Two-component in situ forming supramolecular hydrogels as advanced biomaterials in vitreous body surgery

Polymeric β-CD and poly{(2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt)-co-[6-(acrylamido)-N-adamantylhexaneamide]} are synthesized to build in situ forming hydrogels based on host/guest interactions, so called physical hydrogels. The use of these hydrogels as a potential vitreous body substitute is discussed and recommended. Potential changes in cell morphology and cell vitality of the retinal ganglion cell line RGC-5 are determined. DSC experiments with artificial membrane structures are performed. The analyses show that β-CD overrides the harmful effects of the highly toxic adamantyl-modified polymer. Although the final hydrogel is considered to be biocompatible, the application as a biomaterial has to be reconsidered.     

Identifying true protein complex constituents in interaction proteomics: The example of the DMXL2 protein complex

A typical high-sensitivity antibody affinity purification-mass spectrometry experiment easily identifies hundreds of protein interactors. However, most of these are non-valid resulting from multiple causes other than interaction with the bait protein. To discriminate true interactors from off-target recognition, we propose to differentially include an (peptide) antigen during the antibody incubation in the immuno-precipitation experiment. This contrasts the specific antibody-bait protein interactions, versus all other off-target protein interactions. To exemplify the power of the approach, we studied the DMXL2 interactome. From the initial six immuno-precipitations, we identified about 600 proteins. When filtering for interactors present in all anti-DMXL2 antibody immuno-precipitation experiments, absent in the bead controls, and competed off by the peptide antigen, this hit list is reduced to ten proteins, including known and novel interactors of DMXL2. Together, our approach enables the use of a wide range of available antibodies in large-scale protein interaction proteomics, while gaining specificity of the interactions.     

Thermal stability of structurally different viruses with proven or potential relevance to food safety

Aims: To collect comparative data on thermal stability of structurally different viruses with proven or potential relevance to food safety. Methods and Results: Suspensions with poliovirus Sabin1, adenovirus type5, parechovirus1, human norovirus (NoV) GII.4, murine NoV (MNV1) and human influenza A (H1N1) viruses were heated at 56 and 73°C. Infectivity was tested by culture assay for all but human NoV GII.4 that cannot be cultivated in vitro. Time to first log₁₀ reduction (TFL‐value) was calculated based on best fit using the monophasic, biphasic or Weibull models. The Weibull model provided the best fit at 56°C for all viruses except influenza virus. The TFL at 56°C varied between a high of 27 min (parechovirus) to a low of 10 s (adenovirus) and ranked parechovirus > influenza > MNV1 > poliovirus > adenovirus. The monophasic model best described the behaviour of the viruses at 73°C, in which case the TFL was MNV1(62s) > influenza > adenovirus > parechovirus > poliovirus(14s). Conclusions: Viruses do not follow log‐linear thermal inactivation kinetics and the thermostability of parechovirus and influenza virus is similar to that of proven foodborne viruses. Significance and Impact of the Study: Resistant fractions of viruses may remain infectious in thermal inactivation processes and inactivation of newly discovered or enveloped viruses in thermal food preparation processes should not be assumed without further testing.     

Shedding of Vaccine Viruses with Increased Antigenic and Genetic Divergence after Vaccination of Newborns with Monovalent Type 1 Oral Poliovirus Vaccine

For the final stages in the eradication of poliovirus type 1 (P1), the World Health Organization advocates the selective use of monovalent type 1 oral poliovirus vaccine (mOPV1). To compare the immunogenicity of mOPV1 with that of trivalent OPV (tOPV) in infants, a study was performed in Egypt in 2005. Newborns were vaccinated with mOPV1 or tOPV immediately after birth and were challenged with mOPV1 after 1 month. Vaccination with mOPV1 at birth resulted in significantly higher seroconversion against P1 viruses and lower excretion of P1 viruses than vaccination with tOPV. Intratypic differentiation of the viruses shed by the newborns revealed the presence of remarkably high numbers of antigenically divergent (AD) P1 isolates, especially in the mOPV1 study group. The majority of these AD P1 isolates (71%) were mOPV1 challenge derived and were shed by newborns who did not seroconvert to P1 after the birth dose. Genetic characterization of the viruses revealed that amino acid 60 of the VP3 region was mutated in all AD P1 isolates. Isolates with substitution of residue 99 of the VP1 region had significantly higher numbers of nonsynonymous mutations in the VP1 region than isolates without this substitution and were preferentially shed in the mOPV1 study group. The widespread use of mOPV1 has proven to be a powerful tool for fighting poliovirus circulation in the remaining areas of endemicity. This study provides another justification for the need to achieve high vaccination coverage in order to prevent the circulation of AD strains.Polioviruses are the causative agents of human poliomyelitis and belong to the genus Enterovirus in the family Picornaviridae. The virus is transmitted primarily by the fecal-oral route and replicates in the human intestinal tract. The virus may also be transmitted through respiratory droplets and may replicate for a short period in the upper respiratory tract and tonsillar tissue. From either site of primary replication, the virus may invade the central nervous system and cause paralysis following infection and destruction of motor neurons. Three serologically different types of poliovirus can be distinguished (poliovirus type 1 [P1], P2, and P3), and only limited cross-protection exists between serotypes (35).In 1988, the World Health Assembly passed a resolution to eradicate wild poliovirus globally. A worldwide vaccination campaign with the trivalent oral poliovirus vaccine (tOPV) was launched by the World Health Organization (WHO). This vaccine contains the three attenuated poliovirus vaccine strains developed by Albert Sabin in the proportion of 10:1:6 for P1, P2, and P3, respectively. These OPV strains have been selected to replicate successfully in the human intestinal tract but not in the cells of the central nervous system. In addition to a strong humoral response, these strains generate strong intestinal immunity (12). Sabin type 1 is considered to be the most stable of the three attenuated poliovirus serotypes (19). This strain has 54 mutations compared to the parental Mahoney strain, of which 6 are primarily responsible for attenuation. Sabin type 2 has two major determinants of attenuation, and Sabin type 3 has three determinants of attenuation (11, 32). Upon replication in the human intestinal tract, the sites of attenuation can mutate, which results in reversion of the Sabin strains toward a parental neurovirulent phenotype. Also as a consequence of replication in the host, antibodies are produced that recognize the antigenic sites of the Sabin strains (42). This immunogenic pressure could favor the selection of antigenically divergent (AD) viruses with substituted residues in parts of these antigenic sites. AD Sabin viruses might circulate among a population for a long period and evolve into vaccine-derived polioviruses (VDPVs; with differences of >1% from the prototype Sabin viruses in the VP1 region) capable of causing outbreaks. These viruses might escape current diagnostic screening methods, and the risk for generation of these viruses should be reduced as much as possible (1, 9, 16).The tOPV vaccination campaigns have been very successful, since the number of countries with endemic wild poliovirus circulation decreased from >125 in 1988 to 4 in 2006, and wild type 2 poliovirus has likely been eradicated since 1999 (5). The tOPV vaccine, however, is known to be less immunogenic against type 1 and 3 polioviruses. After tOPV administration, the superior replicative capacity of the P2 vaccine strain interferes with effective replication of the other two serotype viruses in the human intestine (30). To eradicate wild P1 as well, vaccination with monovalent type 1 oral poliovirus vaccine (mOPV1) was introduced in the remaining countries where poliovirus is endemic, since this vaccine is more immunogenic for type 1 than the tOPV (4, 20).In 2005/2006, a clinical study was conducted in Egypt to compare the immunogenicity of mOPV1 with that of the tOPV in newborns (15). Newborns were vaccinated with mOPV1 or tOPV as soon as possible after birth and were challenged with mOPV1 4 weeks later. Vaccination with mOPV1 at birth resulted in a higher humoral and mucosal protection against P1 at day 28 than vaccination with tOPV at birth.In line with the recommendations of the WHO Polio Laboratory Network, we determined the antigenic characters of all the viruses shed by the newborns of the Egyptian study by using an intratypic differentiation (ITD) enzyme-linked immunosorbent assay (ELISA). The outcome of this analysis, an unexpectedly high percentage of AD isolates, prompted further investigation. To determine the possible presence of VDPVs and to gain insight into the genetic and antigenic evolution of the mOPV1 and tOPV isolates shed by the newborns in this study, we determined the sequences of the capsid regions of these isolates. We looked for correlates with antigenic change and rates of mutagenesis in the viruses and compared the evolution rates of the viruses shed by vaccinees of both study groups. We also linked the serological data collected during the study to the excretion of Sabin 1 isolates.     

Single-Molecule Microscopy Reveals Membrane Microdomain Organization of Cells in a Living Vertebrate

It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.     

Bioproduction and characterization of a pH responsive self‐assembling peptide

Peptide P₁₁‐4 (QQRFEWEFEQQ) was designed to self‐assemble to form β‐sheets and nematic gels in the pH range 5–7 at concentrations ≥12.6 mM in water. This self‐assembly is reversibly controlled by adjusting the pH of the solvent. It can also self‐assemble into gels in biological media. This together with its biocompatibility and biodegradability make P₁₁‐4 an attractive building block for the fabrication of nanoscale materials with uses in, for example, tissue engineering. A limitation to large‐scale production of such peptides is the high cost of solid phase chemical synthesis. We describe expression of peptide P₁₁‐4 in the bacterium Escherichia coli from constructs carrying tandem repeats of the peptide coding sequence. The vector pET31b+ was used to express P₁₁‐4 repeats fused to the ketosteroid isomerase protein which accumulates in easily recoverable inclusion bodies. Importantly, the use of auto‐induction growth medium to enhance cell density and protein expression levels resulted in recovery of 2.5 g fusion protein/L culture in both shake flask and batch fermentation. Whole cell detergent lysis allowed recovery of inclusion bodies largely composed of the fusion protein. Cyanogen bromide cleavage followed by reverse phase HPLC allowed purification of the recombinant peptide with a C‐terminal homoserine lactone (rP₁₁‐4(hsl)). This recombinant peptide formed pH dependent hydrogels, displayed β‐structure measured by circular dichroism and fibril formation observed by transmission electron microscopy. Biotechnol. Bioeng. 2009;103: 241–251. © 2009 Wiley Periodicals, Inc.     

Transmission of Novel Influenza A(H1N1) in Households with Post-Exposure Antiviral Prophylaxis

Background

Despite impressive advances in our understanding of the biology of novel influenza A(H1N1) virus, little is as yet known about its transmission efficiency in close contact places such as households, schools, and workplaces. These are widely believed to be key in supporting propagating spread, and it is therefore of importance to assess the transmission levels of the virus in such settings.

Methodology/Principal Findings

We estimate the transmissibility of novel influenza A(H1N1) in 47 households in the Netherlands using stochastic epidemic models. All households contained a laboratory confirmed index case, and antiviral drugs (oseltamivir) were given to both the index case and other households members within 24 hours after detection of the index case. Among the 109 household contacts there were 9 secondary infections in 7 households. The overall estimated secondary attack rate is low (0.075, 95%CI: 0.037–0.13). There is statistical evidence indicating that older persons are less susceptible to infection than younger persons (relative susceptibility of older persons: 0.11, 95%CI: 0.024–0.43. Notably, the secondary attack rate from an older to a younger person is 0.35 (95%CI: 0.14–0.61) when using an age classification of ≤12 versus >12 years, and 0.28 (95%CI: 0.12–0.50) when using an age classification of ≤18 versus >18 years.

Conclusions/Significance

Our results indicate that the overall household transmission levels of novel influenza A(H1N1) in antiviral-treated households were low in the early stage of the epidemic. The relatively high rate of adult-to-child transmission indicates that control measures focused on this transmission route will be most effective in minimizing the total number of infections.     

Inactivation of Caliciviruses

The viruses most commonly associated with food- and waterborne outbreaks of gastroenteritis are the noroviruses. The lack of a culture method for noroviruses warrants the use of cultivable model viruses to gain more insight on their transmission routes and inactivation methods. We studied the inactivation of the reported enteric canine calicivirus no. 48 (CaCV) and the respiratory feline calicivirus F9 (FeCV) and correlated inactivation to reduction in PCR units of FeCV, CaCV, and a norovirus. Inactivation of suspended viruses was temperature and time dependent in the range from 0 to 100°C. UV-B radiation from 0 to 150 mJ/cm² caused dose-dependent inactivation, with a 3 D (D = 1 log₁₀) reduction in infectivity at 34 mJ/cm² for both viruses. Inactivation by 70% ethanol was inefficient, with only 3 D reduction after 30 min. Sodium hypochlorite solutions were only effective at >300 ppm. FeCV showed a higher stability at pH <3 and pH >7 than CaCV. For all treatments, detection of viral RNA underestimated the reduction in viral infectivity. Norovirus was never more sensitive than the animal caliciviruses and profoundly more resistant to low and high pH. Overall, both animal viruses showed similar inactivation profiles when exposed to heat or UV-B radiation or when incubated in ethanol or hypochlorite. The low stability of CaCV at low pH suggests that this is not a typical enteric (calici-) virus. The incomplete inactivation by ethanol and the high hypochlorite concentration needed for sufficient virus inactivation point to a concern for decontamination of fomites and surfaces contaminated with noroviruses and virus-safe water.