Crosslinking of phenylboronic acid receptors as a means of glucose selective holographic detection

A holographic sensor for the detection of glucose has been developed that is based on a hydrogel film containing phenylboronic acid receptors. Changes to the replay wavelength of the hologram were used to characterise the swelling and de-swelling behaviour of the hydrogel matrix upon receptor-ligand binding. The effect of introducing a fixed positive charge into the polymer matrix by modification of the hydrogel with a quaternary amine group (3-acrylamidopropyl)trimethylammonium chloride (ATMA), was investigated for a range of sugars and the alpha-hydroxy acid, lactate, at physiological pH. The quaternary amine-modified hydrogel matrix was found to contract in the presence of glucose, whereas, it was minimally responsive to other saccharides. The selectivity of the sensor for glucose compared to lactate was also significantly improved compared to the unmodified film. A crosslinking mechanism is proposed to explain the enhanced selectivity to glucose.     

The IKKα-dependent NF-κB p52/RelB noncanonical pathway is essential to sustain a CXCL12 autocrine loop in cells migrating in response to HMGB1

HMGB1 is a chromatin architectural protein that is released by dead or damaged cells at sites of tissue injury. Extracellular HMGB1 functions as a proinflammatory cytokine and chemoattractant for immune effector and progenitor cells. Previously, we have shown that the inhibitor of NF-κB kinase (IKK)β- and IKKα-dependent NF-κB signaling pathways are simultaneously required for cell migration to HMGB1. The IKKβ-dependent canonical pathway is needed to maintain expression of receptor for advanced glycation end products, the ubiquitously expressed receptor for HMGB1, but the target of the IKKα non-canonical pathway was not known. In this study, we show that the IKKα-dependent p52/RelB noncanonical pathway is critical to sustain CXCL12/SDF1 production in order for cells to migrate toward HMGB1. Using both mouse bone marrow-derived macrophages and mouse embryo fibroblasts (MEFs), it was observed that neutralization of CXCL12 by a CXCL12 mAb completely eliminated chemotaxis to HMGB1. In addition, the HMGB1 migration defect of IKKα KO and p52 KO cells could be rescued by adding recombinant CXCL12 to cells. Moreover, p52 KO MEFs stably transduced with a GFP retroviral vector that enforces physiologic expression of CXCL12 also showed near normal migration toward HMGB1. Finally, both AMD3100, a specific antagonist of CXCL12's G protein-coupled receptor CXCR4, and an anti-CXCR4 Ab blocked HMGB1 chemotactic responses. These results indicate that HMGB1-CXCL12 interplay drives cell migration toward HMGB1 by engaging receptors of both chemoattractants. This novel requirement for a second receptor-ligand pair enhances our understanding of the molecular mechanisms regulating HMGB1-dependent cell recruitment to sites of tissue injury.     

Genetic Inactivation of Poliovirus Infectivity by Increasing the Frequencies of CpG and UpA Dinucleotides within and across Synonymous Capsid Region Codons

Replicative fitness of poliovirus can be modulated systematically by replacement of preferred capsid region codons with synonymous unpreferred codons. To determine the key genetic contributors to fitness reduction, we introduced different sets of synonymous codons into the capsid coding region of an infectious clone derived from the type 2 prototype strain MEF-1. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased sharply with increased frequencies of the dinucleotides CpG (suppressed in higher eukaryotes and most RNA viruses) and UpA (suppressed nearly universally). Replacement of MEF-1 capsid codons with the corresponding codons from another type 2 prototype strain (Lansing), a randomization of MEF-1 synonymous codons, increased the %G+C without increasing CpG, and reductions in the effective number of codons used had much smaller individual effects on fitness. Poliovirus fitness was reduced to the threshold of viability when CpG and UpA dinucleotides were saturated within and across synonymous codons of a capsid region interval representing only ∼9% of the total genome. Codon replacements were associated with moderate decreases in total virion production but large decreases in the specific infectivities of intact poliovirions and viral RNAs. Replication of codon replacement viruses, but not MEF-1, was temperature sensitive at 39.5°C. Synthesis and processing of viral intracellular proteins were largely unaltered in most codon replacement constructs. Replacement of natural codons with synonymous codons with increased frequencies of CpG and UpA dinucleotides may offer a general approach to the development of attenuated vaccines with well-defined antigenicities and very high genetic stabilities.Diversification of genomic sequences is constrained in all biological systems. At the level of primary sequences, the range of variability in coding regions is restricted by the codon usage bias (CUB), whereby a subset of synonymous codons are preferentially used in translation (24, 53, 69). The intensity of the CUB and the specific set of preferred codons vary widely across biological systems (39). Intertwined with the CUB is the suppression of the dinucleotides CpG and TpA (or UpA in RNA viruses) in the genomes of higher eukaryotes (4, 7, 26, 61) and many of their RNA viruses and small DNA viruses (28, 49). Variation in the primary sequences of RNA virus genomes is further constrained by requirements to maintain essential secondary and higher-order structures (42, 54, 68).We previously described the modulation of the replicative fitness of the Sabin type 2 oral poliovirus vaccine (OPV) strain (Sabin 2) by systematically changing the CUB in the capsid region, replacing the naturally occurring preferred codons with an unpreferred synonymous codon (isocodon) for each of nine amino acids (8). We called our approach “codon deoptimization” to contrast with the process of codon optimization, which is frequently used to maximize expression of foreign proteins in heterologous host systems (1, 27, 70). Apart from its potential application to development of improved poliovirus vaccines (8, 13, 38), experimental investigations of codon deoptimization directly test the relationships between replicative fitness, the extent of CUB, and the intensity of CpG and UpA suppression. As a model system for such studies, polioviruses offer several favorable properties, including (i) intrinsically high error rates for the poliovirus RNA-dependent RNA polymerase (2, 14, 16, 65), (ii) very high evolution rates (25), (iii) short generation times (8 to 10 h) and large progeny yields of prototype polioviruses, and (iv) well-developed reverse genetics (9).In this report, we extend our codon deoptimization strategy to the type 2 wild poliovirus prototype strain MEF-1. As before, we restricted our replacement of synonymous codons to the capsid coding region, which encodes two of the defining properties of polioviruses, namely, (i) the capacity to bind the CD155 poliovirus receptor (PVR) (23) and (ii) the poliovirus type-specific neutralizing antigenic sites (35). No changes were made to the flanking 5′-untranslated region and noncapsid region sequences, as they contain essential secondary structural elements (42, 54, 68) and are frequently exchanged out by recombination during circulation of poliovirus in human populations (20, 30, 32). MEF-1 was selected because of its high fitness level (hence, its use as the type 2 component of the inactivated poliovirus vaccine [IPV]) and because of its neurovirulence for humans (15), for nontransgenic mice (52), and for transgenic mice expressing the PVR (71). Type 2 polioviruses were selected first for study because the Sabin 2 OPV strain is most frequently associated with vaccine-associated paralytic poliomyelitis in contacts of OPV recipients (57, 59), with prolonged excretion of immunodeficiency-associated vaccine-derived polioviruses (VDPVs) (10, 31, 60), and with the emergence of circulating VDPVs in areas of low OPV coverage (10, 31).Consistent with our previous findings, the fitness of MEF-1 decreased in proportion to the total number of synonymous replacement codons. Fitness was reduced most efficiently by increasing the frequencies of CpG and UpA dinucleotides within and across synonymous codons. Saturation of CpG and UpA in a small capsid interval (representing only ∼9% of the genome) reduced fitness to the threshold of viability, even though the MEF-1 amino acid sequence was unaltered. The most prominent biological effect of deoptimization of codon usage and the large-scale incorporation of CpG and UpA was a sharp reduction in virus specific infectivities. In contrast, translation and processing of viral proteins and yields of intact virus particles with native antigenicities were reduced only moderately by increased CpG and UpA frequencies. Codon deoptimization with concurrent increases in the frequencies of CpG and/or UpA dinucleotides in RNA virus genomes may provide a novel general approach to the rational design of improved attenuated vaccines with predictable and stable genetic properties.     

Natural type 3/type 2 intertypic vaccine-related poliovirus recombinants with the first crossover sites within the VP1 capsid coding region

Background

Ten uncommon natural type 3/type 2 intertypic poliovirus recombinants were isolated from stool specimens from nine acute flaccid paralysis case patients and one healthy vaccinee in China from 2001 to 2008.

Principal Findings

Complete genomic sequences revealed their vaccine-related genomic features and showed that their first crossover sites were randomly distributed in the 3′ end of the VP1 coding region. The length of donor Sabin 2 sequences ranged from 55 to 136 nucleotides, which is the longest donor sequence reported in the literature for this type of poliovirus recombination. The recombination resulted in the introduction of Sabin 2 neutralizing antigenic site 3a (NAg3a) into a Sabin 3 genomic background in the VP1 coding region, which may have been altered by some of the type 3-specific antigenic properties, but had not acquired any type 2-specific characterizations. NAg3a of the Sabin 3 strain seems atypical; other wild-type poliovirus isolates that have circulated in recent years have sequences of NAg3a more like the Sabin 2 strain.

Conclusions

10 natural type 3/type 2 intertypic VP1 capsid-recombinant polioviruses, in which the first crossover sites were found to be in the VP1 coding region, were isolated and characterized. In spite of the complete replacement of NAg3a by type 2-specific amino acids, the serotypes of the recombinants were not altered, and they were totally neutralized by polyclonal type 3 antisera but not at all by type 2 antisera. It is possible that recent type 3 wild poliovirus isolates may be a recombinant having NAg3a sequences derived from another strain during between 1967 and 1980, and the type 3/type 2 recombination events in the 3′ end of the VP1 coding region may result in a higher fitness.     

Estimating the extent of vaccine-derived poliovirus infection

Background

Eight outbreaks of paralytic polio attributable to circulating vaccine-derived poliovirus (cVDPV) have highlighted the risks associated with oral poliovirus vaccine (OPV) use in areas of low vaccination coverage and poor hygiene. As the Polio Eradication Initiative enters its final stages, it is important to consider the extent to which these viruses spread under different conditions, so that appropriate strategies can be devised to prevent or respond to future cVDPV outbreaks.

Methods and Findings

This paper examines epidemiological (temporal, geographic, age, vaccine history, social group, ascertainment), and virological (type, genetic diversity, virulence) parameters in order to infer the numbers of individuals likely to have been infected in each of these cVDPV outbreaks, and in association with single acute flaccid paralysis (AFP) cases attributable to VDPVs. Although only 114 virologically-confirmed paralytic cases were identified in the eight cVDPV outbreaks, it is likely that a minimum of hundreds of thousands, and more likely several million individuals were infected during these events, and that many thousands more have been infected by VDPV lineages within outbreaks which have escaped detection.

Conclusions

Our estimates of the extent of cVDPV circulation suggest widespread transmission in some countries, as might be expected from endemic wild poliovirus transmission in these same settings. These methods for inferring extent of infection will be useful in the context of identifying future surveillance needs, planning for OPV cessation and preparing outbreak response plans.     

Synthesis and characterization of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives: Part 3. New potent non-competitive metabotropic glutamate receptor 2/3 antagonists

A series of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives was evaluated as non-competitive mGluR2/3 antagonists. Replacement of the (2-aryl)-ethynyl-moiety in 8-position with smaller less lipophilic substituents produced compounds inhibiting the binding of [3H]-LY354740 to rat mGluR2 with low nanomolar affinity and consistent functional effect at both mGluR2 and mGluR3. These compounds were able to reverse LY354740-mediated inhibition of field excitatory postsynaptic potentials in the rat dentate gyrus and in vivo activity could be demonstrated by reversal of the LY354740-induced hypoactivity in mice after oral administration.     

Calibration of multiple poliovirus molecular clocks covering an extended evolutionary range

We have calibrated five different molecular clocks for circulating poliovirus based upon the rates of fixation of total substitutions (K(t)), synonymous substitutions (K(s)), synonymous transitions (A(s)), synonymous transversions (B(s)), and nonsynonymous substitutions (K(a)) into the P1/capsid region (2,643 nucleotides). Rates were determined over a 10-year period by analysis of sequences of 31 wild poliovirus type 1 isolates representing a well-defined phylogeny derived from a common imported ancestor. Similar rates were obtained by linear regression, the maximum likelihood/single-rate dated-tip method, and Bayesian inference. The very rapid K(t) [(1.03 +/- 0.10) x 10(-2) substitutions/site/year] and K(s) [(1.00 +/- 0.08) x 10(-2)] clocks were driven primarily by the A(s) clock [(0.96 +/- 0.09) x 10(-2)], the B(s) clock was approximately 10-fold slower [(0.10 +/- 0.03) x 10(-2)], and the more stochastic K(a) clock was approximately 30-fold slower [(0.03 +/- 0.01) x 10(-2)]. Nonsynonymous substitutions at all P1/capsid sites, including the neutralizing antigenic sites, appeared to be constrained by purifying selection. Simulation of the evolution of third-codon positions suggested that saturation of synonymous transitions would be evident at 10 years and complete at approximately 65 years of independent transmission. Saturation of synonymous transversions was predicted to be minimal at 20 years and incomplete at 100 years. The rapid evolution of the K(t), K(s), and A(s) clocks can be used to estimate the dates of divergence of closely related viruses, whereas the slower B(s) and K(a) clocks may be used to explore deeper evolutionary relationships within and across poliovirus genotypes.     

Evolution of circulating wild poliovirus and of vaccine-derived poliovirus in an immunodeficient patient: a unifying model

We determined nucleotide sequences of the VP1 and 2AB genes and portions of the 2C and 3D genes of two evolving poliovirus lineages: circulating wild viruses of T geotype and Sabin vaccine-derived isolates from an immunodeficient patient. Different regions of the viral RNA were found to evolve nonsynchronously, and the rate of evolution of the 2AB region in the vaccine-derived population was not constant throughout its history. Synonymous replacements occurred not completely randomly, suggesting the need for conservation of certain rare codons (possibly to control translation elongation) and the existence of unidentified constraints in the viral RNA structure. Nevertheless the major contribution to the evolution of the two lineages came from linear accumulation of synonymous substitutions. Therefore, in agreement with current theories of viral evolution, we suggest that the majority of the mutations in both lineages were fixed as a result of successive sampling, from the heterogeneous populations, of random portions containing predominantly neutral and possibly adverse mutations. As a result of such a mode of evolution, the virus fitness may be maintained at a more or less constant level or may decrease unless more-fit variants are stochastically generated. The proposed unifying model of natural poliovirus evolution has important implications for the epidemiology of poliomyelitis.