Extracellular Vesicles from Infected Cells Are Released Prior to Virion Release

Here, we have attempted to address the timing of EV and virion release from virally infected cells. Uninfected (CEM), HIV-1-infected (J1.1), and human T cell leukemia virus-1 (HTLV-1)-infected (HUT102) cells were synchronized in G0. Viral latency was reversed by increasing gene expression with the addition of serum-rich media and inducers. Supernatants and cell pellets were collected post-induction at different timepoints and assayed for extracellular vesicle (EV) and autophagy markers; and for viral proteins and RNAs. Tetraspanins and autophagy-related proteins were found to be differentially secreted in HIV-1- and HTLV-1-infected cells when compared with uninfected controls. HIV-1 proteins were present at 6 h and their production increased up to 24 h. HTLV-1 proteins peaked at 6 h and plateaued. HIV-1 and HTLV-1 RNA production correlated with viral protein expression. Nanoparticle tracking analysis (NTA) showed increase of EV concentration over time in both uninfected and infected samples. Finally, the HIV-1 supernatant from the 6-h samples was found not to be infectious; however, the virus from the 24-h samples was successfully rescued and infectious. Overall, our data indicate that EV release may occur prior to viral release from infected cells, thereby implicating a potentially significant effect of EVs on uninfected recipient cells prior to subsequent viral infection and spread

Extracellular Vesicle Activation of Latent HIV-1 Is Driven by EV-Associated c-Src and Cellular SRC-1 via the PI3K/AKT/mTOR Pathway

HIV-1 is a global health crisis that has infected more than 37 million people. Latent reservoirs throughout the body are a major hurdle when it comes to eradicating the virus. In our previous study, we found that exosomes, a type of extracellular vesicle (EV), from uninfected cells activate the transcription of HIV-1 in latent infected cells, regardless of combination antiretroviral therapy (cART). In this study, we investigated the specific mechanism behind the EV activation of latent HIV-1. We found that phosphorylated c-Src is present in EVs of various cell lines and has the ability to activate downstream proteins such as EGFR, initiating a signal cascade. EGFR is then able to activate the PI3K/AKT/mTOR pathway, resulting in the activation of STAT3 and SRC-1, culminating in the reversal of HIV-1 latency. This was verified by examining levels of HIV-1 TAR, genomic RNA and HIV-1 Gag p24 protein in cell lines and primary cells. We found that EVs containing c-Src rescued HIV-1 despite the presence of inhibitors, validating the importance of EV-associated c-Src in latent HIV-1 activation. Lastly, we discovered an increased recruitment of p300 and NF-κB in the nucleus of EV-treated infected cells. Collectively, our data suggest that EV-associated c-Src is able to activate latent HIV-1 via the PI3K/AKT/mTOR pathway and SRC-1/p300-driven chromatin remodeling. These findings could aid in designing new strategies to prevent the reactivation of latent HIV-1 in patients under cART

Extracellular Vesicles in HTLV-1 Communication: The Story of an Invisible Messenger

Human T-cell lymphotropic virus type 1 (HTLV-1) infects 5–10 million people worldwide and is the causative agent of adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) as well as other inflammatory diseases. A major concern is that the most majority of individuals with HTLV-1 are asymptomatic carriers and that there is limited global attention by health care officials, setting up potential conditions for increased viral spread. HTLV-1 transmission occurs primarily through sexual intercourse, blood transfusion, intravenous drug usage, and breast feeding. Currently, there is no cure for HTLV-1 infection and only limited treatment options exist, such as class I interferons (IFN) and Zidovudine (AZT), with poor prognosis. Recently, small membrane-bound structures, known as extracellular vesicles (EVs), have received increased attention due to their potential to carry viral cargo (RNA and proteins) in multiple pathogenic infections (i.e., human immunodeficiency virus type I (HIV-1), Zika virus, and HTLV-1). In the case of HTLV-1, EVs isolated from the peripheral blood and cerebral spinal fluid (CSF) of HAM/TSP patients contained the viral transactivator protein Tax. Additionally, EVs derived from HTLV-1-infected cells (HTLV-1 EVs) promote functional effects such as cell aggregation which enhance viral spread. In this review, we present current knowledge surrounding EVs and their potential role as immune-modulating agents in cancer and other infectious diseases such as HTLV-1 and HIV-1. We discuss various features of EVs that make them prime targets for possible vehicles of future diagnostics and therapies

Design of a Secure Wireless Home Automation System with an Open Home Automation Bus (OpenHAB 2) Framework

There is rapid interest growing in the use of smart, connected devices. The developing world market for smart technology is evolving to adopt and adapt to the interconnected world of devices leading to the Internet of Things (IoT) everywhere. This research paper presents the design, development, and deployment of a prototype for the secure wireless home automation system with OpenHAB 2. We employed the use of two (2) high-performance microcontrollers, namely, the Arduino Mega 2560, interfaced with a 16-channel relay, and Raspberry Pi Model B, running the OpenHAB software. The Raspberry Pi functioned as the server to develop a prototype of an automated smart home that is remotely controllable from both a web application and an Android mobile app. In designing a wireless controlled switch for home appliances, two security procedures were implemented, namely, the token-based JSON Web Token (JWT) interface and Advanced Encryption Standard (AES) procedures for authentication and data encryption. Our system delivered a home automation system that leverages on the power of the latest version of OpenHAB to maximize productivity and overall home security while making it adaptable to the management of individual devices. When tested, both the developed hardware and software modules performed extremely well to meet the goal of a secured home automation system. Industry-standard penetration testing tools and frameworks, including Aircrack-ng, were utilized; wireless network audit began with a full sweep of the wireless frequencies with excellent results. It also ensures the efficient use of energy in the home as devices are intelligently controlled from both mobile and web applications. The results of the design and implementation of the additional layer for the security of the OpenHAB framework provide various theoretical and practical implications for home automation

Total Serum Magnesium Levels and Calcium-To-Magnesium Ratio in Sickle Cell Disease

Background and Objectives: Imbalance of calcium/magnesium ratio could lead to clinical complications in sickle cell disease (SCD). Low levels of magnesium have been associated with sickling, increased polymerization and vaso-occlusion (VOC) in sickle cell due to cell dehydration. The K-Cl cotransport plays a very important role in sickle cell dehydration and is inhibited by significantly increasing levels of magnesium. The study evaluated total serum magnesium levels and computed calcium/magnesium ratio in SCD patients and “healthy” controls. Materials and Methods: The study was a case-control cross-sectional one, involving 120 SCD patients (79 Haemoglobin SS (HbSS)and 41 Haemoglobin SC (HbSC)) at the steady state and 48 “healthy” controls. Sera were prepared from whole blood samples (n = 168) and total magnesium and calcium measured using a Flame Atomic Absorption Spectrometer (Variant 240FS manufactured by VARIAN Australia Pty Ltd., Melbourne, VIC, Australia). Calcium/magnesium ratios were calculated in patients and the controls. Results: The prevalence of hypomagnesemia and hypocalcaemia among the SCD patients was observed to be 39.17% and 52.50% respectively, higher than the controls (4.17% and 22.92%, for hypomagnesemia and hypocalcaemia, respectively). Level of magnesium was significantly lower in the SCD patients compared to their healthy counterparts (p = 0.002). The magnesium level was further reduced in the HbSS patients but not significantly different from the HbSC patients (p = 0.584). calcium/magnesium ratio was significantly higher in the SCD patients (p = 0.031). Although calcium/magnesium ratio was higher in the HbSC patients compared to those with the HbSS genotype, the difference was not significant (p = 0.101). Conclusion: The study shows that magnesium homeostasis are altered in SCD patients, and their levels are lower in HbSS patients. Although calcium/magnesium ratio is significantly higher in SCD patients compared with controls, there is no significant difference between patients with HbSS and HbSC genotypes. Magnesium supplementation may be required in sickle cell patients

Total Serum Magnesium Levels and Calcium-To-Magnesium Ratio in Sickle Cell Disease

Background and objectives: Imbalance of calcium/magnesium ratio could lead to clinical complications in sickle cell disease (SCD). Low levels of magnesium have been associated with sickling, increased polymerization and vaso-occlusion (VOC) in sickle cell due to cell dehydration. The K-Cl cotransport plays a very important role in sickle cell dehydration and is inhibited by significantly increasing levels of magnesium. The study evaluated total serum magnesium levels and computed calcium/magnesium ratio in SCD patients and "healthy" controls. Materials and methods: The study was a case-control cross-sectional one, involving 120 SCD patients (79 Haemoglobin SS (HbSS)and 41 Haemoglobin SC (HbSC)) at the steady state and 48 "healthy" controls. Sera were prepared from whole blood samples (n = 168) and total magnesium and calcium measured using a Flame Atomic Absorption Spectrometer (Variant 240FS manufactured by VARIAN Australia Pty Ltd., Melbourne, VIC, Australia). Calcium/magnesium ratios were calculated in patients and the controls. Results: The prevalence of hypomagnesemia and hypocalcaemia among the SCD patients was observed to be 39.17% and 52.50% respectively, higher than the controls (4.17% and 22.92%, for hypomagnesemia and hypocalcaemia, respectively). Level of magnesium was significantly lower in the SCD patients compared to their healthy counterparts (p = 0.002). The magnesium level was further reduced in the HbSS patients but not significantly different from the HbSC patients (p = 0.584). calcium/magnesium ratio was significantly higher in the SCD patients (p = 0.031). Although calcium/magnesium ratio was higher in the HbSC patients compared to those with the HbSS genotype, the difference was not significant (p = 0.101). Conclusion: The study shows that magnesium homeostasis are altered in SCD patients, and their levels are lower in HbSS patients. Although calcium/magnesium ratio is significantly higher in SCD patients compared with controls, there is no significant difference between patients with HbSS and HbSC genotypes. Magnesium supplementation may be required in sickle cell patients

An Omics Approach to Extracellular Vesicles from HIV-1 Infected Cells

Human Immunodeficiency Virus-1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS), infecting nearly 37 million people worldwide. Currently, there is no definitive cure, mainly due to HIV-1′s ability to enact latency. Our previous work has shown that exosomes, a small extracellular vesicle, from uninfected cells can activate HIV-1 in latent cells, leading to increased mostly short and some long HIV-1 RNA transcripts. This is consistent with the notion that none of the FDA-approved antiretroviral drugs used today in the clinic are transcription inhibitors. Furthermore, these HIV-1 transcripts can be packaged into exosomes and released from the infected cell. Here, we examined the differences in protein and nucleic acid content between exosomes from uninfected and HIV-1-infected cells. We found increased cyclin-dependent kinases, among other kinases, in exosomes from infected T-cells while other kinases were present in exosomes from infected monocytes. Additionally, we found a series of short antisense HIV-1 RNA from the 3′ LTR that appears heavily mutated in exosomes from HIV-1-infected cells along with the presence of cellular noncoding RNAs and cellular miRNAs. Both physical and functional validations were performed on some of the key findings. Collectively, our data indicate distinct differences in protein and RNA content between exosomes from uninfected and HIV-1-infected cells, which can lead to different functional outcomes in recipient cells