The αGal Epitope of the Histo-Blood Group Antigen Family Is a Ligand for Bovine Norovirus Newbury2 Expected to Prevent Cross-Species Transmission

Among Caliciviridae, the norovirus genus encompasses enteric viruses that infect humans as well as several animal species, causing gastroenteritis. Porcine strains are classified together with human strains within genogroup II, whilst bovine norovirus strains represent genogroup III. Various GI and GII human strains bind to carbohydrates of the histo-blood group family which may be shared among mammalian species. Genetic relatedness of human and animal strains as well as the presence of potentially shared ligands raises the possibility of norovirus cross-species transmission. In the present study, we identified a carbohydrate ligand for the prototype bovine norovirus strain Bo/Newbury2/76/UK (NB2). Attachment of virus-like particles (VLPs) of the NB2 strain to bovine gut tissue sections showed a complete match with the staining by reagents recognizing the Galα1,3 motif. Alpha-galactosidase treatment confirmed involvement of a terminal alpha-linked galactose. Specific binding of VLPs to the αGal epitope (Galα3Galβ4GlcNAcβ-R) was observed. The binding of Galα3GalαOMe to rNB2 VLPs was characterized at atomic resolution employing saturation transfer difference (STD) NMR experiments. Transfection of human cells with an α1,3galactosyltransferase cDNA allowed binding of NB2 VLPs, whilst inversely, attachment to porcine vascular endothelial cells was lost when the cells originated from an α1,3galactosyltransferase KO animal. The αGal epitope is expressed in all mammalian species with the exception of the Hominidaea family due to the inactivation of the α1,3galactosyltransferase gene (GGTA1). Accordingly, the NB2 carbohydrate ligand is absent from human tissues. Although expressed on porcine vascular endothelial cells, we observed that unlike in cows, it is not present on gut epithelial cells, suggesting that neither man nor pig could be infected by the NB2 bovine strain.     

Investigation of antimicrobial activity of photothermal therapeutic gold/copper sulfide core/shell nanoparticles to bacterial spores and cells

Background

Au/CuS core/shell nanoparticles (NPs) were designed as a new type of transducer agent for photothermal therapy (PTT), with attractive features of easy preparation, low cost and small size for targeting. This paper studied for the first time the intrinsic antimicrobial activity of Au/CuS NPs to B. anthracis spores and cells in addition to its PTT effect.

Results

It was found that Au/CuS NPs were highly efficient in inactivating B. anthracis cells, but not effective to the spores. Treatment with NPs at ~0.83 μM for 30 min achieved a 7 log reduction in viable cells. The antimicrobial effect was both NPs concentration and treatment time dependent. SEM imaging and the efflux of DNA test demonstrated the damage of cell membrane after NPs treatment, yet further research is necessary to fully understand the precise inactivation mechanism.

Conclusions

The Au/CuS NPs had strong antimicrobial activity to B. anthracis cells, which showed a great potential to be an effective antimicrobial agent to bacterial cells.

     

A Novel Prime and Boost Regimen of HIV Virus-Like Particles with TLR4 Adjuvant MPLA Induces Th1 Oriented Immune Responses against HIV

HIV virus-like particles (VLPs) present the HIV envelope protein in its native conformation, providing an ideal vaccine antigen. To enhance the immunogenicity of the VLP vaccine, we sought to improve upon two components; the route of administration and the additional adjuvant. Using HIV VLPs, we evaluated sub-cheek as a novel route of vaccine administration when combined with other conventional routes of immunization. Of five combinations of distinct prime and boost sequences, which included sub-cheek, intranasal, and intradermal routes of administration, intranasal prime and sub-cheek boost (IN+SC) resulted in the highest HIV-specific IgG titers among the groups tested. Using the IN+SC regimen we tested the adjuvant VesiVax Conjugatable Adjuvant Lipid Vesicles (CALV) + monophosphoryl lipid A (MPLA) at MPLA concentrations of 0, 7.5, 12.5, and 25 μg/dose in combination with our VLPs. Mice that received 12.5 or 25 μg/dose MPLA had the highest concentrations of Env-specific IgG2c (20.7 and 18.4 μg/ml respectively), which represents a Th1 type of immune response in C57BL/6 mice. This was in sharp contrast to mice which received 0 or 7.5 μg MPLA adjuvant (6.05 and 5.68 μg/ml of IgG2c respectively). In contrast to IgG2c, MPLA had minor effects on Env-specific IgG1; therefore, 12.5 and 25 μg/dose of MPLA induced the optimal IgG1/IgG2c ratio of 1.3. Additionally, the percentage of germinal center B cells increased significantly from 15.4% in the control group to 31.9% in the CALV + 25 μg MPLA group. These mice also had significantly more IL-2 and less IL-4 Env-specific CD8⁺ T cells than controls, correlating with an increased percentage of Env-specific central memory CD4⁺ and CD8⁺ T cells. Our study shows the strong potential of IN+SC as an efficacious route of administration and the effectiveness of VLPs combined with MPLA adjuvant to induce Env specific Th1-oriented HIV-specific immune responses.     

Structural basis for broad detection of genogroup II noroviruses by a monoclonal antibody that binds to a site occluded in the viral particle

Human noroviruses are genetically and antigenically highly divergent. Monoclonal antibodies raised in mice against one kind of norovirus virus-like particle (VLP), however, were found to have broad recognition. In this study, we present the crystal structure of the antigen-binding fragment (Fab) for one of these broadly reactive monoclonal antibodies, 5B18, in complex with the capsid-protruding domain from a genogroup II genotype 10 (GII.10) norovirus at 3.3-Å resolution and, also, the cryo-electron microscopy structure of the GII.10 VLP at ∼10-Å resolution. The GII.10 VLP structure was more similar in overall architecture to the GV.1 murine norovirus virion than to the prototype GI.1 human norovirus VLP, with the GII.10 protruding domain raised ∼15 Å off the shell domain and rotated ∼40° relative to the GI.1 protruding domain. In the crystal structure, the 5B18 Fab bound to a highly conserved region of the protruding domain. Based on the VLP structure, this region is involved in interactions with other regions of the capsid and is buried in the virus particle. Despite the occluded nature of the recognized epitope in the VLP structure, enzyme-linked immunosorbent assay (ELISA) binding suggested that the 5B18 antibody was able to capture intact VLPs. Together, the results provide evidence that the norovirus particle is capable of extreme conformational flexibility, which may allow for antibody recognition of conserved surfaces that would otherwise be buried on intact particles.     

Identification of Cross-Reactive Norovirus CD4+ T Cell Epitopes

Immune responses and the components of protective immunity following norovirus infection in humans are poorly understood. Although antibody responses following norovirus infection have been partially characterized, T cell responses in humans remain largely undefined. In contrast, T cells have been shown to be essential for viral clearance of mouse norovirus (MNV) infection. In this paper, we demonstrate that CD4⁺ T cells secrete gamma interferon (IFN-γ) in response to stimulation with MNV virus-like particles (VLPs) after MNV infection, supporting earlier reports for norovirus-infected mice and humans. Utilizing this model, we immunized mice with alphavirus vectors (Venezuelan equine encephalitis [VEE] virus replicon particles [VRPs]) expressing Norwalk virus (NV) or Farmington Hills virus (FH) virus-like particles to evaluate T cell epitopes shared between human norovirus strains. Stimulation of splenocytes from norovirus VRP-immunized mice with overlapping peptides from complete libraries of the NV or FH capsid proteins revealed specific amino acid sequences containing T cell epitopes that were conserved within genoclusters and genogroups. Immunization with heterologous norovirus VRPs resulted in specific cross-reactive IFN-γ secretion profiles following stimulation with NV and FH peptides in the mouse. Identification of unique strain-specific and cross-reactive epitopes may provide insight into homologous and heterologous T cell-mediated norovirus immunity and provide a platform for the study of norovirus-induced cellular immunity in humans.Norovirus infection is characterized by the induction of both humoral and cellular immune responses. Humoral immunity in humans following norovirus infection has been described in detail for a limited number of norovirus strains (8, 10, 12, 17, 18, 29). Humans mount specific antibody responses to the infecting strain, which bear complex patterns of unique and cross-reactive, yet undefined, epitopes to other strains within or across genogroups (23, 29). Short-term immunity following homologous norovirus challenge has been documented, but long-term immunity remains controversial (16, 25). Furthermore, no studies to date have demonstrated cross-protection following heterologous norovirus challenge (30). While some susceptible individuals can become reinfected with multiple norovirus strains throughout their lifetimes, the mechanism of short-term protection and the impact of previous exposures on susceptibility to reinfection remain largely unknown.The role of T cells in controlling norovirus infection also remains largely undefined. A single comprehensive study detailing immune responses in genogroup II Snow Mountain virus-infected individuals revealed that CD4⁺ TH1 cells can be stimulated by virus-like particles (VLPs) to secrete gamma interferon (IFN-γ) and interleukin-2 (IL-2) (17). Furthermore, heterologous stimulation from VLPs derived from different norovirus strains within but not across genogroups also induced significant IFN-γ secretion compared to that for uninfected individuals (17). A follow-up study with genogroup I Norwalk virus (NV)-infected individuals confirmed high T cell cross-reactivity within a genogroup as measured by IFN-γ secretion (18). Further, vaccination of humans with VLPs also results in short-term IFN-γ production (27).Because norovirus infection studies in humans are confounded by previous exposure histories, the use of inbred mice maintained in pathogen-free environments allows for the study of norovirus immune responses in a naive background. While mice cannot be infected with human norovirus strains, VLP vaccines expressing norovirus structural proteins induce immune responses that can be measured and studied (14, 20). Mice immunized orally or intranasally with VLP vaccines in the presence of adjuvant similarly induced CD4⁺ IFN-γ responses in Peyer''s patches and spleen (22, 26). Induction of CD8⁺ T cells and secretion of the TH2 cytokine IL-4 were separately noted; however, it is unclear if these responses were influenced by VLPs or the coadministered vaccine adjuvants (22, 26). Further, coadministration of alphavirus adjuvant particles with multivalent norovirus VLP vaccine, including or excluding mouse norovirus (MNV) VLPs, resulted in significantly reduced MNV loads following MNV challenge (21). Multivalent VLP vaccines induced robust receptor-blocking antibody responses to heterologous human strains not included in the vaccine composition (20, 21). Moreover, natural infection with MNV supports a role for T cell immunity in viral clearance and protection (5).To advance our understanding of the scope of the cellular immune response within and between strains, we immunized mice with Venezuelan equine encephalitis (VEE) virus replicon particles (VRPs) expressing norovirus VLPs derived from the Norwalk virus (GI.1-1968) (1) or Farmington Hills virus (FH) (GII.4-2002) (19) strains and analyzed splenocytes for cytokine secretion, epitope identification, and heterologous stimulation. The data presented here indicate that the major capsid proteins of genogroup I and II noroviruses contain robust T cell epitopes that cross-react with related strains in the mouse yet also occur within regions of known variation, especially among the GII.4 noroviruses.     

Preparation of CoS nanoparticles-cisplatin bio-conjugates and investigation of their effects on SH-SY5Y neuroblastoma cell line

Neuroblastoma is one of the most widely seen under the age of 15 tumors that occur in the adrenal medulla and sympathetic ganglia. Cisplatin, an antineoplastic drug, is a Platinum-based compound and is known to inhibit the proliferation of neuroblastoma cells. Effective applications of nanoparticles in biomedical areas such as biomolecular, antimicrobial detection and diagnosis, tissue engineering, theranostics, biomarking, drug delivery, and anti-cancer have been investigated in many studies. This study aims to prepare the bioconjugates of CoS (cobalt sulfide) nanoparticles (NPs) with cisplatin combination groups and to evaluate their effects on the neuroblastoma cell line. Nanoparticle synthesis was done using the green synthesis technique using Punica granatum plant extract. The size and shape of CoS NPs were characterized by SEM, FT-IR, and XRD. Zeta potential was confirmed by the DLS study. For this purpose, the SH-SY5Y neuroblastoma cell line was cultured in a suitable cell culture medium. Cisplatin 5 µg and different concentrations (Cisplatin + CoS NPs bioconjugates (5, 10, 25, 50, 75 μg) doses were applied to SH-SY5Y neuroblastoma cell lines for 24 h. TAC, TOS and MTT tests were performed 24 h after the application. According to the MTT test results, cisplatin and CoS NP combinations reduced the proliferation of neuroblastoma cells by 78 to 57% compared to the cisplatin control. From the findings obtained; the most effective Bio-conjugate group was Cisplatin 5 μg/mL + CoS 75 μg/mL.     

Inactivation of a Norovirus by High-Pressure Processing

Murine norovirus (strain MNV-1), a propagable norovirus, was evaluated for susceptibility to high-pressure processing. Experiments with virus stocks in Dulbecco's modified Eagle medium demonstrated that at room temperature (20°C) the virus was inactivated over a pressure range of 350 to 450 MPa, with a 5-min, 450-MPa treatment being sufficient to inactivate 6.85 log₁₀ PFU of MNV-1. The inactivation of MNV-1 was enhanced when pressure was applied at an initial temperature of 5°C; a 5-min pressure treatment of 350 MPa at 30°C inactivated 1.15 log₁₀ PFU of virus, while the same treatment at 5°C resulted in a reduction of 5.56 log₁₀ PFU. Evaluation of virus inactivation as a function of treatment times ranging from 0 to 150 s and 0 to 900 s at 5°C and 20°C, respectively, indicated that a decreasing rate of inactivation with time was consistent with Weibull or log-logistic inactivation kinetics. The inactivation of MNV-1 directly within oyster tissues was demonstrated; a 5-min, 400-MPa treatment at 5°C was sufficient to inactivate 4.05 log₁₀ PFU. This work is the first demonstration that norovirus can be inactivated by high pressure and suggests good prospects for inactivation of nonpropagable human norovirus strains in foods.     

Lidocaine block of cardiac sodium channels

Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 μM). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 μM, at a negative holding potential where inactivation was completely removed, to approximately 10 μM, at a depolarized holding potential where inactivation was nearly complete. Lidocaine block showed prominent use dependence with trains of depolarizing pulses from a negative holding potential. During the interval between pulses, repriming of I (Na) displayed two exponential components, a normally recovering component (τless than 0.2 s), and a lidocaine-induced, slowly recovering fraction (τ approximately 1-2 s at pH 7.0). Raising the lidocaine concentration magnified the slowly recovering fraction without changing its time course; after a long depolarization, this fraction was one-half at approximately 10 μM lidocaine, just as expected if it corresponded to drug-bound, inactivated channels. At less than or equal to 20 μM lidocaine, the slowly recovering fraction grew exponentially to a steady level as the preceding depolarization was prolonged; the time course was the same for strong or weak depolarizations, that is, with or without significant activation of I(Na). This argues that use dependence at therapeutic levels reflects block of inactivated channels, rather than block of open channels. Overall, these results provide direct evidence for the “modulated-receptor hypothesis” of Hille (1977) and Hondeghem and Katzung (1977). Unlike tetrodotoxin, lidocaine shows similar interactions with Na channels of heart, nerve, and skeletal muscle.     

Heterotypic Humoral and Cellular Immune Responses following Norwalk Virus Infection

Norovirus immunity is poorly understood as the limited data available on protection after infection are often contradictory. In contrast to the more prominent GII noroviruses, GI norovirus infections are less frequent in outbreaks. The GI noroviruses display very complex patterns of heterotypic immune responses following infection, and many individuals are highly susceptible to reinfection. To study the immune responses and mechanisms of GI.1 persistence, we built structural models and recombinant virus-like particles (VLPs) of five GI strains: GI.1-1968, GI.1-2001, GI.2-1999, GI.3-1999, and GI.4-2000. Structural models of four GI genotype capsid P domain dimers suggested that intragenotype structural variation is limited, that the GI binding pocket is mostly preserved between genotypes, and that a conserved, surface-exposed epitope may allow for highly cross-reactive immune responses. GI VLPs bound to histo-blood group antigens (HBGAs) including fucose, Lewis, and A antigens. Volunteers infected with GI.1-1968 (n = 10) had significant increases between prechallenge and convalescent reactive IgG for all five GI VLPs measured by enzyme immunoassay. Potential cross-neutralization of GI VLPs was demonstrated by convalescent-phase serum cross-blockade of GI VLP-HBGA interaction. Although group responses were significant for all GI VLPs, each individual volunteer demonstrated a unique VLP blockade pattern. Further, peripheral blood mononuclear cells (PBMCs) were stimulated with each of the VLPs, and secretion of gamma interferon (IFN-γ) was measured. As seen with blockade responses, IFN-γ secretion responses differed by individual. Sixty percent responded to at least one GI VLP, with only two volunteers responding to GI.1 VLP. Importantly, four of five individuals with sufficient PBMCs for cross-reactivity studies responded more robustly to other GI VLPs. These data suggest that preexposure history and deceptive imprinting may complicate PBMC and B-cell immune responses in some GI.1-1968-challenged individuals and highlight a potential complication in the design of efficacious norovirus vaccines.Noroviruses are the second-most important cause of severe viral gastroenteritis in young children and cause approximately 20% of endemic familial diarrheal disease and traveler''s diarrhea in all ages (reviewed in references 45 and 70). Noroviruses are genetically grouped into five different genogroups (GI to GV). GI and GII genogroups are responsible for the majority of human infections and are subdivided into more than 25 different genotypes (for example, GI.1 is genogroup I genotype 1). Most norovirus outbreaks are caused by the GII.4 genotype (65). Although genogroup I strains are associated with fewer reported outbreaks, they are frequently identified in environmental samples and in children (7, 21, 33, 58, 74, 82). The severity of norovirus disease is usually moderate although infection can be especially virulent, even fatal, in the elderly (14, 24, 31, 38, 46, 67). An effective vaccine would be particularly advantageous to vulnerable older populations, food handlers, child and health care providers, and military personnel. One major obstacle to norovirus vaccine development is the lack of understanding of the extensive antigenic relationships between heterogenic norovirus family members and of how this antigenic heterogeneity affects host protective immunity. Norovirus heterogeneity can be examined through sequence, structural, ligand binding, and host immune studies.Structurally, noroviruses are ∼38-nm icosahedral viruses with an ∼7.5 kb single-stranded, positive-sense RNA genome that encodes three large open reading frames (ORFs). ORF1 encodes the replicase polyprotein, while ORFs 2 and 3 encode the major and minor capsid proteins, respectively. The ORF2 major capsid protein sequence can vary by up to 60% between genogroups and by ∼20 to 30% between the genotypes (91). Expression of the major capsid protein (ORF2) in baculovirus and Venezuelan equine encephalitis (VEE) results in formation of virus-like particles (VLPs) composed of 180 copies of the monomeric protein (72). The monomer is structurally divided into the shell domain (S) that forms the structural core of the particle and the protruding domain (P) that protrudes away from the core. The P domain is further subdivided into the P1 subdomain (residues 226 to 278 and 406 to 520) and the P2 subdomain (residues 279 to 405) (72). P2 represents the most exposed surface of the viral particle and determines interaction with both potential neutralizing antibody recognition sites and putative cellular receptors, the histo-blood group antigens (HBGAs) (13, 16, 54, 57).The P domain has been shown to independently form dimers and P particles comprised of 12 monomers (85). Dimers and P particles share structural and HBGA binding similarities with the VLP generated with the same monomers (9, 85, 87). Three norovirus-HBGA binding profiles have been identified: (i) those that bind A/B and/or H epitopes, (ii) those that bind Lewis and/or H epitopes, and (iii) those that do not bind any available HBGA (86). Elegant structural analyses of Norwalk virus VLPs in complex with synthetic HBGAs identified a highly conserved binding site within the G1 noroviruses and predicted that structural constraints within the GI strains would restrict HBGA binding patterns to either a terminal Gal-Fuc or GalNAc (18, 88).Norwalk virus (NV; GI.1-1968) is the prototypic GI strain and typically infects individuals who encode a functional FUT2 α-1,2-fucosyltransferase enzyme resulting in expression of HBGAs on mucosal surfaces (secretor-positive phenotype) (53). Individuals who do not encode a functional FUT2 enzyme have a secretor-negative phenotype, do not express ABH HBGAs on mucosal surfaces, and are resistant to NV infection. Outbreak investigations have confirmed the association between HBGA expression and norovirus infection for some GI and GII strains (37, 39, 43, 49, 89). It remains likely that enzymes other than FUT2 may function as norovirus susceptibility factors because secretor-negative individuals have low-level norovirus-reactive antibodies (49, 52, 53) and can become infected after challenge with a GII.2 strain (52); in addition, some norovirus strains bind to FUT2-independent HBGAs in vitro (35, 54, 79).Early challenge studies (reviewed in reference 50) suggested that short-term protective immunity may occur following NV challenge (96). Demonstration of long-term protective immunity has been more complex. One early rechallenge study found that 50% of NV-challenged volunteers experienced repeat infections after ∼3 years while the other 50% remained well initially and after repeated challenge (69). Whether these volunteers remained disease free because of acquired immunity or genetic resistance could not be ascertained (69). However, contemporary norovirus challenge studies suggest that an early mucosal IgA response is associated with protection from NV infection (53). Further, strong gamma interferon (IFN-γ) secretion from CD4⁺ T cells (52) was identified in some uninfected GII.2-1976-challenged volunteers.In the absence of additional rechallenge studies, the most compelling evidence for a long-term protective immune response comes from the growing number of reports from around the world indicating that periods of “high norovirus activity” correlated with the emergence of new GII.4 strains (1, 10, 42, 66, 75, 90). Subsequently, the years following the high activity were characterized by decreased numbers of outbreaks, indicating that herd immunity may be an important regulator of GII.4 noroviruses (54, 80, 81). Clearly, the molecular basis for differential protective immunity/susceptibility following repeat norovirus infection is complex and a major challenge for the field.In this report, we compare the VLP phenotypes of the prototypical norovirus strain NV to an extant GI.1 strain isolated 33 years after NV and to a panel of VLPs representing strains GI.2, GI.3, and GI.4. In the results, we evaluate sequence conservation, carbohydrate (CHO) binding patterns, and antigenic relatedness at the antibody and T-cell levels. In contrast to earlier predictions (19), these data suggest that the GI noroviruses can bind many different HBGAs and that individuals infected with norovirus usually mount robust B- and T-cell responses against homologous strains. Surprisingly, some individuals appear to preferentially mount immune responses against heterologous GI strains.     

Nasal Immunization of Mice with Human Papillomavirus Type 16 Virus-Like Particles Elicits Neutralizing Antibodies in Mucosal Secretions

To specifically induce a mucosal antibody response to purified human papillomavirus type 16 (HPV16) virus-like particles (VLP), we immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital secretions were measured by enzyme-linked immunosorbent assay (ELISA). Systemic immunizations alone induced HPV16 VLP-specific IgG in serum and, to a lesser extent, in genital secretions but no secretory IgA. Oral immunization, even in the presence of CT, was inefficient. However, three nasal immunizations with 5 μg of VLP given at weekly intervals to anesthetized mice induced high (>10⁴) and long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all samples, including IgA and IgG in saliva and genital secretions. CT enhanced the VLP-specific antibody response 10-fold in serum and to a lesser extent in saliva and genital secretions. Nasal immunization of conscious mice compared to anesthetized mice was inefficient and correlated with the absence of uptake of a marker into the lung. However, a 1-μg VLP systemic priming followed by two 5-μg VLP intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG and IgA in secretions, although the titers were lower than in anesthetized mice given three intranasal immunizations. Antibodies in serum, saliva, and genital secretions of immunized mice were strongly neutralizing in vitro (50% neutralization with ELISA titers of 65 to 125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols that induced significant titers of neutralizing IgG and secretory IgA in mucosal secretions in mice may be relevant to genital HPV VLP-based human vaccine trials.     

Aptamer–Au NPs conjugates-enhanced SPR sensing for the ultrasensitive sandwich immunoassay

The goal of this work is to explore the amplification effect of aptamer–gold nanoparticles (Au NPs) conjugates for ultrasensitive detection of large biomolecules by surface plasmon resonance (SPR). A novel sandwich immunoassay is designed to demonstrate the amplification effect of aptamer–Au NPs conjugates by using human immunoglobulin E (IgE) as model analyte. Human IgE, captured by immobilized goat anti-human IgE on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 1 ng/ml after anti-human IgE aptamer–Au NPs conjugates is used as amplification reagent. Meanwhile, the non-specific adsorption of aptamer–Au NPs conjugates on goat anti-human IgE is confirmed by SPR spectroscopy and then it is minimized by treating aptamer–Au NPs conjugates with 6-mercaptohexan-1-ol (MCH). These results confirm that aptamer–Au NPs conjugates is a powerful sandwich element and an excellent amplification reagent for SPR-based sandwich immunoassay.     

Propofol Treatment Inhibits Constitutive Apoptosis in Human Primary Neutrophils and Granulocyte-Differentiated Human HL60 Cells

Apoptosis regulation is essential for neutrophil homeostasis. We previously demonstrated that a process involving glycogen synthase kinase (GSK)-3β determines neutrophil apoptosis. As for this apoptotic process, an overdose of propofol (2,6-Diisopropylphenol; 25 μg/ml or 140 μM) also causes GSK-3β-mediated macrophage apoptosis; however, the early deactivation of GSK-3β with low-dose propofol has been shown. Therefore, we hypothesize that low-dose propofol may induce neutrophil survival via GSK-3β inactivation. Following in vitro culture, the therapeutic concentration of propofol (10 μg/ml or 56 μM) treatment decreased constitutive apoptosis in isolated human primary neutrophils and in granulocyte-differentiated HL60 cells after all-trans retinoic acid (1 μM) treatment. The inactivation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT and the activation of GSK-3β results in myeloid cell leukemia 1 (Mcl-1) down-regulation, the loss of the mitochondrial transmembrane potential, and caspase-3 activation in these cells, which is accompanied by apoptosis. Notably, propofol treatment attenuates these effects in a PI3-kinase-regulated manner. We found that propofol initiates PI3-kinase/AKT-mediated GSK-3β inactivation and Mcl-1 stabilization, rescuing the constitutive apoptosis in primary neutrophils and granulocyte-differentiated acute promyelocytic leukemia HL60 cells.     

Detection of Phosphatidylcholine-Coated Gold Nanoparticles in Orthotopic Pancreatic Adenocarcinoma using Hyperspectral Imaging

Nanoparticle uptake and distribution to solid tumors are limited by reticuloendothelial system systemic filtering and transport limitations induced by irregular intra-tumoral vascularization. Although vascular enhanced permeability and retention can aid targeting, high interstitial fluid pressure and dense extracellular matrix may hinder local penetration. Extravascular diffusivity depends upon nanoparticle size, surface modifications, and tissue vascularization. Gold nanoparticles functionalized with biologically-compatible layers may achieve improved uptake and distribution while enabling cytotoxicity through synergistic combination of chemotherapy and thermal ablation. Evaluation of nanoparticle uptake in vivo remains difficult, as detection methods are limited. We employ hyperspectral imaging of histology sections to analyze uptake and distribution of phosphatidylcholine-coated citrate gold nanoparticles (CGN) and silica-gold nanoshells (SGN) after tail-vein injection in mice bearing orthotopic pancreatic adenocarcinoma. For CGN, the liver and tumor showed 26.5±8.2 and 23.3±4.1 particles/100μm² within 10μm from the nearest source and few nanoparticles beyond 50μm, respectively. The spleen had 35.5±9.3 particles/100μm² within 10μm with penetration also limited to 50μm. For SGN, the liver showed 31.1±4.1 particles/100μm² within 10μm of the nearest source with penetration hindered beyond 30μm. The spleen and tumor showed uptake of 22.1±6.2 and 15.8±6.1 particles/100μm² within 10μm, respectively, with penetration similarly hindered. CGH average concentration (nanoparticles/μm²) was 1.09±0.14 in the liver, 0.74±0.12 in the spleen, and 0.43±0.07 in the tumor. SGN average concentration (nanoparticles/μm²) was 0.43±0.07 in the liver, 0.30±0.06 in the spleen, and 0.20±0.04 in the tumor. Hyperspectral imaging of histology sections enables analysis of phosphatidylcholine-coated gold-based nanoparticles in pancreatic tumors with the goal to improve nanotherapeutic efficacy.     

硫化铜纳米粒子在肿瘤成像与治疗中的功能研究进展

硫化铜是一种二价铜的硫化物,可以作为半导体材料,化学式为CuS,呈黑褐色,溶解度极低。硫化铜纳米粒子(Copper sulfide nanoparticles, CuS NPs)是纳米尺度大小的硫化铜。近年来,CuS NPs因其结构的可塑性,良好的光热稳定性、生物相容性、突出的光热及光声转换性能,成为了当今纳米材料医学领域的研究热点,在肿瘤诊断和治疗领域中引起了广泛关注。CuS NPs本身可通过介质鳌合金属离子合成多功能纳米粒子,实现肿瘤多模式诊断,并且在光热治疗研究中体现出突出的治疗效果。本文综述了近几年CuS NPs在肿瘤诊断与治疗方面的研究进展,总结肿瘤治疗中的应用研究方法,对CuS NPs在生物医学领域应用中存在的问题进行分析,为解决实际操作过程所遇到的问题提供参考。     

Silver Nanoparticles Decrease the Viability of Cryptosporidium parvum Oocysts

Oocysts of the waterborne protozoan parasite Cryptosporidium parvum are highly resistant to chlorine disinfection. We show here that both silver nanoparticles (AgNPs) and silver ions significantly decrease oocyst viability, in a dose-dependent manner, between concentrations of 0.005 and 500 μg/ml, as assessed by an excystation assay and the shell/sporozoite ratio. For percent excystation, the results are statistically significant for 500 μg/ml of AgNPs, with reductions from 83% for the control to 33% with AgNPs. For Ag ions, the results were statistically significant at 500 and 5,000 μg/ml, but the percent excystation values were reduced only to 66 and 62%, respectively, from 86% for the control. The sporozoite/shell ratio was affected to a greater extent following AgNP exposure, presumably because sporozoites are destroyed by interaction with NPs. We also demonstrated via hyperspectral imaging that there is a dual mode of interaction, with Ag ions entering the oocyst and destroying the sporozoites while AgNPs interact with the cell wall and, at high concentrations, are able to fully break the oocyst wall.     

Absolute Humidity Influences the Seasonal Persistence and Infectivity of Human Norovirus

Norovirus (NoV) is one of the main causative agents of acute gastroenteritis worldwide. In temperate climates, outbreaks peak during the winter season. The mechanism by which climatic factors influence the occurrence of NoV outbreaks is unknown. We hypothesized that humidity is linked to NoV seasonality. Human NoV is not cultivatable, so we used cultivatable murine norovirus (MNV) as a surrogate to study its persistence when exposed to various levels of relative humidity (RH) from low (10% RH) to saturated (100% RH) conditions at 9 and 25°C. In addition, we conducted similar experiments with virus-like particles (VLPs) from the predominant GII-4 norovirus and studied changes in binding patterns to A, B, and O group carbohydrates that might reflect capsid alterations. The responses of MNV and VLP to humidity were somewhat similar, with 10 and 100% RH exhibiting a strong conserving effect for both models, whereas 50% RH was detrimental for MNV infectivity and VLP binding capacity. The data analysis suggested that absolute humidity (AH) rather than RH is the critical factor for keeping NoV infectious, with an AH below 0.007 kg water/kg air being favorable to NoV survival. Retrospective surveys of the meteorological data in Paris for the last 14 years showed that AH average values have almost always been below 0.007 kg water/kg air during the winter (i.e., 0.0046 ± 0.0014 kg water/kg air), and this finding supports the fact that low AH provides an ideal condition for NoV persistence and transmission during cold months.