Phospho-eIF4E stimulation regulates coronavirus entry by selective expression of cell membrane-residential factors.

The eukaryotic translation initiation factor eIF4E can regulate cellular translation via phosphorylation on serine 209. In a recent study, by two rounds of TMT relative quantitative proteomics, we found that phosphorylated eIF4E (p-eIF4E) favors the translation of selected mRNAs, and the encoded proteins are mainly involved in ECM-receptor, focal adhesion, and PI3K-Akt signaling. The current paper is focused on the relationship between p-eIF4E and the downstream host cell proteins, and their presumed effect on efficient entry of PEDV. We found that the depletion of membrane-residential factor TSPAN3, CD63, and ITGB2 significantly inhibited viral invasion of PEDV, and reduced the entry of pseudotyped particles PEDV-pp, SARS-CoV-pp, and SARS-CoV-2-pp. The specific antibodies of TSPAN3, CD63, and ITGB2 blocked the adsorption of PEDV into host cells. Moreover, we detected that eIF4E phosphorylation was increased at 1 h after PEDV infection, in accordance with the expression of TSPAN3, CD63, and ITGB2. Similar trends appeared in the intestines of piglets in the early stage of PEDV challenge. Compared with Vero cells, S209A-Vero cells in which eIF4E cannot be phosphorylated showed a decrease of invading PEDV virions. MNK kinase inhibitor blocked PEDV invasion, as well as reduced the accumulation of TSPAN3, CD63, and ITGB2. Further study showed that the ERK-MNK pathway was responsible for the regulation of PEDV-induced early phosphorylation of eIF4E. This paper demonstrates for the first time the connections among p-eIF4E stimulation and membrane-residential host factors. Our findings also enrich the understanding of the biological function of phosphorylated eIF4E during the viral life cycle.IMPORTANCEThe eukaryotic translation initiation factor eIF4E can regulate cellular translation via phosphorylation. In our previous study, several host factors susceptible to a high level of p-eIF4E were found to be conducive to viral infection by coronavirus PEDV. The current paper is focused on cell membrane-residential factors, which are involved in signal pathways that are sensitive to phosphorylated eIF4E. We found that the ERK-MNK pathway was activated, which resulted in the stimulation of phosphorylation of eIF4E in early PEDV infection. Phospho-eIF4E promoted the viral invasion of PEDV by upregulating the expression of host factors TSPAN3, CD63, and ITGB2 at the translation level rather than at the transcription level. Moreover, TSPAN3, CD63, or ITGB2 facilitates the efficient entry of coronavirus SARS-CoV, SARS-CoV-2, and HCoV-OC43. Our findings broaden our insights into the dynamic phosphorylation of eIF4E during the viral life cycle, and provide further evidence that phosphorylated eIF4E regulates selective translation of host mRNA.

Comprehensive proteomic analysis of HCoV-OC43 virions and virus-modulated extracellular vesicles.

Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies. While numerous studies have explored the importance of host proteins in coronavirus spread, information about their presence in mature virions is limited. In this study, we developed a protocol to highly enrich mature HCoV-OC43 virions and characterize them by proteomics. Recognizing that cells release extracellular vesicles whose content is modulated by viruses, and given our ability to separate virions from these vesicles, we also analyzed their protein content in both uninfected and infected cells. We uncovered 69 unique cellular proteins associated with virions including 31 high-confidence hits. These proteins primarily regulate RNA metabolism, enzymatic activities, vesicular transport, cell adhesion, metabolite interconversion, and translation. We further discovered that the virus had a profound impact on exosome composition, incorporating 47 novel cellular proteins (11 high confidence) and excluding 92 others (61 high confidence) in virus-associated extracellular vesicles compared to uninfected cells. Moreover, a dsiRNA screen revealed that 11 of 18 select targets significantly impacted viral yields, including proteins found in virions or extracellular vesicles. Overall, this study provides new and important insights into the incorporation of numerous host proteins into HCoV-OC43 virions, their biological significance, and the ability of the virus to modulate extracellular vesicles. IMPORTANCE: In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles.

Metastasizing aneurysmal dermatofibroma initially diagnosed as angiosarcoma confirmed by CD63::PRKCD fusion gene detection with nanopore sequencing.

Dermatofibroma (DF) is a benign tumor that forms pedunculated lesions ranging in size from a few millimeters to 2 cm, usually affecting the extremities and trunks of young adults. Histopathologically, DF is characterized by the storiform proliferation of monomorphic fibroblast-like spindle cells. In addition to neoplastic cells, secondary elements such as foamy histiocytes, Touton-type giant cells, lymphoplasmacytes, and epidermal hyperplasia are characteristic histological features. Several histological variants, including atypical, cellular, aneurysmal, and lipidized variants, have been reported; cases with variant histologies are sometimes misdiagnosed as sarcomas. We present a case of metastasizing aneurysmal DF that was initially diagnosed as an angiosarcoma on biopsy. A 26-year-old woman was referred to our hospital with a gradually enlarging subcutaneous mass in her lower left leg. Positron emission tomography-computed tomography revealed high fluorodeoxyglucose uptake not only in the tumor but also in the left inguinal region. On biopsy, ERG and CD31-positive atypical spindle cells proliferated in slit-like spaces with extravasation, leading to the diagnosis of angiosarcoma. Histology of the wide-resection specimen was consistent with DF, and lymph node metastasis was also observed. Nanopore DNA sequencing detected CD63::PRKCD fusion and copy number gain, although CD63 was not included in the target region of adaptive sampling. This report highlights the importance of recognizing the unusual clinical, radiological, and pathological features of DF to avoid misdiagnosis, and the potential diagnostic utility of nanopore sequencer.

Group A Streptococcus induces lysosomal dysfunction in THP-1 macrophages.

The human-specific bacterial pathogen group A Streptococcus (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous data indicate that GAS can persist in macrophages. In this study, we detail the molecular mechanisms by which GAS survives in THP-1 macrophages. Our fluorescence microscopy studies demonstrate that GAS is readily phagocytosed by macrophages, but persists within phagolysosomes. These phagolysosomes are not acidified, which is in agreement with our findings that GAS cannot survive in low pH environments. We find that the secreted pore-forming toxin Streptolysin O (SLO) perforates the phagolysosomal membrane, allowing leakage of not only protons but also large proteins including the lysosomal protease cathepsin B. Additionally, GAS recruits CD63/LAMP-3, which may contribute to lysosomal permeabilization, especially in the absence of SLO. Thus, although GAS does not inhibit fusion of the lysosome with the phagosome, it has multiple mechanisms to prevent proper phagolysosome function, allowing for persistence of the bacteria within the macrophage. This has important implications for not only the initial response but also the overall functionality of the macrophages, which may lead to the resulting pathologies in GAS infection. Our data suggest that therapies aimed at improving macrophage function may positively impact patient outcomes in GAS infection.

The subtilisin-like protease furin regulates hemin-induced CD63 surface expression on platelets.

Accumulation of free heme B in the plasma can be the result of severe hemolytic events, when the scavenger system for free hemoglobin and heme B is overwhelmed. Free heme B can be oxidized into toxic hemin, which has been proven to activate platelet degranulation and aggregation and promote thrombosis. In the present study we analyzed the effect of hemin on the activation-mediated lysosomal degranulation and CD63 surface expression on platelets using classic flow cytometry and fluorescence microscopy techniques. Classical platelet activators were used as control to distinguish the novel effects of hemin from known activation pathways. CD63 is a tetraspanin protein, also known as lysosomal-associated membrane protein 3 or LAMP-3. In resting platelets CD63 is located within the membrane of delta granules and lysosomes of platelet, from where it is integrated into the platelet outer membrane upon stimulation. We were able to show that hemin like the endogenous platelet activators ADP, collagen or thrombin does provoke CD63 re-localization. Interestingly, only hemin-induced CD63 externalization is dependent on the subtilisin-like pro-protein convertase furin as shown by inhibitor experiments. Furthermore, we were able to demonstrate that hemin induces lysosome secretion, a source of the hemin-mediated CD63 presentation. Again, only the hemin-induced lysosome degranulation is furin dependent. In summary we have shown that the pro-protein convertase furin plays an important role in hemin-mediated lysosomal degranulation and CD63 externalization.

Oncogenic fusion of CD63-BCAR4 contributes cancer stem cell-like properties via ALDH1 activity.

Gene fusions are common somatic alterations in cancers, and fusions with tumorigenic features have been identified as novel drivers of cancer and therapeutic targets. Few studies have determined whether the oncogenic ability of fusion genes is related to the induction of stemness in cells. Cancer stem cells (CSCs) are a subset of cells that contribute to cancer progression, metastasis, and recurrence, and are critical components of the aggressive features of cancer. Here, we investigated the CSC-like properties induced by CD63-BCAR4 fusion gene, previously reported as an oncogenic fusion, and its potential contribution for the enhanced metastasis as a notable characteristic of CD63-BCAR4. CD63-BCAR4 overexpression facilitates sphere formation in immortalized bronchial epithelial cells. The significantly enhanced sphere-forming activity observed in tumor-derived cells from xenografted mice of CD63-BCAR4 overexpressing cells was suppressed by silencing of BCAR4. RNA microarray analysis revealed that ALDH1A1 was upregulated in the BCAR4 fusion-overexpressing cells. Increased activity and expression of ALDH1A1 were observed in the spheres of CD63-BCAR4 overexpressing cells compared with those of the empty vector. CD133 and CD44 levels were also elevated in BCAR4 fusion-overexpressing cells. Increased NANOG, SOX2, and OCT-3/4 protein levels were observed in metastatic tumor cells derived from mice injected with CD63-BCAR4 overexpressing cells. Moreover, DEAB, an ALDH1A1 inhibitor, reduced the migration activity induced by CD63-BCAR4 as well as the sphere-forming activity. Our findings suggest that CD63-BCAR4 fusion induces CSC-like properties by upregulating ALDH1A1, which contributes to its metastatic features.

Combining avidin with CD63 improves basophil activation test accuracy in classifying peanut allergy.

BACKGROUND: Conventional basophil activation tests (BATs) measure basophil activation by the increased expression of CD63. Previously, fluorophore-labeled avidin, a positively-charged molecule, was found to bind to activated basophils, which tend to expose negatively charged granule constituents during degranulation. This study further compares avidin versus CD63 as basophil activation biomarkers in classifying peanut allergy. METHODS: Seventy subjects with either a peanut allergy (N = 47), a food allergy other than peanut (N = 6), or no food allergy (N = 17) were evaluated. We conducted BATs in response to seven peanut extract (PE) concentrations (0.01-10,000 ng/mL) and four control conditions (no stimulant, anti-IgE, fMLP (N-formylmethionine-leucyl-phenylalanine), and anti-FcεRI). We measured avidin binding and CD63 expression on basophils with flow cytometry. We evaluated logistic regression and XGBoost models for peanut allergy classification and feature identification. RESULTS: Avidin binding was correlated with CD63 expression. Both markers discriminated between subjects with and without a peanut allergy. Although small by percentage, an avidin+ /CD63- cell subset was found in all allergic subjects tested, indicating that the combination of avidin and CD63 could allow a more comprehensive identification of activated basophils. Indeed, we obtained the best classification accuracy (97.8% sensitivity, 96.7% specificity) by combining avidin and CD63 across seven PE doses. Similar accuracy was obtained by combining PE dose of 10,000 ng/mL for avidin and PE doses of 10 and 100 ng/mL for CD63. CONCLUSIONS: Avidin and CD63 are reliable BAT activation markers associated with degranulation. Their combination enhances the identification of activated basophils and improves the classification accuracy of peanut allergy.

CD63 + tumor-associated macrophages drive the progression of hepatocellular carcinoma through the induction of epithelial-mesenchymal transition and lipid reprogramming.

BACKGROUND: Tumor-associated macrophages (TAMs) constitute a substantial part of human hepatocellular carcinoma (HCC). The present study was devised to explore TAM diversity and their roles in HCC progression. METHODS: Through the integration of multiple 10 × single-cell transcriptomic data derived from HCC samples and the use of consensus nonnegative matrix factorization (an unsupervised clustering algorithm), TAM molecular subtypes and expression programs were evaluated in detail. The roles played by these TAM subtypes in HCC were further probed through pseudotime, enrichment, and intercellular communication analyses. Lastly, vitro experiments were performed to validate the relationship between CD63, which is an inflammatory TAM expression program marker, and tumor cell lines. RESULTS: We found that the inflammatory expression program in TAMs had a more obvious interaction with HCC cells, and CD63, as a marker gene of the inflammatory expression program, was associated with poor prognosis of HCC patients. Both bulk RNA-seq and vitro experiments confirmed that higher TAM CD63 expression was associated with the growth of HCC cells as well as their epithelial-mesenchymal transition, metastasis, invasion, and the reprogramming of lipid metabolism. CONCLUSIONS: These analyses revealed that the TAM inflammatory expression program in HCC is closely associated with malignant tumor cells, with the hub gene CD63 thus representing an ideal target for therapeutic intervention in this cancer type.

Comparison of the passive mast cell activation test with the basophil activation test for diagnosis of perioperative rocuronium hypersensitivity.

BACKGROUND: Rocuronium is a major cause of perioperative hypersensitivity (POH). Skin tests (STs) and quantification of specific immunoglobulin E antibodies (sIgEs) can yield incongruent results. In such difficult cases, the basophil activation test (BAT) can be helpful. Here, we evaluated the passive mast cell activation test (pMAT) as a substitute of BAT as part of the diagnostic tests for rocuronium allergy. METHODS: Sera from patients with a suspected POH reaction potentially related to rocuronium were included. All patients had a complete diagnostic investigation, including STs, quantification of sIgEs to morphine and rocuronium, and BAT. For execution of pMAT, human mast cells were generated from healthy donor peripheral blood CD34+ progenitor cells and sensitised overnight with patient sera. RESULTS: In total, 90 sera were studied: 41 from ST+sIgE+ patients, 13 from ST-sIgE- patients, 20 from ST+sIgE- patients, and 16 from ST-sIgE+ patients. According to BAT results, patients were further allocated into subgroups. Of the 38 BAT+ patients, 25 (66%) showed a positive pMAT as well. Of the 44 BAT- patients, 43 (98%) also showed a negative pMAT. Mast cells that were not passively sensitised did not respond to rocuronium. CONCLUSIONS: We show that the pMAT, in many cases, can substitute for BAT in the diagnosis of rocuronium hypersensitivity and advance diagnosis in difficult cases with uncertain ST or sIgE results when BAT is not locally available.

Increased circulating platelet-derived extracellular vesicles in severe COVID-19 disease.

Coagulation disturbances are major contributors to COVID-19 pathogenicity, but limited data exist on the involvement of extracellular vesicles (EVs) and residual cells (RCs). Fifty hospitalized COVID-19 patients stratified by their D-dimer levels into high (>1.5 mg/L, n = 15) or low (≤1.5 mg/l, n = 35) and 10 healthy controls were assessed for medium-sized EVs (mEVs; 200-1000 nm) and large EVs/RCs (1000-4000 nm) by high sensitivity flow cytometry. EVs were analyzed for CD61, CD235a, CD45, and CD31, commonly used to detect platelets, red blood cells, leukocytes or endothelial cells, respectively, whilst phosphatidyl serine EVs/RCs were detected by lactadherin-binding implicating procoagulant catalytic surface. Small EV detection (sEVs; 50-200 nm) and CD41a (platelet integrin) colocalization with general EV markers CD9, CD63, and CD81 were performed by single particle interferometric reflectance imaging sensor. Patients with increased D-dimer exhibited the highest number of RCs and sEVs irrespective of cell origin (p < .05). Platelet activation, reflected by increased CD61+ and lactadherin+ mEV and RC levels, associated with coagulation disturbances. Patients with low D-dimer could be discriminated from controls by tetraspanin signatures of the CD41a+ sEVs, suggesting the changes in the circulating platelet sEV subpopulations may offer added prognostic value during COVID progression.

What is the context? Coronavirus disease 19 (COVID-19) frequently leads to blood clotting disturbances, including thromboses.Particles smaller than cells, extracellular vesicles (EVs), and residual cells (RCs) affect blood clotting, but data on their role and diagnostic utility in COVID-19 are sparse.What is new? In this study, we assessed 50 hospitalized COVID-19 patients and 10 healthy controls for their different EV subpopulations and residual cells (50­4000 nm).Blood clotting marker D-dimer, which is elevated in severe COVID-19 infection, was used to characterize disease severity and stratify the patient subgroups. Fifteen patients (30%) with high D-dimer (>1.5 mg/L) were compared to controls, and 35 patients with lower D-dimer (≤1.5 mg/mL).The most topical state-of-the-art methods for detection of EV subpopulations, that is, high sensitivity flow cytometry (hsFCM) and single particle interferometric reflectance imaging sensor (SP-IRIS), were used with markers indicative of platelet, red blood cell, leukocyte or endothelial cells. The subpopulations differentiated by platelet and tetraspanin signatures by hsFCM and SP-IRIS, respectively.The main findings are Patients with high D-dimer systematically exhibited the highest number of platelet EVs in all subpopulations (p < .05).Small EVs subpopulations (differentiated by the tetraspanin signatures) could discriminate patients with low D-dimer (p < .001) from healthy controls.Differences between the two D-dimer groups were seen in the platelet-derived (large and medium EVs and RCs), RBC-derived mEVs and l EVs and RCs, and lactadherin-positive large EVs and RCs (p < .05).What is the impact? Platelet activation, reflected by increased EVs was associated with blood clotting disturbances. Small EVs signatures revealed changes in the EV subpopulations in association with blood clotting during COVID-19. Such signatures may enable identification of severely ill patients before the increase in coagulation is evident by coagulation parameters, for example, by high D-dimer.

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer-Associated Stellate Cell Small Extracellular Vesicles.

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography-tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1-like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.

Degron tagging of BleoR and other antibiotic-resistance genes selects for higher expression of linked transgenes and improved exosome engineering.

Five antibiotic resistance (AR) genes have been used to select for transgenic eukaryotic cell lines, with the BleoR, PuroR, HygR, NeoR, and BsdR cassettes conferring resistance to zeocin, puromycin, hygromycin, geneticin/G418, and blasticidin, respectively. We recently demonstrated that each AR gene establishes a distinct threshold of transgene expression below which no cell can survive, with BleoR selecting for the highest level of transgene expression, nearly ∼10-fold higher than in cells selected using the NeoR or BsdR markers. Here, we tested the hypothesis that there may be an inverse proportionality between AR protein function and the expression of linked, transgene-encoded, recombinant proteins. Specifically, we fused each AR protein to proteasome-targeting degron tags, used these to select for antibiotic-resistant cell lines, and then measured the expression of the linked, recombinant protein, mCherry, as a proxy marker of transgene expression. In each case, degron-tagged AR proteins selected for higher mCherry expression than their cognate WT AR proteins. ER50BleoR selected for the highest level of mCherry expression, greater than twofold higher than BleoR or any other AR gene. Interestingly, use of ER50BleoR as the selectable marker translated to an even higher, 3.5-fold increase in the exosomal loading of the exosomal cargo protein, CD63/Y235A. Although a putative CD63-binding peptide, CP05, has been used to decorate exosome membranes in a technology known as "exosome painting," we show here that CP05 binds equally well to CD63-/- cells, WT 293F cells, and CD63-overexpressing cells, indicating that CP05 may bind membranes nonspecifically. These results are of high significance for cell engineering and especially for exosome engineering.

A shared, stochastic pathway mediates exosome protein budding along plasma and endosome membranes.

Exosomes are small extracellular vesicles of ∼30 to 150 nm that are secreted by all cells, abundant in all biofluids, and play important roles in health and disease. However, details about the mechanism of exosome biogenesis are unclear. Here, we carried out a cargo-based analysis of exosome cargo protein biogenesis in which we identified the most highly enriched exosomal cargo proteins and then followed their biogenesis, trafficking, and exosomal secretion to test different hypotheses for how cells make exosomes. We show that exosome cargo proteins bud from cells (i) in exosome-sized vesicles regardless of whether they are localized to plasma or endosome membranes, (ii) ∼5-fold more efficiently when localized to the plasma membrane, (iii) ∼5-fold less efficiently when targeted to the endosome membrane, (iv) by a stochastic process that leads to ∼100-fold differences in their abundance from one exosome to another, and (v) independently of small GTPase Rab27a, the ESCRT complex-associated protein Alix, or the cargo protein CD63. Taken together, our results demonstrate that cells use a shared, stochastic mechanism to bud exosome cargoes along the spectrum of plasma and endosome membranes and far more efficiently from the plasma membrane than the endosome. Our observations also indicate that the pronounced variation in content between different exosome-sized vesicles is an inevitable consequence of a stochastic mechanism of small vesicle biogenesis, that the origin membrane of exosome-sized extracellular vesicles simply cannot be determined, and that most of what we currently know about exosomes has likely come from studies of plasma membrane-derived vesicles.

LAMP3/CD63 Expression in Early and Late Endosomes in Human Vaginal Epithelial Cells Is Associated with Enhancement of HSV-2 Infection.

Herpes simplex virus 2 (HSV-2) is a lifelong sexually transmitted virus that disproportionately infects women through heterosexual transmission in the vaginal tract. The vaginal epithelium is known to be highly susceptible to HSV-2 infection; however, the cellular mechanism of HSV-2 uptake and replication in vaginal epithelium has not been extensively studied. Previously, we observed that lysosomal-associated membrane protein-3 (LAMP3/CD63) was among the highly upregulated genes during HSV-2 infection of human vaginal epithelial cell line VK2, leading us to posit that LAMP3/CD63 may play a role in HSV-2 infection. Consequently, we generated two gene-altered VK2-derived cell lines, a LAMP3-overexpressed (OE) line and a LAMP3 knockout (KO) line. The wild-type VK2 and the LAMP3 OE and KO cell lines were grown in air-liquid interface (ALI) cultures for 7 days and infected with HSV-2. Twenty-four hours postinfection, LAMP3 OE cells produced and released significantly higher numbers of HSV-2 virions than wild-type VK2 cells, while virus production was greatly attenuated in LAMP3 KO cells, indicating a functional association between LAMP3/CD63 expression and HSV-2 replication. Fluorescence microscopy of HSV-2-infected cells revealed that HSV-2 colocalized with LAMP3 in both early endosomes and lysosomal compartments. In addition, blocking endosomal maturation or late endosomal/lysosomal fusion using specific inhibitors resulted in reduced HSV-2 replication in VK2 cells. Similarly, LAMP3 KO cells exhibited very low viral entry and association with endosomes, while LAMP3 OE cells demonstrated large amounts of virus that colocalized with LAMP3/CD63 in endosomes and lysosomes. IMPORTANCE Collectively, these results showed that HSV-2 is taken up by human vaginal epithelial cells through an endosomal-lysosomal pathway in association with LAMP3, which plays a crucial role in the enhancement of HSV-2 replication. These findings provide the basis for the future design of antiviral agents for prophylactic measures against HSV-2 infection.

New iron export pathways acting via holo-ferritin secretion.

Ferritin is a spherical nanocage protein for iron storage, composed of 24 light- or heavy-polypeptide chain subunits. A single ferritin molecule can carry up to 4500 iron atoms in its core, which plays an important role in suppressing intracellular iron toxicity. Serum ferritin levels are used as a marker for the total amount of iron stored in the body. Most serum ferritin is iron-free (apo-ferritin) and it is unclear how ferritin is released from cells. Ferritin is secreted into serum via extracellular vesicles (EVs) or the secretory autophagy pathway but not via the classical endoplasmic reticulum (ER)-to-Golgi secretion pathway. We recently discovered that the level of tetraspanin CD63, a common EV marker, is post-transcriptionally regulated by the intracellular iron level and both CD63 and ferritin expression is induced by iron loading. Ferritin is incorporated into CD63(+)-EVs through the ferritin-specific autophagy adapter molecule, NCOA4, and then secreted from cells. EV production differs drastically depending on cell type and physiological conditions. Extracellular matrix detached cells express pentaspanin prominin 2 and prominin 2(+)-EVs secrete ferritin independently of NCOA4 trafficking. Ferritin is tightly bound to iron in EVs and functions as an iron-carrier protein in the extracellular environment. Cells can suppress ferroptosis by secreting holo-ferritin, which reduces intracellular iron concentration. However, this exposes the neighboring cells receiving the secreted holo-ferritin to a large excess of iron. This results in cellular toxicity through increased generation of reactive oxygen species (ROS). Here we review the machinery by which ferritin is incorporated into EVs and its role as an intercellular communication molecule.

Iron links endogenous and exogenous nanoparticles.

Current progress in biology and medical science is based on the observation at the level of nanometers via electron microscopy and computation. Of note, the size of most cells in higher species exists in a limited range from 5 to 50 µm. Recently, it was demonstrated that endogenous extracellular nanoparticles play a role in communication among various cellular types in a variety of contexts. Among them, exosomes in serum have been established as biomarkers for human diseases by analyzing the cargo molecules. No life on the earth can survive without iron. However, excess iron can be a risk for carcinogenesis in rodents and humans. Nano-sized molecules may cause unexpected bioeffects, including carcinogenesis, which is a process to establish cellular iron addiction with ferroptosis-resistance. Asbestos and carbon nanotubes are the typical examples, leading to carcinogenesis by the alteration of iron metabolism. Recently, we found that CD63, one of the representative markers of exosomes, is under the regulation of iron-responsive element/iron-regulatory protein system. This is a safe strategy to share excess iron in the form of holo-ferritin between iron-sufficient and -deficient cells. On the other hand, damaged cells may secrete holo-ferritin-loaded exosomes as in the case of macrophages in ferroptosis after asbestos exposure. These holo-ferritin-loaded exosomes can cause mutagenic DNA damage in the recipient mesothelial cells. Thus, there is an iron link between exogenous and endogenous nanoparticles, which requires further investigation for better understanding and the future applications.

Mapping high-grade glioma immune infiltration to 5-ALA fluorescence levels: TCGA data computation, classical histology, and digital image analysis.

PURPOSE: Resection of high-grade gliomas has been considerably improved by 5-aminolevulinic acid (5-ALA). However, not all neurobiological properties of 5-ALA are fully understood. Specifically, potential differences in immune infiltration have not been conclusively examined, despite recent reports that immune cells might play a role. Thus, we here provide a systematic mapping of immune infiltration of different 5-ALA fluorescence levels. METHODS: Tumor-associated macrophages (CD68, CD163), cytotoxic T cells (CD8), and regulatory T cells (FoxP3) were quantified via three methods. First, data from The Cancer Genome Atlas (TCGA) of 172 patients was examined for correlations between 5-ALA fluorescence-related mRNA expression signatures and immune markers. Second, as classical histology, 508 stained slides from 39 high-grade glioma patients were analysed semi-quantitatively by two independent reviewers, generating 1016 data points. Third, digital image analysis was performed with automated scanning and algorithm-based cell quantification. RESULTS: TCGA mRNA data from 172 patients showed a direct, significant correlation between 5-ALA signatures and immune markers (p < 0.001). However, we were not able to confirm this finding in the here studied initial set of 39 patient histologies where we found a comparable immune infiltration in different fluorescence levels. Digital image analysis correlated excellently with standard histology. CONCLUSION: With mapping the immune infiltration pattern of different 5-ALA categories, we are adding fundamental basic insights to the field of 5-ALA and glioma biology. The observation that a significant correlation in TCGA data did not fully translate to detectable differences in immune infiltration in first histology data warrants further investigation in larger cohorts.

Macroporous Epoxy-Based Monoliths Functionalized with Anti-CD63 Nanobodies for Effective Isolation of Extracellular Vesicles in Urine.

Extracellular vesicles (EVs) have enormous potential for the implementation of liquid biopsy and as effective drug delivery means, but the fulfilment of these expectations requires overcoming at least two bottlenecks relative to their purification, namely the finalization of reliable and affordable protocols for: (i) EV sub-population selective isolation and (ii) the scalability of their production/isolation from complex biological fluids. In this work, we demonstrated that these objectives can be achieved by a conceptually new affinity chromatography platform composed of a macroporous epoxy monolith matrix functionalized with anti-CD63 nanobodies with afflux of samples and buffers regulated through a pump. Such a system successfully captured and released integral EVs from urine samples and showed negligible unspecific binding for circulating proteins. Additionally, size discrimination of eluted EVs was achieved by different elution approaches (competitive versus pH-dependent). The physical characteristics of monolith material and the inexpensive production of recombinant nanobodies make scaling-up the capture unit feasible and affordable. Additionally, the availability of nanobodies for further specific EV biomarkers will allow for the preparation of monolithic affinity filters selective for different EV subclasses.

[An overview of iron metabolism and new insights into the role of ferritin secretion].

For approximately 4 billion years after the birth of life, iron, the fourth most abundant metal on earth, has played a critical role in redox reactions. Iron is a trace metal that is required for life activity, and no organism can survive without it. However, it has recently been discovered that iron causes life-threatening toxicity as organisms live longer. Ten years have passed since the discovery of iron-dependent cell death (ferroptosis), and the interest in iron is growing rapidly in various fields. Many diseases, such as neurodegenerative diseases and cancers, have been linked to abnormalities in iron metabolism, and completely new treatment methods based on iron regulation have been developed. In this article, we will assess the secretory mechanism ferritin, an iron storage protein that we recently discovered, as well as the role of secreted ferritin in diseases.

Heparanase-1 is upregulated by hepatitis C virus and favors its replication.

BACKGROUND & AIMS: Over time, chronic HCV infection can lead to hepatocellular carcinoma (HCC), a process that involves changes to the liver extracellular matrix (ECM). However, the exact mechanisms by which HCV induces HCC remain unclear. Therefore, we sought to investigate the impact of HCV on the liver ECM, with a focus on heparanase-1 (HPSE). METHODS: HPSE expression was assessed by quantitative reverse-transcription PCR, immunoblotting and immunofluorescence in liver biopsies infected or not with HCV, and in 10-day-infected hepatoma Huh7.5 cells. Cell lines deficient for or overexpressing HPSE were established to study its role during infection. RESULTS: HCV propagation led to significant HPSE induction, in vivo and in vitro. HPSE enhanced infection when exogenously expressed or supplemented as a recombinant protein. Conversely, when HPSE expression was downregulated or its activity blocked, HCV infection dropped, suggesting a role of HPSE in the HCV life cycle. We further studied the underlying mechanisms of such observations and found that HPSE favored HCV release by enhancing CD63 synthesis and exosome secretion, but not by stimulating HCV entry or genome replication. We also showed that virus-induced oxidative stress was involved in HPSE induction, most likely through NF-κB activation. CONCLUSIONS: We report for the first time that HCV infection is favored by HPSE, and upregulates HPSE expression and secretion, which may result in pathogenic alterations of the ECM. LAY SUMMARY: Chronic hepatitis C virus (HCV) infection can lead to hepatocellular carcinoma development in a process that involves derangement of the extracellular matrix (ECM). Herein, we show that heparanase-1, a protein involved in ECM degradation and remodeling, favors HCV infection and is upregulated by HCV infection; this upregulation may result in pathogenic alterations of the ECM.