Intervention and Improved Well-Being of Basic Science Researchers During the COVID 19 Era: A Case Study.

The coronavirus disease-19 (COVID-19) pandemic has affected individuals of all categories, irrespective of their geographical locations, professions, gender, or race. As a result of full or partial lock-down and stay-at-home orders, the well-being and productivity of individuals were severely affected. Since basic science research requires laboratory experiments, the work-from-home strategy hurt their productivity. In addition, the combination of decreased productivity and staying at home is likely to compromise their well-being by causing stress and anxiety. In this case study, a strategy was developed to engage researchers through listening and learning, motivation, and empowerment, using regular virtual sessions. Through these virtual sessions, research work was prioritized and coordinated, from idea conception to writing research papers and grant proposals. Perceived stress scores (PSS) and COVID-19-related stress (COVID-SS) scores were measured to evaluate general and COVID-19-induced stress, respectively, every month from March to July 2020 during the COVID-19 era. The result showed a significant improvement in both the PSS and the COVID-SS scores of the intervention group compared to the control group. In addition, while there was no/minimal change in PSS and COVID-SS scores from March to subsequent months until July for the control group, the intervention groups showed significant and consistent improvement in both scores in the intervention group. Overall, the intervention strategy showed improved well-being for basic science researchers, which was also consistent with their improved productivity during the COVID-19 era.

Circulatory Astrocyte and Neuronal EVs as Potential Biomarkers of Neurological Dysfunction in HIV-Infected Subjects and Alcohol/Tobacco Users.

The diagnosis of neurocognitive disorders associated with HIV infection, alcohol, and tobacco using CSF or neuroimaging are invasive or expensive methods, respectively. Therefore, extracellular vesicles (EVs) can serve as reliable noninvasive markers due to their bidirectional transport of cargo from the brain to the systemic circulation. Hence, our objective was to investigate the expression of astrocytic (GFAP) and neuronal (L1CAM) specific proteins in EVs circulated in the plasma of HIV subjects, with and without a history of alcohol consumption and tobacco smoking. The protein expression of GFAP (p < 0.01) was significantly enhanced in plasma EVs obtained from HIV-positive subjects and alcohol users compared to healthy subjects, suggesting enhanced activation of astrocytes in those subjects. The L1CAM expression was found to be significantly elevated in cigarette smokers (p < 0.05). However, its expression was not found to be significant in HIV subjects and alcohol users. Both GFAP and L1CAM levels were not further elevated in HIV-positive alcohol or tobacco users compared to HIV-positive nonsubstance users. Taken together, our data demonstrate that the astrocytic and neuronal-specific markers (GFAP and L1CAM) can be packaged in EVs and circulate in plasma, which is further elevated in the presence of HIV infection, alcohol, and/or tobacco. Thus, the astroglial marker GFAP and neuronal marker L1CAM may represent potential biomarkers targeting neurological dysfunction upon HIV infection and/or alcohol/tobacco consumption.

Repurposing Antiviral Protease Inhibitors Using Extracellular Vesicles for Potential Therapy of COVID-19.

In January 2020, Chinese health agencies reported an outbreak of a novel coronavirus-2 (CoV-2) which can lead to severe acute respiratory syndrome (SARS). The virus, which belongs to the coronavirus family (SARS-CoV-2), was named coronavirus disease 2019 (COVID-19) and declared a pandemic by the World Health Organization (WHO). Full-length genome sequences of SARS-CoV-2 showed 79.6% sequence identity to SARS-CoV, with 96% identity to a bat coronavirus at the whole-genome level. COVID-19 has caused over 133,000 deaths and there are over 2 million total confirmed cases as of April 15th, 2020. Current treatment plans are still under investigation due to a lack of understanding of COVID-19. One potential mechanism to slow disease progression is the use of antiviral drugs to either block the entry of the virus or interfere with viral replication and maturation. Currently, antiviral drugs, including chloroquine/hydroxychloroquine, remdesivir, and lopinavir/ritonavir, have shown effective inhibition of SARS-CoV-2 in vitro. Due to the high dose needed and narrow therapeutic window, many patients are experiencing severe side effects with the above drugs. Hence, repurposing these drugs with a proper formulation is needed to improve the safety and efficacy for COVID-19 treatment. Extracellular vesicles (EVs) are a family of natural carriers in the human body. They play a critical role in cell-to-cell communications. EVs can be used as unique drug carriers to deliver protease inhibitors to treat COVID-19. EVs may provide targeted delivery of protease inhibitors, with fewer systemic side effects. More importantly, EVs are eligible for major aseptic processing and can be upscaled for mass production. Currently, the FDA is facilitating applications to treat COVID-19, which provides a very good chance to use EVs to contribute in this combat.

Extracellular Vesicles in Smoking-Mediated HIV Pathogenesis and their Potential Role in Biomarker Discovery and Therapeutic Interventions.

In the last two decades, the mortality rate in people living with HIV/AIDS (PLWHA) has decreased significantly, resulting in an almost normal longevity in this population. However, a large portion of this population still endures a poor quality of life, mostly due to an increased inclination for substance abuse, including tobacco smoking. The prevalence of smoking in PLWHA is consistently higher than in HIV negative persons. A predisposition to cigarette smoking in the setting of HIV potentially leads to exacerbated HIV replication and a higher risk for developing neurocognitive and other CNS disorders. Oxidative stress and inflammation have been identified as mechanistic pathways in smoking-mediated HIV pathogenesis and HIV-associated neuropathogenesis. Extracellular vesicles (EVs), packaged with oxidative stress and inflammatory agents, show promise in understanding the underlying mechanisms of smoking-induced HIV pathogenesis via cell-cell interactions. This review focuses on recent advances in the field of EVs with an emphasis on smoking-mediated HIV pathogenesis and HIV-associated neuropathogenesis. This review also provides an overview of the potential applications of EVs in developing novel therapeutic carriers for the treatment of HIV-infected individuals who smoke, and in the discovery of novel biomarkers that are associated with HIV-smoking interactions in the CNS.

Proteomic Profiling of Exosomes Derived from Plasma of HIV-Infected Alcohol Drinkers and Cigarette Smokers.

Abuse of alcohol and tobacco could exacerbate HIV pathogenesis by transferring materials through exosomes (small nanovesicles). Exosomes present a stable and accessible source of information concerning the health and/or disease status of patients, which can provide diagnostic and prognostic biomarkers for myriad conditions. Therefore, we aimed to study the specific exosomal proteins that are altered in both HIV-infected subjects and alcohol/tobacco users. Exosomes were isolated from plasma of the following subjects: a) HIV-negative subjects (healthy), b) HIV-positive subjects (HIV), c) HIV-negative alcohol drinkers (drinkers), d) HIV-negative tobacco smokers (smokers), e) HIV-positive drinkers (HIV + drinkers), and f) HIV-positive smokers (HIV + smokers). Quantitative proteomic profiling was then performed from these exosomes. Sixteen proteins were significantly altered in the HIV group, ten in drinkers, four in HIV + drinkers, and fifteen in smokers compared to healthy subjects. Only one protein, fibulin-1 (FBLN1), was significantly altered in HIV + smokers. Interestingly, hemopexin was not significantly altered in drinkers or HIV patients but was significantly altered in HIV + drinkers. Further, our study is the first to show properdin expression in plasma exosomes, which was decreased in HIV + smokers and HIV + drinkers compared to HIV patients. The present findings suggest that hemopexin and properdin show potential as markers for physiological effects that may arise in HIV-infected individuals who abuse alcohol and tobacco. Graphical abstract This study presents a proteomic analysis of plasma-derived exosomes from HIV-infected alcohol drinkers and smokers. Among the proteins altered due to drug-abuse, hemopexin and properdin were of highest significance. These proteins can be potential biomarkers for co-morbid conditions associated with drug abuse in HIV-patients.

Circulating Extracellular Vesicles Containing Xenobiotic Metabolizing CYP Enzymes and Their Potential Roles in Extrahepatic Cells Via Cell-Cell Interactions.

The cytochrome P450 (CYP) family of enzymes is known to metabolize the majority of xenobiotics. Hepatocytes, powerhouses of CYP enzymes, are where most drugs are metabolized into non-toxic metabolites. Additional tissues/cells such as gut, kidneys, lungs, blood, and brain cells express selective CYP enzymes. Extrahepatic CYP enzymes, especially in kidneys, also metabolize drugs into excretable forms. However, extrahepatic cells express a much lower level of CYPs than hepatocytes. It is possible that the liver secretes CYP enzymes, which circulate via plasma and are eventually delivered to extrahepatic cells (e.g., brain cells). CYP circulation likely occurs via extracellular vesicles (EVs), which carry important biomolecules for delivery to distant cells. Recent studies have revealed an abundance of several CYPs in plasma EVs and other cell-derived EVs, and have demonstrated the role of CYP-containing EVs in xenobiotic-induced toxicity via cell-cell interactions. Thus, it is important to study the mechanism for packaging CYP into EVs, their circulation via plasma, and their role in extrahepatic cells. Future studies could help to find novel EV biomarkers and help to utilize EVs in novel interventions via CYP-containing EV drug delivery. This review mainly covers the abundance of CYPs in plasma EVs and EVs derived from CYP-expressing cells, as well as the potential role of EV CYPs in cell-cell communication and their application with respect to novel biomarkers and therapeutic interventions.

Extracellular Vesicles: A Possible Link between HIV and Alzheimer's Disease-Like Pathology in HIV Subjects?

The longevity of people with HIV/AIDS has been prolonged with the use of antiretroviral therapy (ART). The age-related complications, especially cognitive deficits, rise as HIV patients live longer. Deposition of beta-amyloid (Aß), a hallmark of Alzheimer's disease (AD), has been observed in subjects with HIV-associated neurocognitive disorders (HAND). Various mechanisms such as neuroinflammation induced by HIV proteins (e.g., Tat, gp120, Nef), excitotoxicity, oxidative stress, and the use of ART contribute to the deposition of Aß, leading to dementia. However, progressive dementia in older subjects with HIV might be due to HAND, AD, or both. Recently, extracellular vesicles (EVs)/exosomes, have gained recognition for their importance in understanding the pathology of both HAND and AD. EVs can serve as a possible link between HIV and AD, due to their ability to package and transport the toxic proteins implicated in both AD and HIV (Aß/tau and gp120/tat, respectively). Given that Aß is also elevated in neuron-derived exosomes isolated from the plasma of HIV patients, it is reasonable to suggest that neuron-to-neuron exosomal transport of Aß and tau also contributes to AD-like pathology in HIV-infected subjects. Therefore, exploring exosomal contents is likely to help distinguish HAND from AD. However, future prospective clinical studies need to be conducted to compare the exosomal contents in the plasma of HIV subjects with and without HAND as well as those with and without AD. This would help to find new markers and develop new treatment strategies to treat AD in HIV-positive subjects. This review presents comprehensive literatures on the mechanisms contributing to Aß deposition in HIV-infected cells, the role of EVs in the propagation of Aß in AD, the possible role of EVs in HIV-induced AD-like pathology, and finally, possible therapeutic targets or molecules to treat HIV subjects with AD.

Potential neuroprotective role of astroglial exosomes against smoking-induced oxidative stress and HIV-1 replication in the central nervous system.

INTRODUCTION: HIV-1-infected smokers are at risk of oxidative damage to neuronal cells in the central nervous system by both HIV-1 and cigarette smoke. Since neurons have a weak antioxidant defense system, they mostly depend on glial cells, particularly astrocytes, for protection against oxidative damage and neurotoxicity. Astrocytes augment the neuronal antioxidant system by supplying cysteine-containing products for glutathione synthesis, antioxidant enzymes such as SOD and catalase, glucose for antioxidant regeneration via the pentose-phosphate pathway, and by recycling of ascorbic acid. Areas covered: The transport of antioxidants and energy substrates from astrocytes to neurons could possibly occur via extracellular nanovesicles called exosomes. This review highlights the neuroprotective potential of exosomes derived from astrocytes against smoking-induced oxidative stress, HIV-1 replication, and subsequent neurotoxicity observed in HIV-1-positive smokers. Expert opinion: During stress conditions, the antioxidants released from astrocytes either via extracellular fluid or exosomes to neurons may not be sufficient to provide neuroprotection. Therefore, we put forward a novel strategy to combat oxidative stress in the central nervous system, using synthetically developed exosomes loaded with antioxidants such as glutathione and the anti-aging protein Klotho.

Specific packaging and circulation of cytochromes P450, especially 2E1 isozyme, in human plasma exosomes and their implications in cellular communications.

Cytochrome P450 (CYP) enzymes metabolize the majority of xenobiotics and are mainly found in hepatic and some extra-hepatic cells. However, their presence and functional role in exosomes, small extracellular vesicles that are secreted from various cells into extracellular fluids including plasma, is unknown. In this study, we analyzed the expression and biological activity of CYP enzymes in human plasma exosomes. First, we optimized isolation of plasma exosomes and characterized them for their physical properties and quality. The results showed that the purity of exosomes (<200 nm) improved upon prior filtration of plasma using a 0.22 micron filter. We then analyzed the relative level of exosomal CYP mRNAs, proteins, and enzyme activity. The results showed that the relative level of CYP enzymes in exosomes is higher than in plasma, suggesting their specific packaging in exosomes. Of the seven CYP enzymes tested, the mRNA of CYP1B1, CYP2A6, CYP2E1, and CYP3A4 were detectable in exosomes. Interestingly, the relative level of CYP2E1 mRNA was >500-fold higher than the other CYPs. The results from the Western blot showed detectable levels of CYP1A1, CYP1B1, CYP2A6, CYP2E1, and CYP3A4. Our results also demonstrated that exosomal CYP2E1 and CYP3A4 show appreciable activity relative to their respective positive controls (CYP-induced baculosomes). Our results also showed that CYP2E1 is expressed relatively higher in plasma exosomes than hepatic and monocytic cells and exosomes derived from these cells. In conclusion, this is the first evidence of the specific packaging and circulation of CYP enzymes, especially CYP2E1, in human plasma exosomes. The findings have biological and clinical significance in terms of their implications in cellular communications and potential use of plasma exosomal CYPs as biomarkers.