We also discussed the challenges and prospects of exosome-based drug delivery systems in cancer therapy.Over the years, there has been a high demand for developing new safe and effective drug carriers for cancer therapy. Emerging studies have indicated that exosomes can serve as potent therapeutic carriers since they offer low immunogenicity, high stability, innate and acquired targetability, and the stimulation of anti-cancer immune responses. Yet, the development of exosome-based drug delivery systems remains challenging due to their heterogeneity, low yield, and limited drug loading efficiency. Herein, we summarized the current application of exosomes derived from different cells as drug carriers in anti-cancer therapy  Cancer is a major public health problem and a leading cause of death worldwide. In 2020, 19.3 million new cancer cases and approximately 10.0 million cancer-related deaths were reported . SurgeryRecently, immunotherapy has become very popular in the treatment of cancer therapy. Immune checkpoint blockade (ICB), such as antibodies against programmed death receptor (PD)-1, PD ligand (PD-L)-1, and cytotoxic T-lymphocyte antigen 4 (CTLA-4), has shown to be very promising in treating cancer patients. However, immune checkpoint inhibitors fail to control tumor progression in many patients due to the immunosuppressive microenvironment in tumor tissues induced by cancer cells, immune cells, and tumor-associated stromal cells . Thus, net al. designed polymeric nanoparticles combined with an aptamer specifically recognizing human epidermal growth factor receptor 2 (Her 2), which could directly deliver docetaxel to cancer cells ovexxpressing Her 2 in vitro and in vivo  higher bioavailability ; 4) low accumulative toxicity in normal tissues; 5) selectively delivering anti-cancer drugs into cancer cells via ligand-receptor interaction or endocytosis to overcome drug resistance mediated by P-glycoprotein or other multidrug resistance-associated proteins  low immunogenicity; 2) low clearance of reticuloendothelial system proteins ; 6) the This review discussed the application, challenges, and prospects of exosome-based drug delivery systems in cancer therapy.Exosomes were first reported by Johnstone in 1987, who discovered a vesicle formation during the maturation process of reticulocytes . A few yet al. demonstrated that glioblastoma stem cell-derived exosomes enhance the self-renewal ability of glioma cells in vitro and in vivo , and docetaxel, respectively) exerted stronger proliferation-inhibiting effects on lung and breast cancer compared with the free drugs  could increase drug stability and cellular uptake ability, and in turn, reduce tumor growth in lung cancer-bearing mice compared with the free CEL , MMP9, vimentin, and N-cadherin (in vivo.RNA interference (RNAi) refers to the process of homologous mRNA-specific silencing induced by exogenous or endogenous double-stranded RNA. Compared to the small molecules mentioned above, RNAi exerts more prominent anti-cancer effects by selectively inhibiting pivotal genes controlling proliferation, migration, invasion, differentiation, or other malignant biological behaviors of cancer cells. Gene therapy through RNAi showed targeted advantage and few adverse reactions h et al.  loaded Tcadherin . Howeverin vivo and in vitro, the tissue distribution of these exosomes in vivo needs to be further investigated. Radha et al. found that, after administration of DIR-labelled milk-derived exosomes by oral gavage in mice, the biodistribution of these exosomes within the liver, lung, kidney, spleen, and brain was uniform , thus enhancing tumor targetability. Dan et al. co-incubated CD44 specific binding ligand hyaluronan with milk-derived exosomes to deliver doxorubicin (DOX) and found that these exosomes could target CD44 positive cancer cells to induce apoptosis  are cells isolated from multiple biological tissues such as adult bone marrow, adipose tissues, placenta, and umbilical cord. et al. demonstrated that MSC-derived exosomal miR-223 could protect neuronal cells from apoptosis via the PTEN-PI3K/Akt signaling pathway, and, in turn, suppress the progression of Alzheimer’s diseases  expression in breast cancer to inhibit angiogenesis in tumor tissues, thereby suppressing tumor growth in mice with breast cancer -2 expression and suppress the proliferation of breast cancer cells -DARPin could produce exosomes rich in LAMP2-DARPin. After being loaded with DOX, these modified exosomes could selectively target Her 2 positive breast cancer cells and, in turn, exert their cytotoxicity  is a small-molecule binding protein with a high affinity to Her 2. Hosna toxicity . Moreoveal cells .Despite the abilities of genetically engineered MSCs to endow their derived exosomes with targetability, the pro-cancer effects of these exosomes should not be ignored and need to be further elucidated before clinical application.Mammalian cells, including HEK293, HT-1080, and NS0 cells, represent important manufacturing platforms in bioengineering and are widely used for the production of recombinant therapeutic proteins and vaccines. Among them, HEK293 cells provide attractive advantages for the development of recombinant proteins or adenovirus productions, such as the human-like post-translational modification of protein molecules to support a better biological activity and high transfection efficiency to produce high-quality recombinant proteins. Recent studies have shown that exosomes derived from HEK293 cells have great potential to deliver anti-cancer drugs.et al. demonstrated that HEK293 cell-derived exosomes carrying miR-204-5p could decrease the expression of miR-204-5p targeted genes RAB22A and Bcl-2 and inhibit proliferation of colorectal cancer, breast cancer, lung cancer, gastric cancer, and glioma  could promote tumor progression by activating downstream pathways, such as phosphatidylinositol 3-kinase-AKT, RAS-mitogen-activated protein kinase, and vascular endothelial growth factor (VEGF) signaling . Zhang epression .et al. found that HEK293 cell-derived exosomes transferring siRNA-c-MET could significantly inhibit the expression level of c-MET and reverse cisplatin resistance of gastric cancer  is one of the mechanisms of cisplatin resistance in gastric cancer. Zhang c cancer .et al. infected HEK293 cells with LAMP-DARPin chimeric gene and obtained their derived exosomes. Consequently, exosomes carrying siRNA-TPD52 could target Her 2 positive breast cancer cells and obviously suppress oncogene TPD52 expression  overexpressing lung cancer stem cells and significantly decrease stemness-associated SOX expression to attenuate stemness phenotypic features of these cells via delivering siRNA  and purified their derived exosomes, which when loaded with anti-cancer drug 5-Fluorouracil (5-Fu) could directly target Her 2 positive colon cancer cells and inhibit the growth of colon cancer in mice  molecules; thus, they have an important role in initiating, regulating, and maintaining the immune responses as well as participating in the immunotherapy of cancer.et al. demonstrated that exosomes isolated from cryopreserved umbilical cord blood mononuclear cell-derived DCs (cryo CBMDCs) and pre-treated with lung cancer cell lysates could induce proliferation of allogeneic T-cells to exert greater cytotoxic activity against lung cancer cells compared with untreated cryo CBMDCs-derived exosomes , derived exosomes could significantly promote proliferation of CD8+ and CD4+ T cells, thus enhancing the cytotoxicity of T cells to melanoma cells  and E749-57 peptide, could promote cytotoxic activity and interferon (IFN)-γ excretion of CD8+ T cells, thereby significantly inhibiting the growth of cervical cancer -treated DCs could greatly activate T cells, stimulate cell proliferation, and increase the CTL/Treg ratio in tumor tissues, thus resulting in an obvious inhibition of tumor growth in melanoma-bearing mice , could be modified onto the surface of DC-derived exosomes. After being loaded with DOX, these exosomes could efficiently deliver DOX to cancer cells overexpressing PTK7 and thus exert their anti-cancer effects  cells  could be specifically delivered to tumor tissues and exert significantly anti-cancer effects in vivo  cells . Similar in vivo .DC-derived exosomes as drug carriers can improve the immunosuppressive microenvironment of tumor tissues through activation and increasing proportion of proliferating T cells and can exert cytotoxicity by delivering anti-cancer drugs, thus are considered to have great potential in the treatment of cancer.et al. reported that ovarian cancer cell-derived exosomes loaded with triptolide could accumulate in tumor tissues and inhibit ovarian cancer in mice  plasmid into gastric cancer cells to acquire TRIM3-overexpressing exosomes and found that these exosomes could downregulate the expression levels of OCT4, SOX2, and N-cadherin of gastric cancer cells, thereby inhibiting the tumor growth in mice  exhibited obvious targetability to surfactant protein C overexpressing lung cancer cells. After being loaded with miRNA-126, these exosomes showed an obvious suppression of tumor growth in lung cancer-bearing mice -derived exosomes is very low (et al. demonstrated that breast cancer cells produce fewer exosomes compared with normal cells in the same number per unit time (The yield of exosomes may vary in accordance with the type of donor cells. Tian very low . Moreovenit time . Compareet al. compared the loading efficiency of milk-derived exosomes with siRNA between chemical transfection and electroporation and found that the loading efficiency of siRNA into exosomes via chemical transfection was 30% while it was 35% through electroporation (et al. discovered that the loading efficiency of macrophages-derived exosomes with PTX when using co-incubation, electroporation, and sonication is different; the loading efficiency of sonication was 28.29%, while co-incubation and electroporation were 1.44 and 5.3%, respectively (et al. demonstrated that glioblastoma stem cell or glioma cell-derived exosomes loaded with DOX in the same condition exhibited different loading efficiency with 19.7 and 7.86%, respectively (At present, many methods have been used to load anti-cancer drugs into exosomes, including co-incubation, electroporation, and chemical transfection . Howeverporation . Kim et ectively . Moreoveectively . TherefoAlthough there are significant challenges and difficulties in applying exosome-based drug delivery systems, this natural vesicle has great potential in the biomedical field, especially in anti-cancer therapy. Yet, certain problems need to be solved before they can be used in clinical practice. Thus, the scientific community should further focus on the exploration of these problems.


Despite increased testing efforts and the deployment of vaccines, COVID-19 cases and death toll continue to rise at record rates. Health systems routinely collect clinical and non-clinical information in electronic health records (EHR), yet little is known about how the minimal or intermediate spectra of EHR data can be leveraged to characterize patient SARS-CoV-2 pretest probability in support of interventional strategies.We modeled patient pretest probability for SARS-CoV-2 test positivity and determined which features were contributing to the prediction and relative to patients triaged in inpatient, outpatient, and telehealth/drive-up visit-types. Data from the University of Washington (UW) Medicine Health System, which excluded UW Medicine care providers, included patients predominately residing in the Seattle Puget Sound area, were used to develop a gradient-boosting decision tree (GBDT) model. Patients were included if they had at least one visit prior to initial SARS-CoV-2 RT-PCR testing between January 01, 2020 through August 7, 2020. Model performance assessments used area-under-the-receiver-operating-characteristic (AUROC) and area-under-the-precision-recall (AUPR) curves. Feature performance assessments used SHapley Additive exPlanations (SHAP) values. The generalized pretest probability model using all available features achieved high overall discriminative performance . Performance among inpatients  was higher than telehealth/drive-up testing  or outpatient testing . The two-week test positivity rate in patient ZIP code was the most informative feature towards test positivity across visit-types. Geographic and sociodemographic factors were more important predictors of SARS-CoV-2 positivity than individual clinical characteristics.Recent geographic and sociodemographic factors, routinely collected in EHR though not routinely considered in clinical care, are the strongest predictors of initial SARS-CoV-2 test result. These findings were consistent across visit types, informing our understanding of individual SARS-CoV-2 risk factors with implications for deployment of testing, outreach, and population-level prevention efforts. As of January 20, 2021, over 96 million confirmed cases of COVID-19 and over 2 million COVID-19-related deaths have been reported worldwide . Health A review of published diagnostic and prognostic models for COVID-19 revealed substantial gaps in knowledge about factors contributing to individual outcomes . In the While seemingly obvious, geographic features of recent COVID-19 cases are not well-studied with regards to the spectrum of patient-level clinical features. Spatial analysis of COVID-19 incidence often used county-level or state-level aggregate or deidentified datasets to minimize reidentification risks , 19. In To better understand the value of routinely-collected EHR data, we examined the contribution of clinical, sociodemographic, and geographic features towards individualized risk of COVID-19 infection.We developed a retrospective cohort to assess the relative contributions of patient demographics, medical history information, and spatial factors to predict initial SARS-CoV-2 test results. We stratified the analysis based on the health system visit type  and characterized model performance using all features as well as limited subsets of features. The University of Washington Institutional Review Board approved this study as minimal risk and waived consent requirements. All analyses were performed in a HIPAA-compliant compute environment. Our approach used an Observational Medical Outcomes Partnership (OMOP) structured dataset  for tranThe CONSORT diagram depicting study inclusion/exclusion criteria is shown in Patients were classified as test-positive or test-negative based on the lab-confirmed outcome of their first recorded SARS-CoV-2 RT-PCR test. Those with inconclusive results or missing essential features were omitted. We converted clinical EHR data into continuous, categorical, or binary-valued features and merged rare categories. For example, patient occupation information was categorized to 2018 Census Occupation Codes groups . Highly We identified patient residential ZIP code from billing records and linked them to the American Community Survey’s 2018 ZIP Code Tabulation Area (ZCTA) to identify population density (person per sq. mile) and median household income. For each patient, as a proxy for disease prevalence near the patient’s home, we computed the positivity rate of all test results from the same ZIP code during the preceding two weeks. For the first test from each ZIP code, we treated this feature as missing.Ultimately, 186 features were available for analysis. Further details on the processing steps are provided in the We developed a binary classifier to predict the outcome of a patient’s initial SARS-CoV-2 RT-PCR test. To develop this model, we split data into three fixed partitions: training, validation, and testing . we first withheld a random 20% of the dataset for final model performance assessment . From the remaining 80%, we randomly subsampled data in a 4:1 fashion into training and validation subsets, respectively.Our primary models were GBDT implemented using the LightGBM library . GBDT haOur primary analysis characterized overall model performance across all visit types. Primary metrics for model performance were AUROC curves, which compare model sensitivity versus specificity, and AUPR curves, which compare positive predictive value (PPV) to sensitivity. These complementary metrics offer insight into the expected false positive rate of the model in contexts where positive cases are rare. For all model evaluations, we report mean AUROC and 95% confidence intervals computed using 1000 bootstrap resamples of the evaluation set. Imputation of missing data was not performed as GBDT natively handles missingness as potentially informative, appropriate for a clinical setting in which the choice to order a test reflects clinical judgment.To characterize model performance across clinical contexts, we stratified patients by visit type . To quantify the extent to which a given feature contributed positively or negatively to model prediction in these clinical settings, we computed SHAP values . Using aminimal , 2) intermediate , or 3) full . Table A1 in Finally, we examined the likely impact of data accessibility on model use in these settings by training variants using subsets of features representative of those expected to be available in inpatient, outpatient, or community testing scenarios. The intention of these feature sets was to represent the information likely to be accessible for real-word application of the model in that clinical environment. To do this, we first grouped the features into categories , and then associated these categories with one of three feature sets: 1) https://github.com/microsoft/sars-cov2-pretest-probability.To validate our choice of GBDT as a primary modeling strategy, we performed comparisons with logistic regression using mean imputation to account for missing features  were non-White ((n = 2522) tested positive for SARS-CoV-2 at the time of first testing, with observed differences by race, ethnicity, occupation status, and geography.Ultimately, records for 63 996 patients were available for analysis. Similar numbers of included patients were referred for testing from inpatient, outpatient, and telehealth visits. Data preprocessing is summarized in on-White . ApproxiThe primary model, fitted across all patients, achieved high discriminatory performance  for prediction of positive versus negative first-test result . This moThe feature “two-week test positivity in ZIP” was the most informative, with high recent positivity rates increasing positive predictions. Although age was also informative, the relationship between age and predicted test positivity was not monotonic, as depicted by the mottled coloring along the x-axis. Fig A2 in minimal set  maintained useful discriminatory power (AUROC > 0.7)  .In this study, we demonstrate that routinely collected EHR data can be used to build predictive models of initial SARS-CoV-2 RT-PCR test results with high discriminative performance across visit types. Overall, while a combination of both sociodemographic and clinical features yielded the highest predictive performance, geographic, demographic, and socioeconomic features were more strongly associated with initial SARS-CoV-2 test positivity than clinical characteristics across all visit types examined. Recent test positivity rate in home ZIP code was the most informative single factor. We also found that models using only a minimal set of variables, commonly available in most test settings, retained useful discriminative power. These findings demonstrate that effective risk-stratification is possible in drive-up test settings and at the population level, where individual clinical data  are not typically available.Unlike prior studies –13, thisA unique aspect of this study was the derived feature “two-week test positivity in ZIP,” which emerged as the most important single feature across all visit types and contributed more to the prediction than clinical data. This feature is a useful proxy for recent disease prevalence in the area and its relative importance in this context is consistent with the known epidemiology of community-based transmission , 26. We Other geographically derived factors also featured prominently within the list of important model features. Most notable was the feature “median household income in ZIP”, with lower income being associated with higher pretest probability of positivity. Alongside the presence of commercial health insurance and occupation status, these features point to the importance of including socioeconomic factors in models of test positivity. High population density areas have experienced high exposure risks and transmission levels, which may in part be due to high-density group occupancy , 28. WhiThe relative importance of geographic features in our model is key because these features are typically available in telehealth/drive-up test settings. Our finding that model discrimination varied by treatment setting was unsurprising but is important. Data availability , reasons for testing, and underlying health status vary greatly between inpatient, outpatient, and telehealth settings. Indeed, approximately half of the total cohort did not have a clinical history meriting antecedent vitals and laboratory studies. Researchers and practitioners considering implementation of such models should consider treatment setting carefully when evaluating their application and potential performance across these distinct clinical settings. Using this approach, we demonstrate that data routinely available to testing facilities, hospital systems, and public health officials can be usefully derived using readily available EHR data without recourse to other disparate sources . HeighteWhile clinical measures, such as vitals and laboratory measures, were not the focus of this study, these measures did contribute measurably to the predictive performance of models in some settings and warrant examination. Several clinical markers predictive of SARS-CoV-2 positivity in this study have previously been associated with severe COVID-19, including decreased white blood cell count , 37, 38,In the United States, COVID-19 vaccination strategies have prioritized social determinants among their guiding principles . These eThis study has some limitations. Data analyzed were drawn from a clinical care database wherein patient referral for testing was influenced by regional guidelines that impact broader generalizability . For exaIn conclusion, our study demonstrates that routinely collected non-clinical features in EHR contribute significantly to prediction of initial SARS-CoV-2 test positivity across a variety of visit types and clinical testing scenarios. The key role of sociodemographic features in the outcome of SARS-CoV-2 testing has implications not only for prediction of individual test positivity but also for objective deployment of testing, outreach, and population-level prevention efforts.S1 Appendix(DOCX)Click here for additional data file.S1 Checklist(DOCX)Click here for additional data file.


Triple-negative breast cancer (TNBC) is the most aggressive and heterogeneous cancer subtypes. High rates of metastasis, poor prognosis, and drug resistance are the major problems associated with TNBC. The current chemotherapeutics eliminate only the bulk tumor cells (non-BCSCs) and do not affect breast cancer stem cells (BCSCs). The BCSCs which are left behind after chemotherapy is reported to promote recurrence and metastasis of TNBC. Death receptor-5 (DR-5) is exclusively expressed in TNBCs and mediates the extrinsic pathway of apoptosis. DR-5, therefore, can be exploited for targeted drug delivery and to induce apoptosis. Gamma-secretase mediated Notch signaling in BCSCs regulates its proliferation, differentiation, and metastasis. The endogenous ligand, Delta-like ligand 4 (DLL4), is reported to activate this Notch signaling in TNBC. Blocking this signaling pathway using both gamma-secretase inhibitors (GSIs) and DLL4 monoclonal antibody (mAb) may produce synergistic benefits. Further, the GSIs  suffer from poor bioavailability and off-target side effects such as diarrhea, suppression of lymphopoiesis, headache, hypertension, fatigue, and ventricular dysfunctions. In this hypothesis, we discuss Solid lipid nanoparticles (SLNs) based drug delivery systems containing GSIs and surface modified with DR-5 and DLL4 monoclonal antibodies (mAb) to effectivity target and treat TNBC. The delivery system is designed to deliver the drug cargo precisely to TNBCs through its DR-5 receptors and hence expected to reduce the off-target side effects of GSIs. Further, DLL4 mAb and GSIs are expected to act synergistically to block the Notch signal mediated BCSCs proliferation, differentiation, and metastasis. In the EMT process, the epithelial BCSCs cells lose their inter-cellular adherence by gaining invasion and migration capabilities.8 There is a need, therefore, to eliminate BCSCs in addition to non-BCSCs.Triple-negative breast cancer (TNBC) is characterized by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2).11 The above findings has prompted scientists to evaluate possible benefits of gamma-secretase inhibitors (GSIs) in TNBC to control growth, prevent self-renewal, and suppress drug resistance of TNBCs.16 Some studies suggest that, although GSIs, like DAPT, LY-411575, RO4929097, MK0752 are not significantly cytotoxic, several studies indicate that they will be useful in potentiating the cytotoxic effects of other anticancer agents and helpful in eliminating BCSCs. However, the GSIs are associated with severe off-target side effects such as diarrhea, suppression of lymphopoiesis, headache, hypertension, fatigue and ventricular dysfunctions, which limit their clinical use.19 Targeted delivery of GSIs to TNBCs could be one of the strategies to overcome this problem.20The Notch signaling pathway is a fundamental regulator of angiogenesis in non-BCSCS and self-renewal & maintenance in BCSCs.21 DLL4 binding to Notch1/Notch4 receptors ensures the cleavage of the Notch intracellular domain (NICD) by the enzyme gamma-secretase. The NICD translocates to the nucleus and activates the expression of notch target genes involved in angiogenesis, apoptosis, metastasis, and chemoresistance.23 DLL4 and gamma-secretase enzyme are highly expressed in TNBC cells and are, therefore, considered as unique targets of TNBC.25Activation of Notch1 and 4 receptors by delta-like ligand 4 (DLL4) endogenous ligand in TNBC results in aberrant activation of Notch signaling.27 Besides, GSIs also potentiate the effect of chemotherapeutic agents by inhibiting the genes involved in chemoresistance (Hes and Hey).29 Existing evidence also suggests that the inhibition of DLL4 mediated activation of Notch receptors by anti-DLL4 mAb also produce anti-angiogenic, proapoptotic and chemo-sensitizing effects on TNBC cells.32 It was recently reported that a combination of DLL4 mAb with GSIs has synergetic proapoptotic effects.33Recent studies have reported that GSIs are useful in the eradication of BCSCs, inhibition of EMT, angiogenesis, and tumor growth.35 Besides, activation of DR-5 leads to activation of extrinsic apoptotic signaling and hence proapoptotic effects.36Death receptor 5 (DR-5) is a member of tumor necrosis factor receptor superfamily, overexpressed explicitly on the surface of TNBC cells. Therefore, it can be used as a target for effective drug delivery in TNBCs.37 Also, SLNs offer several advantages such as improved stability of the drug, higher entrapment efficacy, and biocompatibility over other nanoparticle-based delivery systems. SLNs with appropriate stealth properties will have reduced clearance and improved drug cargo delivery to the tumor site through enhanced permeation and retention (EPR) effect.39 However, accumulating evidence suggest that the EPR effect alone` is not sufficient to achieve site-specific delivery of drug cargo to the tumor site. Alternatively, unique cancer surface proteins have been targeted using monoclonal antibodies (mAbs) to improve target-specific delivery of drug cargo.40Solid liquid nanoparticles (SLNs) are widely used for targeted drug delivery to minimize off-target effects and improve the bioavailability of drugs.TNBC targeted GSI-SLNs surface modified with DR-5, and DLL4 mAbs can be an effective strategy to treat TNBC.The SLN delivery system will deliver the GSIs drug cargo precisely to TNBCs through membrane DR-5 receptors and hence reduce the off-target side effects of GSIs. DLL4 mAb and GSIs are expected to act synergistically to block the Notch signal mediated BCSCs proliferation, differentiation, and metastasis. Further, when combined conventional chemotherapy, the formulation will effectively eliminate both BCSCs and non-BCSCs and, thus, help in achieving the complete cure of TNBC.42 Notch signaling is activated by the binding of transmembrane ligands  to the Notch receptors present on the cell surface. This binding results in the proteolytic invasion of Notch by a presenilin-dependent gamma-secretase complex to release of NICD, which later translocates to the nucleus and heterodimerizes with a transcription factor, CSL (suppressor of hairless), and activates various target genes  pathway.50 The Notch-mediated activation of all these factors promotes cell proliferation, angiogenesis, resistance to apoptosis, and self-renewal of BCSCs.Notch signaling is one of the critical pathological pathways implicated in TNBCs. This pathway regulates the expression of target genes such as HES1, HEY2, MYC, CCND1, HES4, NRAR, etc., and involved in BCSCs proliferation, differentiation, and apoptosis.et genes .43-45 Se51 Ding et al demonstrated that anti-DR-5 mAb-mediated delivery of dacarbazine (DTIC) nanoparticles show an improved antitumor and proapoptotic activity when compared to the bulk drug.52 Tummala et al, report that, gold nanoparticles of oxaliplatin conjugating with anti-DR-5 mAb show enhanced anticancer activity and site-specific delivery of drug cargo.53 Zhang et al reported that anti-DR-5 (Zaptuzumab) antibody-drug conjugate show increased therapeutic efficacy and safety when compared to naked antibody Zaptuzumab treatment.54Surface modified SLNs with DR-5 mAb expected to bind selectively to DR-5 receptors present on TNBC and therefore help in active targeting through receptor-mediated endocytosis, further binding with DR-5 may also initiate the extrinsic pathway of apoptosis and hence proapoptotic effects.55 In TNBC, DLL4 binding to the Notch receptors promotes the transcription activation of genes regulating tumor angiogenesis and growth.56 DLL4 mediated activation of Notch signaling, therefore, improves vascular function and promotes tumor growth.57 Jia et al demonstrated that a humanized anti-DLL4 mAb inhibits breast tumor growth in an MDA-MB-231 xenograft model in mice. The results indicate that anti-DLL4 mAb prevents tumor growth by blocking the DLL4-Notch signaling pathway.58 Hoey et al report that anti-DLL4 treatment blocks the Notch pathway mediated expression of anti-apoptotic genes (HSPA6 and BIRC3) and hence sensitize the tumor cells towards chemotherapy.59The expression of Notch ligand (DLL4) in tumor vasculature is high as compared to healthy tissues.60 Wang et al successfully developed antibody-drug conjugates of Monomethyl auristatin E and anti-DLL4 antibody to promote tumor cell death and to achieve site-specific delivery.61Yen et al establish that the combination of anti-DLL4 antibody and paclitaxel decrease BCSCs frequency and retard the tumor relapse in paclitaxel-resistant TNBCs.63 Accumulating pieces of evidence suggest that, the GSIs are associated with clinical limitations such as poor bioavailability and off-target side effects include diarrhea, suppression of lymphopoiesis, headache, hypertension, fatigue, and ventricular dysfunctions.63 To overcome these limitations, researchers focused on nanocarriers based drug delivery which improves bioavailability and delivers the drug cargo specifically to tumor sites.64Gamma-secretase enzyme regulates tumor-promoting Notch signaling in TNBC by controlling NICD levels. Inhibition of this enzyme through GSIs, therefore, a promising strategy.15 Kang et al establish that the concurrent treatment of GSIs with anti-DLL4 increases the anticancer and proapoptotic efficiency of GSIs in gastrointestinal cancer. The combined therapy of GSIs with anti-DLL4 boosted the expression of BAX and P53 and reduced the expression of Bcl-2. On the other hand, naïve GSI only enhanced the expression of BAX and P53, suggesting that the reduced Bcl-2 expression had a significant function in synergistic antitumor and proapoptotic effects.33Mamaeva et al developed a glucose functionalized Mesoporous silica nanoparticles of GSI (DAPT). They report that the developed nanoformulation efficiently delivered the drug to TNBC and reduced the BCSCs population by inhibiting the Notch signaling pathway.66 Pindiprolu et al reported that, SLNs of niclosamide improve the anticancer efficiency by increasing the site-specific delivery of niclosamide to the TNBC.67 Wang et al successfully demonstrate the improved anticancer efficacy of Curcumin SLNs in SKBR3 cells as compared to naïve curcumin.68Drug delivery using SLNs is an accepted approach for targeted drug delivery to improve efficacy and reduce off-target side effects. SLNs as a nanocarrier, enhance the bioavailability, and provide control release. Besides, SLNs also modulate the release kinetics, minimize systemic toxicity and increase the therapeutic efficacy of chemotherapeutic agents.69 Kosmides et al developed nanoparticles surface modified with two diﬀerent mAbs (anti-PD-1 and anti-CTLA-4 mAb), which concurrently impede the inhibitory PD-L1 signal and activate T cells through the 4-1BB co-stimulatory pathway.70 The combination of mAbs on the surface of nanoparticles has significantly increased their efficacy when compared to individual mAbs.70 Chen et al developed dual antibodies (anti-CD44 and anti-CD133) conjugated transretinoic acid-loaded poly(lactide-co-glycolide)-lecithin-PEG nanoparticles to target cancer stem cells (CSCs). They report that dual-targeted nanoparticles effectively inhibit the tumor growth and eradicate the CSC population.71Dominguez et al successfully conjugated anti-RNEU and anti-CD40 antibodies on the surface of PLA-(poly dl-lactic acid)-biodegradable nanoparticles. They establish that dual antibody conjugated nanoformulation boosts the antitumor response and also results in complete tumor eradication.In this article, we propose to prepare DR-5, and DLL-4 mAbs functionalized SLNs of GSIs to enhance their bioavailability, provide TNBC specific delivery, and to reduce off-target side effects. Further, anti-DLL-4 mAb and GSIs may synergistically act to eliminate the resistant BCSCs of TNBC. Besides, when combined with anticancer chemotherapeutics, the formulation may enhance their overall anticancer efficacy.The study does not involve any human subjects and/or animals.The authors confirm that this article content has no conflict of interest.The authors would like to thank the Department of Science and Technology- Fund for Improvement of Science and Technology Infrastructure in Universities and Higher Educational Institutions (DST-FIST), New Delhi, for their infrastructure support to our department (Grant No. SR/FST/LSI-574/2013). MK acknowledges AICTE-NDF (Application No.- 52198) for her SRF.


Correction to: Eur J Hybrid Imaginghttps://doi.org/10.1186/s41824-019-0060-8The original publication of this article , and who underwent clinically indicated a 123I-MIBG…Correct: Heart failure patients who received an ICD for primary prevention, according to prevailing guidelines , and who underwent a 123I-MIBG…Page 3Incorrect: For retrospective analysis of clinically acquired data anonymously handled, the institutional review board waived the need for written patient informed consentCorrect: For retrospective analysis of clinically acquired data anonymously handled, the institutional review board waived the need for written patient informed consent. For a subgroup of patients, the 123I-MIBG scintigraphy was performed under a prospective study (NCT01940081) and the patients provided written informed consent.


Following the introduction of live-attenuated rotavirus vaccines in many countries, a notable reduction in deaths and hospitalisations associated with diarrhoea in children <5 years of age has been reported. There is growing evidence to suggest that live-attenuated vaccines also provide protection against other infections beyond the vaccine-targeted pathogens. These so called off-target effects of vaccination have been associated with the tuberculosis vaccine Bacille Calmette Guérin (BCG), measles, oral polio and recently salmonella vaccines, and are thought to be mediated by modified innate and possibly adaptive immunity. Indeed, rotavirus vaccines have been reported to provide greater than expected reductions in acute gastroenteritis caused by other enteropathogens, that have mostly been attributed to herd protection and prior underestimation of rotavirus disease. Whether rotavirus vaccines also alter the immune system to reduce non targeted gastrointestinal infections has not been studied directly. Here we review the current understanding of the mechanisms underlying off-target effects of vaccines and propose a mechanism by which the live-attenuated neonatal rotavirus vaccine, RV3-BB, could promote protection beyond the targeted pathogen. Finally, we consider how vaccine developers may leverage these properties to improve health outcomes in children, particularly those in low-income countries where disease burden and mortality is disproportionately high relative to developed countries. Diarrhoea, pneumonia and malaria remain the leading causes of death among children under the age of five years . ApproxiNotably, diarrhoeal disease associated with rotavirus infection accounts for an estimated 128,500 under-five deaths annually, despite increasing rollout of rotavirus vaccines . RotavirBoth epidemiological and immunological studies suggest that certain vaccines exhibit immuno-modulatory functions that induce beneficial off-target effects against unrelated pathogens. Whilst limited data are available to explain this phenomenon, non-specific effects are in part thought to originate from epigenetic reprogramming of innate cells that subsequently lead to altered innate memory. Strong evidence on non-specific effects has been reported from live-attenuated vaccines, notably Bacille Calmette Guérin (BCG) and measles vaccines ,12,13,14Contrary to the traditional view that vaccines induce specific immunity to a specific pathogen, evidence has emerged over the past decades that suggests vaccines can also induce protection against non-targeted pathogens. This phenomenon has been termed ‘non-specific effects’, ‘heterologous effects’ or ‘off-target effects’ of vaccination, and from herein this article will use the latter term. Both epidemiological and immunological studies have demonstrated protective off-target effects from live vaccines whereby they reduce susceptibility to non-targeted pathogens. By contrast, non-live vaccines such as whole cell Diphtheria-Tetanus-Pertusis (DTP) may have been associated with negative effects such as slightly increased morbidity and mortality from non-targeted pathogens, particularly observed in low income-high mortality settings ,15,16. TDespite the concept of off-target effects of vaccines attracting attention from scientists worldwide, the precise immunological mechanisms associated with these effects remain unclear. Nevertheless, several vaccine effect studies have investigated both innate and adaptive immune components as potential mediators of off-target effects. BCG is a widely used anti-tuberculosis vaccine and a well-studied model of vaccine-induced off-target effects. BCG-induced off-target effects have been widely investigated using murine models by comparing immune responses following heterologous antigen challenge between BCG naïve and BCG-vaccinated mice. Earlier studies by Spencer et al. (1976) reported increased capacity of BCG-stimulated macrophages in mice in reducing viral titres of influenza A virus compared with controls. A study by Starr et al. further demonstrated that a single dose of BCG vaccine administered intradermally or intraperitoneally protected newborn mice from herpes simplex virus type 2 in a controlled murine model . It has FH) cell development and B-cell maturation, thus enhancing a broad range of antibody responses against an array of pathogens  live-attenuated vaccine and RotaTeq® , a live-attenuated pentavalent  bovine-human reassortant vaccine, are currently the most widely used rotavirus vaccines - and Rotasiil®, G1, G2, G3, G4, G9  have been introduced into the National Immunisation program in India with introductions planned in Benin (Rotavac®) and Democratic Republic of Congo and Burkino Faso (Rotasiil®). The Lanzhou Lamb (G10P [® (G1P[8]) are available in the market for use in China and Vietnam, respectively. Over 80 countries have incorporated Rotarix® into their immunisation programmes, 16 countries have introduced RotaTeq® and 10 countries have introduced both Rotarix® and RotaTeq® into their routine immunisation schedules. Whilst both vaccines are administered to infants from 6 weeks of age, Rotarix® is given in two doses and RotaTeq® is administered in a three-dose schedule [Early observations that children with secondary exposure to rotavirus were protected from moderate-to-severe rotavirus-associated gastroenteritis paved the way for rotavirus vaccine development . The firsception ,73,74. Cvaccines ,6. Rotavmb (G10P ) and Rotschedule ,  reported that Rotarix prevented 93% of severe rotavirus diarrhoea cases and 52% of severe all-cause diarrhoea cases in low-mortality settings. In high mortality settings, Rotarix prevented 58% of severe rotavirus diarrhoea cases and 27% of severe all-cause diarrhoea cases . UnderpeThe persistence of a high rotavirus disease burden in early life requires development of better vaccines that can be administered as early as the first week of life. The current licenced rotavirus vaccines are administered from 6 weeks of age, therefore missing the opportune window when the neonatal immune system is more susceptible to infections ,83,84. FRotavirus vaccines are routinely administered with the first dose at 6 weeks of age. Developed from a human neonatal rotavirus strain, RV3-BB vaccine is an ideal candidate for a neonatal administration strategy, with the first dose administered at or soon after birth. RV3-BB phase II clinical trials conducted in both a high-income, low-child mortality country  and a low-middle income, high-child mortality country  have shown that RV3-BB is immunogenic and well tolerated when administered as a three-dose neonatal or a routine infant schedule ,84,88,89Clearly, the concept of off-target effects of vaccines continues to evolve and important lessons are yet to be unmasked. However, there are important questions that remain to be addressed in order to utilise this intrinsic feature of vaccines to inform vaccine design. To date, limited studies have analysed the putative off-target beneficial effects of live-attenuated rotavirus vaccines either from an epidemiological or mechanistic point of view ,91,92. MWhile the current understanding of mechanisms of vaccine-induced off-target effects is largely drawn from BCG studies, it would be intriguing to investigate the possible genes and signalling pathways that may be involved in influencing innate and non-targeted adaptive responses following rotavirus vaccination. Animal model studies by Lee et al. 2018) demonstrated that a cold-adapted live-attenuated influenza vaccine (CAIV) provided immediate protection against respiratory syncytial virus (RSV) infection. They further reported that TL3/TL7 signalling pathways were essential for these observed rapid beneficial off-target effects ,94,95,96 demonstrEfforts to improve rotavirus vaccine effectiveness in low-income countries include development of next generation oral rotavirus vaccines, exploration of vaccine adjuvanticity, alternative vaccine administration schedules and alternative routes of vaccine administration such as injectable subunit or inactivated rotavirus vaccines. It is hoped that these alternative options will address some of the challenges associated with oral vaccination in children in low-income countries including oral vaccine interference, negative impact of enteropathy and burden of enteric infections. However, when considering these alternative approaches the potential for impact on non-specific effects currently provided by an oral live-attenuated rotavirus vaccine should be assessed ,80Neonates have unique immune systems that are susceptible to a range of infections largely due to limited experience and exposure to microbes. The neonatal immunological milieu favours inhibition of T helper 1 (Th1) and pro-inflammatory responses that further impair innate and adaptive responses to neonatal diseases ,102. FurA lack of an appropriate research model to study off-target effects in neonates and infants has presented a challenge to its further understanding. Human neonatal trials are often impractical and ethically challenging thereby limiting interrogation of immune responses in early life. Neonatal mouse models provide a practical means of investigating off-target effects and vaccine interventions, however, inherent variations between humans and mice restrain the direct extrapolation of findings to humans . UmbilicUmbilical cord blood samples can be leveraged to provide useful insights into the immune ontogeny and the suspected vaccine influence in inducing off-target effects of vaccines . A compaHow long do vaccine-induced off-target effects last?To what extent do external factors such as maternal vaccine exposure, birth method and vaccine type influence occurrence of off-target effects?What mechanistic approaches can separate vaccine-induced off-target effects versus environmentally induced off-target effects ?At what age does sex influence the occurrence of off-target effects?Where adequate evidence on sex interaction with vaccine-induced effects is gathered, what is the practicability of developing vaccines tailored to specific sex or about interindividual variations that occur naturally?In the context of a neonatal live-attenuated RV3-BB vaccine, can co-administration of RV3-BB with known off-target inducers such as BCG affect induction of anticipated off-target effects?Important mechanistic questions that require further attention include:As the hunt for improved knowledge and understanding of vaccine-induced off-target effects continues, interpretation of findings must take into consideration the potential cofounders such as the geographical or epidemiological setting, maternal vaccine or pathogen exposure, biological factors such as sex, disease burden to infections such as norovirus, cytomegalovirus and vaccine timing . AttribuIn summary, live-attenuated rotavirus vaccines have the potential to induce beneficial off-target effects which could be clinically significant when administered to the immunologically naïve neonate. The live-attenuated neonatal rotavirus vaccine, RV3-BB, administered from birth offers potential to provide early protection against severe rotavirus disease but also infection from other pathogens via its off-target effects on the immune system of the newborn.


Coronavirus disease 2019 (COVID-19) often presents asymptomatically or milder inchildren compared to adults. The role of young children in the transmission ofsevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains largelyunknown. In the Netherlands, the first action of loosening the partial lockdownthat had been implemented to reduce SARS-CoV-2 transmission was the reopening ofprimary schools on 1 May 2020. We subsequently conducted a prospective cohortstudy among healthcare workers (HCWs) with primary school-attending childrenversus HCWs without children living at home. We tested each HCW three times forSARS-CoV-2 from May 20 to June 15 2020 at 1-week intervals. In total, 832nasopharyngeal swabs were taken from 283 HCWs with primary school-attendingchildren living at home and 864 nasopharyngeal swabs from 285 HCWs withoutchildren living at home. All nasopharyngeal swabs tested negative forSARS-CoV-2. In our region with a low population density and low SARS-CoV-2prevalence, reopening of primary schools did not lead to an increase ininfections. The results of this study may serve as an example for theimplementation of regional strategies to reduce SARS-CoV-2 transmission incountries with large variations in both population density and SARS-CoV-2prevalence. Coronavirus disease 2019 (COVID-19), the disease caused by severe acute respiratorysyndrome coronavirus 2 (SARS-CoV-2), often presents asymptomatically or milder inchildren compared to adults . An analSupplementary material)  . ToanswThis cohort study started after a period of active case finding among HCWs at theUniversity Medical Center Groningen (UMCG). In the northern Netherlands, the firstcase of COVID-19 was diagnosed in the last week of February 2020 . As of 1The UMCG is the sole tertiary care centre in the northern part of the Netherlandssupplying care for the provinces of Groningen, Friesland and Drenthe, a populationof approximately 1.7 million inhabitants. As of 10 March 2020, HCWs of the UMCG wereroutinely tested by the occupational health service when showing symptoms compatiblewith COVID-19. If transmission within a department was likely, asymptomatic HCWs onthe department were also tested. The number of HCWs tested and the numbers ofpositive and negative results were recorded.Supplementary material). Each participant was tested three times, at1-week intervals. If symptoms compatible with COVID-19 occurred between two testingmoments, an extra test was scheduled . A baseline questionnaire was filled outprior to the first testing moment. An additional questionnaire regarding dailycontacts, travel history and symptoms was filled out every testing day .The BackToSchool-study was a prospective cohort study among UMCG employees. Arecruiting advertisement was posted in the daily digital newsletter. HCWs wereeligible for inclusion if they were 18 years or older, had at least one primaryschool-attending child (study group) or had no children living at home (controlgroup). An exclusion criterion was a previous positive test result for SARS-CoV-2for the participant or their family members. Only one HCW per family could beenrolled. After reopening of primary schools on 11 May, from 20 May to 15 June 2020,participants were tested for SARS-CoV-2 by real-time polymerase chain reaction(RT-PCR) on nasopharyngeal and throat swabs  and the estimation of a difference between groupsof 3%. Statistical analyses were performed using IBM SPSS 23.0.Supplementary Figure 1). The epidemic started in the south of theNetherlands and before it had reached the northern provinces, the partial lockdownwas introduced country-wide.After reopening primary schools, we found no increased SARS-CoV-2 incidence amongHCWs compared to previous weeks. Nor did we find a difference in SARS-CoV-2incidences between HCWs with primary school-attending children versus HCWs withoutchildren living at home. In fact, no infections were detected at all. To put thesefindings in perspective, the epidemic in the Netherlands evolved from the beginningof March, peaked in April and stabilised at low frequency in May and June . However, the cumulative prevalence in ourregion until July 21 2020 was 91/100,000 inhabitants, compared to the Dutch total of299/100,000 inhabitants  and of 0.5% (41/7703) for the three northern provinces . We did The majority of positive cases in the UMCG were UMCG-HCWs (69%). By very early andactive testing of all symptomatic HCWs, and excluding those with a positive testfrom working, we were able to reduce transmission of SARS-CoV-2 in our hospital.This service was promptly extended to all HCWs in critical professions in theprovinces of Groningen and Drenthe, in cooperation with the Municipal HealthServices and regional laboratories. In this collaboration, we also offered testingto symptomatic family members of HCWs, before the nationwide screening wasinitiated. This contributed amongst many other factors to a very low reproductivenumber in the northern Netherlands.A cross-sectional study conducted in the southern province of Noord-Brabant showedthat 6% out of 1353 symptomatic HCWs tested positive for SARS-CoV-2 and that themajority only experienced mild symptoms . It isoThe findings of this study suggest that reopening primary schools in areas with a lowpopulation density and low SARS-CoV-2 incidences will not cause disproportionalSARS-CoV-2 transmission in this area. However, it is important to state that ourstudy does not exclude that in another epidemiological context, with a higherincidence, introduction of positive cases into schools could have led to enhancedtransmission of SARS-CoV-2.Click here for additional data file.Supplemental material, sj-docx-1-bji-10.1177_17571774211012469 for Effect ofschool reopening on SARS-CoV-2 incidence in a low-prevalence region: ProspectiveSARS-CoV-2 testing in healthcare workers with primary school-attending childrenversus without children living at home by Melvin Frie, Lisa M Havinga, JannekeWiersema-Buist, Charlotte G Veldman, Marjan JT de Vries, Lilli Rurenga-Gard,Alex W Friedrich and Marjolein Knoester in Journal of Infection Prevention


Leishmania infantum, Anaplasma phagocytophilum, Rickettsia spp., Toxoplasma gondii or Chlamydophila felis. No pharyngeal or rectal swab tested positive for SARS-CoV-2 RNA on real time reverse transcription-polymerase chain reaction (rRT-PCR). Our data show that SARS-CoV-2 did infect stray cats in Lombardy during the COVID-19 pandemic, but with lower prevalence than found in owned cats. This should alleviate public concerns about stray cats acting as SARS-CoV-2 carriers. Cats are susceptible to infection with severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). Whilst a number of studies have been performed worldwide on owned cats, limited data are available on stray, colony or shelter cats. We investigated SARS-CoV-2 infection in a stray cat population before and during human outbreaks of SARS-CoV-2 in cities in the Lombardy region in northern Italy, a high endemic region for SARS-CoV-2, using serological and molecular methods. A cohort of different samples were collected from 241 cats, including frozen archived serum samples from 136 cats collected before the 2019 coronavirus disease (COVID-19) pandemic and serum, pharyngeal and rectal swab samples from 105 cats collected during the SARS-CoV-2 outbreak. All pre-pandemic samples tested seronegative for antibodies against the nucleocapsid of SARS-CoV-2 using indirect enzyme linked immunosorbent assay (ELISA) test, while one serum sample collected during the pandemic was seropositive. No serological cross-reactivity was detected between SARS-CoV-2 antibodies and antibodies against feline enteric (FECV) and infectious peritonitis coronavirus (FIPC), Feline Immunodeficiency Virus (FIV), Feline Calicivirus (FCV), Feline Herpesvirus-1 (FHV-1), Feline Parvovirus (FPV),  In December 2019, human cases of coronavirus disease 2019 (COVID- 19), caused by the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), were first reported in Wuhan, China .As already reported during the 2003 SARS-CoV-1 outbreak , cats arDomestic cats were also sporadically reported to be naturally infected by SARS-CoV-2 ,10,11,12Experimentally ,6,7,8 anWhilst a number of studies have been performed on owned cats worldwide in the last year ,22,23,24Leishmania infantum in cats [Stray colony free-ranging and shelter cats are very common in Italy and often have close contact with human populations. These animals are an important sentinel for local disease trends and for the detection of the emergence of new pathogens and infectious diseases, such as that caused by  in cats  or for f in cats ,27. ActiIn this study, we investigated the serological and molecular presence and prevalence of SARS-CoV-2 infection in a cohort of stray colony and shelter cats living in cities in Lombardy region, northern Italy using serological and molecular techniques, to better understand the epidemiological roles of these cats in SARS-CoV-2 infection.Samples were collected from a total of 241 cats mainly from two cities  and surrounding areas, in Lombardy region, northern Italy. Samples were collected pre- and during COVID-19 pandemic, with the cutoff between the two periods defined as the date of first detection of SARS-CoV-2 RNA in the environment—as found in sewage samples from Milan city on 18 December 2019 .Leishmania infantum and co-infections  and Feline Leukemia virus (FeLV)) in stray colony cats [From the pre-pandemic period we analyzed 136 frozen archived serum samples, 89 from free-ranging stray colony cats and 47 from abandoned shelter cats, collected before December 2019. These samples had mostly been collected for previously published epidemiological studies, including one on ony cats . Some saony cats . Some ofPandemic samples were collected until February 2021 from 105 cats, 77 free-ranging stray colony cats and 28 abandoned shelter cats. These consisted of paired serum samples, oro-pharyngeal and rectal swabs from 75 stray colony cats and 23 shelter cats. In seven cats, rectal and oro-pharyngeal swabs were not collected, while in six cats, serum was not collected, giving a total of 99 serum samples available for serological analysis and 98 paired rectal and oro-pharyngeal swabs to be analyzed by molecular methodsDeep oro-pharyngeal swabs were collected by insertion and rotation of sterile swabs into the posterior pharynx and tonsillar areas. Rectal swabs were collected by inserting the swab 1.5 cm past the anal opening and rotating the swab gently. Swab samples were stored at −20 °C and shipped within 24 h to the Experimental Zooprophylactic Institute (IZS) of Sicily, where real time reverse transcription-polymerase chain reaction (rRT-PCR) analysis was performed to determine the presence of SARS-CoV-2 RNA. Whole blood samples (2.5 mL) were drawn from jugular vein into plain collection tubes to obtain serum after centrifugation, which was aliquoted and stored at −20 °C prior to being sent for enzyme-linked immunosorbent assay (ELISA) antibody tests at IZS of Sicily. All samples were collected when the cats were under general anesthesia before performing the neutering surgery during trap, neuter and release (TNR) sterilization programs that were part of a national program to control stray pet populations under Italian National Law (law no. 281/1991).Full signalment details, including breed, sex, age, clinical status, province in which the cat was trapped and clinical status for each cat  were collected when available.The protocols for the study and animal welfare were reviewed and approved by the Animal Welfare Bioethical Committee of the University of Milan . Frozen archived serum samples were collected from cats following protocols already approved by Animal Welfare Bioethical Committee of the University of Milan and were collected with the consent of legal representatives of the stray colony or shelter cats.® ELISA, SARS-CoV-2 Double Antigen Multi-species, IDvet, Grabels, France), following the manufacture’s instruction and as previously used and described [Antibody presence against the nucleocapsid of SARS-CoV-2 antigen was tested by double antigen indirect enzyme linked immunosorbent assay (ID-Screenescribed . The kitFor each sample the “Sample/Positive control” (S/P) ratio was calculated and expressed as a percentage (S/P%) (1):Based on the manufacturer’s ELISA cut-off, samples with S/P% less than or equal to 50% were considered negative, between 50% and 60% are considered as doubtful. Greater than or equal to 60% were considered as positive.® Viral RNA Xpress Kit  according to the manufacturer’s instructions. The RNA was tested for SARS-CoV-2 RNA in assay RT-PCR to check for the presence of E, N2 and RdRp gene sequences . A volume as 5 µL of extracted RNA, was employed in a 20 μL reaction test contained, 10 μL of 2× Brilliant one step RT-PCR reaction buffer  and 3.2 mM magnesium sulphate). Primer and probe sequences were employed at concentrations and conditions optimized, respectively, according to the Centers for Diseases Control (CDC) protocol and Corman paper [The deep oropharyngeal swabs and rectal swabs were kept on cool-packs until arrival in the laboratory. RNA was extracted from clinical samples employing the Mag-Bindan paper . The proThe demographic and clinical data of cats surveyed in the study are summarized in All 136 sera collected pre-pandemic were seronegative by indirect ELISA.Out of the 99 sera collected in cats during the pandemic, one (1.0%) was ELISA positive with a S/P% of 200%. Tests were performed in triplicate and repeated three times with similar results and the mean OD of the sample was 1.74 with a standard deviation of 0.085. The three replicates were performed by two operators using different batches of the kit.® Combo Plus FeLV Ag/FIV Ab, IDEXX Laboratories, Europe), seronegative at immunofluorescence antibody test (IFAT) for IgG against FCoV and seropositive at IFAT for IgG against Leishmania infantum. All available data for this cat are reported in Additional analyzes were performed on archived frozen aliquots of this serum sample showing it to be seronegative for retrovirus infection by FIV and FeLV at rapid ELISA kit  cat sampled during the pandemic was positive for antibodies against SARS-CoV-2, while none tested positive by RT-PCR. None of the 136 pre-pandemic samples tested positive for SARS-CoV-2 antibodies.Until now, limited data have been available on SARS-CoV-2 infection in stray cats. To the author’s knowledge, only two studies have investigated the infection in a limited number of stray or shelter cats and both studies were performed in China. No specific antibodies were detected using double-antigen sandwich ELISA in 21 street cats in Wuhan, China . AnotherResults from this survey of SARS-CoV-2 infection in stray colony and shelter cats living in areas where viral transmission was active in the human population confirms field observations that cats can seroconvert under the normal conditions ,12,18,24Owned cats from Lombardy region were previously surveyed  using plOur serological results suggest that the ELISA positive cat had been exposed to the virus, although viral RNA detection was negative, as reported in previous studies performed in cats in China ,19,20 anAlthough it is not possible to identify how stray cats become infected with SARS-CoV-2, it is reasonable to speculate that these infections are probably due to contact with a SARS-CoV-2 polluted environment or wastewater contaminated with SARS-CoV-2 RNA as found in Milan city in northern Italy . It is pThe first laboratory-confirmed Italian human COVID-19 case was identified in Lombardy on February 20, 2020 , howeverLeishmania infantum, Anaplasma phagocytophilum, Rickettsia spp, Toxoplasma gondii, Chlamydophila felis, FIV, FCV, FHCV-1 and FPV and found no cross reactivity with SARS-CoV-2 ELISA in any of these samples. Collectively these results show that the ELISA test used in our study has good specificity without cross-reaction with common pathogens or feline coronaviruses present in free-ranging or abandoned stray un-owned feline populations.Both enteric (FECV) and FIP (FIPV) coronavirus seropositive sera showed no cross-reactivity with the SARS-CoV-2 serological test used in this study, as previously shown in studies using the receptor-binding domain (RBD) protein ELISA ,21, IFATLeishmania infantum, a protozoal infection that is sporadically found in stray cat populations in Lombardy region in northern Italy and, in particular, in stray cats from Milan city where the incidence has been increasing in epidemiological surveys since 2010 [Previous case reports have raised the question of whether SARS-CoV-2 infection may exacerbate existing disorders in cats  or, convnce 2010 ,35,36.Our study has some limitations. Surveillance systems relying on a single serological test could result in antigenic cross-reactivity and diagnostic errors; therefore, as a rule, two different tests should be used for confirmation of SARS-CoV-2 serological results. We tested the seropositive sample thrice using three different kits of the same ELISA test and this resulted in the same OD, confirming that no diagnostic errors occurred in analyzing this sample. In addition, we tested serum samples seropositive for different bacterial, protozoal and virus pathogens and found no cross-reaction with SARS-CoV-2 ELISA which was negative in all these samples. However, we did not additionally investigate our samples by other serological methods such as IFAT or VNT, in order to confirm the antibody status and assess the level of neutralizing antibodies in the ELISA seropositive cat. In a previous study the IFAT confirmed the antibody-positive status of the ELISA pre-screened serum samples, but neutralizing antibodies were only detected in 33.3% (2/6) of the ELISA- and IFAT-positive serum samples and titers of the VNT test did not correspond to the OD values of the ELISA test . This haResults from ELISA tests in human samples are influenced by the type of antigen, with whole spike protein or receptor binding domain of spike antigens giving better specificity and sensitivity when compared to whole virus, membrane or nucleocapsid proteins . A recenAnother limitation relates to the characteristics of the investigated feline population. Anamnestic and clinical data on free-ranging stray and shelter cats are inaccurate and this limited our understanding of the potential source of SARS-CoV-2 infection in the seropositive cat. We report that SARS-CoV-2 can be transmitted to stray cats, but we cannot know if this happened through contact with people carrying COVID-19 or from environmental contamination or contact with other infected cats.This study provides serological evidence for limited SARS-CoV-2 infection in stray cats in Lombardy region of northern Italy, one of the worst affected Italian regions. The negative molecular results showed that these cats are free of infections and the very low seroprevalence indicates that free-ranging stray or shelter cats play a limited role in transmission of SARS-CoV-2 during the pandemic. Collectively, these results showed that there is no indication of SARS-CoV-2 circulating in stray cats. As recently reported for owned cats , at this


MicroRNAs (miRNAs) are small, non-coding RNAs that function as endogenous gene silencers. Soon after the discovery of miRNAs, a subset of brain-enriched and brain-specific miRNAs were identified and significant advancements were made in delineating miRNA function in brain development. However, understanding the molecular mechanisms that regulate miRNA biogenesis in normal and diseased brains has become a prevailing challenge. Besides transcriptional regulation of miRNA host genes, miRNA processing intermediates are subjected to multifaceted regulation by canonical miRNA processing enzymes, RNA binding proteins (RBPs) and epitranscriptomic modifications. Further still, miRNA activity can be regulated by the sponging activity of other non-coding RNA classes, namely circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs). Differential abundance of these factors in neuronal and glial lineages partly underlies the spatiotemporal expression and function of lineage-specific miRNAs. Here, we review the continuously evolving understanding of the regulation of neuronal and glial miRNA biogenesis at the transcriptional and posttranscriptional levels and the cooperativity of miRNA species in targeting key mRNAs to drive lineage-specific development. In addition, we review dysregulation of neuronal and glial miRNAs and the detrimental impacts which contribute to developmental brain disorders. MicroRNAs (miRNAs) are a class of small, non-coding RNAs 20–24 nt) that suppress mRNA targets through mRNA degradation and translational repression . Mature 0–24 nt tDespite the large volume of discoveries that document the essential roles of miRNAs in brain development and function, molecular mechanisms that precisely regulate miRNA biogenesis in neuronal and glial development and thus impact normal and diseased brains have just begun to unfold. How distinct miRNAs converge on downstream molecular networks to advance neuronal development is a prevailing research question under active investigation. Given the increasingly appreciated functional importance of glia in modulating neuronal function, uncovering the role of glia-expressed miRNAs in neuron–glia interaction/communication has become a frontier field of research.Here we provide an up-to-date and comprehensive review of the recent advancements and outstanding questions regarding mechanisms that control miRNA biogenesis and functional abundance in neuronal and glial lineage development, as well as how diverse neural miRNAs converge on common downstream molecular networks. In addition, we highlight recent discoveries regarding important roles of miRNAs in neuron–glia communication, malfunction of miRNAs in neurodevelopmental diseases and therapeutic potential of miRNAs in various brain disorders. Decades of investigation have established a canonical miRNA biogenesis pathway beginning with the transcription of a miRNA host gene by RNA polymerase II (RNAPII) to produce a primary miRNA (pri-miRNA) transcript . Pri-miRDrosha in mouse NPCs resulted in a loss of multipotency status and induced precocious neuronal differentiation, which was phenocopied by depletion of DGCR8 [Mounting evidence has demonstrated the crucial roles of the canonical miRNA biogenesis enzymes in proper brain development and function. Conditional knockout of of DGCR8 . Dicer dof DGCR8 , mature of DGCR8 ,31,32, oof DGCR8 , microglof DGCR8  and astrof DGCR8 ,36. Deveof DGCR8 . In matuof DGCR8 ,39, poteof DGCR8 . Thus, dAlternative non-canonical pathways for miRNA biogenesis exist, which can bypass steps of the aforementioned canonical pathway. Mechanisms for both Drosha-independent and Dicer-independent miRNA biogenesis have been reviewed by Yang and Lai . MirtronThe mature miR-124 miR-124-3p) is one of the most well-characterized pro-neurogenic miRNAs, which governs various aspects of neuronal development and function, including neurogenesis, neuronal network assembly and synaptic plasticity ,51,52,53p is one SCP1 gene in HeLa cells [The abundant pool of miR-124 generated in the CNS targets numerous well-defined repressors of neuronal differentiation, including but not limited to RhoG, PAX3 and BAF53a ,57,58. OLa cells . Thus, a+/GFAP+) during lineage differentiation [An early study found that in vitro overexpression of miR-124 duplexes and inhibition by anti-miR-124 2′-O-methyl oligonucleotides in murine neural precursors had no significant effects on the neuron/astrocyte ratio  upon differentiation in vitro, compared to control. These effects on cell differentiation were not observed when either duplex was delivered separately [Despite the fact that miR-124 deletion alone failed to significantly impact neuron-glia fate in cultured neural precursors , miR-124parately , highligparately , allowinAnalysis of multiple independent miRNA studies establishes the repressor element 1 (RE-1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) complex as another anti-neural transcriptional hub  at whichDistinct miRNAs species govern the development and function of various glial cell types in the brain. In oligodendroglia (OL), which are responsible for CNS myelination and providing structural and metabolic support for neurons ,78,79, mIt should be noted that many well-characterized pro-neurogenic miRNAs were recently detected in OPCs and their decline is crucial for OL development, represented by miR-9 . The expNumerous miRNAs that control neuronal development are derived from multigene families, represented by miR-9, -128, -125, and -124 . In contmiR-124-1  and miR-124-2  have been annotated, while miR-124-3 is intergenic. In the human genome, only miR-124-2 (8q12.3) is annotated as an intronic miR of a long-noncoding pri-miRNA (lnc-pri-miR) [miR-124-1 (8p23.1) and miR-124-3 (20q13.33) are intergenic. As a multigene family with paralogous miRNAs located in distant regions and/or different chromosomes, the miR-124 paralogous genes likely arose from a non-local gene duplication event during vertebrate evolution [The exemplary miR-124 multigene family undergoes cell type- and developmental stage-specific regulation of miRNA paralogs to drive neuronal differentiation. Both human and mouse miR-124 are encoded by three paralogous genes, giving rise to pri-miR-124-1, -2 and -3 ,94. In tpri-miR) , while mvolution . Despitevolution ,101, indRncr3−/−) in mice results in increased neuronal apoptosis, axonal mis-sprouting and an overall smaller brain size [In mouse embryonic stem cells (mESCs), pri-miR-124-1 is the predominantly expressed pri-miR-124 paralog . The expain size , demonstain size , which, ain size . Whether-) [As miRNA host genes are primarily transcribed by RNAPII, pri-miRNAs are generally capped and polyadenylated  Figure A. Import-) . Process-) ,106,107.Ozsolak and colleagues reported that the promoters of RNAPII-transcribed miRNAs share the same features as protein-coding gene promoters, namely a CpG island and eukaryotic core promoter elements including a TATA box, TFIIB recognition element (BRE), initiator (Inr) and downstream promoter element (DPE)  Figure A. Of notAs the majority of pri-miRNAs harbor a 5′ cap, these transcripts are also candidates for cap analysis of gene expression sequencing (CAGE-seq) , which iThe significance of miRNA host transcript analyses remains dubious as the correlation between expression of miRNA host transcripts and embedded intragenic miRNAs remains unclear. Early human and mouse miRNA studies across different organs revealed frequent coordination between intronic miRNAs and host gene mRNAs ,114. MorTranscriptional regulation of neuronal miRNAs and the functional impact on brain development has been increasingly recognized. Co-operation of the DNA methyl-CpG-binding protein 2 (MeCP2) and the transcription factor Sox2 in stem cells has been reported to control miR-137 expression in adult neural stem cells, providing an intriguing cross talk between miRNA and chromatin modification-mediated epigenetic regulation in adult neurogenesis . In addiAatk and Mest, respectively [Sox9, which in turn suppresses Sox9 and drives OL terminal differentiation [Glial miRNA host genes are also subjected to transcriptional regulation. One recent report demonstrated that the well-characterized transcription factor Sox10 enhances production of miR-335 and miR-338, two miRNAs known to play critical roles in OL and myelin development . The preectively . Sox10 bntiation .6-methyladenosine (m6A) modifications of pri-miRNAs to enhance DGCR8 recognition and efficient processing by the Microprocessor complex [6A is not dependent on genomic position as m6A modifications were found on both inter- and intragenic pri-miRNAs [In recent years, a growing number of studies have investigated specific pri-miRNA characteristics for Microprocessor substrate recognition and cleavage ,123,124.cis motifs within pri-miRNAs which influence Microprocessor efficiency have been identified, namely UG at the 5′ basal junction of the stem-loop and UGUG/GUG in the apical loop [trans acting factors which may bind to pri-miRNAs to modulate cleavage by Microprocessor. Some trans acting co-factors, which enhance processing of specific pri-miRNAs, include SRp20 (SRSF3), QKI, p72 (DDX17), KSRP, hnRNP A1, BRCA1, FUS and SF2/ASF [Several cal loop   are single-stranded, closed loop RNA structures, produced by non-canonical ‘back-splicing’ that covalently links a downstream splice donor site to an upstream splice acceptor site ,143. TheA second class of non-coding RNAs with the potential to regulate miRNA function/abundance are long noncoding RNAs (lncRNAs). Defined as RNAs more than 200 nucleotides in length without protein-coding capacity, lncRNAs are highly expressed in the brain with known roles in transcriptional and posttranscriptional regulation of coding genes ,151,152.Abnormalities in neuronal development underlie the pathogenesis of numerous neuropsychiatric diseases, including schizophrenia, major depression, autism and fragile X intellectual disability ,155,156.Beyond neurodevelopmental disorders, the role of embryonic and adult neurogenesis in neurodegenerative disorders is increasingly recognized. In Parkinson’s disease (PD), malfunction of PD-related genes affects neural stem cell proliferation and maintenance . Thus, aPerturbations in miR-124 are implicated in several neuropsychiatric and neurodegenerative diseases, including schizophrenia, Alzheimer’s disease (AD), PD, hypoxic-ischemic encephalopathy, Huntington’s disease, and ischemic stroke . The humRncr3−/− mice, homozygous for deletion of the miR-124-1 host transcript, exhibit abnormal front and hind limb clasping, commonly observed in mouse models of neurodegenerative disorders [−/−Rncr3 mice, decreased expression of mature miR-124 has been reported in a mouse model of and in the frontal cortex of subjects with behavioral variant frontotemporal dementia (bvFTD) [isorders . In line (bvFTD) . Decreas (bvFTD) . In huma (bvFTD) . In huma (bvFTD) ,180. Mor (bvFTD) . A compr (bvFTD) . Taken tMIR137 Host Gene was found to affect alternative splicing and biogenesis of miR-137, which contributes to the risk for SCZ [MiR-137 is another brain-enriched miRNA that governs differentiation and maturation of the nervous system and is implicated in various major mental illnesses . Aberran for SCZ . In gene for SCZ . Mechani for SCZ . In addi for SCZ ,187. Mor for SCZ . It is increasingly appreciated that defects in both neurons and glia underlie the pathogenesis of brain diseases. Deficits in myelinogenesis and neuronal function contribute to distinct aspects of the complex etiology of SCZ ,189. IntBesides myelin–neuron interactions, communication between microglia and astrocytes with neurons modulates neuronal cell death, neurogenesis and synaptic interactions during normal brain development, as well as neuroimmune-responses in various brain diseases ,192,193.Finally, exciting discoveries suggest therapeutic potentials of EV miRNAs derived from neural cells. NPCs are known to have potent therapeutic effects in neurological disorders through EV secretion. In a recent report, miR-21a was found to be highly enriched in NPC-originated EVs, which promoted neuronal differentiation but not gliogenesis . In addiThe miRNA species mentioned in this review, the brain cell types in which they are expressed, their roles in neural cell development, and their relevance to various brain disease are summarized in In comparison to the well-documented mechanisms of miRNA action and the expression profiles of brain miRNAs produced by the canonical miRNA biogenesis pathway, our current understanding of the molecular mechanisms that regulate miRNA expression during neural development and miRNA dysregulation in various human brain disorders are still in their infancy. The new advancements regarding the multifaceted regulation of neuronal and glial miRNAs in healthy and diseased brains, reviewed here, highlight the importance of further delineating fundamental rules that govern miRNA production and activity in normal and diseased brains. The complex regulation of miRNAs provides spatial and temporal divergence of miRNA-dependent gene regulation to accommodate sophisticated brain function as well as fragility to human brain diseases caused by miRNA dysregulation. By contrast, the emerging examples of lncRNAs/circRNAs in soaking multiple miRNAs derived from distinct genes may provide a mechanism for functional re-organization of miRNA activity that converge on downstream targets, especially in neuronal somatodendritic compartments. Nonetheless, the current prevailing challenge is delineating how miRNAs converge on a vast number of downstream mRNA targets to advance neuronal and glial differentiation, especially considering the emerging roles of miRNAs in neuron–glia communication. Finally, therapeutic potentials of pro-neurogenic miRNAs have become an intriguing area of active research that may provide promising outcomes.


A 26-year-old man with symptomatic SARS-CoV-2 infection developed a sudden-onset acute testicular pain. The echo-doppler images showed massive testicular infarction, so orchiectomy was performed. On gross examination, the surgical specimen showed complete testicular necrosis and diffuse thickening of the testicular coverings. Under the microscope, a severe obliterative arteritis was evidenced. SARS-CoV-2 spike antibody was detected by immunohistochemistry in the arterial endothelium. Electron microscopy displayed intracytoplasmic spiky viral particles in endothelial cells. The patient was treated with corticoids and was asymptomatic at last contact. Individual host reactivity against SARS-CoV-2 is largely unpredictable and responsible for most symptoms and sequelae of this infection. Vasculitis is a systemic response to the virus with variable qualitative and quantitative clinical manifestations. Here, we present an extreme case of massive unilateral testicular necrosis occurring in the recovery phase of SARS-CoV-2 infection in a young man, a manifestation which represents a previously non reported clinical complication of this infection.A 26-year-old man with symptomatic SARS-CoV-2 infection, including mild dyspnea and fever, developed a left testicular pain two days after the acute symptomatic infection was vanished . No antecedents of any systemic disease were recorded in the anamnesis and analytical data, other than those specifically related to the SARS-CoV-2, were within the normal limits. No signs of coagulopathy were detected. The echo-doppler study of the testis at that time showed mild hypoperfusion. Testicular pain was then thought to be secondary to orchitis and symptomatic therapy with empiric antibiotic coverage was started. Pain, however, did not recover and 3 days after a second echo-doppler showed a complete testicular infarction A. Left oGrossly, the testicle  showed a massive infarction and diffuse thickening of the spermatic cord and testicular coverings C. A segmThe intravascular lymphocyte population associated with endothelial damage included both CD4 and CD8 cells. Interstitial and perivascular infiltrates were composed of a mixture of macrophages, and T and B cells. Immunohistochemistry against the SARS-CoV-2 spike protein  showed positive endothelial reaction F. MoreovCOVID-19-associated vasculopathy has been well documented in almost any organ . DiffuseThe present case recalls the attention of general practitioners and urologists when facing testicular pain in the context of SARS-CoV-2 infection.


Briefly, hippocampal and septal tissue were harvested from embryonic day 19 (E19) Sprague-Dawley rats, digested with 0.1% trypsin, and an equal number of cells from each region plated onto coverslips coated with poly-D-lysine and laminin at a final density of 300 cells/mm2. We use immunostaining with validated primary antibodies and a fluorescent binding assay, together with confocal microscopy, to determine the structure of cholinergic synapses that are 1) native, 2) mammalian, 3) CNS derived, 4) comprised of physiological synaptic partners, and 5) developmentally mature. RESULTS/ANTICIPATED RESULTS: After DIV21, co-cultures maintained a healthy morphology. A subpopulation of neurons strongly expressed the cholinergic markers vesicular ACh transporter (vAChT), choline acetyltransferase (ChAT), and the high-affinity choline transporter (ChT1), whereas most neurons lacked vAChT expression and were presumably glutamatergic or GABAergic. The percentage of cholinergic neurons in the co-culture attained up to ~5-7%, depending on conditions such as embryo age at dissection or ratio of septal to hippocampal cells. We also report on cholinergic synapse structure by examining postsynaptic markers (excitatory and inhibitory) and staining for nicotinic acetylcholine receptor subunits. DISCUSSION/SIGNIFICANCE OF IMPACT: Primary septal-hippocampal co-cultured neurons have not been exploited extensively in the field, perhaps due to the difficulty in maintaining such cultures for extended periods. Here, we optimized an in vitro septal-hippocampal co-culture system, a powerful tool to comprehensively analyze central cholinergic synapse formation and dysfunction.OBJECTIVES/GOALS: Septal cholinergic innervation to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer’s disease. To understand the molecular events underlying this loss, we optimized a primary septal-hippocampal co-culture system that facilitates study of central cholinergic synapses. METHODS/STUDY POPULATION: We developed an optimized


Reverse high-freedom shape mutations of diversified microstructures were created from simple initial shapes and identified without evident fatigue. We further confirmed the biocompatibility, cell adhesion, and tunable mechanics of the as-prepared hydrogels. Benefiting from the high-efficiency two-photon polymerization (TPP), nanometer feature size (<200 nm), and flexible digitalized modeling technique, many more micro/nanoscale hydrogel robots or machines have become obtainable in respect of future interdisciplinary applications.Rapid fabricating and harnessing stimuli-responsive behaviors of microscale bio-compatible hydrogels are of great interest to the emerging micro-mechanics, drug delivery, artificial scaffolds, nano-robotics, and lab chips. Herein, we demonstrate a novel femtosecond laser additive manufacturing process with smart materials for soft interactive hydrogel micro-machines. Bio-compatible hyaluronic acid methacryloyl was polymerized with hydrophilic diacrylate into an absorbent hydrogel matrix under a tight topological control through a 532 nm green femtosecond laser beam. The proposed hetero-scanning strategy modifies the hierarchical polymeric degrees inside the hydrogel matrix, leading to a controllable surface tension mismatch. Strikingly, these programmable stimuli-responsive matrices mechanized hydrogels into robotic applications at the micro/nanoscale (<300 × 300 × 100 μm The development of modern robotics/sensors , micro/nThese smart hydrogel devices would ideally possess the embeddable volume and the reconfigurable morphology to be applied ,10. The Many additive manufacturing methods have been demonstrated with the four-dimensional (4D) time-dependent shape reconfiguration to obtain controllable stimuli-responsive behavior . Four-diAmong the existing macroscopic 4D products and fabrications with a relative millimeter or sub-millimeter resolution, the TPP using the femtosecond laser as a light source captures roaring attention. As is known, the primary-stage products of TPP are mostly stationary without controllable stimuli-inspired properties or artificial shape morphing. We recently reported a carbon nanotube-doped hydrogel with swelling-to-shrinkage behavior using two-photon polymerization (TPP) , where tMicro/nanostructures fabricated via TPP are formed through high-density covalent bonding networks. The solidified structures, generally, cannot undergo large transformations such as soft/elastomer materials. Recently, the smart properties of hydrogels have been improved. For example, the traditional actuation uses a residual stress-driven method for thermal shape deformation ,31. SomeHerein, to obtain direct fabricating and temporally controlling micro/nanoscale quasi-arbitrary 3D geometries without filling other functional particles, we tentatively perform the modified TPP  on the bInterestingly, the optical double-frequency technique is adopted here for generating the 532 nm green femtosecond laser beam during TPP fabrication for the first time. The double-frequency crystal is inserted in the path to decrease the optical power and wavelength simultaneously. We also use a micro-mechanics platform to characterize the mechanical behaviors of the TPP-fabricated hydrogels. A surface tension analysis expounds the mechanics’ theoretical rationales during the controllable shape morphing.Hyaluronic acid methacryloyl , 2-hydroxy-2-methylpropiophenone 2) transformed the 1064 nm wavelength femtosecond laser beam from a Ti:Sapphire femtosecond laser  into a green 532 nm beam  with acceleration voltages of 5 KV were pre-coated with indium tin oxide semiconductor (ITO) film for electric conduction. The SEM software analyzed the size and spatial resolution. The imaging spectrometer  reflected the fluorescence images of hydrogel structures with a high-resolution scientific complementary metal oxide semiconductor . The software of island-320 set the color of fluorescence images. A CCD camera installed on the digitalized inverted microscope  recorded the dynamic responsiveness. The minimum time interval of each video was a single sec. The external water stimuli were realized by dropping deionized water onto the sample or blowing moisture from a humidifier to investigate the responsive behavior.2 tip radius and 5 nN resolution. Young’s Modulus of the solidified samples was measured using FT-S100-TP of 2 × 2 µm2 tip radius and at the same resolution.The pH variation was realized by adding the diluted hydrochloric acid into in-solution hydrogels . A lightBy laser scanning inside the photoresist b, the smThe experimental observation confirmed the desirable mobility of the photoresist and highly effective two-photon absorption at a mild laser power. The photon-induced cross-linking reaction was confined at the submicron voxel . All comAn ultrafine feature size was observable in the CCD or SEM images  contributed to the stimuli responsiveness. Therefore, we changed the optical power and spacing width formed during scanning to display the resilient shape deformation ability.In nature, many plants use water sorption and desorption for motion or reversible shape morphing. To mimic this behavior at a microscale, we fabricated the water-swelling hydrogel. The polymeric matrix consisted of permanent covalent carbon bonds in polymer materials, and various chemical functional groups could collaborate with outside-applied stimuli for judicious motion. As seen in Here, the water molecules worked as the triggering condition, and the molecular force captured water to swell or shrink by heating to recover. The volume ratio shrank over 200% in evaporation, demonstrating a high water retention . The repFurthermore, we fabricated the tadpole-shaped hydrogel, which swung its tail shape using the swelling effect seen in , where tThen, we fabricated a smart two-layer structure a, and thBesides the humidity or light stimuli for responsiveness, the heating process also led to a self-bending action similar to an artificial muscle . The uniThe heterostructure consisted of solidified hydrogel nanowires, and the smooth spacing resulted in a divergence in shrinkage behavior. This divergence caused residual stress at the molecular level and caused the inward-direction contractile surface tension to accumulate. As seen in In this study, we succeeded in developing a composite hydrogel material sensitive to a water/light/heat environment with a 532 nm femtosecond laser TPP. Compared to the mainstream optics/electron beam mask-projected stereolithography, the proposed two-photon polymerization held several advantages. An ultrafine feature size was obtained by staking the nanoscale voxel of the TPP system. The conventional macroscopic signal-triggered patterns or structures were miniaturized to a three-dimensional micron/nanoscale. The nonlinear characteristics of the fabrication processes still offered a sub-micron writing resolution, which is of great interest to micron-robotics, nano-drivers, and wearable sensors. Meanwhile, the stimuli-responsive photoresist contained no metal or alloy to improve biocompatibility. The controllable behaviors of the micro/nanostructures were being fatigue-free, environment-inspired, and quickly responsive, promising broad applications in micron actuators, sensors, micro-robotics, and biomimetic fields.


Immune-mediated necrotizing myopathy, a new subgroup of inflammatory myopathies, usually begins with subacute onset of symmetrical proximal muscle weakness. A 35-year-old male presented with severe asymmetric iliopsoas atrophy and low back pain with a previous history of left lower extremity weakness. Although his first left lower extremity weakness occurred 12 years ago, he did not receive a clear diagnosis. Magnetic resonance imaging of both thigh muscles showed muscle edema and contrast enhancement in patch patterns, and the left buttock and thigh muscles were more atrophied compared to the right side. Serum creatine kinase levels were elevated, and serologic testings were all negative. Genetic testing using a targeted gene-sequencing panel for neuromuscular disease including myopathy identified no pathogenic variants. Muscle biopsy on the right vastus lateralis showed scattered myofiber necrosis with phagocytosis and an absence of prominent inflammatory cells, consistent with seronegative necrotizing myopathy. Thus, unusual asymmetric muscle weakness and atrophy can be a manifestation of inflammatory myopathy. Immune-mediated necrotizing myopathy (IMNM) is a new subgroup of inflammatory myopathies, distinguished by myositis-specific-antibodies (MSAs), including anti-signal recognition particles (anti-SRP) and anti-3-hydroxy-3-methylglutaryl coenzyme A reductase (anti-HMGCR) ,2,3. TheElectromyography and serologic testing are commonly performed in patients with proximal muscle weakness and elevated creatinine phosphokinase (CPK) levels. Negative serological findings, along with electromyography suggesting myopathy, can cause confusion in diagnosis. A patient’s clinical features may be nonspecific, and accurate pathological diagnosis and genetic testing play an important role in diagnosis. We present a case in which a seronegative patient with histology-proven IMNM developed prominent asymmetric proximal muscle atrophy in the stepwise subacute disease course.A 35-year-old man was referred to the neuromuscular disease clinic of our institution from a local clinic due to severe left iliopsoas atrophy. He visited the local spine clinic due to a recent weakness in the left proximal leg and lower back pain. Lumbar magnetic resonance imaging (MRI) was performed and showed severe left iliopsoas atrophy but no definite spinal lesions . He deniWhen he visited our clinic, an initial physical examination revealed proximal motor weakness in the left leg, especially in the left hip flexion and adduction strength, using the grade 2 Medical Research Council (MRC) scale. There was no weakness in other extremities. He had no sensory disturbance and other neurologic examinations were normal. The laboratory examination showed an elevated CPK 4254 IU/L and AST/ALT 76/103 IU/L levels. Myositis-specific-antibodies including anti-SRP and anti-HMGCR were negative upon serologic testing. Anti-tRNA synthetase antibodies such as anti-Jo-1, non-specific myositis-associated antibodies such as anti-RNP antibodies and anti-SSA/SSB (Ro/La) were all negative. An MRI of both thigh muscles indicated that the left buttock and thigh muscles were more atrophied compared to the right side, and muscle edema and contrast enhancement were observed in patch patterns in both thigh muscles . MalignaMuscle biopsy on the right vastus lateralis was performed and showed myofiber necrosis with phagocytosis and absence of prominent inflammatory cells, consistent with necrotizing myopathy . A diagnIMNM has been characterized by subacute onset of symmetrical proximal muscle weakness, elevated CPK levels, and myopathic findings of electromyography ,6. In a ANO5 (LGMD2L), DYSF (LGMD2B), SGCA (LGMD2D), SGCB (LGMD2E), SGCD (LGMD2F), SGCE (myoclonus-dystonia syndrome), and SGCG (LGMD2C), and no pathogenic variant was detected. Therefore, it is important to analyze the pathological findings and differential diagnosis of genetic diseases when the patient has atypical IMNM findings and is seronegative.The patient was diagnosed with antibody-negative IMNM, as he displayed proximal muscle weakness, increased muscle enzyme levels, myopathic-patterned electromyogram, no myositis-specific autoantibodies and muscle biopsies showing scattered necrotic fibers with macrophage predominant, paucilymphocytic infiltrates ,3. Some Seronegative IMNM was characterized in 2011, and it is difficult to estimate its current prevalence and incidence . StudiesThis case report describes a clinical feature of a stepwise subacute course of prominent asymmetric muscle weakness and atrophy, which was ultimately diagnosed as seronegative IMNM. Therefore, physicians should pay attention to unusual asymmetric muscle weakness or atrophy, which can be a manifestation of inflammatory myopathies. When the patient has atypical IMNM findings and is seronegative, it is important to analyze the pathological findings and differential diagnosis of inherited muscular diseases.