Frontiers in microbiology, 2015 Extracellular vesicles from infected cells: potential for direct pathogenesis Infections that result in natural or manmade spread of lethal biological agents are a concern and require national and focused preparedness. In this manuscript, as part of an early diagnostics and pathogen treatment strategy, we have focused on extracellular vesicles (EVs) that arise following infections. Although the field of biodefense does not currently have a rich resource in EVs literature, none the less, similar pathogens belonging to the more classical emerging and non-emerging diseases have been studied in their EV/exosomal contents and function. These exosomes are formed in late endosomes and released from the cell membrane in almost every cell type in vivo. These vesicles contain proteins, RNA, and lipids from the cells they originate from and function in development, signal transduction, cell survival, and transfer of infectious material. The current review focuses on how different forms of infection exploit the exosomal pathway and how exosomes can be exploited artificially to treat infection and disease and potentially also be used as a source of vaccine. Virally-infected cells can secrete viral as well as cellular proteins and RNA in exosomes, allowing viruses to cause latent infection and spread of miRNA to nearby cells prior to a subsequent infection. In addition to virally-infected host cells, bacteria, protozoa, and fungi can all release small vesicles that contain pathogen-associated molecular patterns, regulating the neighboring uninfected cells. Examples of exosomes from both virally and bacterially infected cells point toward a re-programming network of pathways in the recipient cells. Finally, many of these exosomes contain cytokines and miRNAs that in turn can effect gene expression in the recipient cells through the classical toll-like receptor and NFκB pathway. Therefore, although exosomes do not replicate as an independent entity, they however facilitate movement of infectious material through tissues and may be the cause of many pathologies seen in infected hosts.

POSTER Neurology, 2015 Detection of Human T-cell Lymphotropic Virus Type I proteins in exosomes from HAM/TSP patient CSF by novel Nanotrap® technology OBJECTIVE: To assess the presence of viral proteins in the CSF of patients with HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP) BACKGROUND: There has been increasing evidence for the role of microvesicles (MV) in CNS inflammation and viral disease. HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neuroinflammatory disease that affects a subset of virus-infected infected individuals. This disorder is immunopathologically mediated since virus -specific immune cells can be found in both the CSF and in the CNS of patients although HTLV-I virus has been difficult to isolate in CNS resident cells. The possibility of transfer of viral proteins via MV from viral reservoirs to uninfected cells in the absence of virus is an intriguing mechanism by which this can occur. Recently, HTLV-1 tax proteins have been shown in exosomes (EX) from HTLV-1 infected cell lines (Jaworksi et al 2014). Therefore, we examined if similar MV were present in the CNS of HAM/TSP patients. DESIGN/METHODS: HTLV-1 infected and uninfected cell line supernatants were used for isolation of EX and MV by a novel Nanotrap® (NT) technology. These NTs are hydrogel particles consisting of high affinity aromatic baits surrounded by a sieving shell. HTLV-I viral proteins were assessed by Western blot analysis. HTLV-I virions were detected by RT-qPCR. In addition, EXs were isolated using this EX-NT from CSF of HAM/TSP patients and MS patients as controls. RESULTS: Exosomes were successfully isolated specifically from HTLV-1 infected cell lines that contained HTLV-I tax protein but lacked HTLV-I virions. Moreover, CSF from HAM/TSP patients and not from MS, also demonstrated exosomes that were HTLV-I tax Western blot positive. CONCLUSIONS: These results suggest the possibility that HTLV-I protein present in virus-free CSF can be a potential source of antigen in an inflammatory neuropathological disease.

PLOS Neglected Tropical Diseases, 2013 Use of a Novel Chagas Urine Nanoparticle Test for Diagnosis of Congenital Chagas Disease Detection of congenital T. cruzi transmission is considered one of the pillars of control programs of Chagas disease. Congenital transmission accounts for 25% of new infections with an estimated 15,000 infected infants per year. Current programs to detect congenital Chagas disease in Latin America utilize microscopy early in life and serology after 6 months. These programs suffer from low sensitivity by microscopy and high loss to follow-up later in infancy. We developed a Chagas urine nanoparticle test (Chunap) to concentrate, preserve and detect T. cruzi antigens in urine for early, non-invasive diagnosis of congenital Chagas disease.

PLOS Neglected Tropical Diseases, July 2014 The Use of NanoTrap® Particles as a Sample Enrichment Method to Enhance the Detection of Rift Valley Fever Virus Rift Valley Fever Virus (RVFV) is a zoonotic virus that is not only an emerging pathogen but is also considered a biodefense pathogen due to the threat it may cause to public health and national security. The current state of diagnosis has led to misdiagnosis early on in infection. Here we describe the use of a novel sample preparation technology, NanoTrap® particles, to enhance the detection of RVFV. Previous studies demonstrated that NanoTrap® particles lead to both 100 percent capture of protein analytes as well as an improvement of more than 100-fold in sensitivity compared to existing methods. Here we extend these findings by demonstrating the capture and enrichment of viruses.

Pathogens and Diesease 2014 The use of Nanotrap® particles for emerging and infectious disease diagnostics Detection of early infectious disease may be challenging due to the low copy number of organisms present. To overcome this limitation and rapidly measure low concentrations of the pathogen, we developed a novel technology: Nanotrap® particles, which are designed to capture, concentrate, and protect biomarkers from complex biofluids. Nanotrap® particles are thermoresponsive hydrogels that are capable of antigen capture through the coupling of affinity baits to the particles. Here, we describe recent findings demonstrating that Nanotrap® particles are able to capture live infectious virus, viral RNA, and viral proteins. Capture is possible even in complex mixtures such as serum and allows the concentration and protection of these analytes, providing increased performance of downstream assays. The Nanotrap® particles are a versatile sample preparation technology that has far reaching implications for biomarker discovery and diagnostic assays.

PLoS ONE 2014 The Use of Nanotrap® Particles Technology in Capturing HIV-1 Virions and Viral Proteins from Infected Cells HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap® technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap® particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap® particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB). Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients' serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap® particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a platform to enhance HIV-1 detection by concentrating viral proteins and infectious virions from infected samples.

Journal of Virology, 2013 Novel Neuroprotective GSK-3β Inhibitor Restricts Tat-Mediated HIV-1 Replication ​ The implementation of new antiretroviral therapies targeting transcription of early viral proteins in postintegrated HIV-1 can aid in overcoming current therapy limitations. Using high-throughput screening assays, we have previously described a novel Tat-dependent HIV-1 transcriptional inhibitor named 6-bromoindirubin-3′-oxime (6BIO). The screening of 6BIO derivatives yielded unique compounds that show potent inhibition of HIV-1 transcription. We have identified a second-generation derivative called 18BIOder as an inhibitor of HIV-1 Tat-dependent transcription in TZM-bl cells and a potent inhibitor of GSK-3β kinase in vitro. Structurally, 18BIOder is half the molecular weight and structure of its parental compound, 6BIO. More importantly, we also have found a different GSK-3β complex present only in HIV-1-infected cells. 18BIOder preferentially inhibits this novel kinase complex from infected cells at nanomolar concentrations. Finally, we observed that neuronal cultures treated with Tat protein are protected from Tat-mediated cytotoxicity when treated with 18BIOder. Overall, our data suggest that HIV-1 Tat-dependent transcription is sensitive to small-molecule inhibition of GSK-3β.

PLOS ONE, 2015 Use of Nanotrap® particles in the enhanced detection of rift valley fever virus nucleoprotein Rift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus that has a detrimental effect on both livestock and human populations. While there are several diagnostic methodologies available for RVFV detection, many are not sensitive enough to diagnose early infections. Furthermore, detection may be hindered by high abundant proteins such as albumin. Previous findings have shown that Nanotrap particles can be used to significantly enhance detection of various small analytes of low abundance. We have expanded upon this repertoire to show that this simple and efficient sample preparation technology can drastically improve the detection of the RVFV nucleoprotein (NP), the most abundant and widely used viral protein for RVFV diagnostics.

Journal of Molecular Biology, 2013 Effect of Mimetic CDK9 Inhibitors on HIV-1-Activated Transcription Potent anti-retroviral therapy has transformed HIV-1 infection into a chronic manageable disease; however, drug resistance remains a common problem that limits the effectiveness and clinical benefits of this type of treatment. The discovery of viral reservoirs in the body, in which HIV-1 may persist, has helped to explain why therapeutic eradication of HIV-1 has proved so difficult. In the current study, we utilized a combination of structure-based analysis of cyclin/CDK complexes with our previously published Tat peptide derivatives. We modeled the Tat peptide inhibitors with CDKs and found a particular pocket that showed the most stable binding site (Cavity 1) using in silico analysis. Furthermore, we were able to find peptide mimetics that bound to similar regions using in silico searches of a chemical library, followed by cell-based biological assays. Using these methods, we obtained the first-generation mimetic drugs and tested these compounds on HIV-1 long terminal repeat-activated transcription. Using biological assays followed by similar in silico analysis to find second-generation drugs resembling the original mimetic, we found the new targets of Cavity 1 and Cavity 2 regions on CDK9. We examined the second-generation mimetic against various viral isolates and observed a generalized suppression of most HIV-1 isolates. Finally, the drug inhibited viral replication in humanized mouse models of Rag2−/−γc−/− with no toxicity to the animals at tested concentrations. Our results suggest that it may be possible to model peptide inhibitors into available crystal structures and further find drug mimetics using in silico analysis.

Analytica Chimica Acta, 2012 Comparison of three different enrichment strategies for serum low molecular weight protein identification using shotgun proteomics approach Serum low-molecular weight (LMW) proteins potentially contain useful biological information and their identification can be used to discover novel potential biomarkers. Given the high complexity of serum samples, in the last years several different prefractionation and enrichment strategies have been developed. In this study three different methods, i.e. hydrogel nanoparticles, Proteominer(®) peptide ligand affinity beads and Sartorius Vivaspin(®) centrifugal ultrafiltration device, were compared and evaluated in order to select the best strategy for the enrichment and prefractionation of LMW proteins. A shotgun proteomics approach was adopted, with in-solution proteolytic digestion of the whole protein mixture and determination of the resulting peptides by nanoHPLC coupled with a high-resolution Orbitrap LTQ-XL mass spectrometer. Data analysis, focusing on the LMW proteome (MW ≤ 40 kDa), has shown that the hydrogel nanoparticles performed better in enriching the LMW protein profiles, with 115 proteins identified against 93 and 95 for Proteominer(®) beads and Sartorius Vivaspin(®) device, respectively.

Journal of Proteome Research, May 29, 2012 Over the past few years, mass spectrometry has emerged as a technology to complement and potentially replace standard immunoassays in routine clinical core laboratories. Application of mass spectrometry to protein and peptide measurement can provide advantages including high sensitivity, the ability to multiplex analytes, and high specificity at the amino acid sequence level. In our previous study, we demonstrated excellent reproducibility of mass spectrometry-selective reaction monitoring (MS-SRM) assays when applying standardized standard operating procedures (SOPs) to measure synthetic peptides in a complex sample, as lack of reproducibility has been a frequent criticism leveled at the use of mass spectrometers in the clinical laboratory compared to immunoassays. Furthermore, an important caveat of SRM-based assays for proteins is that many low-abundance analytes require some type of enrichment before detection with MS. This adds a level of complexity to the procedure and the potential for irreproducibility increases, especially across different laboratories with different operators. The purpose of this study was to test the interlaboratory reproducibility of SRM assays with various upfront enrichment strategies and different types of clinical samples (representing real-world body fluids commonly encountered in routine clinical laboratories). Three different, previously published enrichment strategies for low-abundance analytes and a no-enrichment strategy for high-abundance analytes were tested across four different laboratories using different liquid chromatography-SRM (LC-SRM) platforms and previously developed SOPs. The results demonstrated that these assays were indeed reproducible with coefficients of variation of less than 30% for the measurement of important clinical proteins across all four laboratories in real world samples.

Journal of The American Chemical Society, 2011 Multifunctional Core-Shell Nanoparticles: Discovery of Previously Invisible Biomarkers Many low-abundance biomarkers for early detection of cancer and other diseases are invisible to mass spectrometry because they exist in body fluids in very low concentrations, are masked by high-abundance proteins such as albumin and immunoglobulins, and are very labile. To overcome these barriers, we created porous, buoyant, core–shell hydrogel nanoparticles containing novel high affinity reactive chemical baits for protein and peptide harvesting, concentration, and preservation in body fluids. Poly(N-isopropylacrylamide-co-acrylic acid) nanoparticles were functionalized with amino-containing dyes via zero-length cross-linking amidation reactions. Nanoparticles functionalized in the core with 17 different (12 chemically novel) molecular baits showed preferential high affinities (KD < 10–11 M) for specific low-abundance protein analytes. A poly(N-isopropylacrylamide-co-vinylsulfonic acid) shell was added to the core particles. This shell chemistry selectively prevented unwanted entry of all size peptides derived from albumin without hindering the penetration of non-albumin small proteins and peptides. Proteins and peptides entered the core to be captured with high affinity by baits immobilized in the core. Nanoparticles effectively protected interleukin-6 from enzymatic degradation in sweat and increased the effective detection sensitivity of human growth hormone in human urine using multiple reaction monitoring analysis. Used in whole blood as a one-step, in-solution preprocessing step, the nanoparticles greatly enriched the concentration of low-molecular weight proteins and peptides while excluding albumin and other proteins above 30 kDa; this achieved a 10,000-fold effective amplification of the analyte concentration, enabling mass spectrometry (MS) discovery of candidate biomarkers that were previously undetectable.