Prostate specific antigen (PSA) test is the most common, clinically validated test for the diagnosis of prostate cancer (PCa). While neoplastic lesions of the prostate may cause aberrant levels of ...PSA in the blood, the quantitation of free or complexed PSA poorly discriminates cancer patients from those developing benign lesions, often leading to invasive and unnecessary surgical procedures. Microenvironmental acidity increases exosome release by cancer cells. In this study we evaluated whether acidity, a critical phenotype of malignancy, could influence exosome release and increase the PSA expression in nanovesicles released by PCa cells. To this aim, we exploited Nanoparticle Tracking Analysis (NTA), an immunocapture-based ELISA, and nanoscale flow-cytometry. The results show that microenvironmental acidity induces an increased release of nanovesicles expressing both PSA and the exosome marker CD81. In order to verify whether the changes induced by the local selective pressure of extracellular acidity may correspond to a clinical pathway we used the same approach to evaluate the levels of PSA-expressing exosomes in the plasma of PCa patients and controls, including subjects with benign prostatic hypertrophy (BPH). The results show that only PCa patients have high levels of nanovesicles expressing both CD81 and PSA. This study shows that tumor acidity exerts a selective pressure leading to the release of extracellular vesicles that express both PSA and exosome markers. A comparable scenario was shown in the plasma of prostate cancer patients as compared to both BPH and healthy controls. These results suggest that microenvironmental acidity may represent a key factor which determines qualitatively and quantitatively the release of extracellular vesicles by malignant tumors, including prostate cancer. This condition leads to the spill-over of nanovesicles into the peripheral blood of prostate cancer patients, where the levels of tumor biomarkers expressed by exosomes, such as PSA-exosomes, may represent a novel, non-invasive clinical tool for the screening and early diagnosis of prostate cancer.
•PSA prostate-specific antigen (PSA) is a prostate cancer (PCa) specific marker.•Microenvironmental acidity increases exosome release by cancer cells.•Acidity induces expression of PSA by exosomes.•PCa patients had high levels of PSA-expressing exosomes.•Tumor acidity exerts a selective pressure leading to expression of PSA by exosomes, leading in turn to the spilling over of PSA-expressing exosomes in the blood of PCa patients.•The use of plasmatic PSA-exosomes may be a novel, non-invasive clinical tool for screening and early diagnosis of prostate cancer.
HIV-1 encodes a viral protease that is essential for the maturation of infectious viral particles. While protease inhibitors are effective antiretroviral agents, recent studies have shown that ...prematurely activating, rather than inhibiting, protease function leads to the pyroptotic death of infected cells, with exciting implications for efforts to eradicate viral reservoirs. Despite 40 years of research into the kinetics of protease activation, it remains unclear exactly when protease becomes activated. Recent reports have estimated that protease activation occurs minutes to hours after viral release, suggesting that premature protease activation is challenging to induce efficiently. Here, monitoring viral protease activity with sensitive techniques, including nanoscale flow cytometry and instant structured illumination microscopy, we demonstrate that the viral protease is activated within cells prior to the release of free virions. Using genetic mutants that lock protease into a precursor conformation, we further show that both the precursor and mature protease have rapid activation kinetics and that the activity of the precursor protease is sufficient for viral fusion with target cells. Our finding that HIV-1 protease is activated within producer cells prior to release of free virions helps resolve a long-standing question of when protease is activated and suggests that only a modest acceleration of protease activation kinetics is required to induce potent and specific elimination of HIV-infected cells.
HIV-1 protease inhibitors have been a mainstay of antiretroviral therapy for more than 2 decades. Although antiretroviral therapy is effective at controlling HIV-1 replication, persistent reservoirs of latently infected cells quickly reestablish replication if therapy is halted. A promising new strategy to eradicate the latent reservoir involves prematurely activating the viral protease, which leads to the pyroptotic killing of infected cells. Here, we use highly sensitive techniques to examine the kinetics of protease activation during and shortly after particle formation. We found that protease is fully activated before virus is released from the cell membrane, which is hours earlier than recent estimates. Our findings help resolve a long-standing debate as to when the viral protease is initially activated during viral assembly and confirm that prematurely activating HIV-1 protease is a viable strategy to eradicate infected cells following latency reversal.
Zwitterion polymers with strong antifouling properties have been suggested as the prime alternative to polyethylene glycol (PEG) for drug nanocarriers surface coating. It is believed that PEG coating ...shortcomings, such as immune responses and incomplete protein repellency, could be overcome by zwitterionic polymers. However, no systematic study has been conducted so far to complete a comparative appraisal of PEG and zwitterionic-coating effects on nanoparticles (NPs) stealthness, cell uptake, cell barrier translocation and biodistribution in the context of nanocarriers brain targeting.
Core-shell polymeric particles with identical cores and a shell of either PEG or poly(2-methacryloyloxyethyl phosphorylcholine (PMPC) were prepared by impinging jet mixer nanoprecipitation. NPs with similar size and surface potential were systematically compared using in vitro and in vivo assays. NPs behavior differences were rationalized based on their protein-particles interactions.
PMPC-coated NPs were significantly more endocytosed by mouse macrophages or brain resident macrophages compared to PEGylated NPs but exhibited the remarkable ability to cross the blood-brain barrier in in vitro models. Nanoscale flow cytometry assays showed significantly more adsorbed proteins on PMPC-coated NPs than PEG-coated NPs. In vivo, distribution in zebrafish larvae, showed a strong propensity for PMPC-coated NPs to adhere to the vascular endothelium, while PEG-coated NPs were able to circulate for a longer time and escape the bloodstream to penetrate deep into the cerebral tissue.
The stark differences between these two types of particles, besides their similarities in size and surface potential, points towards the paramount role of surface chemistry in controlling NPs fate likely via the formation of distinct protein corona for each coating.
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Prostate Specific Antigen (PSA) fails to discriminate between benign prostatic hyperplasia (BPH) and Prostate Cancer (PCa), resulting in large numbers of unnecessary biopsies and missed cancer ...diagnoses. Nanovesicles called exosomes are directly detectable in patient plasma and here we explore the potential use of plasmatic exosomes expressing PSA (Exo-PSA) in distinguishing healthy individuals, BPH, and PCa. Exosomes were obtained from plasma samples of 80 PCa, 80 BPH, and 80 healthy donors (CTR). Nanoparticle Tracking Analysis (NTA), immunocapture-based ELISA (IC-ELISA), and nanoscale flow-cytometry (NSFC), were exploited to detect and characterize plasmatic exosomes. Statistical analysis showed that plasmatic exosomes expressing both CD81 and PSA were significantly higher in PCa as compared to both BPH and CTR, reaching 100% specificity and sensitivity in distinguishing PCa patients from healthy individuals. IC-ELISA, NSFC, and Exo-PSA consensus score (EXOMIX) showed 98% to 100% specificity and sensitivity for BPH-PCa discrimination. This study outperforms the conventional PSA test with a minimally invasive widely exploitable approach.
The ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification ...of EVs from biofluids, such purified EV samples will also contain non-EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation.
We compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non-target EVs in the final EV preparation.
To determine enrichment of prostate derived EVs relative to non-target MPs, anti-PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody-based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein.
While attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches.
HIV-1 protease inhibitors are an essential component of antiretroviral therapy. However, drug resistance is a pervasive issue motivating a persistent search for novel therapies. Recent reports found ...that when protease activates within the host cell’s cytosol, it facilitates the pyroptotic killing of infected cells. This has led to speculation that promoting protease activation, rather than inhibiting it, could help to eradicate infected cells and potentially cure HIV-1 infection. Here, we used a nanoscale flow cytometry-based assay to characterize protease resistance mutations and polymorphisms. We quantified protease activity, viral concentration, and premature protease activation and confirmed previous findings that major resistance mutations generally destabilize the protease structure. Intriguingly, we found evidence that common polymorphisms in the hinge domain of protease can influence its susceptibility to premature activation. This suggests that viral heterogeneity could pose a considerable challenge for therapeutic strategies aimed at inducing premature protease activation in the future.
This review focuses on the emerging field of flow virometry, the study and characterization of individual viral particles using flow cytometry instruments and protocols optimized for the detection of ...nanoscale events. Flow virometry faces considerable technical challenges including minimal light scattering by small viruses that complicates detection, coincidental detection of multiple small particles due to their high concentrations, and challenges with sample preparation including the inability to easily "wash" samples to remove unbound fluorescent antibodies. We will discuss how the field has overcome these challenges to reveal novel insights into viral biology.
The HIV envelope glycoprotein (Env) is a trimeric protein that facilitates viral binding and fusion with target cells. As the sole viral protein on the HIV surface, Env is important both for immune ...responses to HIV and in vaccine designs. Targeting Env in clinical applications is challenging due to its heavy glycosylation, high genetic variability, conformational camouflage, and its low abundance on virions. Thus, there is a critical need to better understand this protein. Flow virometry (FV) is a useful methodology for phenotyping the virion surface in a high-throughput, single virion manner. To demonstrate the utility of FV to characterize Env, we stained HIV virions with a panel of 85 monoclonal antibodies targeting different regions of Env. A broad range of antibodies yielded robust staining of Env, with V3 antibodies showing the highest quantitative staining. A subset of antibodies tested in parallel on viruses produced in CD4+ T cell lines, HEK293T cells, and primary cells showed that the cellular model of virus production can impact Env detection. Finally, in addition to being able to highlight Env heterogeneity on virions, we show FV can sensitively detect differences in Env conformation when soluble CD4 is added to virions before staining.
The study of tumor exosomes has gained relevance in the last decades due to their potential use for therapeutic and diagnostic application. Although there is extensive knowledge of exosome biology, ...some biological samples like tumor-derived exosomes have been difficult to characterize due to their complexity and heterogeneity. This distinctive feature makes difficult the identification of specific exosome subpopulations with a shared molecular signature that could allow for targeting of exosomes with therapeutic and diagnostic potential use in cancer patients. Nanoscale flow cytometry has lately emerged as an alternative tool that can be adapted to the study of nanoparticles, such as exosomes. However, the physicochemical properties of these particles are an important issue to consider as nanoparticles need the application of specific settings which differ from those used in conventional flow cytometry of cells. Therefore, in the last few years, one of the main aims has been the optimization of technical and experimental protocols to improve exosome analysis. In this chapter, we discuss several aspects of cytometric systems with a special emphasis in technical considerations of samples and equipment.
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