Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens.

Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by fusogenic proteins that form a complex between membranes to drive rearrangements needed for fusion. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens, but do not structurally or functionally resemble classical viral fusogens.

A novel operator-independent noninvasive device for assessing arterial reactivity.

Background: Endothelial dysfunction is associated with increased risk of cardiovascular disease (CVD). Currently available noninvasive methods of measuring endothelial function have limitations. We tested a novel device that provides an automated measurement of the difference between baseline and post-ischemic, hyperemia-induced, brachial arterial compliance, a phenomenon known to be endothelium-dependent.

An Assessment of Serological Assays for SARS-CoV-2 as Surrogates for Authentic Virus Neutralization.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and has since caused a global pandemic resulting in millions of cases and deaths. Diagnostic tools and serological assays are critical for controlling the outbreak, especially assays designed to quantitate neutralizing antibody levels, considered the best correlate of protection. As vaccines become increasingly available, it is important to identify reliable methods for measuring neutralizing antibody responses that correlate with authentic virus neutralization but can be performed outside biosafety level 3 (BSL3) laboratories.

Fatal Multisystem Inflammatory Syndrome in Adult after SARS-CoV-2 Natural Infection and COVID-19 Vaccination.

We describe a fatal case of multisystem inflammatory syndrome in an adult with onset 22 days after a second dose of mRNA coronavirus disease vaccine. Serologic and clinical findings indicated severe acute respiratory syndrome coronavirus 2 infection occurred before vaccination. The immunopathology of this syndrome, regardless of vaccination status, remains poorly understood.

Rapid and Flexible Platform To Assess Anti-SARS-CoV-2 Antibody Neutralization and Spike Protein-Specific Antivirals.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is ongoing and has shown the community that flexible methods for rapidly identifying and screening candidate antivirals are needed. Assessing virus-neutralizing activity of human serum to monitor population immunity and response to infection and vaccination is key to pandemic control. We developed a virus neutralization platform strategy that relies only on bioinformatic and genetic information of the virus of interest.

Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol.

The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus requires reliable assays for studying viral entry mechanisms which remains poorly understood. This knowledge is important for the development of therapeutic approaches to control SARS-CoV-2 infection by permitting the screening for neutralizing antibodies and other agents that can block infection. This is particularly important for patients who are at high risk for severe outcomes related to COVID-19.

Middle East respiratory coronavirus (MERS-CoV) spike (S) protein vesicular stomatitis virus pseudoparticle neutralization assays offer a reliable alternative to the conventional neutralization assay in human seroepidemiological studies.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel zoonotic coronavirus that was identified in 2012. MERS-CoV infection in humans can result in an acute, severe respiratory disease and in some cases multi-organ failure; the global mortality rate is approximately 35 %. The MERS-CoV spike (S) protein is a major target for neutralizing antibodies in infected patients.

Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity.

Evaluation of potential immunity against the novel severe acute respiratory syndrome (SARS) coronavirus that emerged in 2019 (SARS-CoV-2) is essential for health, as well as social and economic recovery. Generation of antibody response to SARS-CoV-2 (seroconversion) may inform on acquired immunity from prior exposure, and antibodies against the SARS-CoV-2 spike protein receptor binding domain (S-RBD) are speculated to neutralize virus infection. Some serology assays rely solely on SARS-CoV-2 nucleocapsid protein (N-protein) as the antibody detection antigen; however, whether such immune responses correlate with S-RBD response and COVID-19 immunity remains unknown.

Practicality and importance of selected endothelial dysfunction measurement techniques: review.

The measurement of endothelial dysfunction (ED) has importance in that it indicates the presence of coronary artery disease (Kuvin et al. in J Am Coll Cardiol 38(7):1843-1849, 2001) in addition to acting as a predictor of future adverse events (Halcox et al. in Circulation 106:653-658, 2002).

Rapid Sequencing of Multiple RNA Viruses in Their Native Form.

Long-read nanopore sequencing by a MinION device offers the unique possibility to directly sequence native RNA. We combined an enzymatic poly-A tailing reaction with the native RNA sequencing to (i) sequence complex population of single-stranded (ss)RNA viruses in parallel, (ii) detect genome, subgenomic mRNA/mRNA simultaneously, (iii) detect a complex transcriptomic architecture without the need for assembly, (iv) enable real-time detection. Using this protocol, positive-ssRNA, negative-ssRNA, with/without a poly(A)-tail, segmented/non-segmented genomes were mixed and sequenced in parallel.

Modulation of endoplasmic reticulum stress and the unfolded protein response in cancerous and noncancerous cells.

Objectives: The bio-field array is a device that generates a dielectrophoretic electromagnetic field when placed in a hypotonic saline solution and a direct current of approximately 3 A is applied. It is known that cell physiology is guided by bioelectrical properties, and there is a significant growth inhibition in cancerous (MDA-MB-231) cells that are grown in media that has been reconstituted with the saline that has been exposed to the bio-field array direct current dielectrophoretic electromagnetic field, alternatively there is no growth inhibition noted in noncancerous cells (MCF-10A) when grown in the bio-field array direct current dielectrophoretic electromagnetic field treated versus control media.

Methods: To examine the basis for selective growth inhibition in human breast carcinoma, we employed cell death assays, cell cycle assays, microarray analysis and reverse transcription-quantitative polymerase chain reaction.

Single-Vector, Single-Injection Recombinant Vesicular Stomatitis Virus Vaccines Against High-Containment Viruses.

There are many avenues for making an effective vaccine against viruses. Depending on the virus these can include one of the following: inactivation of whole virions; attenuation of viruses; recombinant viral proteins; non-replication-competent virus particles; or surrogate virus vector systems such as vesicular stomatitis virus (VSV). VSV is a prototypic enveloped animal virus that has been used for over four decades to study virus replication, entry, and assembly due to its ability to replicate to high titers in a wide variety of mammalian and insect cells.

Rab43 regulates the sorting of a subset of membrane protein cargo through the medial Golgi.

To evaluate the role of cytoplasmic domains of membrane-spanning proteins in directing trafficking through the secretory pathway, we generated fluorescently tagged VSV G tsO45 with either the native G tail (G) or a cytoplasmic tail derived from the chicken AE1-4 anion exchanger (G(AE)). We previously showed that these two proteins progressed through the Golgi with distinct kinetics. To investigate the basis for the differential sorting of G and G(AE), we analyzed the role of several Golgi-associated small GTP-binding proteins and found that Rab43 differentially regulated their transport through the Golgi.

Inhibition of melanocortin 1 receptor slows melanoma growth, reduces tumor heterogeneity and increases survival.

Melanoma risk is increased in patients with mutations of melanocortin 1 receptor (MC1R) yet the basis for the increased risk remains unknown. Here we report in vivo evidence supporting a critical role for MC1R in regulating melanoma tumor growth and determining overall survival time. Inhibition of MC1R by its physiologically relevant competitive inhibitor, agouti signaling protein (ASIP), reduced melanin synthesis and morphological heterogeneity in murine B16-F10 melanoma cells.

Bioelectrodynamics: A New Patient Care Strategy for Nursing, Health, and Wellness.

Bioelectrodynamics is an interdisciplinary subject that offers a pathway for nursing to develop a new patient care strategy in health care. The application of bioenergy to living organisms has the potential to advance medical science in the areas of prevention, cancer, wound care, pain, and many other chronic diseases.

Kinetically distinct sorting pathways through the Golgi exhibit different requirements for Arf1.

To investigate the role of cytoplasmic sequences in directing transmembrane protein trafficking through the Golgi, we analyzed the sorting of VSV tsO45 G fusions with either the native G cytoplasmic domain (G) or an alternative cytoplasmic tail derived from the chicken AE1-4 anion exchanger (G(AE) ). At restrictive temperature G(AE) and G accumulated in the ER, and upon shifting the cells to permissive temperature both proteins folded and underwent transport through the Golgi. However, G(AE) and G did not form hetero-oligomers upon the shift to permissive temperature and they progressed through the Golgi with distinct kinetics.

Single injection recombinant vesicular stomatitis virus vaccines protect ferrets against lethal Nipah virus disease.

Background: Nipah virus (NiV) is a highly pathogenic zoonotic agent in the family Paramyxoviridae that is maintained in nature by bats. Outbreaks have occurred in Malaysia, Singapore, India, and Bangladesh and have been associated with 40 to 75% case fatality rates. There are currently no vaccines or postexposure treatments licensed for combating human NiV infection.

Inhibition of type I interferon-mediated antiviral action in human glioma cells by the IKK inhibitors BMS-345541 and TPCA-1.

The nuclear factor-kappa B (NFκB) signal transduction pathway plays an important role in immunity, inflammation, cell growth, and survival. Since dysregulation of this pathway results in high, constitutive NFκB activation in various cancers and immune disorders, the development of specific drugs to target this pathway has become a focus for treating these diseases. NFκB regulates various aspects of the cellular response to interferon (IFN).

Rapid screening for entry inhibitors of highly pathogenic viruses under low-level biocontainment.

Emerging viruses including Nipah, Hendra, Lujo, and Junin viruses have enormous potential to spread rapidly. Nipah virus, after emerging as a zoonosis, has also evolved the capacity for human-to-human transmission. Most of the diseases caused by these pathogens are untreatable and require high biocontainment conditions.

Use of replication restricted recombinant vesicular stomatitis virus vectors for detection of antigen-specific T cells.

Detection of antigen-specific T cells at the single-cell level by ELISpot or flow cytometry techniques employing intracellular cytokine staining (ICS) is now an indispensable tool in many areas of immunology. When precisely mapped, optimal MHC-binding peptide epitopes are unknown, these assays use antigen in a variety of forms, including recombinant proteins, overlapping peptide sets representing one or more target protein sequences, microbial lysates, lysates of microbially-infected cells, or gene delivery vectors such as DNA expression plasmids or recombinant vaccinia or adenoviruses expressing a target protein of interest. Here we introduce replication-restricted, recombinant vesicular stomatitis virus (VSV) vectors as a safe, easy to produce, simple to use, and highly effective vector for genetic antigen delivery for the detection of human antigen-specific helper and cytotoxic T cells.

Utilization of fluorescently-labeled tetracysteine-tagged proteins to study virus entry by live cell microscopy.

Viruses exploit cellular machinery to gain entry and initiate their replication cycle within host cells. The development of methods to visualize virus entry in live cells has provided new insights to the cellular processes involved in virus entry and the intracellular locations where viral payloads are deposited. The use of fluorescently labeled virus and high-resolution microscopy is currently the method of choice to study virus entry in live cells.

The protease-sensitive loop of the vesicular stomatitis virus matrix protein is involved in virus assembly and protein translation.

To study the contribution of the protease-sensitive loop of the VSV M protein in virus assembly we recovered recombinant VSV (rVSV) with mutations in this region and examined virus replication. Mutations in the highly conserved LXD motif (aa 123-125) resulted in reduced virion budding, reduced virus titers and enhanced M protein exchange with M-ribonucleocapsid complexes (M-RNPs), suggesting that the mutant M proteins were less tightly associated with RNP skeletons. In addition, viral protein synthesis began to decrease at 4h post-infection (hpi) and was reduced by ~80% at 8 hpi for the mutant rVSV-D125A.