Age-dependent Powassan virus lethality is linked to glial cell activation and divergent neuroinflammatory cytokine responses in a murine model.

Powassan virus (POWV) is an emergent tick-borne flavivirus that causes fatal encephalitis in the elderly and long-term neurologic sequelae in survivors. How age contributes to severe POWV encephalitis remains an enigma, and no animal models have assessed age-dependent POWV neuropathology. Inoculating C57BL/6 mice with a POWV strain (LI9) currently circulating in Ixodes ticks resulted in age-dependent POWV lethality 10-20 dpi. POWV infection of 50-week-old mice was 82% fatal with lethality sequentially reduced by age to 7.1% in 10-week-old mice. POWV LI9 was neuroinvasive in mice of all ages, causing acute spongiform CNS pathology and reactive gliosis 5-15 dpi that persisted in survivors 30 dpi. High CNS viral loads were found in all mice 10 dpi. However, by 15 dpi, viral loads decreased by 2-4 logs in 10- to 40-week-old mice, while remaining at high levels in 50-week-old mice. Age-dependent differences in CNS viral loads 15 dpi occurred concomitantly with striking changes in CNS cytokine responses. In the CNS of 50-week-old mice, POWV induced Th1-type cytokines (IFNγ, IL-2, IL-12, IL-4, TNFα, IL-6), suggesting a neurodegenerative pro-inflammatory M1 microglial program. By contrast, in 10-week-old mice, POWV-induced Th2-type cytokines (IL-10, TGFß, IL-4) were consistent with a neuroprotective M2 microglial phenotype. These findings correlate age-dependent CNS cytokine responses and viral loads with POWV lethality and suggest potential neuroinflammatory therapeutic targets. Our results establish the age-dependent lethality of POWV in a murine model that mirrors human POWV severity and long-term CNS pathology in the elderly. IMPORTANCE: Powassan virus is an emerging tick-borne flavivirus causing lethal encephalitis in aged individuals. We reveal an age-dependent POWV murine model that mirrors human POWV encephalitis and long-term CNS damage in the elderly. We found that POWV is neuroinvasive and directs reactive gliosis in all age mice, but at acute stages selectively induces pro-inflammatory Th1 cytokine responses in 50-week-old mice and neuroprotective Th2 cytokine responses in 10-week-old mice. Our findings associate CNS viral loads and divergent cytokine responses with age-dependent POWV lethality and survival outcomes. Responses of young mice suggest potential therapeutic targets and approaches for preventing severe POWV encephalitis that may be broadly applicable to other neurodegenerative diseases. Our age-dependent murine POWV model permits analysis of vaccines that prevent POWV lethality, and therapeutics that resolve severe POWV encephalitis.

Optimizing Antimicrobial Peptide Design: Integration of Cell-Penetrating Peptides, Amyloidogenic Fragments, and Amino Acid Residue Modifications.

The escalating threat of multidrug-resistant pathogens necessitates innovative approaches to combat infectious diseases. In this study, we examined peptides R23FS*, V31KS*, and R44KS*, which were engineered to include an amyloidogenic fragment sourced from the S1 protein of S. aureus, along with one or two cell-penetrating peptide (CPP) components. We assessed the antimicrobial efficacy of these peptides in a liquid medium against various strains of both Gram-positive bacteria, including S. aureus (209P and 129B strains), MRSA (SA 180 and ATCC 43300 strains), and B. cereus (strain IP 5832), and Gram-negative bacteria such as P. aeruginosa (ATCC 28753 and 2943 strains) and E. coli (MG1655 and K12 strains). Peptides R23FS*, V31KS*, and R44KS* exhibited antimicrobial activity comparable to gentamicin and meropenem against all tested bacteria at concentrations ranging from 24 to 48 µM. The peptides showed a stronger antimicrobial effect against B. cereus. Notably, peptide R44KS* displayed high efficacy compared to peptides R23FS* and V31KS*, particularly evident at lower concentrations, resulting in significant inhibition of bacterial growth. Furthermore, modified peptides V31KS* and R44KS* demonstrated enhanced inhibitory effects on bacterial growth across different strains compared to their unmodified counterparts V31KS and R44KS. These results highlight the potential of integrating cell-penetrating peptides, amyloidogenic fragments, and amino acid residue modifications to advance the innovation in the field of antimicrobial peptides, thereby increasing their effectiveness against a broad spectrum of pathogens.

Powassan Viruses Spread Cell to Cell during Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes.

Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the northeastern United States, with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted within as little as 15 min of a tick bite and enter the central nervous system (CNS) to cause encephalitis (10% of cases are fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV-infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of cell to cell spread, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9 and -LI41 and lineage I POWV-LB productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower-chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-ß (IFN-ß) and proteins of the IFN-stimulated gene family (ISGs), with delayed IFN-ß secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion, a subset of POWV-infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes, and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments. IMPORTANCE We isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells, and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculation of VeroE6 cells with POWV-containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell-to-cell spread. POWV-LI9 and -LI41 and lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower-chamber pericytes, suggesting a mechanism for POWV transmission across the blood-brain barrier (BBB). POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.

Enhancing the Antimicrobial Properties of Peptides through Cell-Penetrating Peptide Conjugation: A Comprehensive Assessment.

Combining antimicrobial peptides (AMPs) with cell-penetrating peptides (CPPs) has shown promise in boosting antimicrobial potency, especially against Gram-negative bacteria. We examined the CPP-AMP interaction with distinct bacterial types based on cell wall differences. Our investigation focused on AMPs incorporating penetratin CPP and dihybrid peptides containing both cell-penetrating TAT protein fragments from the human immunodeficiency virus and Antennapedia peptide (Antp). Assessment of the peptides TAT-AMP, AMP-Antp, and TAT-AMP-Antp revealed their potential against Gram-positive strains (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), and Bacillus cereus). Peptides TAT-AMP and AMP-Antp using an amyloidogenic AMP from S1 ribosomal protein Thermus thermophilus, at concentrations ranging from 3 to 12 µM, exhibited enhanced antimicrobial activity against B. cereus. TAT-AMP and TAT-AMP-Antp, using an amyloidogenic AMP from the S1 ribosomal protein Pseudomonas aeruginosa, at a concentration of 12 µM, demonstrated potent antimicrobial activity against S. aureus and MRSA. Notably, the TAT-AMP, at a concentration of 12 µM, effectively inhibited Escherichia coli (E. coli) growth and displayed antimicrobial effects similar to gentamicin after 15 h of incubation. Peptide characteristics determined antimicrobial activity against diverse strains. The study highlights the intricate relationship between peptide properties and antimicrobial potential. Mechanisms of AMP action are closely tied to bacterial cell wall attributes. Peptides with the TAT fragment exhibited enhanced antimicrobial activity against S. aureus, MRSA, and P. aeruginosa. Peptides containing only the Antp fragment displayed lower activity. None of the investigated peptides demonstrated cytotoxic or cytostatic effects on either BT-474 cells or human skin fibroblasts. In conclusion, CPP-AMPs offer promise against various bacterial strains, offering insights for targeted antimicrobial development.

Binding of the Andes Virus Nucleocapsid Protein to RhoGDI Induces the Release and Activation of the Permeability Factor RhoA.

Andes virus (ANDV) nonlytically infects pulmonary microvascular endothelial cells (PMECs), causing acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). In HPS patients, virtually every PMEC is infected; however, the mechanism by which ANDV induces vascular permeability and edema remains to be resolved. The ANDV nucleocapsid (N) protein activates the GTPase RhoA in primary human PMECs, causing VE-cadherin internalization from adherens junctions and PMEC permeability. We found that ANDV N protein failed to bind RhoA but coprecipitates RhoGDI (Rho GDP dissociation inhibitor), the primary RhoA repressor that normally sequesters RhoA in an inactive state. ANDV N protein selectively binds the RhoGDI C terminus (residues 69 to 204) but fails to form ternary complexes with RhoA or inhibit RhoA binding to the RhoGDI N terminus (residues 1 to 69). However, we found that ANDV N protein uniquely inhibits RhoA binding to an S34D phosphomimetic RhoGDI mutant. Hypoxia and vascular endothelial growth factor (VEGF) increase RhoA-induced PMEC permeability by directing protein kinase Cα (PKCα) phosphorylation of S34 on RhoGDI. Collectively, ANDV N protein alone activates RhoA by sequestering and reducing RhoGDI available to suppress RhoA. In response to hypoxia and VEGF-activated PKCα, ANDV N protein additionally directs the release of RhoA from S34-phosphorylated RhoGDI, synergistically activating RhoA and PMEC permeability. These findings reveal a fundamental edemagenic mechanism that permits ANDV to amplify PMEC permeability in hypoxic HPS patients. Our results rationalize therapeutically targeting PKCα and opposing protein kinase A (PKA) pathways that control RhoGDI phosphorylation as a means of resolving ANDV-induced capillary permeability, edema, and HPS. IMPORTANCE HPS-causing hantaviruses infect pulmonary endothelial cells (ECs), causing vascular leakage, pulmonary edema, and a 35% fatal acute respiratory distress syndrome (ARDS). Hantaviruses do not lyse or disrupt the endothelium but dysregulate normal EC barrier functions and increase hypoxia-directed permeability. Our findings reveal a novel underlying mechanism of EC permeability resulting from ANDV N protein binding to RhoGDI, a regulatory protein that normally maintains edemagenic RhoA in an inactive state and inhibits EC permeability. ANDV N sequesters RhoGDI and enhances the release of RhoA from S34-phosphorylated RhoGDI. These findings indicate that ANDV N induces the release of RhoA from PKC-phosphorylated RhoGDI, synergistically enhancing hypoxia-directed RhoA activation and PMEC permeability. Our data suggest inhibiting PKC and activating PKA phosphorylation of RhoGDI as mechanisms of inhibiting ANDV-directed EC permeability and therapeutically restricting edema in HPS patients. These findings may be broadly applicable to other causes of ARDS.

Multiple Antimicrobial Effects of Hybrid Peptides Synthesized Based on the Sequence of Ribosomal S1 Protein from Staphylococcus aureus.

The need to develop new antimicrobial peptides is due to the high resistance of pathogenic bacteria to traditional antibiotics now and in the future. The creation of synthetic peptide constructs is a common and successful approach to the development of new antimicrobial peptides. In this work, we use a simple, flexible, and scalable technique to create hybrid antimicrobial peptides containing amyloidogenic regions of the ribosomal S1 protein from Staphylococcus aureus. While the cell-penetrating peptide allows the peptide to enter the bacterial cell, the amyloidogenic site provides an antimicrobial effect by coaggregating with functional bacterial proteins. We have demonstrated the antimicrobial effects of the R23F, R23DI, and R23EI hybrid peptides against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa, Escherichia coli, and Bacillus cereus. R23F, R23DI, and R23EI can be used as antimicrobial peptides against Gram-positive and Gram-negative bacteria resistant to traditional antibiotics.

Is It Possible to Create Antimicrobial Peptides Based on the Amyloidogenic Sequence of Ribosomal S1 Protein of P. aeruginosa?

The development and testing of new antimicrobial peptides (AMPs) represent an important milestone toward the development of new antimicrobial drugs that can inhibit the growth of pathogens and multidrug-resistant microorganisms such as Pseudomonas aeruginosa, Gram-negative bacteria. Most AMPs achieve these goals through mechanisms that disrupt the normal permeability of the cell membrane, which ultimately leads to the death of the pathogenic cell. Here, we developed a unique combination of a membrane penetrating peptide and peptides prone to amyloidogenesis to create hybrid peptide: "cell penetrating peptide + linker + amyloidogenic peptide". We evaluated the antimicrobial effects of two peptides that were developed from sequences with different propensities for amyloid formation. Among the two hybrid peptides, one was found with antibacterial activity comparable to antibiotic gentamicin sulfate. Our peptides showed no toxicity to eukaryotic cells. In addition, we evaluated the effect on the antimicrobial properties of amino acid substitutions in the non-amyloidogenic region of peptides. We compared the results with data on the predicted secondary structure, hydrophobicity, and antimicrobial properties of the original and modified peptides. In conclusion, our study demonstrates the promise of hybrid peptides based on amyloidogenic regions of the ribosomal S1 protein for the development of new antimicrobial drugs against P. aeruginosa.

Unique Interferon Pathway Regulation by the Andes Virus Nucleocapsid Protein Is Conferred by Phosphorylation of Serine 386.

Andes virus (ANDV) causes hantavirus pulmonary syndrome (HPS) and is the only hantavirus shown to spread person to person and cause a highly lethal HPS-like disease in Syrian hamsters. The unique ability of ANDV N protein to inhibit beta interferon (IFNß) induction may contribute to its virulence and spread. Here we analyzed IFNß regulation by ANDV N protein substituted with divergent residues from the nearly identical Maporal virus (MAPV) N protein. We found that MAPV N fails to inhibit IFNß signaling and that replacing ANDV residues 252 to 296 with a hypervariable domain (HVD) from MAPV N prevents IFNß regulation. In addition, changing ANDV residue S386 to the histidine present in MAPV N or the alanine present in other hantaviruses prevented ANDV N from regulating IFNß induction. In contrast, replacing serine with phosphoserine-mimetic aspartic acid (S386D) in ANDV N robustly inhibited interferon regulatory factor 3 (IRF3) phosphorylation and IFNß induction. Additionally, the MAPV N protein gained the ability to inhibit IRF3 phosphorylation and IFNß induction when ANDV HVD and H386D replaced MAPV residues. Mass spectroscopy analysis of N protein from ANDV-infected cells revealed that S386 is phosphorylated, newly classifying ANDV N as a phosphoprotein and phosphorylated S386 as a unique determinant of IFN regulation. In this context, the finding that the ANDV HVD is required for IFN regulation by S386 but dispensable for IFN regulation by D386 suggests a role for HVD in kinase recruitment and S386 phosphorylation. These findings delineate elements within the ANDV N protein that can be targeted to attenuate ANDV and suggest targeting cellular kinases as potential ANDV therapeutics.IMPORTANCE ANDV contains virulence determinants that uniquely permit it to spread person to person and cause highly lethal HPS in immunocompetent hamsters. We discovered that ANDV S386 and an ANDV-specific hypervariable domain permit ANDV N to inhibit IFN induction and that IFN regulation is directed by phosphomimetic S386D substitutions in ANDV N. In addition, MAPV N proteins containing D386 and ANDV HVD gained the ability to inhibit IFN induction. Validating these findings, mass spectroscopy analysis revealed that S386 of ANDV N protein is uniquely phosphorylated during ANDV infection. Collectively, these findings reveal new paradigms for ANDV N protein as a phosphoprotein and IFN pathway regulator and suggest new mechanisms for hantavirus regulation of cellular kinases and signaling pathways. Our findings define novel IFN-regulating virulence determinants of ANDV, identify residues that can be modified to attenuate ANDV for vaccine development, and suggest the potential for kinase inhibitors to therapeutically restrict ANDV replication.

Antimicrobial and Amyloidogenic Activity of Peptides Synthesized on the Basis of the Ribosomal S1 Protein from Thermus Thermophilus.

Controlling the aggregation of vital bacterial proteins could be one of the new research directions and form the basis for the search and development of antibacterial drugs with targeted action. Such approach may be considered as an alternative one to antibiotics. Amyloidogenic regions can, like antibacterial peptides, interact with the "parent" protein, for example, ribosomal S1 protein (specific only for bacteria), and interfere with its functioning. The aim of the work was to search for peptides based on the ribosomal S1 protein from T. thermophilus, exhibiting both aggregation and antibacterial properties. The biological system of the response of Gram-negative bacteria T. thermophilus to the action of peptides was characterized. Among the seven studied peptides, designed based on the S1 protein sequence, the R23I (modified by the addition of HIV transcription factor fragment for bacterial cell penetration), R23T (modified), and V10I (unmodified) peptides have biological activity that inhibits the growth of T. thermophilus cells, that is, they have antimicrobial activity. But, only the R23I peptide had the most pronounced activity comparable with the commercial antibiotics. We have compared the proteome of peptide-treated and intact T. thermophilus cells. These important data indicate a decrease in the level of energy metabolism and anabolic processes, including the processes of biosynthesis of proteins and nucleic acids. Under the action of 20 and 50 µg/mL R23I, a decrease in the number of proteins in T. thermophilus cells was observed and S1 ribosomal protein was absent. The obtained results are important for understanding the mechanism of amyloidogenic peptides with antimicrobial activity and can be used to develop new and improved analogues.

Amyloidogenic Propensities of Ribosomal S1 Proteins: Bioinformatics Screening and Experimental Checking.

Structural S1 domains belong to the superfamily of oligosaccharide/oligonucleotide-binding fold domains, which are highly conserved from prokaryotes to higher eukaryotes and able to function in RNA binding. An important feature of this family is the presence of several copies of the structural domain, the number of which is determined in a strictly limited range from one to six. Despite the strong tendency for the aggregation of several amyloidogenic regions in the family of the ribosomal S1 proteins, their fibril formation process is still poorly understood. Here, we combined computational and experimental approaches for studying some features of the amyloidogenic regions in this protein family. The FoldAmyloid, Waltz, PASTA 2.0 and Aggrescan programs were used to assess the amyloidogenic propensities in the ribosomal S1 proteins and to identify such regions in various structural domains. The thioflavin T fluorescence assay and electron microscopy were used to check the chosen amyloidogenic peptides' ability to form fibrils. The bioinformatics tools were used to study the amyloidogenic propensities in 1331 ribosomal S1 proteins. We found that amyloidogenicity decreases with increasing sizes of proteins. Inside one domain, the amyloidogenicity is higher in the terminal parts. We selected and synthesized 11 amyloidogenic peptides from the Escherichia coli and Thermus thermophilus ribosomal S1 proteins and checked their ability to form amyloids using the thioflavin T fluorescence assay and electron microscopy. All 11 amyloidogenic peptides form amyloid-like fibrils. The described specific amyloidogenic regions are actually responsible for the fibrillogenesis process and may be potential targets for modulating the amyloid properties of bacterial ribosomal S1 proteins.

Structural model of amyloid fibrils for amyloidogenic peptide from Bgl2p-glucantransferase of S. cerevisiae cell wall and its modifying analog. New morphology of amyloid fibrils.

We performed a comparative study of the process of amyloid formation by short homologous peptides with a substitution of aspartate for glutamate in position 2 - VDSWNVLVAG (AspNB) and VESWNVLVAG (GluNB) - with unblocked termini. Peptide AspNB (residues 166-175) corresponded to the predicted amyloidogenic region of the protein glucantransferase Bgl2 from the Saccharomyces cerevisiae cell wall. The process of amyloid formation was monitored by fluorescence spectroscopy (FS), electron microscopy (EM), tandem mass spectrometry (TMS), and X-ray diffraction (XD) methods. The experimental study at pH3.0 revealed formation of amyloid fibrils with similar morphology for both peptides. Moreover, we found that the morphology of fibrils made of untreated ammonia peptide is not mentioned in the literature. This morphology resembles snakes lying side by side in the form of a wave without intertwining. Irrespective of the way of the peptide preparation, the rate of fibril formation is higher for AspNB than for GluNB. However, preliminary treatment with ammonia highly affected fibril morphology especially for AspNB. Such treatment allowed us to obtain a lag period during the process of amyloid formation. It showed that the process was nucleation-dependent. With or without treatment, amyloid fibrils consisted of ring-like oligomers with the diameter of about 6nm packed either directly ring-to-ring or ring-on-ring with a slight shift. We also proposed the molecular structure of amyloid fibrils for two studied peptides.

Hypoxia induces permeability and giant cell responses of Andes virus-infected pulmonary endothelial cells by activating the mTOR-S6K signaling pathway.

Andes virus (ANDV) is a South American hantavirus that causes a highly lethal hantavirus pulmonary syndrome (HPS) characterized by hypoxia, thrombocytopenia, and vascular leakage leading to acute pulmonary edema. ANDV infects human pulmonary microvascular and lymphatic endothelial cells (MECs and LECs, respectively) and nonlytically enhances the permeability of interendothelial cell adherence junctions in response to vascular endothelial growth factor (VEGF). Recent findings also indicate that ANDV causes the formation of giant endothelial cells. Here, we demonstrate that hypoxic conditions alone enhance permeability and giant cell responses of ANDV-infected MECs and LECs through activation of the mTOR signaling pathway. In contrast to infection of cells with nonpathogenic Tula virus (TULV), we observed that exposure of ANDV-infected MECs and LECs to hypoxic conditions resulted in a 3- to 6-fold increase in monolayer permeability and the formation of giant cells 3× to 5× normal size. ANDV infection in combination with hypoxic conditions resulted in the enhancement of hypoxia-inducible factor 1α (HIF1α)-directed VEGF A, angiopoietin 4, and EGLN3 transcriptional responses. Constitutive mTOR signaling induces the formation of giant cells via phosphorylation of S6K, and mTOR regulates hypoxia and VEGF A-induced cellular responses. We found that S6K was hyperphosphorylated in ANDV-infected, hypoxia-treated MECs and LECs and that rapamycin treatment for 1 h inhibited mTOR signaling responses and blocked permeability and giant cell formation in ANDV-infected monolayers. These findings indicate that ANDV infection and hypoxic conditions enhance mTOR signaling responses, resulting in enhanced endothelial cell permeability and suggest a role for rapamycin in therapeutically stabilizing the endothelium of microvascular and lymphatic vessels during ANDV infection.

Controlled oxide films formation by nanosecond laser pulses for color marking.

A technology of laser-induced coloration of metals by surface oxidation is demonstrated. Each color of the oxide film corresponds to a technologic chromacity coefficient, which takes into account the temperature of the sample after exposure by sequence of laser pulses with nanosecond duration and effective time of action. The coefficient can be used for the calculation of laser exposure regimes for the development of a specific color on the metal. A correlation between the composition of the films obtained on the surface of stainless steel AISI 304 and commercial titanium Grade 2 and its color and chromacity coordinates is shown.

Andes virus infection of lymphatic endothelial cells causes giant cell and enhanced permeability responses that are rapamycin and vascular endothelial growth factor C sensitive.

Hantaviruses primarily infect endothelial cells (ECs) and nonlytically cause vascular changes that result in hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Acute pulmonary edema during HPS may be caused by capillary leakage and failure of lymphatic vessels to clear fluids. Uniquely regulated lymphatic ECs (LECs) control fluid clearance, although roles for lymphatics in hantavirus disease remain undetermined. Here we report that hantaviruses productively infect LECs and that LEC infection by HPS causing Andes virus (ANDV) and HFRS causing Hantaan virus (HTNV) are inhibited by α(v)ß(3) integrin antibodies. Although α(v)ß(3) integrins regulate permeabilizing responses directed by vascular endothelial growth factor receptor 2 (VEGFR2), we found that only ANDV-infected LECs were hyperpermeabilized by the addition of VEGF-A. However, VEGF-C activation of LEC-specific VEGFR3 receptors blocked ANDV- and VEGF-A-induced LEC permeability. In addition, ∼75% of ANDV-infected LECs became viable mononuclear giant cells, >4 times larger than normal, in response to VEGF-A. Giant cells are associated with constitutive mammalian target of rapamycin (mTOR) activation, and we found that both giant LECs and LEC permeability were sensitive to rapamycin, an mTOR inhibitor, and VEGF-C addition. These findings indicate that ANDV uniquely alters VEGFR2-mTOR signaling responses of LECs, resulting in giant cell and LEC permeability responses. This suggests that ANDV infection alters normal LEC and lymphatic vessel functions which may contribute to edematous fluid accumulation during HPS. Moreover, the ability of VEGF-C and rapamycin to normalize LEC responses suggests a potential therapeutic approach for reducing pulmonary edema and the severity of HPS following ANDV infection.

Formation of truncated peptide by-products via sequence-specific formyl group transfer from Trp(For) residues to Nα in the course of Boc-SPPS.

(N(In))-Formyl protective group of tryptophan has been introduced as a base/nucleophile-labile protective group. It has long been known that a free Nα-amino group of the peptide can serve as a nucleophile: an irreversible formyl N(In) → NH(2) transfer is consistently observed when deformylation is performed last on an otherwise deprotected peptide that possesses free Nα-amino group. Obviously, this particular side reaction should be expected any time free amino group is exposed to Trp(For), but, at the best of our knowledge, has never been reported in the course of Boc-SPPS. In the present communication, we describe a set of appropriately designed model experiments that permitted to detect the title side reaction both in solution and in solid-phase reactions. We observed intermolecular formyl group transfer with a model compound, Trp(For)-NH(2). Importantly, we also observed this migration on solid support with the rate roughly estimated to be up to 1% of residues per minute. We also observed that the formyl-group transfer reaction occurred in a sequence-dependent manner and was suppressed to a non-detectable level using 'in situ neutralization' technique. Because this side reaction is sequence dependent, there might be situations when the rate of the formation of Nα -formyl termination by-products is significant. In other cases, the Nα -For truncated by-products would not contaminate the final peptide significantly but still could be a source of microheterogeneity.

Establishment of a CPER reverse genetics system for Powassan virus defines attenuating NS1 glycosylation sites and an infectious NS1-GFP11 reporter virus.

Powassan virus (POWV) is an emerging tick-borne Flavivirus that causes lethal encephalitis and long-term neurologic damage. Currently, there are no POWV therapeutics, licensed vaccines, or reverse genetics systems for producing infectious POWVs from recombinant DNA. Using a circular polymerase extension reaction (CPER), we generated recombinant LI9 (recLI9) POWVs with attenuating NS1 protein mutations and a recLI9-split-eGFP reporter virus. NS1 proteins are highly conserved glycoproteins that regulate replication, spread, and neurovirulence. POWV NS1 contains three putative N-linked glycosylation sites that we modified individually in infectious recLI9 mutants (N85Q, N208Q, and N224Q). NS1 glycosylation site mutations reduced replication kinetics and were attenuated, with 1-2 log decreases in titer. Severely attenuated recLI9-N224Q exhibited a 2- to 3-day delay in focal cell-to-cell spread and reduced NS1 secretion but was lethal when intracranially inoculated into suckling mice. However, footpad inoculation of recLI9-N224Q resulted in the survival of 80% of mice and demonstrated that NS1-N224Q mutations reduce POWV neuroinvasion in vivo. To monitor NS1 trafficking, we CPER fused a split GFP11-tag to the NS1 C-terminus and generated an infectious reporter virus, recLI9-NS1-GFP11. Cells infected with recLI9-NS1-GFP11 revealed NS1 trafficking in live cells and the novel formation of large NS1-lined intracellular vesicles. An infectious recLI9-NS1-GFP11 reporter virus permits real-time analysis of NS1 functions in POWV replication, assembly, and secretion and provides a platform for evaluating antiviral compounds. Collectively, our robust POWV reverse genetics system permits analysis of viral spread and neurovirulence determinants in vitro and in vivo and enables the rational genetic design of live attenuated POWV vaccines. IMPORTANCE Our findings newly establish a mechanism for genetically modifying Powassan viruses (POWVs), systematically defining pathogenic determinants and rationally designing live attenuated POWV vaccines. This initial study demonstrates that mutating POWV NS1 glycosylation sites attenuates POWV spread and neurovirulence in vitro and in vivo. Our findings validate a robust circular polymerase extension reaction approach as a mechanism for developing, and evaluating, attenuated genetically modified POWVs. We further designed an infectious GFP-tagged reporter POWV that permits us to monitor secretory trafficking of POWV in live cells, which can be applied to screen potential POWV replication inhibitors. This robust system for modifying POWVs provides the ability to define attenuating POWV mutations and create genetically attenuated recPOWV vaccines.

VEGFR2 and Src kinase inhibitors suppress Andes virus-induced endothelial cell permeability.

Hantaviruses predominantly infect human endothelial cells and, in the absence of cell lysis, cause two diseases resulting from increased vascular permeability. Andes virus (ANDV) causes a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). ANDV infection enhances the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF) by increasing signaling responses directed by the VEGFR2-Src-VE-cadherin pathway, which directs adherens junction (AJ) disassembly. Here we demonstrate that inhibiting pathway-specific VEGFR2 and Src family kinases (SFKs) blocks ANDV-induced endothelial cell permeability. Small interfering RNA (siRNA) knockdown of Src within ANDV-infected endothelial cells resulted in an ∼70% decrease in endothelial cell permeability compared to that for siRNA controls. This finding suggested that existing FDA-approved small-molecule kinase inhibitors might similarly block ANDV-induced permeability. The VEGFR2 kinase inhibitor pazopanib as well as SFK inhibitors dasatinib, PP1, bosutinib, and Src inhibitor 1 dramatically inhibited ANDV-induced endothelial cell permeability. Consistent with their kinase-inhibitory concentrations, dasatinib, PP1, and pazopanib inhibited ANDV-induced permeability at 1, 10, and 100 nanomolar 50% inhibitory concentrations (IC(50)s), respectively. We further demonstrated that dasatinib and pazopanib blocked VE-cadherin dissociation from the AJs of ANDV-infected endothelial cells by >90%. These findings indicate that VEGFR2 and Src kinases are potential targets for therapeutically reducing ANDV-induced endothelial cell permeability and, as a result, capillary permeability during HPS. Since the functions of VEGFR2 and SFK inhibitors are already well defined and FDA approved for clinical use, these findings rationalize their therapeutic evaluation for efficacy in reducing HPS disease. Endothelial cell barrier functions are disrupted by a number of viruses that cause hemorrhagic, edematous, or neurologic disease, and as a result, our findings suggest that VEGFR2 and SFK inhibitors should be considered for regulating endothelial cell barrier functions altered by additional viral pathogens.

The C-terminal 42 residues of the Tula virus Gn protein regulate interferon induction.

Hantaviruses primarily infect the endothelial cell lining of capillaries and cause two vascular permeability-based diseases. The ability of pathogenic hantaviruses to regulate the early induction of interferon determines whether hantaviruses replicate in endothelial cells. Tula virus (TULV) and Prospect Hill virus (PHV) are hantaviruses which infect human endothelial cells but fail to cause human disease. PHV is unable to inhibit early interferon (IFN) responses and fails to replicate within human endothelial cells. However, TULV replicates successfully in human endothelial cells, suggesting that TULV is capable of regulating cellular IFN responses. We observed a >300-fold reduction in the IFN-stimulated genes (ISGs) MxA and ISG56 following TULV versus PHV infection of endothelial cells 1 day postinfection. Similar to results with pathogenic hantaviruses, expressing the TULV Gn protein cytoplasmic tail (Gn-T) blocked RIG-I- and TBK1-directed transcription from IFN-stimulated response elements (ISREs) and IFN-ß promoters (>90%) but not transcription directed by constitutively active IFN regulatory factor-3 (IRF3). In contrast, expressing the PHV Gn-T had no effect on TBK1-induced transcriptional responses. Analysis of Gn-T truncations demonstrated that the C-terminal 42 residues of the Gn-T (Gn-T-C42) from TULV, but not PHV, inhibited IFN induction >70%. These findings demonstrate that the TULV Gn-T inhibits IFN- and ISRE-directed responses upstream of IRF3 at the level of the TBK1 complex and further define a 42-residue domain of the TULV Gn-T that inhibits IFN induction. In contrast to pathogenic hantavirus Gn-Ts, the TULV Gn-T lacks a C-terminal degron domain and failed to bind tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3), a TBK1 complex component required for IRF3 activation. These findings indicate that the nonpathogenic TULV Gn-T regulates IFN induction but accomplishes this via unique interactions with cellular TBK1 complexes. These findings fundamentally distinguish nonpathogenic hantaviruses, PHV and TULV, and demonstrate that IFN regulation alone is insufficient for hantaviruses to cause disease. Yet regulating the early IFN response is necessary for hantaviruses to replicate within human endothelial cells and to be pathogenic. Thus, in addition to IFN regulation, hantaviruses contain discrete virulence determinants which permit them to be human pathogens.

The Role of Emotional Information in Banner Blindness.

Modern theories in affective science postulate that emotional stimuli can affect subject's attention. Emotional stimuli can guide and capture visual attention, which may be related to evolutional importance of quick reactions for emotional objects in the real life. The study examined the influence of valence and arousal of advertisement on the banner blindness phenomenon-ignoring the advertisement or interface details similar to it on the website. In two experiments participants were asked to find the information on the website, where different banners were placed. In the first experiment banners had the same valence, but different arousal. In the second experiment, the banners had different valence, but equal arousal. Contrary to the classical studies in affective science, we found that banners with neutral valence were recognized better as compared to negative and positive ones. The results are discussed in terms of user experience contributing to banner blindness occurrence.

The Manifestation of Incidental Findings in Different Experimental Visual Search Paradigms.

Background: Incidental findings are items of visual search that are potentially of significance, but were not the main object of the initial search. They have been previously widely discussed in the field of radiology. However, the underlying perceptual mechanisms of such phenomenon are still unclear. Objective: The current study aims to examine incidental findings in different paradigms of visual search in order to reveal their primary perceptual aspects. Design: Two behavioral visual search experiments were conducted. The mixed hybrid search task model was used in the first experiment, while the subsequent search miss effect was employed in the second experiment. The task was to find targets among distractors, according to given instructions. Stimuli material consisted of images of real-life objects that were randomly distributed across the screen for each trial. Results: Accuracy and reaction time of the participants were analyzed in both experiments. Similar effects were observed for both parameters. Specific targets in the first experiment and typical targets in the second experiment were found significantly faster and more accurately in comparison to categorical and atypical targets. Moreover, this tendency did not depend on the order of target identification. Hence, the prevalence of the targets was revealed to be the primary factor in the case of incidental findings. Conclusion: The study revealed the emergence of incidental findings in both experiments. Typical or specific targets were detected significantly more accurately, compared to atypical or categorical targets. Subsequent search misses were not detected, suggesting that target prevalence could be a crucial factor that is specific for incidental findings.