as upstream regulators of mRNAs. By integrating RNA-sequencing with genotype data from autistic brains, we assessed expression quantitative trait loci of circRNAs (circQTLs) that  Autism spectrum disorder (ASD) is a highly pervasive neurodevelopmental and heritable complex disorder, which is characterized by limited social communication, restricted and repetitive interests or behaviors . Previoucis-eQTL) or distant (trans-eQTL) genes, providing a connection between genetics and regulatory mechanisms of gene expression [cis-eQTL SNPs often reside within or close to promoter regions of their nearby genes (cis-eGenes) and mediate gene expression through directly affecting the corresponding transcription factor binding process [cis-eQTLs, the regulatory mechanisms underlying trans-eQTLs are less known because of the relatively weaker effects on gene expression for trans-actions than for cis-actions and the multiple-testing burden [cis-effects on gene expression, trans-genetic effects were reported to explain more than two times the variability in gene expression [trans-effect analyses may miss in cis-effect analyses [trans-eQTL effects [trans-eQTLs is that trans-eQTLs indirectly affect the expression of distant genes (trans-eGenes) through regulating the expression of genes near the trans-eQTLs (cis-eGenes) [trans-eQTL is also a cis-eQTLs, wherein the corresponding cis-eGene acts as a mediator of the trans-eQTL. Investigation of trans-genetic effects and the causal inference for the corresponding eQTL-associated networks will allow us to better understand complex trait genetics.By integrating genome-wide genotyping with transcriptome profiling, expression quantitative trait loci (eQTL) analysis identifies single nucleotide polymorphisms (SNPs) that affect the expression levels of local  . In suchtrans through mediating the activities of miRNAs or RNA binding proteins (RBPs), with the common miRNA/RBP target sites of circRNAs and the target genes [trans-eGenes. If this is possible, we may generate circQTL-circRNA-trans-eGene propagation paths in autistic brains and shed light on the functional consequences of the genetic loci and the etiology of this complex disease.CircRNAs are an emerging class of RNAs, which are formed by pre-mRNA back-splicing with a structure of covalently closed loops and endogenously expressed as single-strand, non-polyadenylated circular molecules , 16. Altet genes , 16. Hentrans-eQTLs and infer cis-mediators of the identified circQTLs  and causal effects between circQTLs and ASD diagnosis. Our findings will help to unveil the gap between genetic variation and phenotypic changes in ASD. The designed framework was also applied to the replication analysis of circQTLs (trans-QTLs) in schizophrenia (SCZ), supporting the framework for future analyses of eQTLs in neuropsychiatric disease or other complex traits.There are two challenges to achieve the abovementioned goals. First, eQTL analysis requires transcriptomic and genomic data from the same samples with a large sample size. Second, large-scale circRNA study is often hampered by the limitation of the intrinsic circRNA characteristic of lacking polyadenylated tails. The newly released Synapse database , 18 comphttp://www.synapse.org) with permission under the accession number syn4587609. The examined circRNAs (1060 circRNAs) were extracted from our previous study [The rRNA-depleted paired-end RNA-seq data  and the us study . The genus study  upon reqP values regardless of the distribution of circRNA expression and statistical test, a permutation test was conducted to estimate the empirical P value (empP) for each uncorrected circQTL SNP by performing 10,000 permutations with randomly sampling labels of the circRNA expression matrix. The empP value for each uncorrected circQTL P value (PcircQTL) was calculated as empP = q value for each circRNA using Storey’s method [P values of all circRNAs. A circRNA was defined as a circQTL-containing circRNA if the q value of the circRNA was less than 0.05. For each circQTL-containing circRNA, only the associated circQTLs with empP < 0.005 were retained. We further performed conditional and joint (COJO) analysis based on GCTA-COJO [We limited our analysis to SNPs in ±200 kb nucleotides of each back-splice site and evaluated circQTLs by testing the correlations between the imputed genotype dosages and covariate-adjusted circRNA expression using Matrix eQTL with an additive linear model   value for each uncorrected circQTL SNP by performing 10,000 permutations with randomly sampling labels of the expression matrix of remote genes. The trans-effect of a circQTL on the expression of a trans-eGene was determined if uncorrected trans-eQTL P value was less than 10−4 and empP value was less than 0.005 simultaneously (Supplementary Methods). The trans-effects of non-circQTLs on the expression of remote genes were also examined using the same procedures mentioned above.We examined whether a circQTLs was also a https://cran.r-project.org/web/packages/mediation/index.html (Supplementary Methods). For the partial correlation analysis [trans-eGenes with controlling for the circQTL (trans-eQTL) SNPs. The residuals after adjusting for the circQTLs would be significantly correlated (post-adjustment P < 0.05), if there was a causal relationship between circRNA expression and trans-eGene expression . The CIT package [https://cran.r-project.org/web/packages/cit/index.html. A CIT-passing circQTL-trans-eGene pair (CIT P < 0.05 and FDR (q) <0.05) represents that the circQTL has a causal effect on the diagnosis status (ASD vs. non-ASD or SCZ vs. non-SCZ) through the trans-eGene expression (Supplementary Methods). The FDR (q) values were further estimated using CIT-provided permutation test with default parameters.We conducted mediations analysis using the mediation package  downloadanalysis , we calc package  was downMBNL3 via in vitro perturbation of circHOMER1a or miR-641 in normal human astrocyte cells (Supplementary Methods). The primer, Dsi-RNA, miRNA mimic, and miRNA inhibitor sequences used in this study were listed in Supplementary Table We performed qRT-PCR analyses to examine the correlations between the expression of circHOMER1a, miR-641, and trans-eQTLs affecting the expression of distant genes (trans-eGenes)  Fig. . We colles) Fig. . The exaes) Fig.  were extons Fig. . To deteP values larger than 0.05 (Supplementary Methods). This result suggests that circQTLs located in the flanking sequences of back-splice sites may contribute to circRNA formation.We then studied the relationship between circQTLs and circRNA formation. We found that the majority of the independent circQTLs  were identified to be also trans-eQTLs affecting the expression of 8270 trans-eGenes , suggesting that circQTLs were more likely to be also trans-eQTLs as compared with non-circQTLs.We next examined whether the identified circQTLs were also TLs Fig. . We alsotrans-effect of circQTLs on the expression of trans-eGenes. In addition to the direct effect of circQTLs on trans-eGene expression, we hypothesized that in some cases the expression of trans-eGenes were indirectly regulated through the expression of circRNAs near the circQTLs  axes passed the mediation test , the proportion of mediation of the circQTL-trans-eGene association by the circRNAs was more than 30% , whereas such a percentage was significantly reduced from 39% to 6% if the circRNA expression and trans-eGene expression were uncorrelated  on circRNA expression were positively correlated with the magnitude of circRNA-trans-eGene correlation of expression profile, implying a causal relationship between circRNA expression and trans-eGene expression. We thus speculated that there was a residual correlation between circRNA expression and trans-eGene expression after repressing out the variance attributable to the circQTL SNP. We conducted a partial correlation analysis by performing Spearman correlation coefficient between circRNA expression and trans-eGene expression with adjustment for the circQTLs. The residuals after removing the variance due to the circQTLs would be uncorrelated, if the SNPs had independent associations with circRNA expression and trans-eGene expression  estimated by the mediation test were more strongly correlated with the post-adjustment P values than with the correlation P values before SNP adjustment , were in the cases wherein the circQTL-trans-eGene associations were mediated by the expression of circRNAs. Considering all circQTLs  on circRNA expression were positively correlated with the magnitude of circRNA-trans-eGene correlation of expression profile, regardless of whether the circQTL SNPs were controlled . Although none of the circQTLs was significantly ASD-related GWAS sites (P < 5 × 10−8), 168 circQTLs were located within the ASD-associated SNP clumps. Regarding the circRNAs of the identified circQTL-circRNA pairs, the back-splice sites (donor or acceptor sites) of 106 circRNAs overlapped with the ASD-associated SNP clumps  have identified numerous high-confidence genetic risk loci for ASD . To deteS-FUSION  (“FUSIONtrans-effects of circQTLs on trans-eGene expression might explain the association between the circQTLs and diagnosis status (ASD vs. non-ASD) and infer the direction of association between circQTLs, trans-eGene expression, and ASD diagnosis  involved in the 2070 circQTL-trans-eGene pairs may be implicated in ASD. We examined enrichment analysis for the genes previously implicated in ASD from Simons Foundation Autism Research Institutive (SFARI) [trans-eGene pairs in the SCZ samples  , the hig (SFARI) , and Aut (SFARI) . Indeed, (SFARI) , genes w (SFARI) , RBFOX1  (SFARI) , and ELA (SFARI) ), and di (SFARI)  that wer (SFARI) . The CIT (SFARI) ) compare (SFARI) . These otrans-eGene axes with significant mediation effects of circQTLs on trans-eGene expression through circRNA expression , 10 circRNAs, and 118 trans-eGenes  were associated with the 101 ASD-relevant genes. Particularly, of the 101 ASD-relevant genes, 47 were DEGs in ASD and 11 were SFARI genes (five were top SFARI genes), suggesting the regulatory role of the corresponding circQTL-circRNA-trans-eGene axes in ASD brain. Similarly, in SCZ, 263 circQTL-circRNA-trans-eGene-SCZ diagnosis propagation paths that passed both CIT and MPT were conducted  had identified 8103 circQTL-circRNA-ion Fig. . By inteirs Fig. , left wixes Fig. , middle,trans-eGene axes -seq data-supported RNA interactomes (extracted from ENCORI [trans-eGene pairs (Supplementary Methods). Second, we extracted the miRNA expression data [trans-eGene axes were retained, if the circRNA expression was significantly correlated with the trans-eGene expression (P < 0.05 by Spearman’s correlation test). Third, we reevaluated the above correlations by performing a partial correlation analysis to control for the miRNA expression. If the miRNAs acted as a mediator for the circRNA-trans-eGene associations, the residuals after adjusting for the miRNA expression would be unrelated. We thus identified 117 circRNA-miRNA-trans-eGene axes passing the abovementioned partial correlation test, which were associated with 26 circRNAs (4 were DE-circRNAs or involved in the DE-circRNA module), 71 miRNAs (6 DE-miRNAs), and 68 trans-eGenes (49 ASD-relevant genes)  to searRTarBase  or DIANARTarBase  , which was previously demonstrated to play an important regulatory role in synaptic gene expression and cognitive flexibility . Spearmaher Fig. . We examNL3 Fig. b–f. The 641 Fig. , reflectcis-regulated expression of nearby circRNAs and trans-regulated expression of distant genes simultaneously. We asked whether in some cases the effects of trans-eQTLs on trans-eGene expression were mediated by the circRNA expression. We first performed the mediation analysis (a linear model) and suggested that a considerable percentage of the identified circQTL-trans-eGene associations were mediated by the corresponding circRNA expression. We found that the mediation effects of the circQTLs (trans-eQTLs) on circRNA expression significantly increased with increasing significance levels of the correlation of expression profile between circRNAs and trans-eGenes  to explain the relationships between an eQTL, an eGene, and a trait , 30. Forsis Fig. . AccordiP < 5 × 10−8) in the SCZ GWAS, only 93 significantly ASD-related SNPs were detected in the ASD GWAS  on trans-eGene expression, in which the corresponding trans-eGenes included 21 SFARI genes and 14 DEGs in ASD  explained by a circQTL for each DEG, we observed that the r2 values ranged from 0.14 to 0.38  and the miRNAs may act as mediators for the circRNA-trans-eGene associations . One of the 12 axes (circHOMER1a-miR-641-MBNL3) involved a psychiatric disease-associated circRNA (circHOMER1a), which was previously reported to be important in regulating synaptic gene expression and cognitive flexibility [MBNL3 and the mediation effect of miR-641 on the circHOMER1a-miR-641-MBNL3 association. Such a correlation pattern is similar to the previously validated interactions between circCSNK1G3, miR-181b/d, and CDK1/CDC25A in PC-3 cells [MBNL3 is known as a splicing factor, which can regulate numerous alternative polyadenylation events [Using the miRNA expression data from the same samples examined in this analysis, we identified 12 circQTL-circRNA-miRNA-ths Fig. , whereinxibility . Spearma-3 cells . While t-3 cells , 16, the-3 cells , 45. Of n events ; meanwhin events . The funtrans-genetic effects of circQTLs and inferring the corresponding causal relations in diseases. Our replication analysis in SCZ samples also supports the potential of the provided framework for future investigation of eQTL-trait relations in other complex diseases. Integration of the relevance of the individual internal modules  of the identified axes to ASD (or SCZ) provides a helpful dataset for further investigating the cryptic regulatory mechanisms underlying these neuropsychiatric diseases (Supplementary Fig. According to a large brain sample size of both RNA-seq data and the corresponding genotype data from ASD cases and controls, this study is the first report, to the best of our knowledge, for systematically investigating Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8


Sick leave due to musculoskeletal pain is common in the workforce. Time use in physical behaviors at work such as sitting, standing, low- (LIPA) and moderate-to-vigorous physical activity (MVPA) may impact on sick leave due to pain. However, studies addressing this relationship using technical measures of physical behaviors are scarce. The aim was to investigate the association between time-use compositions of physical behavior at work and sick leave trajectories due to musculoskeletal pain over one year.We analyzed data of 981 workers in a Danish cohort (DPHACTO 2012-2014). We assessed physical behaviors at work at baseline using thigh-worn accelerometers, and classified behaviors at work as sitting, standing, LIPA, and MVPA. Over 1 year follow-up, workers reported sick leave days due to musculoskeletal pain using text messages at 4-week intervals (14 waves). We used Latent class growth analysis to distinguish sub-groups with different trajectories of sick leave. We analyzed associations between time-use in physical behaviors and sick leave trajectories using multinomial regression analysis with adjustment for age, gender, BMI, smoking, and accelerometry-measured physical activity during leisure. Compositional data analysis was used to account for the co-dependency of different behaviors.We identified four distinct trajectories of sick leave due to pain over one year as follows: no sick days , few days-increasing trajectory (19%), some days-decreasing trajectory (3%), and some days-increasing trajectory (2%). Spending more time in sitting relative to the other behaviors was negatively associated with few days-increasing trajectory of sick leave (p > 0.001), while time in LIPA was positively associated with some days-increasing trajectory of sick leave (p = 0.001). Reallocating 60 min/day from sitting to other behaviors at work predicted a 22% increased likelihood of few days-increasing trajectory of sick-leave. In contrast, reallocating 30 min/day from LIPA to other behaviors at work predicted a 57% decreased likelihood for some days-increasing trajectory.We found that compositions with more sitting relative to the other behaviors had lower odds for the trajectory with increasing sick leave due to pain, while compositions with more LIPA had higher odds. This may have implications for prevention of pain-related sick leave in workers.


Social relationships are crucial for well-being of older adults with cognitive impairment (CI), however, evidence is lacking on how social relationships may influence symptom experience and functioning among those living with CI. This study aimed to identify subgroups of older adults with CI with distinct symptom and functioning profiles and to examine the association between latent class membership and social relationships. The sample included 927 older adults who were screened as having moderate or severe CI from wave 2 of the National Social Life, Health and Aging Project. Symptom  and functioning  variables were used to identify subgroups. Latent profile analyses identified five distinct groups: most (51.7%) belonged to the low-symptom-high-functioning; few (7%) belonged to the high-symptom-low-functioning; while two groups had average symptom burden, one group (25%) had frequent urinary incontinence and normal daily functioning (poor-urinary-functioning), and the other group (5%) had normal urinary functioning and the worst daily functioning (worst-daily-functioning); interestingly, 13% belonged to high-symptom-normal-functioning group. Multinomial logistic regression modeling showed that, among social networks, support, strain, and engagement, members in groups with worse symptom burden and daily functioning were significantly more likely to have social strain, after adjusting for covariates . Only severe CI was associated with worst-daily-functioning . Interventions that ameliorate social strain may benefit symptom management and promote independent daily living among older adults with CI.