The virus responsible for the coronavirus viral pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Emerging SARS-CoV-2 variants caused by distinctive mutations within the viral spike glycoprotein of SARS-CoV-2 are considered the cause for the rapid spread of the disease and make it challenging to treat SARS-CoV-2. The manufacturing of appropriate efficient vaccines and therapeutics is the only option to combat this pandemic. Nanomedicine has enabled the delivery of nucleic acids and protein-based vaccines to antigen-presenting cells to produce protective immunity against the coronavirus. Nucleic acid-based vaccines, particularly mRNA nanotechnology vaccines, are the best prevention option against the SARS-CoV-2 pandemic worldwide, and they are effective against the novel coronavirus and its multiple variants. This review will report on progress made thus far with SARS-CoV-2 vaccines and beyond employing nanotechnology-based nucleic acid vaccine approaches. Coronaries family . No. No63]. There are currently two types of nanovaccines: protective (prophylactic) and therapeutic nanovaccines . ProtectNanoparticles can present antigens to effector cells such as macrophages and dendritic cells and modulate the uptake of antigens. Furthermore, they are highly degradable and easy to synthesise and manufacture, as the mature mRNA manufacturing process and formulation platform can produce various vaccines within very short periods . NanovacThe mRNA nanotech SARS-CoV-2 vaccine candidates use mRNA encoding the full-length spike protein of SARS-CoV-2 to elicit protective immunity against coronaviruses. Nanotech DNA candidates for the SARS-CoV-2 vaccine make use of a small fragment of bacterial DNA plasmid that encodes the virus' spike protein  and nanoex vivo loading of dendritic cell (labour intensive and expensive) [NCT00833781, NCT00381212, and NCT00672191) and bare viral antigenic mRNA (NCT02413645). A phase 1 study (NCT05414786) is currently recruiting participants in South Africa and Rwanda to evaluate the safety and immunogenicity of the eOD-GT8 60mer mRNA vaccine (mRNA-1644). Currently, on the clinical trial website, there are 7 HIV clinical trials for the HIV vaccine based on mRNA technology. Among numerous studies, nanoformulations  are widely utilised in the delivery of HIV mRNA vaccine delivery [According to data from The Joint United Nations Programme on HIV/AIDS (UNAIDS), there were approximately 38.4 million HIV-positive individuals worldwide in 2021 . Currentpensive) . Clinicadelivery .+ T-cells [NCT02410733) [The mRNA cancer vaccine has been proposed to be able to take the place of other traditional vaccination platforms for cancer immunotherapy due to its high potency, safe administration, quick development potential, and affordable production . The tum T-cells . Nanomat T-cells . The nan T-cells . The A l2410733) . The dis2410733) . There a2410733) . To impr2410733) .In the immediate aftermath of the SARS-CoV-2 pandemic, nanovaccines may be effective in combatting many other infectious diseases, such as the Middle East respiratory syndrome (MERS-CoV), HIV, malaria, and Zika virus, which still require effective and affordable vaccines. In the future, we are more likely to see novel nanotechnologies being used to reformulate old vaccines since nanocarrier-based delivery systems can get the vaccine to target areas, unlike conventional vaccines. The future epidemic is more likely to be confronted with new nanotechnologies due to advances in the field of nanotechnology. Nanovaccines have thus far presented themselves as effective and best to use to tackle this pandemic since there is an urgent need to develop effective and safe vaccines and therapeutics against the rising virus variants. They have demonstrated high efficacy and minimal life-threatening side effects. The greatest prophylactic approach against the SARS-CoV-2 pandemic has been nucleic acid-based vaccines, notably mRNA nanotech vaccines, which are effective against both novel coronavirus and its numerous variants. These nucleic acid-based vaccines are currently being administered worldwide in the fight against SARS-CoV-2. Nanovaccines have presented more advantages compared to traditional vaccines in the fight against SARS-CoV-2.


Telehealth has the potential to improve access to and quality of care for low-income patients with chronic conditions such as diabetes -4. This Our study population included Medicaid-enrolled patients with a diagnosis of diabetes, aged 18-64 years, who were attributed to the C3 ACO, had at least 1 primary care visit to a C3 FQHC within the last 18 months, and otherwise met Healthcare Effectiveness Data and Information Set (HEDIS) denominator criteria for patients with diabetes . Our data source was the 2019-2021 Electronic Data Warehouse, which stores electronic health record (EHR) and claims data from C3; this included data from 11 FQHCs after excluding FQHCs with invalid EHR telehealth identifiers.Study outcomes included three quality-of-care diabetes HEDIS measures  and a primary care engagement measure (number of FQHC visits). Our treatment group included FQHCs with high telehealth use during COVID-19 , and our comparison group included FQHCs with lower telehealth use during COVID-19 . Telehealth included real-time video and audio-only visits delivered directly to remote patients.Our unit of analysis was the patient-month. We used a difference-in-differences approach with linear probability models and negative binomial models to examine changes in patient-level outcomes for patients at high- versus low-telehealth FQHCs before (March 2019-February 2020) versus after (April 2020-March 2021) the rapid adoption of telehealth. All models adjusted for patient age, sex, and clinical risk score, applied month and FQHC fixed effects, used robust variance estimators, and applied inverse probability of treatment weights based on propensity scores to balance on 16 patient-level baseline covariates.P values were 2-tailed, and statistical significance was set at P=.05. Analyses were performed using Stata 17.0 (StataCorp).Boston University’s institutional review board deemed the study exempt because we used secondary deidentified data.P<.001) increase in rates of annual retinal exams and a relative rate increase of 1.5  in the number of visits among patients with diabetes, although overall visit rates fell in both groups during COVID-19. Telehealth was further associated with a 5.6 percentage point decrease  in the HEDIS blood pressure control measure and with no statistical change in the blood glucose control measure (P=.97).Of the 11,503 diabetic patient-months included in the study, the mean age was 48.1 years and 56.3% (n=6474) were female, with 27.3% (n=3142) being non-Hispanic White, 10.7% (n=1235) being non-Hispanic Black, and 53.4% (n=6139) being Hispanic based on self-reported race/ethnicity . High teThis study indicates that high FQHC-level telehealth use was associated with more sustained engagement in care for patients with diabetes at FQHCs in Massachusetts, with mixed effects on quality of care measures. Telehealth availability likely helped to partially mitigate the overall decrease in visit rates during COVID-19 by improving care access, while telehealth visits may have also provided the opportunity to encourage patients to return for in-person retinal screenings . In contThe results have important implications. First, telehealth has the potential to improve some measured quality, which suggests that incorporating telehealth into care delivery models and supporting telehealth sustainability through telehealth reimbursement may benefit patients with diabetes. Second, providers must simultaneously consider how to best adapt quality measures and underlying data reporting to ensure that quality-reliant payment models validly capture the quality of care under new delivery modalities, as measures such as blood pressure control may not be validly captured during telehealth encounters.This research is not without limitations. Limitations include potential measurement error in the data, unmeasured confounding due to simultaneous implementation of telehealth and other service capacity changes during COVID-19, lack of subgroup analyses that explore heterogeneity in effects, and FQHC-level assignment of exposure. Nonetheless, based on this study, Medicaid reimbursement policies and care delivery models that support the use of telehealth as part of chronic disease care may be associated with better care engagement for low-income patients with diabetes.


Cell, Chien et al.1 published their new findings on severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) penetrating through the mucosal barrier of respiratory by hijacking the cilia and microvilli of nasal epithelial cells, and elucidated the specific mechanism in detail using electron microscopy as well as phosphoproteomics. This study fully explained the way of SARS-CoV-2 breaking through the mucus barrier to infect nasal epithelial cells, and provided a novel direction for the blocking treatment of respiratory tract infections.Recently in 3 Upper airway is the major site of replication for SARS-CoV-2 and variant of concerns (VOCs) such as Omicron. Taking the nasal airway as an example, it is primarily composed of ciliated, basal, and mucus-secreting goblet cells. Epithelial cells are first covered by the periciliary layer (PCL), which contains mucin to prevent viral invasion. There is an additional mucus layer above the PCL, which traps pathogens and other particles preventing them from reaching the lungs.5 The mechanism by which SARS-CoV-2 breaks through the natural PCL barrier remains unclear.The culprit of coronavirus disease 2019 (COVID-19) pandemic, SARS-CoV-2, binds to angiotensin-converting enzyme 2 (ACE2) on host cell membrane with its spike protein (SP). The SP is then cleaved by host transmembrane serine protease 2 (TMPRSS2). Conformational changes of spike protein assist in bringing viral membrane closer to host cell membrane and subsequently facilitating their fusion, then viral genome is released into the host cell for replication.Chien et al. aimed to determine the SARS-CoV-2 entry mechanism. To achieve this aim, they cultured primary human nasal epithelial cells (HNEs) in air-liquid interface (ALI) to induce the differentiation into nasal epithelial organoids. The authors inoculated SARS-CoV-2 D614G to nasal epithelial organoids at gradient MOI . Immunofluorescence results showed that ciliated epithelial cells were the main target cells of infection. ACE2 and TMPRSS2 on motile cilia mediated viral invasion. More importantly, a small number (~3%) of ciliated epithelial cells were infected at early stage (~24 h post-infection (hpi)), while infected HNEs surged to ~80% at 48 hpi. This phenomenon suggested that SARS-CoV-2 infection of airway epithelial cells may occur in a two-step process.Further experiments verified the two-step approach of infection. Treatment of HNEs with StcE, a kind of selective protease capable of disrupting mucin network, promoted initial viral invasion. This result demonstrated that mucin layer efficiently protects ALI-cultured HNEs from SARS-CoV-2 infection, which explains the delay of viral spread. Immunofluorescence and transmission electron microscopy (TEM) also revealed that virus particles were attached to cilia at 6 hpi, and 6% of cilia-attached viral particles were submerged in the PCL. Cilia depletion, using CEP83 shRNA, significantly inhibited SARS-CoV-2, respiratory syncytial virus (RSV), and human parainfluenza virus (PIV) infections. Blocking the retrograde trafficking of ciliary dynein, with the inhibitor Ciliobrevin D also effectively reduced positive ratio of ciliated cells at 48 hpi. These results suggested that cilia assisted SARS-CoV-2 in breaking through the mucosal barrier and invading the nasal epithelial cells.Chien et al. further investigated the SARS-CoV-2 infection surge by monitoring virus replication and efflux at 24 and 48 hpi using immunofluorescence, TEM, and scanning electron microscope (SEM). Fluorescently labelled N protein was observed in cytoplasm at 6 hpi. From 24 hpi onwards, fluorescently labelled viral S and N protein colocalized with phalloidin labelled microvilli. A large number of newly generated virus particles accumulated in microvilli. Some large vesicles in cytoplasm, containing multiple progeny virus, were also driven near the microvilli. Further observations revealed that SARS-CoV-2 infection induced formation of dome-like alienated structures of microvilli, as well as a significant increase in highly extended microvilli. The phosphorylation level of the actin-binding protein ezrin (EZR), which is involved in microvilli formation, and the protein level of the microvilli core protein, EBP50, were significantly increased after infection. Before and after SARS-CoV-2 infection, using inhibitor NSC-668394 and SB-633825 targeting EZR and LOK/STK10 to treat HNEs, respectively, it was found that the inhibitor pretreatment had no effect. However, inhibitors administration at 18 hpi significantly reduced the number of infected cells at 48 hpi. It was concluded that following SARS-CoV-2 infection, the associated mediators involved in microvilli assembly process are over-activated, resulting in microvilli elongation and branching. Viral progeny shed by means of the alienated microvilli.Chien et al. then cultured primary HNEs derived from patients with primary ciliary dyskinesia (PCD) and observed that at the later stage SARS-CoV-2 only infected localized areas rather than full coverage, forming positive plaques. The mucus flow triggered by external force increased the infected area and number of positive cells. This showed that mucus flow, based on ciliary beating, is important for the spread of viral progeny. To investigate the molecular mechanism by which SARS-CoV-2 infection affects microvilli biogenesis, Chien et al. used global phosphoproteomics and kinase set enrichment analysis (KSEA) to identify 72 over-activated kinases. Among them, the kinases involved in the regulation of microvilli and cytoskeleton were significantly activated, such as PAK1/4, AKT serine/threonine kinase (AKT1/2), mitogen-activated protein kinase P38 Alpha (p38), mitogen-activated protein kinase 3 (ERK1), and Rho-associated coiled-coil containing protein kinase 1 (ROCK1). Treatment of HNEs with PAK1/4 kinase inhibitor significantly reduced EZR phosphorylation in microvilli and effectively reduced infected ratio at 48 hpi. It has been suggested that PAK1/4 could be therapeutic targets for blocking SARS-CoV-2 spreading. The authors also compared the spatial and temporal infection patterns of SARS-CoV-2 and other VOCs in nasal epithelial organoids. In the Omicron group, 40% infected cells were observed at 24 hpi, which significantly advanced the transmission period of the viral progeny.During natural evolution, humans have formed three immune barriers against pathogens, such as viruses and bacteria. The emergence of SARS-CoV-2 poses a severe challenge to the host’s antiviral immune function. Although there have been many reports on the mechanism of SARS-CoV-2 replication within host cells, the mechanism of SARS-CoV-2 penetrating respiratory mucus barrier remains unclear.This question was adequately addressed by Chien et al. using ALI-cultured nasal epithelial organoids for SARS-CoV-2 challenge. Their results showed that initially, only a small number of SARS-CoV-2 could penetrate the PCL through the airway cilia for efficient infection in trace amounts. Subsequently, SARS-CoV-2 hijacks the microvilli of infected cells to rearrange by promoting the activation of multiple kinases such as PAK1/4. Progeny virus exits via microvilli and spreads laterally to other areas via mucociliary transport (Fig. Chien et al. have revealed the specific steps and molecular mechanism of SARS-CoV-2 breaking the mucosal barrier at the early stage of airway infection through solid data. It is not difficult to find that the core of SARS-CoV-2 escape from mucociliary clearance is still the interaction between viral S protein and ACE2 receptor on the membrane of airway epithelial ciliary. Therefore, blocking or knocking down ACE2 receptors that perform non-essential physiological functions is also well worth trying as a blocking therapeutic strategy. Next researchers should further explore the physiological function of ACE2 on motile cilia and try to develop low-toxicity inhaled biologics to attenuate the escape effect of SARS-CoV-2 on mucociliary clearance by reducing the localization of ACE2 on motile cilia of airway epithelial cells. Alternatively, the design of liposome- or exosome-based low-irritation engineered vesicles capable of blocking cilia-ACE2 and residing in the airway mucosa for long periods of time is a worthwhile strategy to try. Combined with topical administration of kinase inhibitors to the nasal mucosa to modulate the function of microvilli, as mentioned by the authors, it may be possible to achieve treatment of different stages of SARS-CoV-2 infection.


An incidental finding from this scan revealed diffuse cardiac uptake, necessitating further diagnostic procedures to exclude the possibility of cardiac amyloidosis. In the subsequent 99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid (DPD) scan and SPECT images, no perceptible cardiac uptake was observed at all. Upon retrospective review of the patient’s medical records, she received 1000 mg of ferric carboxymaltose for iron-deficient anemia the day before the 99mTc-HDP bone scan. Therefore, it was assumed that the diffuse and temporary cardiac activity was due to the transient iron overload. We present and share these bone scan images in order to avoid possible future misinterpretation of cardiac amyloidosis.In this presented case, a 77-year-old woman with an implanted prosthesis and ongoing knee pain underwent a bone scan using


High-grade Glioma (WHO classification 2016 grade III and IV) shows elevated levels of CXCR4 ligand expression in the affected tumoral cells. Healthy and non-affected organ cells express low-level CXCR4 ligands density. We performed [68Ga] Ga-Pentixafor (Pars-Cixafor™) PET/CT in a patient with high-grade Glioma (anaplastic oligodendroglioma WHO grade III) with no other documented medical condition and history. In addition to the Pentixafor-avid tumor remnant in the PET/CT images, we observed mild symmetrical bilateral uptake in the fibro glandular tissue of the breasts and moderate CXCR4(Pentixafor) avidity in both adrenal glands without any discernable pathology and abnormal density changes in the CT component of the study. Attention should be paid to the interpreting [68Ga] Ga-Pentixafor PET/CT examination and its normal uptakes and variants.[ Information regarding potential pitfalls and various normal variants once interpreting [68Ga] Ga-Pentixafor PET/CT are limited. [Case Report68Ga] Ga-CXCR4 PET/CT.  A Thirty-three-year-old woman with a history of treated anaplastic oligodendroglioma (WHO III) in the left occipito-parietal mass with spread to the lateral ventricle and basal ganglia was referred due to worsening headaches and new-onset seizure. She was referred to our department for  Ga-labeled C-X-C motif receptor4 as a novel radio-ligand using PET/CT has been investigated for tracing various kinds of solid and hematopoietic malignancies .   Ga-CXCR4 PET/CT examination. In contrast to normal adrenal tissue, significantly elevated levels of CXCR4 have been reported in cortisol-producing and aldosterone-producing adenomas, as well as adrenal hyperplasia (8-10). Nevertheless, our patient had no prior history of hypertension, neither relevant family nor drug history. The blood lab data  were within normal limits as well. To sum up, tracer uptake in both breasts and adrenals can be considered normal variants. Thus, attention should be paid to these normal uptakes while interpreting [ The patient was enrolled in a clinical trial of CXCR4-targeted PET/CT in vivo assessment of high-grade gliomas . All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The authors declare that they have no conflict of interest. Written Informed consent was obtained from our patient. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.


The hepatitis B virus core protein (HBcAg) is a highly immunogenic particulate antigen. Nearly all patients with persistent or resolved hepatitis B virus (HBV) infection show seropositivity for hepatitis B core antibody (anti-HBc), which appears in the early stage of infection and is mostly present for life. Traditionally, the anti-HBc is regarded as an evidential serological marker of HBV infections. In the last ten years, several studies revealed the predictive value of quantitative anti-HBc (qAnti-HBc) level in the treatment response and clinical outcome of chronic HBV infections, implying new insights into this classic marker. Overall, qAnti-HBc should be regarded as an indicator of the host’s immune response specific to HBV, which correlates with HBV-related hepatitis activity and liver pathology. This review summarized the latest understanding of the clinical values of qAnti-HBc for differentiating the CHB phase, predicting treatment response, and providing disease prognosis. Moreover, we also discussed the possible mechanism of qAnti-HBc regulation during different courses of HBV infection. The hepatitis B virus (HBV) is one major pathogen causing viral hepatitis in humans and putting people at high risk of death from cirrhosis and liver cancer. The virus is transmitted mainly through mother-to-child transmission, which leads to many people suffering from hepatitis B for the rest of their lives; in addition, it can be transmitted to others through blood or sexual contact . WorldwiIn the clinic, both classic and emerging biomarkers are used to characterize HBV infection status at various dimensions in associating with the HBV life cycle and natural history of HBV infections . SpecifiBoth T cells and serum antibodies could theoretically be potential biomarkers of host anti-HBV immunity. However, virus-specific T cells are typically exhausted in cases of chronic HBV infections and pose challenges in detection ,18. FurtIn chronic HBV infections, individuals in the immune-active phase  with active hepatitis typically exhibit ~10-fold higher levels of qAnti-HBc compared to those in the immune-tolerant or inactive-carrier phases when hepatitis is absent . Severalp < 0.001), with the qAnti-HBc cut-off value for diagnosing moderate and severe inflammation at about 4.5 log10 IU/mL [Liver tissue biopsy is the gold standard for diagnosing liver inflammation but is not commonly used due to its invasive nature . The com10 IU/mL . This st10 IU/mL . Specifi10 IU/mL .Furthermore, a longitudinal study with long-term follow-up (median 19.8 years) in 182 HBeAg-positive children with normal ALT demonstrated that the baseline qAnti-HBc (500 IU/mL) is a strong independent predictor for spontaneous HBeAg seroconversion . This stIn cases where HBsAg clearance is achieved, anti-HBc is often the only detectable marker of a past HBV infection. However, in HBsAg-negative individuals during the late phase of either occult or resolved HBV infections, the qAnti-HBc is typically around 1000-fold lower than that observed in HBsAg-positive cases . In tranThe established relationship between elevated qAnti-HBc and activated hepatitis in HBsAg-positive individuals raises an interesting question about the qAnti-HBc level in individuals co-infected with HBV and other hepatotropic pathogens. However, current understanding on this topic is limited. Some studies have characterized qAnti-HBc in HBV/HDV superinfections. In a study of a small number of patients with ALT elevation, qAnti-HBc was found at lower levels in HDV-superinfected inactive carriers than in untreated CHB patients . MultivaExtensive studies demonstrated that the high level of qAnti-HBc is a strong predictor for HBeAg seroconversion. For HBeAg-positive individuals chronically infected with HBV, HBeAg seroconversion (ESR) often confers a favorable prognosis with reduced incidence of progressive liver inflammation and advanced liver diseases ,54,55. OIn the natural course of chronic HBV infection, similar to that observed in the anti-HBV treatment cohorts, the qAnti-HBc also plays a significant predictive role for ESR. Liu et al. showed the independent association between high levels of qAnti-HBc with spontaneous ESR and suggested it is a surrogate marker of the anti-HBV T-cell activity in retrospective analyses based on the community-based REVEAL-HBV cohort . In addin = 2500), a lower level of qAnti-HBc (<3.0 log10 IU/mL) in HBeAg-negative patients was independently associated with a higher HBsAg seroclearance (53% vs. 19.8%) after a 12-year follow-up. A combination of the qAnti-HBc and HBsAg levels presented improved performance of serological parameters in predicting HBsAg seroclearance in HBeAg-negative CHB patients [Unlike that in HBeAg-positive people, the prognostic significance of qAnti-HBc in HBeAg-negative CHB phases was not fully understood. However, some studies suggested the prognostic direction for this marker in HBeAg-negative populations was different from that in HBeAg-positive patients. In a retrospective analysis based on the REVEAL-HBV cohort . This study found the HBV-ACLF patients had a significantly higher level of anti-HBc than CHB patients (4.95 ± 0.54 vs. 4.47 ± 0.84 log10 IU/mL), and the qAnti-HBc showed a comparable and complementary prognostic accuracy to MELD (model for end-stage liver disease) score in predicting clinical outcome . A high level of qAnti-HBc was associated with less mortality in HBV-ACLF patients . In anotThe well-established relationship between qAnti-HBc and HBeAg seroconversion implies the linkage between activated core-specific immunity in mediating HBeAg loss and virological control. Peripheral high levels of HBsAg and HBeAg are essential viral factors to induce T cell exhaustion and immune tolerance in persistent HBV infections. For HBeAg, the animal study suggested that maternal-derived HBeAg alters hepatic macrophages toward M2 polarization and impaired CD8+ cytotoxic T lymphocyte (CTL) response leading to viral persistence . In CHB The marked contrast in qAnti-HBc between HBsAg-positive populations and individuals with resolved HBV infection , as wellExtensive studies uncovered the new roles of qAnti-HBc in the CHB natural course and treatment process. Although further excavations and validations are still required to be conducted, based on currently available evidence, the clinical significance of qAnti-HBc could be summarized as following points for reference  The qAnti-HBc is a well-validated predictor for spontaneous and treatment-induced HBeAg seroconversion among HBeAg-positive individuals. People with a higher level of qAnti-HBc have more chance of achieving HBeAg seroconversion. The cut-off values from different studies showed a little difference, but 4.0–4.5 log10  IU/mL is possibly preferable for adult CHB patients.(2) For individuals in HBeAg seronegative phase with successful virological control, a persistent low level (maybe <1000 IU/mL) of qAnti-HBc links with a high probability of achieving HBsAg loss.(3) Regardless of HBeAg status or ALT activity, the qAnti-HBc (>4.0 or >4.5 log10 IU/mL) positively correlates with the activated host anti-HBV immune activity and the risk of the presence of significant hepatic inflammation.(4) During the recovery phase of post-HBsAg loss, qAnti-HBc may reflect the non-eradicated HBV residuals, and a relatively higher qAnti-HBc indicates the risks for occult HBV infection or HBV reactivation when receiving rituximab-containing chemotherapy.In conclusion, the qAnti-HBc is a surrogate marker for host anti-HBV immune activity in chronic HBV infections. Elevated qAnti-HBc-associated antiviral immunity is essential to drive immunological control against HBV infection during HBeAg-positive phases but signifies immune-escape hepatitis during HBeAg-negative phases. Wide clinical applications of qAnti-HBc are still challenged by the absence of standardized automatic assays and the well-validated cut-off index for various scenarios . Further


Tumor tissues consist of tumor cells and tumor stroma, which is structured by non-tumor cells and the extracellular matrix. Macrophages are the predominant immune cells in the tumor microenvironment (TME). Based on the intimate interaction between macrophages and tumor cells, macrophages are closely involved in tumor initiation and progression, playing a key role in tumor formation, angiogenesis, metastasis, and immune escape. Extracellular vesicles (EVs) are a group of membrane-enclosed structures secreted by almost all cell types. As crucial mediators of cell-to-cell communication, EVs play a role in various physiological processes and the development of diseases including cancer. According to numerous studies, tumor cell-derived extracellular vesicles (T-EVs) could highly modulate the phenotypes and functions of macrophages, thus promoting tumor development. Herein, we comprehensively introduce the role of T-EVs in regulating the M1/M2 phenotypes and immune functions of macrophages, including cytokine secretion, expression of immune regulatory molecules on the membrane, phagocytosis, and antigen presentation. More importantly, based on the regulatory effects of T-EVs on macrophages, we propose several potential therapeutic approaches that may guide future attempts to increase the effectiveness of cancer therapy. Tumor tissue consists of tumor cells and tumor stroma. Tumor stroma contains the extracellular matrix and various non-tumor cells, including fibroblasts, endothelial cells, and immune cells , forming a complex tumor microenvironment (TME) As the predominant immune cells within the TME Herein, we comprehensively introduce the role of T-EVs in regulating the phenotypes (M1/M2) and immune functions of macrophages, including cytokine secretion, expression of immune regulatory molecules on the membrane, phagocytosis, and antigen presentation. Most importantly, given the critical regulatory effects of T-EVs on macrophages and the TME, there is an urgent need for the suppression or elimination of T-EVs to improve the therapy and prognosis of cancer patients. Herein, we propose several potential therapeutic approaches that may guide future attempts to increase the effectiveness of cancer therapy.EVs are a diverse population of membrane vesicles generated via multiple mechanisms. Based on their formation mechanism and size, EVs are generally categorized as exosomes and microvesicles (MVs). Exosomes (30-150 nm in diameter) originate from the endosomal pathway that involves sorted endosomes and intracellular multivesicular bodies (MVBs) containing intraluminal vesicles (ILVs) EVs inherit biomolecules  from their donor cells, and the level of EV cargos is regulated by a variety of molecules in their donor cells Regulated by the stimuli from the milieu, macrophages are classified into different phenotypes according to their functional orientation. The M1 phenotype and the M2 phenotype are the two major polarization states of macrophages RNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), have different effects on cell growth, proliferation, and metabolism through different pathways miRNAs are the most abundant kind of RNAs encapsulated in EVs Hypoxia is a common characteristic of solid tumors. Hypoxia may increase the production of T-EVs that carry miRNAs, and the miRNAs encapsulated in T-EVs may cause M2-like macrophage polarization. Chen et al. demonstrated that hypoxia increased the level of miR-940 in EVs produced by epithelial ovarian cancer (EOC) cells, and that EV-miR-940 delivered to macrophages encouraged M2-phenotype polarization, hence promoting EOC proliferation and migration + head and neck squamous cell carcinoma (HNSCC) cell-derived EVs and could be carried to macrophages and promote M1 polarization by downregulating PPAR, hence promoting HNSCC radiosensitivity In addition to the pro-tumoral M2 phenotype, specific miRNAs  in T-EVs may polarize macrophages towards the anti-tumor M1 phenotype lncRNAs are RNAs that have a length of over 200 nucleotides and have a restricted potential to encode proteins. There is mounting evidence that lncRNAs have a role in a wide range of biological processes, from development and physiology to pathology, including tumorigenesis and tumor progression circRNAs are a type of non-coding RNA that is generated through a process known as back-splicing, which involves the circularization of exons or introns. Studies have shown that circRNAs could be packaged into EVs and play a key role in the development of tumors. Chen et al. reported that circFARSA in EVs of non-small cell lung cancer cells induced M2 macrophage polarization via PTEN ubiquitination and degradation, which promoted tumor metastasis Studies have shown that T-EVs may act as trafficking vehicles to transport proteins from tumor cells to macrophages, modulating macrophage polarization. Among these proteins are immune checkpoint molecules and other proteins.The term "immune checkpoints" describes a group of ligands present on the surface (membrane) of antigen-presenting cells that bind to specific receptor partners on T cells + T cells and promoted tumor growth + T cells and induce macrophage M2 polarization, which ultimately promoted melanoma cell growth and metastasis T-EVs may inherit inhibitory checkpoint molecules from tumor cells and thus limit T-cell activation as well as polarize macrophages towards pro-tumoral M2 phenotype The blocking of immune checkpoints is an essential component of immunotherapy, the significance of which in treating cancer is well recognized. Ideally, immune checkpoint blockade enables efficient immune responses, and in the meanwhile, maintains sufficient self-tolerance and prevents excessive immune cell activation In addition to immune checkpoint molecules, studies have identified other T-EV proteins that trigger M1 or M2 macrophage polarization. TEK gene-encoded tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (TIE2) is the receptor for angiopoietins and performs essential functions in vascular remodeling during inflammation and tumor angiogenesis Membrane lipids, especially arachidonic acid (AA), are essential for the fusion of EVs and plasma membranes It is commonly accepted that the immune system is comprised of two fundamental components: innate immunity and adaptive immunity. The innate immune system is the body's initial line of defense against pathogens  and tumor cells +) T-EVs could suppress the activation of IRF3 and type I interferon, thereby impairing the host's innate antiviral immunity By regulating macrophages, T-EVs participate in the innate immune response. Epidermal growth factor receptor positive , anti-inflammatory cytokines , chemokines  and angiogenic factors . Other T-EVs, however, inhibit the secretion of pro-inflammatory cytokines and co-stimulatory molecules  and transforming growth factors (TGFs), is also influenced by T-EVs. Sun et al. demonstrated that EV-IRF-2 from CT26 cells induced the release of VEGFC by macrophages Macrophage surface molecules, including antigens, receptors and other molecules mediate the immune responses of macrophages. Studies have shown that T-EVs regulate the levels of immune regulatory molecules on membrane of macrophages to affect immune suppression and disease progression Figure .+ TAM production, which impaired CD8+ T-cell function The PD-L1/PD-1 signaling axis plays a critical role in tumor immunosuppression since it serves as an immune checkpoint and “don't eat me” signal. This axis inhibits T lymphocyte activation and increases the immunological tolerance of tumor cells, hence enabling tumor immune escape CD39 functions as a “molecular switch” that regulates the inflammatory reactions of macrophages Furthermore, T-EVs may regulate the expression of a set of M1  and M2  markers So far, a considerable amount of research has been conducted on PD-1/PD-L1 Phagocytosis is a major characteristic of macrophages. Macrophages engulf and eliminate invading microorganisms and damaged cells, providing an effective immune response and maintaining homeostasis Macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) is of great importance to tumor cell clearance. The stimulation of macrophage-mediated ADCP seems to be a significant mechanism behind the anti-tumor effects of a variety of therapeutic antibodies The phagocytic ability of macrophages is essential for the clearance of EVs from circulation and is stimulated by “eat me” signals  and suppressed by “don't eat me” signals  + macrophages by analyzing the proportion of Ovalbumin+ macrophages. They discovered that gastric cancer cells significantly educated monocytes into PD-1+ macrophages by secreting EVs in vitro and in vivo, and PD-1+ macrophages phagocytosed significantly less ovalbumin in vitro than PD-1- macrophages did The phagocytic ability of macrophages could also be evaluated by the phagocytosis activity of the ovalbumin, yeast, or latex beads-rabbit IgG-FITC complex. Wang et al. evaluated phagocytosis levels of PD-1Capture, endocytosis, and antigen presentation are crucial aspects of macrophage functions, which connect innate and adaptive immunity H cells. The TH cells may activate both cytotoxic T-lymphocytes and antibody-producing B-lymphocytes subsequently. Therefore, inhibiting Ia antigen expression on macrophages may limit both cellular and humoral immune responses. Taylor and Black discovered that plasma membrane-derived vesicles shed by metastatic variants of the murine B16 melanoma significantly inhibited the stimulation of macrophage Ia antigen expression, the earliest stage in the establishment of an immune response According to studies, T-EVs may facilitate macrophage antigen presentation Figure E. The exAs important cellular mediators, T-EVs regulate macrophage phenotypes and functions by transporting various cargos to macrophages, thereby influencing tumor progression and cancer treatment. T-EVs that target and regulate macrophages are considered promising anti-tumor therapeutic targets, either alone or in combination therapies Figure . A promi“Eat me” and “don't eat me” signals together control the process by which macrophages phagocytose tumor cells. Since CD47 can competitively occupy SIRPα, the overexpression of CD47 on T-EVs may disrupt CD47/SIRPα signal and lead to enhanced macrophage-mediated phagocytosis of tumor cells T-EVs inherit tumor antigens from tumor cells, making them a source of tumor antigens that the immune system can identify. Therefore, T-EVs can be employed as tumor vaccines that stimulate immune responses against not only tumor cells but also T-EVs Figure A. Shi etGenerally, a viable method to treat cancer is to use small molecule inhibitors or antibodies that target specific signaling components to stop the inhibiting effects of T-EVs on macrophages. Given the pro-tumor properties of M2 macrophages, inhibiting macrophage M2 polarization and suppressing TAM infiltration with T-EVs that contain effector molecules can be promising therapeutic strategies against tumors Figure B. For invβ5 that specifically binds to Kupffer cells and contributes to liver tropism T-EVs can target macrophages, and integrins are the molecular basis for T-EVs' targeting ability Figure C. AccordThe macrophage-targeting ability of T-EVs could be enhanced when the “don't eat me” and “eat me” signals are smartly used. In a study by Liu et al., matrix metalloproteinase 2 (MMP2) was exploited to regulate PS externalization on the surface of the PEGylated nanoparticles (NPs) in order to achieve tumor-specific macrophage targeting Compared to EVs from other cells, T-EVs have a substantial advantage in blood circulation time Figure D. MacropDespite this, it is still being determined whether native T-EVs can safely serve as drug carriers. The high level of immunosuppressive PD-L1 expression in native T-EVs raises the necessity to consider whether it may promote the progression of tumors Overall, this review summarizes the impact of T-EVs on macrophage phenotype and immune function, along with the underlying molecular mechanisms. More importantly, it presents several treatment strategies based on the regulation of T-EVs on macrophages, which are promising methods for treating cancer. Developing new strategies and combination regimens with enhanced specificity profiles is essential to overcome therapy resistance and eradicate cancer. Further research is needed to fully comprehend the complex interactions between T-EVs and macrophages and how these interactions influence cancer treatment.Supplementary table.Click here for additional data file.


Endoscopic ultrasound-guided gastrojejunostomy (EUS-GJ) is a minimally invasive alternative treatment for gastric and duodenal outlet obstruction. However, technical difficulty arises from bowel motility. To address this, we present a novel technique using an improvised EUS-directed T-anchor to improve safety and feasibility, particularly for patients with ascites or retained intragastric food.A 92-year-old man developed gastric outlet obstruction due to a pancreatic head mass . ThrougEndoscopic ultrasound-guided (EUS) gastrojejunostomy using an improvised EUS-directed T-anchor in a patient with severe gastric outlet obstruction due to a pancreatic head mass.Video 1Endoscopic gastrojejunopexy offers the potential to make EUS-GJ a safer and more reliable procedure by preventing jejunal migration away from the gastric wall and permitting the use of a guidewire to ensure intraluminal placement of the LAMS. Without a dedicated EUS-directed T-anchor system, an endoscopic suture and cinch can be improvised to affix the jejunum to the gastric wall to accomplish this maneuver.Gastrojejunopexy-assisted endoscopic gastrojejunostomy may potentially increase the safety and feasibility of the endoscopic gastrojejunostomy, by minimizing the risk of jejunal migration. Dedicated EUS-directed T-anchors are needed.Endoscopy_UCTN_Code_TTT_1AS_2AB


The self-driven organization of model protocells intohigher-ordernested cytomimetic systems with coordinated structural and functionalrelationships offers a step toward the autonomic implementation ofartificial multicellularity. Here, we describe an endosymbiotic-likepathway in which proteinosomes are captured within membranized alginate/silkfibroin coacervate vesicles by guest-mediated reconfiguration of thehost protocells. We demonstrate that interchange of coacervate vesicleand droplet morphologies through proteinosome-mediated urease/glucoseoxidase activity produces discrete nested communities capable of integratedcatalytic activity and selective disintegration. The self-drivingcapacity is modulated by an internalized fuel-driven process usingstarch hydrolases sequestered within the host coacervate phase, andstructural stabilization of the integrated protocell populations canbe achieved by on-site enzyme-mediated matrix reinforcement involvingdipeptide supramolecular assembly or tyramine–alginate covalentcross-linking. Our work highlights a semi-autonomous mechanism forconstructing symbiotic cell-like nested communities and provides opportunitiesfor the development of reconfigurable cytomimetic materials with structural,functional, and organizational complexity. Alternatively,properties such as wetting and interfacial tension can be utilizedfor the development of physically interactive artificial cell communitiesthat exhibit behaviors such as artificial phagocytosis,43 parasitism,44 andpredation48 to produce nested protocells via mechanisms of engulfment, processing,and functional integration . In general,these mechanisms involve the use of molecularly crowded coacervatedroplets that display a membrane-less interfacial barrier, which facilitatesthe capture of guest protocells by contact-dependent attractive interactionswith the exposed coacervate matrix. As the hybrid constructs are inherentlyunstable due to coacervate droplet–droplet coalescence, recentstudies have used membranized coacervates52 with triggerable membrane dynamicsto control the selective uptake and integration of guest protocellsin response to light irradiation or enzyme-mediated changes in pH.53 Because such approaches are compromised by irreversibledepletion of the membrane building blocks and concomitant structuralinstability, in this paper, we develop a self-driving process of reversiblecoacervate membranization, which offers increased robustness and functionaladaptation in cell-like nested communities and provides an autonomousmechanism for the construction of artificial multicellularity viathe chemical programming of interactive protocell behavior.Synthetic protobiology is concerned withthe construction, reconstitution,and functionalization of individual cell-like entities,49 For example, self-membranization of silk-basedcoacervate droplets produces osmotically inflated semi-permeable coacervatevesicles and modulates the trafficking of low-molecular weight solutes.54 In this paper, we implement the self-drivencapture of proteinosomes within reconfigurable alginate/silk fibroincoacervate vesicles to establish the autonomic integration of thedifferent protocell populations. We show that the capture of singleor binary populations of proteinosomes56 by preformedsingle-chambered coacervate vesicles can be achieved via the reversibleinterchange of the coacervate vesicle and droplet morphologies throughprogrammed urease/glucose oxidase (GOx) activity specifically withinthe proteinosomes. Moreover, sequestration of starch hydrolases withinthe host coacervate phase modulates the self-driving capacity viaan internalized fuel-driven process, thereby establishing primitivelevels of host/guest communication and cooperativity in the endosymbiotic-likepathway. We demonstrate that capture of the proteinosomes into multi-chamberedcoacervate vesicles with increased structural robustness can be achievedby guest- or host-mediated hydrogelation of the coacervate phase usingon-site enzyme-mediated dipeptide supramolecular assembly or covalentcross-linking of tyramine–alginate, respectively. The integratedprotocell populations are exploited as catalytic host–guestconstructs and undergo selective membrane disassembly when challengedby a reducing agent in the external environment. Overall, our workhighlights a semi-autonomous mechanism for constructing symbioticcell-like nested communities capable of self-induced matrix reinforcement,endogenously coupled catalytic reactivity, and selective membranedisintegration and provides opportunities for the development of reconfigurablecytomimetic materials with structural, functional, and organizationalcomplexity.Membranized coacervate-based protocells can be spontaneously generatedthrough coacervate droplet-to-vesicle reconfigurations by employingauxiliary surface reconstruction agents or triggering endogenous structuraltransitions (self-membranization) under nonstoichiometric conditions.Mw ≈ 390 kDa; light chain, Mw ≈26 kDa) was prepared as previously reported 54 and mixed with negativelycharged alginate (Mw = 140–160kDa) to induce coacervation by associative liquid–liquid phaseseparation. Depending on the conditions, near-to-neutral membrane-lesscoacervate droplets, multi-compartmentalized coacervate vesicles,and positively or negatively charged coacervate vesicles were producedand reversibly interchanged by alterations in the alginate [COOH]:CSF[NH2] charge ratio . The coacervate vesicles consisted of single or multiple osmoticallyexpanded water-filled vacuoles that were caged within a continuousand compressed coacervate phase that served as a barrier to macromolecularuptake . Significantly, thepositively charged coacervate vesicles were formed via a self-membranizationprocess arising from the segregation of excess amphiphilic CSF chainsat the coacervate droplet/water interface under non-stoichiometric(charge-mismatched) conditions.54 Consequently,the coacervate vesicles were reversibly reconfigured into membrane-freealginate/CSF coacervate droplets by increasing the degree of alginatedeprotonation by increasing the pH above 8, or adding additional alginateto give alginate: CSF charge ratios above 2:1 . Alternatively, populations of silk-basedcoacervate droplets were transformed into suspensions of positivelycharged coacervate vesicles by decreasing the pH below 6 or addingCSF to change the charge ratio to less than 1:1 .Cationized silk fibroin  intothe positively charged alginate/CSF coacervate vesicles ethyl]polyethyleneglycol  and containedco-encapsulated urease and glucose oxidase (GOx) as endogenous agentsfor increasing (production of NH3) or decreasing (productionof gluconic acid) the pH, respectively, along with encapsulated aqueousgelatin to stabilize the proteinosomes against changes in osmoticpressure. Significantly, in the absence of enzyme activity, binarypopulations of coacervate vesicles  and proteinosomes  were non-interacting over 24 h . The absence of fusion and wettingof the different populations indicated that the coacervate vesiclemembrane was an effective physical barrier to proteinosome uptake.In contrast, the addition of urea to the mixed populations gave risewithin 2 h to an increase in pH from 4 to 8.3 due to urease activitywithin the proteinosomes . Consequently,the coacervate vesicles were transformed into membrane-less coacervatedroplets that at pH 7–8 began to coalesce, adventitiously entrappingsome of the proteinosomes . As thepH increased to 9, spontaneous contact and rearrangement between thecoacervate droplets and proteinosomes resulted in well-defined proteinosome-in-coacervatedroplets . To re-establish the coacervatevesicle membrane, glucose was added to re-acidify the medium by endogenousproteinosome-based GOx activity . Decreases in pH to ca. 7.5 resulted in the formation of multiplevacuoles that continued to expand until they coalesced into a singlewater-filled lumen at pH 6 to produce discrete alginate/CSF coacervatevesicles with the guest proteinosomes expelled from the compressedcoacervate phase into the lumen . The proteinosome captureefficiency was ca. 30% for binary populations prepared at an initialproteinosome:coacervate vesicle number ratio of 1:2. Increasing therelative number of coacervate vesicles increased the capture efficiencyof the two-step pathway to values of 48 and 62% at proteinosome:coacervatevesicle ratios of 1:3 and 1:10, respectively. Non-captured proteinosomeswere often associated with the surface of the coacervate droplets/vesicles.Given the facile and reversiblenature of self-membranization, we developed a self-driving two-stepenzyme-mediated pathway for the chemically induced capture of guestprotocells  analysisof single populationsof positively charged alginate/CSF coacervate vesicles, membrane-lessalginate/CSF coacervate droplets, or enzyme-loaded proteinosomes displayed2D pseudo-color dot plots that were distinguishable due to differencesin granularity and particle size 1f. MixinFigure S11) and starch added to the external environment.Uptake of starch into the coacervate droplets  initiated an endogenous enzyme cascade in whichthe digestion of starch to glucose  within the hydrolase-containing coacervate phase resulted in thediffusive transfer of glucose to the captured proteinosomes. In turn,this resulted in GOx-mediated acidification and reconfiguration intothe proteinosome-in-coacervate vesicles . The addition of aqueous solutions of glucoseand the derivatized dipeptide Fmoc-Ala-Ala-OH to proteinosomes at pH 9 resulted in acidification of the proteinosomeinterior and assembly of a hydrogel network due to protonation ofthe peptide C-terminal carboxylic acid and onset of supramolecularstacking via hydrophobic and π–π interactions.57 LSCM images indicated that the peptide nanofilamentsnucleated within the proteinosomes at approximately pH 7 and thatfurther growth gave rise to the hydrogelation of the proteinosomesand their external environment as the pH decreased to around 6 . Given these observations, we preparedGOx/gelatin/proteinosome-in-coacervate droplets at pH 9 and addedglucose and Fmoc-AA-OH to simultaneously initiate membranization andpeptide assembly, respectively . Sequestrationof Fmoc-AA-OH into the proteinosome-in-coacervate constructs at pH9 had minimal influence on the morphology of the hybrid droplets . Incorporation of the nanofilamentsin the coacervate matrix increased the mechanical robustness of thecoacervate vesicles  and considerablydecreased the fluidity of the constituent coacervate phase as determinedby fluorescence recovery after photo-bleaching (FRAP) experiments. Increasing the pH to 9 regeneratedthe coacervate droplets, indicating that reinforcement of the coacervatematrix by in situ dipeptide self-assembly was reversible .As coacervate droplets prepared by electrostatic complexation areoften dissociated under high ionic strength, we designed two on-boardenzyme-driven mechanisms based on guest- or host-mediated matrix reinforcementto structurally stabilize the proteinosome-in-coacervate vesiclesand thereby extend their functionality under adverse conditions. Toimplement guest-mediated reinforcement of the coacervate matrix, wefirst demonstrated that GOx-containing proteinosomes alone could beused as semi-permeable microscale reactors for initiating the reversibleassembly of a network of peptide nanofilaments S13. The Figure S19) to generate silk-based coacervatescapable of post-assembly covalent cross-linking  and confirmed that alginate-Tyr/CSF positively charged coacervatevesicles and near-neutral coacervate droplets could be readily assembled. Consequently, alginate-Tyr/CSFcoacervate vesicles were prepared at pH 4 and subsequently transformedat pH 9 into coacervate droplets with captured urease/GOx/gelatin-containingproteinosomes and sequestered HRP. Addition of glucose and productionof gluconic acid and hydrogen peroxide over 12 h resulted in membranizationand HRP-mediated oxidative cross-linking of alginate-Tyr, respectively,to produce multi-chambered vesicles comprising a structurally reinforcedcoacervate matrix infiltrated with multiple proteinosome-containingvacuoles . No reconfiguration ofthe coacervate vesicles into coacervate droplets was observed afterthe subsequent addition of urea and urease-mediated alkalinizationof the suspension , indicating thatcross-linking of the alginate-Tyr chains was irreversible under theconditions employed.Alternatively, we implemented a host-mediated processof matrixreinforcement in the proteinosome-in-coacervate vesicles by replacingalginate with a synthesized tyramine-derivatized alginate . Guest-mediated reinforcementof the coacervate membrane domain with peptide nanofilaments enabledsequestered lipase to remain active within the coacervate vesiclesunder high-ionic strength conditions and concurrent processes of membranization, vacuole expansion, dipeptidenanofilament assembly, and alginate-Tyr cross-linking.o-phenylenediamine such that the reconfiguration mechanism (acidification)was coupled to the catalytic production of 2,3-diaminophenazine . Increasing starch concentrationsincreased both the rates of GOx-mediated pH decrease and GOx/HRP-dependentproduct production , indicatingthat diffusive transfer of hydrogen peroxide occurred between theco-trapped proteinosomes. Corresponding LSCM images of individualcoacervate vesicles confirmed the spatially confined catalytic reactivityof the co-located proteinosomes and the dependence on an inter-protocellhydrogen peroxide signaling pathway  and were enzymatically inactive. A 1:1 mixture of the twodifferent protocells was prepared, added to a suspension of alginate/CSFcoacervate vesicles, and then co-captured using an bi-enzyme-mediatedtwo-step pH-oscillation processing pathway. Selective disassemblyof the PEG-NHS-disulfide-cross-linked proteinosomes occurred withinthe lumen over ca. 2 h by the addition of TCEP, resulting in the releaseof FITC-dextran into the water-filled vacuole . While the addition of TCEP at concentrations of 10 mM in the absenceof glucose led to the disassembly of all the disulfide-cross-linkedproteinosomes, the simultaneous addition of glucose and TCEP decreasedthe percentage of the disassembled proteinosomes over 6 h to approximately40% at a glucose concentration of 20 mM . Interestingly,non-captured proteinosomes present in the external environment weredisassembled under these conditions, indicating that the mitigationof TCEP-induced disassembly was more effective when the defense mechanismwas embedded within the coacervate matrix. This was consistent withcontrol experiments undertaken in the absence of a coacervate phase,which showed higher levels of membrane disassembly at the same glucoseand TCEP concentrations . Moreover,changing the spatial location of the enzymes such that GOx/urease-sequesteredcoacervate droplets are mixed with non-enzymatic proteinosomes hasminimal effect on the capture of the proteinosomes after the additionof urea. However, subsequent reconfiguration after the addition ofglucose into single-chambered coacervate vesicles is less efficient,producing multi-compartmentalized vesicles in which the proteinosomesremain attached to the coacervate phase , suggesting that the locus of H+ productioninfluences the reconfiguration pathway.Although capture efficienciesof 62% are achieved in the presenceof a large excess of coacervate vesicles, only 30% of the proteinosomepopulation is sequestered at a proteinosome:coacervate vesicle numberratio of 1:2. Thus, under these conditions, both non-captured andcaptured proteinosomes contribute to the endogenous GOx-mediated decreasein pH associated with reconfiguration of the coacervate droplets intocoacervate vesicles . Consequently, the resultingacidification associated with CO2 production triggers reconfigurationof the coacervate droplets into stabilized coacervate vesicles, whichexpels the captured yeast cells into the aqueous-filled lumen wheredensely packed colonies of viable cells develop over 36 h.It should be possibleto develop our approach to the self-drivencapture of living cells in coacervate vesicles for the developmentof nested cellular bionic systems with high biocompatibility. Forexample, fuel-driven activation of coacervate membranization can beaccomplished by capturing live yeast cells within alginate/CSF coacervatedroplets containing starch hydrolases and adding starch to the externalenvironment to generate glucose for cellular metabolism and growth(58 and offers a step to self-driven artificialmulticellularity in life-like materials. The development of reconfigurablecytomimetic materials with structural, functional, and organizationalcomplexity could provide future opportunities for bottom-up syntheticbiology, soft microbotics, and programmable storage–releasetechnology.Ingeneral, our semi-autonomous mechanism for constructing cell-likenested communities is inspired by the notion of endosymbiosis


What are the efficacy and mechanisms of change of a therapist-guided, internet-delivered emotion regulation therapy for nonsuicidal self-injury among adolescents?In this randomized clinical trial that included 166 adolescents, internet-delivered emotion regulation individual therapy delivered adjunctive to treatment as usual resulted in an 82% reduction in masked assessor-rated nonsuicidal self-injury frequency , a statistically significant difference. Improvements in emotion regulation mediated improvements in nonsuicidal self-injury.A therapist-guided emotion regulation treatment delivered online may overcome common treatment barriers and increase availability of evidence-based psychological treatments for adolescents with nonsuicidal self-injury. This randomized clinical trial examines the effectiveness of internet-delivered emotion regulation individual therapy in addition to usual care for reducing nonsuicidal self-injury among adolescents in Sweden. Nonsuicidal self-injury is prevalent in adolescence and associated with adverse clinical outcomes. Effective interventions that are brief, transportable, and scalable are lacking.To test the hypotheses that an internet-delivered emotion regulation individual therapy for adolescents delivered adjunctive to treatment as usual is superior to treatment as usual only in reducing nonsuicidal self-injury and that improvements in emotion regulation mediate these treatment effects.This 3-site, single-masked, randomized superiority trial enrolled participants from November 20, 2017, to April 9, 2020. Eligible participants were aged between 13 and 17 years and met diagnostic criteria for nonsuicidal self-injury disorder; they were enrolled as a mixed cohort of consecutive patients and volunteers. Parents participated in parallel to their children. The primary end point was at 1 month after treatment. Participants were followed up at 3 months posttreatment. Data collection ended in January 2021.Twelve weeks of therapist-guided, internet-delivered emotion regulation individual therapy delivered adjunctive to treatment as usual vs treatment as usual only.Primary outcome was the youth version of the Deliberate Self-harm Inventory, both self-reported by participants prior to treatment, once every week during treatment, and for 4 weeks posttreatment, and clinician-rated by masked assessors prior to treatment and at 1 and 3 months posttreatment.A total of 166 adolescents  were randomized to internet-delivered emotion regulation therapy plus treatment as usual (84 participants) or treatment as usual only (82 participants). The experimental intervention was superior to the control condition in reducing clinician-rated nonsuicidal self-injury  from pretreatment to 1-month posttreatment. These results were maintained at 3-month posttreatment. Improvements in emotion dysregulation mediated improvements in self-injury during treatment.In this randomized clinical trial, a 12-week, therapist-guided, internet-delivered emotion regulation therapy delivered adjunctive to treatment as usual was efficacious in reducing self-injury, and mediation analysis supported the theorized role of emotion regulation as the mechanism of change in this treatment. This treatment may increase availability of evidence-based psychological treatments for adolescents with nonsuicidal self-injury.NCT03353961ClinicalTrials.gov Identifier:  The first aim was to investigate the efficacy of therapist-guided IERITA delivered adjunctive to treatment as usual (TAU) compared with TAU only. The second aim was to examine emotion regulation as a mechanism of change in IERITA. We expected that IERITA plus TAU, relative to TAU only, would lead to both larger reductions in self-reported and assessor-rated NSSI frequency and greater improvements in the secondary outcomes of emotion dysregulation, global functioning, impulsive self-destructive behaviors, and psychiatric symptoms. Finally, we expected that improvements in emotion regulation would mediate improvements in NSSI during treatment.CONSORT) reporting guidelines. The trial was approved by the Stockholm Regional Ethical Review Board. All participants provided informed consent, with older participants providing written consent and younger participants verbal consent with parental written consent  in Stockholm, Västra Götaland, and Skåne in Sweden. Recruitment took place from November 20, 2017, to April 9, 2020. The primary end point was 1 month after treatment. Participants were followed up 3 months after treatment (ended January 2021). Participants randomized to TAU only were offered IERITA after the 3-month follow-up. We followed the Consolidated Standards of Reporting Trials  who could participate in the parent program. Exclusion criteria for the adolescents included: immediate suicide risk ; diagnosis of psychotic or bipolar I disorder or ongoing (defined as past month) substance use disorder; other primary psychiatric disorder requiring immediate treatment ; insufficient understanding of the Swedish language; life circumstances that could prevent treatment participation or that required immediate intervention; and a clinician-assessed global functioning level corresponding to a Children’s Global Assessment Scale (CGAS)Procedures for being admitted into the trial included an initial telephone screening, consecutive self-reported baseline assessments every 7 days for 4 weeks, and a face-to-face assessment at the clinic. During the face-to-face assessment, a psychologist or psychotherapist administered semi-structured interviews to assess for NSSID and other co-occurring psychiatric disorders  and conducted by an independent researcher in blocks of 4 or 6 per treatment clinic and placed in opaque sealed envelopes. Outcome measures included masked clinician-rated interviews (via telephone) and self-reported measures (via online platform). Psychologists and psychotherapists masked to treatment allocation and independent from the scientific team conducted the clinician-rated follow-up assessments. Details on masking and participant reimbursement are available in eMethods in 23 The DSHI-Y assesses the frequency of the 6 most common forms of NSSI . To optimize reliability and statistical power, the self-reported DSHI-Y, measuring past-week engagement in NSSI, was administered pretreatment, once every week during the treatment period, and for 4 weeks posttreatment (1-month posttreatment served as the primary end point). A clinician-rated version of the DSHI-Y measuring past-month NSSI was administered at pretreatment, 1-month posttreatment (primary end point), and 3-month posttreatment by masked assessors. Secondary outcomes included the masked clinician-rated assessment of global functioning (CGAS)21 and adolescent-reported emotion dysregulation , self-destructive behaviors .27 Self-reported measures were also administered to assess treatment credibility or expectancy, satisfaction, and adverse events in the IERITA plus TAU group. See eMethods and eTable 1 in The primary outcome was self-reported and clinician-rated NSSI frequency as measured by the youth version of the Deliberate Self Harm Inventory (DSHI-Y).17 and feedback from patients and end-user representatives. IERITA is a web-based, acceptance-based behavioral treatment that includes 11 modules for the adolescent participant and 6 modules for the parents delivered over the course of 12 weeks. The modules include text, animations, and interactive scripts  and ensure patient safety. The TAU was delivered in both conditions within regular CAMHS care by community clinicians  and typically involved supportive therapy sessions every 2 weeks. Thus, participants in IERITA plus TAU typically had contact with 2 clinicians (face-to-face contact with a community clinician and online contact with the IERITA therapist). TAU was enhanced by weekly self-rated assessments and as needed follow-ups from the research team  . A zero-inflated negative binomial generalized linear-mixed effects regression model was used to estimate the trend over time for self-reported NSSI counts measured every week by including an interaction term between treatment condition and time while allowing for a random intercept and slope per individual. Treatment condition and study site were included as categorical variables and time (weeks zero to 16) was treated as a continuous variable. Exploratory analyses investigated whether certain relevant TAU or client characteristics moderated treatment effects . Treatment condition and study site were included as categorical variables. For assessor-rated NSSI frequency (DSHI-Y), a zero-inflated negative binomial regression model was fitted to the NSSI counts. For counts on other self-destructive behaviors (BSL-Supplement), zero-inflated Poisson regression models were used. Model comparisons are available in eTable 10 in 29 A detailed description of the mediation analysis, including sensitivity analyses that examined the impact of unmeasured pretreatment mediator-outcome confounding, is available in eMethods in 30 Mixed-effect regression analyses for repeated measures  are valid under the assumption that the data are missing at random  and Mplus version 8.1 (Muthén & Muthén), and statistical significance was set at P < .05.To determine whether change in week-to-week emotion dysregulation (DERS-16) during treatment mediated the overall effect of IERITA on week-to-week change in the self-reported NSSI frequency, we employed a parallel process latent growth curve modeling approach for mediation.A total of 166 participants  were recruited and randomly allocated to IERITA plus TAU (84 participants) or TAU only (82 participants) . Twelve Type and level of TAU (counseling and psychopharmacology) did not differ across conditions during the trial . In addiRatings of treatment credibility, expectancy, and satisfaction with IERITA were moderate to high  (Reduction in self-reported NSSI frequency (DSHI-Y) was larger in IERITA plus TAU vs TAU only from pretreatment to 1-month posttreatment  , panel A14-0.58) . Change 14-0.58) .P < .001; IRR, 0.34; 95% CI, 0.20-0.57). At 1-month posttreatment, participants in IERITA evidenced an 82% reduction in NSSI frequency , whereas those in TAU only evidenced a 47% reduction . At 3-month posttreatment, IERITA plus TAU still showed a superior reduction in NSSI frequency than TAU only  (30-0.96) , panel Bd = 0.52; 95% CI, 0.14-0.89), self-reported self-destructive behaviors , and assessor-rated global functioning  at 1-month posttreatment, as well as 3-month posttreatment . There were no statistically significant differences between the conditions in psychiatric symptoms (DASS-21) at 1-month or 3-month posttreatment  and 3-month post-treatment  did not differ significantly as a function of correctly vs incorrectly guessed treatment allocation. Eleven families revealed the treatment allocation by accident at 1-month posttreatment. Results on primary outcome remained virtually unchanged when the model was rerun without these ratings  was statistically significant   effect (Five participants (6%) reported suicide attempts in the IERITA plus TAU condition during the study period, compared with 9 participants (11%) in the TAU only condition. Further information on adverse events is presented in the eResults in 15Findings of this randomized clinical trial support the efficacy of this IERITA delivered adjunctive to TAU (vs TAU only). IERITA resulted in greater improvements in NSSI frequency (both self-reported and assessor-rated) and emotion dysregulation from pretreatment to 1-month posttreatment than TAU only. Results also revealed superior effects of IERITA plus TAU (vs TAU only) on global functioning and other self-destructive behaviors, with between-group effect sizes in the moderate range. Notably, treatment effects were maintained at 3-month posttreatment, suggesting durability of effects. Finally, week-to-week improvements in emotion dysregulation mediated week-to-week reductions in NSSI frequency in IERITA, providing further support for the theoretical framework underlying this treatment.The conditions did not differ significantly in psychiatric symptoms at 1 or 3 months following treatment, suggesting that IERITA results in improvements in emotion dysregulation and global functioning despite comparable levels of depression and anxiety (relative to TAU). These findings are consistent with the emphasis in IERITA on the acceptance of internal experiences and control of behaviors in the context of emotional distress (vs the control or downregulation of emotions themselves), and suggest that targeting emotion regulation may not be sufficient for the treatment of anxiety and depression among adolescents.This study had several strengths. These include the use of TAU as the control condition, the use of weekly measures of NSSI, inclusion of both self-reported and clinician-administered assessments, integrity of the masked assessments, and the sexual orientation diversity of the sample.The study also has limitations. IERITA was delivered adjunctive to TAU and compared with TAU only. Although this type of augmentation design holds value in the early stages of treatment development, the lack of equipoise between conditions precludes any conclusions regarding the specific effects of IERITA or mechanistic claims of the intervention. Also, not receiving IERITA could constitute a nocebo condition. Furthermore, although our mediation analysis included a sensitivity analysis to assess the impact of unobserved pretreatment confounders, potential posttreatment confounding  was not addressed. Future studies should include an active internet-delivered control condition matched on level of activity and attention from therapist and include additional mediators  and analysis  to test whether the mediator temporally precedes the outcome. Additionally, data cannot speak to whether participants’ ongoing psychopharmacological treatment was appropriately targeted and dosed. However, given that the onset, type, and frequency of counselling and psychopharmacological treatment were equivalent across conditions, it is unlikely that the observed treatment effects were the result of differences in these additional treatment contacts alone. Furthermore, although 39% of participants were self-referred and may have been particularly motivated to engage in an internet-delivered intervention, most participants were clinician-referred and many of the self-referred patients had previous or ongoing contact with CAMHS, suggesting that the sample is clinically representative. Moreover, the lack of data on interrater reliability of the clinician-administered assessments across clinicians and time is an important limitation and precludes determination of the quality of the clinician-rated outcomes , thereby threatening the validity of the findings. Nonetheless, replication of findings across self-reported and clinician-assessed NSSI frequency suggests that findings pertaining to the primary outcome of NSSI were not driven solely by rater differences.32 Moreover, although statistically significant, most treatment effects were in the moderate range, and for some measures, such as self-destructive behaviors, the difference in medians between the 2 conditions was only 1 episode. Future trials could include preregistered thresholds for evaluation of clinical significance.Likewise, the predominantly female sample and focus on adolescents with NSSID limit the generalizability of our findings. Future studies should examine the efficacy of this treatment among self-injuring adolescents without NSSID and with greater gender diversity. Future studies of IERITA should also include longer follow-up periods of at least 1 year. Moreover, at some weeks, the proportion of missing self-reported weekly data was moderate and the wide variety of observed missing data patterns precluded formal sensitivity analysis (treating data as not missing at random). However, intermittent missing observations are common in clinical trials employing weekly measures, and it is often reasonable to assume that these data points are randomly missing.Results of this trial suggest that a 12-week, therapist-guided, IERITA delivered adjunctive to TAU is efficacious in the treatment of NSSI and related factors compared with TAU only. Findings that week-to-week improvements in emotion dysregulation were associated with week-to-week reductions in NSSI frequency during IERITA provide preliminary support for the proposed mechanism of change in IERITA.