Loss of GABARAP mediates resistance to immunogenic chemotherapy in multiple myeloma.

ABSTRACT: Immunogenic cell death (ICD) is a form of cell death by which cancer treatments can induce a clinically relevant antitumor immune response in a broad range of cancers. In multiple myeloma (MM), the proteasome inhibitor bortezomib is an ICD inducer and creates durable therapeutic responses in patients. However, eventual relapse and resistance to bortezomib appear inevitable. Here, by integrating patient transcriptomic data with an analysis of calreticulin (CRT) protein interactors, we found that GABA type A receptor-associated protein (GABARAP) is a key player whose loss prevented tumor cell death from being perceived as immunogenic after bortezomib treatment. GABARAP is located on chromosome 17p, which is commonly deleted in patients with high risk MM. GABARAP deletion impaired the exposure of the eat-me signal CRT on the surface of dying MM cells in vitro and in vivo, thus reducing tumor cell phagocytosis by dendritic cells and the subsequent antitumor T-cell response. Low GABARAP was independently associated with shorter survival in patients with MM and reduced tumor immune infiltration. Mechanistically, we found that GABARAP deletion blocked ICD signaling by decreasing autophagy and altering Golgi apparatus morphology, with consequent defects in the downstream vesicular transport of CRT. Conversely, upregulating autophagy using rapamycin restored Golgi morphology, CRT exposure, and ICD signaling in GABARAPKO cells undergoing bortezomib treatment. Therefore, coupling an ICD inducer, such as bortezomib, with an autophagy inducer, such as rapamycin, may improve patient outcomes in MM, in which low GABARAP in the form of del(17p) is common and leads to worse outcomes.

The JAK-STAT pathway regulates CD38 on myeloma cells in the bone marrow microenvironment: therapeutic implications.

Anti-CD38 monoclonal antibody (MoAb) treatments including daratumumab (DARA) are effective therapies for both newly diagnosed and relapsed multiple myeloma (MM). In this study, we examined the soluble factors that modulate CD38 expression and are associated with sensitivity to DARA-mediated antibody-dependent cellular cytotoxicity (ADCC) in the bone marrow (BM) microenvironment. Importantly, primary BM stromal cell (BMSC) culture supernatant (BMSC-sup) and interleukin-6 (IL-6) downregulated CD38 expression and reduced DARA-mediated ADCC. Both cytokine profiling of the BMSC-sup and genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) knockout screening in MM cell lines identified and validated the JAK-STAT3 signaling pathway mediating CD38 downregulation, whereas the JAK-STAT1 pathway mediated CD38 upregulation. STAT3 knockdown abrogated BMSC-sup- and IL-6-induced CD38 downregulation on MM cell lines. We also confirmed that STAT3 and CD38 is negatively correlated in primary MM cells. To assess potential clinical relevance, pharmacological inhibition of the JAK-STAT pathway on BMSC-sup-induced CD38 downregulation was further examined. JAK inhibitor ruxolitinib inhibited STAT3 phosphorylation in MM cell lines, upregulated CD38 expression in MM cell lines and primary patient MM cells, and augmented DARA-mediated ADCC against MM cell lines. Taken together, our results suggest that CD38 expression on MM cells in the BM microenvironment is regulated by both STAT1 (positively) and STAT3 (negatively), and that inhibition of the JAK-STAT3 pathway represents a novel therapeutic option to enhance CD38 expression and anti-CD38 MoAb-mediated MM cytotoxicity.

Biosynthesis of biocompatibility Ag2Se quantum dots in Saccharomyces cerevisiae and its application.

As fluorescence in the second near-infrared window (NIR-II, 1000-1400 nm) could image deep tissue with high signal-to-noise ratios compared with that in NIR-I (750-900 nm), Ag2Se quantum dots (QDs) with fluorescence in the NIR-II could be ideal fluorophores. Here, we described a biosynthesis method to prepare the Ag2Se QDs by using temporally coupling the irrelated biochemical reactions, whose photoluminescence (PL) emission can reach NIR-II. The nanoparticles were characterized by transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The results showed that the nanoparticles obtained by extracellular purification were Ag2Se QDs with a uniform size of 3.9 ± 0.6 nm. In addition, the fluorescence intensity of Saccharomyces cerevisiae was improved successfully by nearly 4-fold by constructed engineering strain. In particular, the biosynthesis of Ag2Se QDs had good biocompatibility because it was capped by protein. Furthermore, investigating the toxicity of Ag2Se on cells and NIR images of nude mice showed that the Ag2Se synthesized using S. cerevisiae had low toxicity and could be used for in vivo imaging. In this work, the synthesis pathway of biocompatible Ag2Se was broadened and laid a foundation for the enlarged applicability of bioimaging in the biosynthesis of NIR-II QDs.

CRL4 antagonizes SCFFbxo7-mediated turnover of cereblon and BK channel to regulate learning and memory.

Intellectual disability (ID), one of the most common human developmental disorders, can be caused by genetic mutations in Cullin 4B (Cul4B) and cereblon (CRBN). CRBN is a substrate receptor for the Cul4A/B-DDB1 ubiquitin ligase (CRL4) and can target voltage- and calcium-activated BK channel for ER retention. Here we report that ID-associated CRL4CRBN mutations abolish the interaction of the BK channel with CRL4, and redirect the BK channel to the SCFFbxo7 ubiquitin ligase for proteasomal degradation. Glioma cell lines harbouring CRBN mutations record density-dependent decrease of BK currents, which can be restored by blocking Cullin ubiquitin ligase activity. Importantly, mice with neuron-specific deletion of DDB1 or CRBN express reduced BK protein levels in the brain, and exhibit similar impairment in learning and memory, a deficit that can be partially rescued by activating the BK channel. Our results reveal a competitive targeting of the BK channel by two ubiquitin ligases to achieve exquisite control of its stability, and support changes in neuronal excitability as a common pathogenic mechanism underlying CRL4CRBN-associated ID.

Activation of c-Abl Kinase Potentiates the Anti-myeloma Drug Lenalidomide by Promoting DDA1 Protein Recruitment to the CRL4 Ubiquitin Ligase.

Cullin-RING ligase 4 (CRL4), a complex of Cul4 and DDB1, regulates the cell cycle, DNA damage repair, and chromatin replication by targeting a variety of substrates for ubiquitination. CRL4 is also hijacked by viral proteins or thalidomide-derived compounds to degrade host restriction factors. Here we report that the c-Abl non-receptor kinase phosphorylates DDB1 at residue Tyr-316 to recruit a small regulatory protein, DDA1, leading to increased substrate ubiquitination. Pharmacological inhibition or genetic ablation of the Abl-DDB1-DDA1 axis decreases the ubiquitination of CRL4 substrates, including IKZF1 and IKZF3, in lenalidomide-treated multiple myeloma cells. Importantly, panobinostat, a recently approved anti-myeloma drug, and dexamethasone enhance lenalidomide-induced substrate degradation and cytotoxicity by activating c-Abl, therefore providing a mechanism underlying their combination with lenalidomide to treat multiple myeloma.

Selection of Cholesterol-Lowering Lactic Acid Bacteria and its Effects on Rats Fed with High-Cholesterol Diet.

High cholesterol level in serum is a major factor of influence for coronary heart disease. The cholesterol-lowering ability of lactic acid bacteria (LAB) without side effects makes them more and more attractive. Seventy-nine strains of LAB isolated from fermented food were screened in vitro for their ability to assimilate cholesterol. Then, ten strains which exhibited higher ability of cholesterol assimilation were investigated with the characteristics of acidic resistance, bile salt tolerance, and cell adhesion. According to the results, the best strain LP96 was picked out, and used to evaluate its effects on the high-cholesterol diet-fed rats. The results demonstrated that the levels of serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol, and liver TC and TG were reduced significantly in the groups that received the strain LP96 solution compared with the model group, and that the serum high-density lipoprotein cholesterol levels were increased without any significant difference. Furthermore, LP96 also showed good antioxidative activity and improvement of intestinal microbial balance in the rats. Thus, LP96 may be a promising probiotics with potential cholesterol-lowering ability.

Development of a biotinylated broad-specificity single-chain variable fragment antibody and a sensitive immunoassay for detection of organophosphorus pesticides.

Organophosphorus pesticides (OPs) are the most widely used pesticides in agriculture, and OP residues have been broadly reported in food and environmental samples. The aim of this study is to develop a recombinant antibody-based broad-specificity immunoassay for OPs. A phage display library was prepared from a mouse pre-immunized with a generic immunogen of OPs, and a single-chain variable fragment (scFv) antibody was selected. The selected scFv antibody was fused with biotin acceptor domain (BAD) and overexpressed as an inclusion body in Escherichia coli BL21 (DE3). Then, the protein was refolded by stepwise urea gradient dialysis and biotinylated in vitro by E. coli biotin ligase (BirA). Subsequently, the scFv-BAD protein was purified from the biotinylated system with high yield (66.7 mg L(-1)) and confirmed by SDS-PAGE and Western blot. Based on the biotinylated scFv-BAD, a sensitive and broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for detection of OPs was developed. The cross-reactivity (CR) studies demonstrated that the ciELISA described here exhibited the broadest detection spectrum for OPs up to now, and 30 OPs could be determined with 50 % inhibition value (IC50) values ranging from 19.4 to 515.2 ng mL(-1). Moreover, the developed ciELISA was used for the recovery study of the spiked samples and showed satisfactory recoveries. Graphical Abstract Schematic diagram of the development of biotinylated broad-specificity single-chain variable fragment antibody-based immunoassay for organophosphorus pesticides.

Biomimetic synthesis of RPL14B-based CdSe quantum dots for the detection of heavy metal copper ions.

In the present study, an Escherichia coli-expressed yeast ribosomal protein was used as a template for synthesizing RPL14B-based CdSe quantum dots in vitro via the quasi-biosynthesis strategy at low temperature. The synthetic bionic RPL14B-based CdSe quantum dots were characterized using TEM, HRTEM, and EDX spectra, and the results showed that the synthesized quantum dots were CdSe quantum dots with a crystal face spacing of 0.21 and 0.18 nm. The biomimetic method-synthesized quantum dots exhibited the characteristics of a uniform particle size, good dispersion, and strong photobleaching resistance. Moreover, the fluorescence of the RPL14b-based CdSe quantum dots could be specifically quenched using Cu2+ in a linear range of 0.2-10 µM. Finally, these RPL14b-based CdSe quantum dots can be used for the specific detection of heavy metal copper ions in addition to other applications in biological analyses.

Epigenetic regulation of CD38/CD48 by KDM6A mediates NK cell response in multiple myeloma.

Anti-CD38 monoclonal antibodies like Daratumumab (Dara) are effective in multiple myeloma (MM); however, drug resistance ultimately occurs and the mechanisms behind this are poorly understood. Here, we identify, via two in vitro genome-wide CRISPR screens probing Daratumumab resistance, KDM6A as an important regulator of sensitivity to Daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). Loss of KDM6A leads to increased levels of H3K27me3 on the promoter of CD38, resulting in a marked downregulation in CD38 expression, which may cause resistance to Daratumumab-mediated ADCC. Re-introducing CD38 does not reverse Daratumumab-mediated ADCC fully, which suggests that additional KDM6A targets, including CD48 which is also downregulated upon KDM6A loss, contribute to Daratumumab-mediated ADCC. Inhibition of H3K27me3 with an EZH2 inhibitor resulted in CD38 and CD48 upregulation and restored sensitivity to Daratumumab. These findings suggest KDM6A loss as a mechanism of Daratumumab resistance and lay down the proof of principle for the therapeutic application of EZH2 inhibitors, one of which is already FDA-approved, in improving MM responsiveness to Daratumumab.

Publication trends in nutrition research for sarcopenic obesity: A 20-year bibliometric analysis.

BACKGROUND: We used bibliometric methods to evaluate publications on the role of nutrition in sarcopenic obesity and analyzed the current situation and developmental trends over the past 2 decades. METHODS: Publications from 2002 to 2022 related to the role of nutrition in sarcopenic obesity were extracted from the Web of Science Core Collection database. CiteSpace, VOSviewer, and the Bibliometrix R package were applied to build relevant network diagrams. RESULTS: One thousand ninety-four articles from 64 countries were included. The annual number of publications in this field has shown an intense growth trend. The University of Alberta, Yonsei University, and Korea University are the major research institutions. Clinical Nutrition has published the most papers on the role of nutrition in sarcopenic obesity, and the American Journal of Clinical Nutrition is the most co-cited journal. A total of 5834 authors conducted the relevant studies. Yves Boirie has published the most papers in this field, and AJ Cruz-Jentoft is the most co-cited author. CONCLUSION: This is the first bibliometric study of the role of nutrition in sarcopenic obesity. This study systematically summarizes the research hotspots and development directions in this field, and provides a reference for scholars studying the role of nutrition in sarcopenic obesity.

Targeting BCMA in Multiple Myeloma: Advances in Antibody-Drug Conjugate Therapy.

Multiple myeloma (MM) is an incurable cancer of the plasma cells. In the last twenty years, treatment strategies have evolved toward targeting MM cells-from the shotgun chemotherapy approach to the slightly more targeted approach of disrupting important MM molecular pathways to the immunotherapy approach that specifically targets MM cells based on protein expression. Antibody-drug conjugates (ADCs) are introduced as immunotherapeutic drugs which utilize an antibody to deliver cytotoxic agents to cancer cells distinctively. Recent investigations of ADCs for MM treatment focus on targeting B cell maturation antigen (BCMA), which regulates B cell proliferation, survival, maturation, and differentiation into plasma cells (PCs). Given its selective expression in malignant PCs, BCMA is one of the most promising targets in MM immunotherapy. Compared to other BCMA-targeting immunotherapies, ADCs have several benefits, such as lower price, shorter production period, fewer infusions, less dependence on the patient's immune system, and they are less likely to over-activate the immune system. In clinical trials, anti-BCMA ADCs have shown safety and remarkable response rates in patients with relapsed and refractory MM. Here, we review the properties and clinical applications of anti-BCMA ADC therapies and discuss the potential mechanisms of resistance and ways to overcome them.

Trends in parthenolide research over the past two decades: A bibliometric analysis.

Parthenolide (PTL) is a new compound extracted from traditional Chinese medicine. In recent years, it has been proven to play an undeniable role in tumors, autoimmune diseases, and inflammatory diseases. Similarly, an increasing number of experiments have also confirmed the biological mechanism of PTL in these diseases. In order to better understand the development trend and potential hot spots of PTL in cancer and other diseases, we conducted a detailed bibliometric analysis. The purpose of presenting this bibliometric analysis was to highlight and inform researchers of the important research directions, co-occurrence relationships and research status in this field. Publications related to PTL research from 2002 to 2022 were extracted on the web of science core collection (WoSCC) platform. CiteSpace, VOSviewers and R package "bibliometrix" were applied to build relevant network diagrams. The bibliometric analysis was presented in terms of performance analysis (including publication statistics, top publishing countries, top publishing institutions, publishing journals and co-cited journals, authors and co-cited authors, co-cited references statistics, citation bursts statistics, keyword statistics and trend topic statistics) and science mapping (including citations by country, citations by institution, citations by journal, citations by author, co-citation analysis, and keyword co-occurrence). The detailed discussion of the results explained the focus and latest trends from the bibliometric analysis. Finally, the current status and shortcomings of the research field on PTLwere clearly pointed out for reference by scholars.

Prevalence of sarcopenia among patients with hepatocellular carcinoma: A systematic review and meta­analysis.

Sarcopenia is a common condition in patients with hepatocellular carcinoma (HCC). Sarcopenia affects the prognosis of patients with HCC and reduces their quality of life. However, to date, there has been no systematic review and meta-analysis to assess the prevalence of sarcopenia in patients with HCC, to the best of our knowledge. PubMed, Embase, Web of Science and the Cochrane Library were comprehensively screened for relevant literature published from March 2001 to June 2022. A random effect analysis was conducted to pool the incidence rates for each study. Subgroup and meta-regression analyses were used to investigate the latent sources of heterogeneities. The Newcastle-Ottawa Scale was used to estimate the quality of the included studies. The I2 statistic was used to evaluate heterogeneity between studies. In total, 48 studies encompassing 8,959 patients were included in the meta-analysis. The results of the present meta-analysis showed that nearly half (42%) of the patients with HCC had sarcopenia (95% CI, 0.36-0.48). The morbidity of sarcopenia in studies with a high proportion of males (45%) was higher compared with the morbidity observed in studies with a lower proportion of males (37%). In addition, the incidence rate in younger patients (46%) was found to be higher compared with the incidence rate in older patients (39%). In conclusion, the findings in the present systematic review revealed that a large number of patients with HCC suffer from sarcopenia, indicating the necessity of developing screening and intervention measures to improve the outcome in these patients.

MicroRNA­mediated regulation in lung adenocarcinoma: Signaling pathways and potential therapeutic implications (Review).

Lung adenocarcinoma (LUAD) poses a significant global health burden owing to its high incidence rate and unfavorable prognosis, driven by frequent recurrence and drug resistance. Understanding the biological mechanisms underlying LUAD is imperative to developing advanced therapeutic strategies. Recent research has highlighted the role of dysregulated microRNAs (miRNAs) in LUAD progression through diverse signaling pathways, including the Wnt and AKT pathways. Of particular interest is the novel pathological mechanism involving the interaction between competing endogenous RNAs (ceRNAs) and miRNAs. This review critically analyzed the impact of aberrant miRNA expression on LUAD development, shedding light on the associated signaling pathways. It also highlighted the emerging significance of ceRNA­miRNA interactions in LUAD pathogenesis. Elucidating the intricate regulatory networks involving miRNAs and ceRNAs presents a promising avenue for the development of potential therapeutic interventions and diagnostic biomarkers in LUAD. Further research in this area is essential to advance precision medicine approaches and improve patient outcomes.

Effect of Pre-Operative Low Serum Pre-Albumin on Surgical Site Infection in Post-Surgery Subjects: A Systematic Review and Meta-Analysis.

Background: The correlation between pre-operative serum pre-albumin and surgical site infection (SSI) has been the focus of many studies. However, existing literature presents conflicting evidence on this association. Therefore, this meta-analysis was conducted to determine the significance of low serum pre-albumin as a prognostic factor SSI, and to assess the potential utility of pre-albumin in predicting SSI. Methods: A comprehensive literature search and analysis was conducted in PubMed, Web of Science, Cochrane of Library, Scopus, Embase, and Google Scholar databases through August 2022 to identify studies reporting low pre-operative serum pre-albumin levels in patients undergoing surgery and their association with SSIs. The pooled risk estimates were shown in odds ratio with 95% confidence interval. The random effect model was used according to the test of heterogeneity among studies. Subgroup analyses and sensitivity analyses were performed to identify the possible sources of heterogeneity. This meta-analysis was prospectively registered in the PROSPERO database (number: CRD42022376167). Results: Nine studies involving 5,306 patients were eligible. The results demonstrated an association between low pre-operative serum pre-albumin levels and a higher probability of developing SSI (odds ratio [OR], 2.04; 95% confidence interval [CI], 1.28-3.26). Conclusions: Our findings suggest that low serum pre-albumin level may serve as an independent and valuable predictor of SSI. These results provide important insights for clinicians in identifying high-risk patients and implementing preventive measures.

BCMA-targeted bortezomib nanotherapy improves therapeutic efficacy, overcomes resistance, and modulates the immune microenvironment in multiple myeloma.

Bortezomib (BTZ) is a standard-of-care treatment in multiple myeloma (MM); however, adverse side effects and development of resistance limit its long term benefit. To improve target specificity, therapeutic efficacy, and overcome resistance, we designed nanoparticles that encapsulate BTZ and are surface-functionalized with BCMA antibodies (BCMA-BTZ-NPs). We confirmed efficient cellular internalization of the BCMA-BTZ-NPs only in BCMA-expressing MM cells, but not in BCMA-knockout (KO) cells. In addition, BCMA-BTZ-NPs showed target-specific cytotoxicity against MM cell lines and primary tumor cells from MM patients. The BCMA-BTZ-NPs entered the cell through receptor-mediated uptake, which escapes a mechanism of BTZ resistance based on upregulating P-glycoprotein. Furthermore, BCMA-BTZ-NPs induced cell death more efficiently than non-targeted nanoparticles or free BTZ, triggering potent mitochondrial depolarization followed by apoptosis. In BTZ-resistant cells, BCMA-BTZ-NPs inhibited proteasome activity more effectively than free BTZ or non-targeted nanoparticles. Additionally, BCMA-BTZ-NPs enhanced immunogenic cell death and activated the autophagic pathway more than free BTZ. Finally, we found that BCMA-BTZ-NPs selectively accumulated at the tumor site in a murine xenograft model, enhanced tumor reduction, and prolonged host survival. These results suggest BCMA-BTZ-NPs provide a promising therapeutic strategy for enhancing the efficacy of BTZ and establish a framework for their evaluation in a clinical setting.

BRD9 Degradation Disrupts Ribosome Biogenesis in Multiple Myeloma.

PURPOSE: BRD9 is a defining component of the noncanonical SWI/SNF complex, which regulates gene expression by controlling chromatin dynamics. Although recent studies have found an oncogenic role for BRD9 in multiple cancer types including multiple myeloma, its clinical significance and oncogenic mechanism have not yet been elucidated. Here, we sought to identify the clinical and biological impact of BRD9 in multiple myeloma, which may contribute to the development of novel therapeutic strategies. EXPERIMENTAL DESIGN: We performed integrated analyses of BRD9 in vitro and in vivo using multiple myeloma cell lines and primary multiple myeloma cells in established preclinical models, which identified the molecular functions of BRD9 contributing to multiple myeloma cell survival. RESULTS: We found that high BRD9 expression was a poor prognostic factor in multiple myeloma. Depleting BRD9 by genetic (shRNA) and pharmacologic (dBRD9-A; proteolysis-targeting chimera; BRD9 degrader) approaches downregulated ribosome biogenesis genes, decreased the expression of the master regulator MYC, and disrupted the protein-synthesis maintenance machinery, thereby inhibiting multiple myeloma cell growth in vitro and in vivo in preclinical models. Importantly, we identified that the expression of ribosome biogenesis genes was associated with the disease progression and prognosis of patients with multiple myeloma. Our results suggest that BRD9 promotes gene expression by predominantly occupying the promoter regions of ribosome biogenesis genes and cooperating with BRD4 to enhance the transcriptional function of MYC. CONCLUSIONS: Our study identifies and validates BRD9 as a novel therapeutic target in preclinical models of multiple myeloma, which provides the framework for the clinical evaluation of BRD9 degraders to improve patient outcome.

The bHLH-zip transcription factor SREBP regulates triterpenoid and lipid metabolisms in the medicinal fungus Ganoderma lingzhi.

Ganoderic acids (GAs) are well recognized as important pharmacological components of the medicinal species belonging to the basidiomycete genus Ganoderma. However, transcription factors directly regulating the expression of GA biosynthesis genes remain poorly understood. Here, the genome of Ganoderma lingzhi is de novo sequenced. Using DNA affinity purification sequencing, we identify putative targets of the transcription factor sterol regulatory element-binding protein (SREBP), including the genes of triterpenoid synthesis and lipid metabolism. Interactions between SREBP and the targets are verified by electrophoretic mobility gel shift assay. RNA-seq shows that SREBP targets, mevalonate kinase and 3-hydroxy-3-methylglutaryl coenzyme A synthetase in mevalonate pathway, sterol isomerase and lanosterol 14-demethylase in ergosterol biosynthesis, are significantly upregulated in the SREBP overexpression (OE::SREBP) strain. In addition, 3 targets involved in glycerophospholipid/glycerolipid metabolism are upregulated. Then, the contents of mevalonic acid, lanosterol, ergosterol and 13 different GAs as well as a variety of lipids are significantly increased in this strain. Furthermore, the effects of SREBP overexpression on triterpenoid and lipid metabolisms are recovered when OE::SREBP strain are treated with exogenous fatostatin, a specific inhibitor of SREBP. Taken together, our genome-wide study clarify the role of SREBP in triterpenoid and lipid metabolisms of G. lingzhi.

Proteomic profiling of single extracellular vesicles reveals colocalization of SARS-CoV-2 with a CD81/integrin-rich EV subpopulation in sputum from COVID-19 severe patients.

Background: The global outbreak of COVID-19, and the limited availability of clinical treatments, forced researchers around the world to search for the pathogenesis and potential treatments. Understanding the pathogenesis of SARS-CoV-2 is crucial to respond better to the current coronavirus disease 2019 (COVID-19) pandemic. Methods: We collected sputum samples from 20 COVID-19 patients and healthy controls. Transmission electron microscopy was used to observe the morphology of SARS-CoV-2. Extracellular vesicles (EVs) were isolated from sputum and the supernatant of VeroE6 cells, and were characterized by transmission electron microscopy, nanoparticle tracking analysis and Western-Blotting. Furthermore, a proximity barcoding assay was used to investigate immune-related proteins in single EV, and the relationship between EVs and SARS-CoV-2. Result: Transmission electron microscopy images of SARS-COV-2 virus reveal EV-like vesicles around the virion, and western blot analysis of EVs extracted from the supernatant of SARS-COV-2-infected VeroE6 cells showed that they expressed SARS-COV-2 protein. These EVs have the infectivity of SARS-COV-2, and the addition can cause the infection and damage of normal VeroE6 cells. In addition, EVs derived from the sputum of patients infected with SARS-COV-2 expressed high levels of IL6 and TGF-ß, which correlated strongly with expression of the SARS-CoV-2 N protein. Among 40 EV subpopulations identified, 18 differed significantly between patients and controls. The EV subpopulation regulated by CD81 was the most likely to correlate with changes in the pulmonary microenvironment after SARS-CoV-2 infection. Single extracellular vesicles in the sputum of COVID-19 patients harbor infection-mediated alterations in host and virus-derived proteins. Conclusions: These results demonstrate that EVs derived from the sputum of patients participate in virus infection and immune responses. This study provides evidence of an association between EVs and SARS-CoV-2, providing insight into the possible pathogenesis of SARS-CoV-2 infection and the possibility of developing nanoparticle-based antiviral drugs.

Immunomodulatory drugs activate NK cells via both Zap-70 and cereblon-dependent pathways.

Immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide show remarkable antitumor activity in multiple myeloma (MM) via directly inhibiting MM-cell growth in the bone marrow (BM) microenvironment and promoting immune effector cell function. They are known to bind to the ubiquitin 3 ligase CRBN complex and thereby triggering degradation of IKZF1/3. In this study, we demonstrate that IMiDs also directly bind and activate zeta-chain-associated protein kinase-70 (Zap-70) via its tyrosine residue phosphorylation in T cells. IMiDs also triggered phosphorylation of Zap-70 in natural killer (NK) cells. Importantly, increased granzyme-B (GZM-B) expression and NK-cell activity triggered by IMiDs is associated with Zap-70 activation and inhibited by Zap-70 knockdown (KD), independent of CRBN. We also demonstrate a second mechanism whereby IMiDs trigger GZM-B and NK cytotoxicity which is CRBN and IKZF3 mediated, and inhibited or enhanced by KD of CRBN or IKZF3, respectively, independent of Zap-70. Our studies therefore show that IMiDs can enhance NK and T-cell cytotoxicity in (1) ZAP-70-mediated CRBN independent, as well as (2) CRBN-mediated ZAP-70 independent mechanisms; and provide the framework for developing novel therapeutics to activate Zap-70 and thereby enhance T and NK anti-MM cytotoxicity.