Ceramic Bonding Interface under Shear-Compression Stress: Ultra-High-Speed Imaging Contribution.

The aim of this study is to visualize and characterize by ultra-high-speed imaging (UHSI) the failure phenomena at the resin-ceramic bonding interface of lithium disilicate (LiSi2) samples bonded with gold-standard protocol (Monobond Plus [MB]) and the nontoxic one (Monobond Etch & Prime [MEP]) subjected to mechanical loading. Unprecedented frame rate, image resolution, and recording time were reached by using the most advanced UHSI camera. The finite element analysis (FEA) of the proposed mechanical test confirmed that the specific design of our samples enables a combined shear and compression stress state, prone to test the bonding interface while being close to physiological stresses. Ten LiSi2 samples were pretreated by gold standard (MB, n = 5) and self-etching primer (MEP, n = 5). Axial compression loading gradually increased until catastrophic failure was performed. As shown by the FEA, the angle between the bonding interface and load direction leads to shear-compression stresses at the resin-ceramic bonding interface. Failure was recorded by UHSI at 300,000 fps. All recorded images were analyzed to segregate events and isolate the origin of fracture. For the first time, thanks to the image recording setup, it was observed that debonding is the first event before breakage, highlighting that sample fracture occurs by interfacial rupture followed by slippage and cohesive failure of materials. Failure mode could be described as mixed. MEP and MB showed similar results and behavior.

Current practice of French health professionals regarding Japanese encephalitis vaccination.

OBJECTIVE: Japanese encephalitis (JE) is a rare but severe disease. It is a potential threat for people traveling to endemic areas. The risk of developing JE is low (<1%), but the associated case fatality is high (30%). There is no specific treatment for JE, but a vaccine is available. We performed an observational survey to assess practices of French health professionals regarding JE vaccination. METHODS: Standardized questionnaires were sent by email to a sample of French health professionals practicing in vaccination centers. Participation was on a voluntary and anonymous basis. The questionnaires requested socio-demographic details, and included multiple choice questions. RESULTS: The response rate was 38.5%. Most participating health professionals had been working for more than three years in a vaccination center and declared not to be reluctant to perform JE vaccination. Reluctance was mostly based on the vaccine cost and on the difficulty to properly assess the risk for patients. The rapid protocol was largely preferred except in the overseas regions (P<0.05, Fisher's exact test). Traveling to South Asia and backpacking were considered at-risk conditions. Participants proposed the vaccination all year round. Most participants would not have proposed the JE vaccination for the concrete case outlined in the questionnaire. CONCLUSIONS: French health professionals are globally favorable to JE vaccination. However, assessing the risk of exposure is difficult in routine practice.

Constitutive modeling of stress softening and permanent set in a porcine skin tissue: Impact of the storage preservation.

Prior to testing, soft tissues are usually maintained in different media and additives (solution, air, cryopreservant…) under various environment conditions (temperature, storage duration….). In many cases, results from mechanical tests performed on these stored tissues are supposed to be as closed as possible to the fresh ones. In the present work, cyclic tensile tests were performed with increasing values of strain on porcine skin tissues (excised following the Langer's lines) to enhance tissues mechanical nonlinearity such as softening behavior and permanent set. Optical methods were used to follow the in-plane strains evolution. These latest values were used as data to simulate the structural behavior of these heterogeneous materials. The numerical simulation is based on the constitutive pseudo-elastic model accounting for the softening behavior as well as the permanent set. As a result, reliable material parameters were extracted from the experiments/model comparison for each storage solution. The result of this study reveals that preservation conditions must be carefully chosen: at low strain the tissues store in fridge in a saline solution during a short time, or in freezer (-80°C) in water with cryopreservant and the fresh one lead to a similar mechanical response. For larger strain, the freezing (-80°C) in water with cryopreservant is the only procedure for which the tissue recovers its initial behavior.

Advances in Genomics of Entomopathogenic Fungi.

Fungi are the commonest pathogens of insects and crucial regulators of insect populations. The rapid advance of genome technologies has revolutionized our understanding of entomopathogenic fungi with multiple Metarhizium spp. sequenced, as well as Beauveria bassiana, Cordyceps militaris, and Ophiocordyceps sinensis among others. Phylogenomic analysis suggests that the ancestors of many of these fungi were plant endophytes or pathogens, with entomopathogenicity being an acquired characteristic. These fungi now occupy a wide range of habitats and hosts, and their genomes have provided a wealth of information on the evolution of virulence-related characteristics, as well as the protein families and genomic structure associated with ecological and econutritional heterogeneity, genome evolution, and host range diversification. In particular, their evolutionary transition from plant pathogens or endophytes to insect pathogens provides a novel perspective on how new functional mechanisms important for host switching and virulence are acquired. Importantly, genomic resources have helped make entomopathogenic fungi ideal model systems for answering basic questions in parasitology, entomology, and speciation. At the same time, identifying the selective forces that act upon entomopathogen fitness traits could underpin both the development of new mycoinsecticides and further our understanding of the natural roles of these fungi in nature. These roles frequently include mutualistic relationships with plants. Genomics has also facilitated the rapid identification of genes encoding biologically useful molecules, with implications for the development of pharmaceuticals and the use of these fungi as bioreactors.

Insect Immunity to Entomopathogenic Fungi.

The study of infection and immunity in insects has achieved considerable prominence with the appreciation that their host defense mechanisms share many fundamental characteristics with the innate immune system of vertebrates. Studies on the highly tractable model organism Drosophila in particular have led to a detailed understanding of conserved innate immunity networks, such as Toll. However, most of these studies have used opportunistic human pathogens and may not have revealed specialized immune strategies that have arisen through evolutionary arms races with natural insect pathogens. Fungi are the commonest natural insect pathogens, and in this review, we focus on studies using Metarhizium and Beauveria spp. that have addressed immune system function and pathogen virulence via behavioral avoidance, the use of physical barriers, and the activation of local and systemic immune responses. In particular, we highlight studies on the evolutionary genetics of insect immunity and discuss insect-pathogen coevolution.

Effect of sample preservation on stress softening and permanent set of porcine skin.

Skin is a composite material with a complex structure which exhibits a wide range of behaviours such as anisotropy, viscoelasticity, hyperelasticity, plasticity etc. Indeed it remains a great challenge to understand its behaviour as it is involved in many consumer and medical applications. In most studies, experiments are performed in situ or in vitro on fresh tissues but most of the time samples are preserved before testing (fridge, freezer, saline solution etc.). In this paper, the impact of samples conservation on the softening behaviour and on the permanent set is studied in order to select the appropriate conservation protocol. Samples are extracted from several pigs' abdomens (direction parallel to spine) and the mechanical testing consists in loading-unloading uniaxial tension tests instrumented with digital image correlation inducing thus reliable strain measurements in a chosen region of interest. The results of this study revealed that preservation conditions must be carefully chosen; conservation in a saline solution and freezing without any caution alter the irreversible part of the global mechanical behaviour of the tissues.

Not all GMOs are crop plants: non-plant GMO applications in agriculture.

Since tools of modern biotechnology have become available, the most commonly applied and often discussed genetically modified organisms are genetically modified crop plants, although genetic engineering is also being used successfully in organisms other than plants, including bacteria, fungi, insects, and viruses. Many of these organisms, as with crop plants, are being engineered for applications in agriculture, to control plant insect pests or diseases. This paper reviews the genetically modified non-plant organisms that have been the subject of permit approvals for environmental release by the United States Department of Agriculture/Animal and Plant Health Inspection Service since the US began regulating genetically modified organisms. This is an indication of the breadth and progress of research in the area of non-plant genetically modified organisms. This review includes three examples of promising research on non-plant genetically modified organisms for application in agriculture: (1) insects for insect pest control using improved vector systems; (2) fungal pathogens of insects to control insect pests; and (3) virus for use as transient-expression vectors for disease control in plants.

Clinical evaluation for lymphoproliferative disease prompted by finding of IgM warm autoanti-IT in two cases.

Anti-IT is an unusual specificity originally described as a naturally occurring cold agglutinin. The antibody reacts strongly with cord RBCs, weakly with adult I RBCs, and most weakly with the rare adult i RBCs. IgG anti-IT in patients with hemolytic anemia has been associated with Hodgkin's lymphoma. Difficulties in blood grouping tests and the presence of a warm reactive agglutinin in samples from two patients with hemolytic anemia led to further serologic studies and the identification of anti-IT. In both cases, the anti-IT was a rarely encountered IgM warm reactive agglutinin; in one case, the IgG component was also anti-IT, whereas in the second case the IgG antibody was broadly reactive. The unusual serologic finding of anti-IT prompted further clinical evaluation for lymphoproliferative disease in these two patients.

PP2A-Bgamma subunit and KCNQ2 K+ channels in bipolar disorder.

Many bipolar affective disorder (BD) susceptibility loci have been identified but the molecular mechanisms responsible for the disease remain to be elucidated. In the locus 4p16, several candidate genes were identified but none of them was definitively shown to be associated with BD. In this region, the PPP2R2C gene encodes the Bgamma-regulatory subunit of the protein phosphatase 2A (PP2A-Bgamma). First, we identified, in two different populations, single nucleotide polymorphisms and risk haplotypes for this gene that are associated to BD. Then, we used the Bgamma subunit as bait to screen a human brain cDNA library with the yeast two-hybrid technique. This led us to two new splice variants of KCNQ2 channels and to the KCNQ2 channel itself. This unusual K+ channel has particularly interesting functional properties and belongs to a channel family that is already known to be implicated in several other monogenic diseases. In one of the BD populations, we also found a genetic association between the KCNQ2 gene and BD. We show that KCNQ2 splice variants differ from native channels by their shortened C-terminal sequences and are unique as they are active and exert a dominant-negative effect on KCNQ2 wild-type (wt) channel activity. We also show that the PP2A-Bgamma subunit significantly increases the current generated by KCNQ2wt, a channel normally inhibited by phosphorylation. The kinase glycogen synthase kinase 3 beta (GSK3beta) is considered as an interesting target of lithium, the classical drug used in BD. GSK3beta phosphorylates the KCNQ2 channel and this phosphorylation is decreased by Li+.

A phylogenomic approach to reconstructing the diversification of serine proteases in fungi.

Using a phylogenomic approach with 10 fungi of very different virulence and habitat, we determined that there was substantial diversification of subtilase-type proteases early in ascomycete history (with subsequent loss in many lineages) but with no comparable diversification of trypsins. Patterns of intron loss and the degree of divergence between paralogues demonstrated that the proliferation of proteinase K subtilases and subtilisin type subtilases seen in pathogenic ascomycetes (Metarhizium anisopliae, Magnaporthe grisea, Fusarium graminearum) occurred after the basidiomycete/ascomycete split but predated radiation of ascomycete lineages. This suggests that the early ascomycetes had a lifestyle that selected for multiple proteases, whereas the current disparity in gene numbers between ascomycete lineages results from retention of genes in at least some pathogens that have been lost in other lineages (yeasts, Aspergillus nidulans, Neurospora crassa). A similar prevailing trend towards lineage specific gene loss of trypsins in saprophytes and some pathogens suggests that their phylogenetic breadth will have been much wider in early fungi than currently.

Stereochemical elucidation and total synthesis of dihydropacidamycin D, a semisynthetic pacidamycin.

Hydrogenation of the C(4') exocyclic olefin of the pacidamycins has been shown to produce a series of semisynthetic compounds, the dihydropacidamycins, with antimicrobial activity similar to that of the natural products. Elucidation of stereochemistry in the pacidamycins has been completed through a campaign of natural product degradation experiments in combination with the total synthesis of the lowest-molecular weight dihydropacidamycin, dihydropacidamycin D. The stereochemical identities of the tryptophan and two alanine residues contained in pacidamycin D have been shown to be of the natural (S) configuration, and the unique 3-methylamino-2-aminobutyric acid contained in this series of antibiotics has been shown to be of the (2S,3S) configuration. Finally, the stereochemistry obtained by hydrogenation of the C(4')-C(5') exocyclic olefin has been shown to be (R) at the C(4') nucleoside site.

Addressing the stability of C-capped dipeptide efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa.

Synthetic optimization of a biologically labile class of dipeptides that function as efflux pump inhibitors to potentiate the antibacterial agent levofloxacin in Pseudomonas aeruginosa has led to the discovery of a related series of compounds that are completely stable in a variety of biological matrices. Other than the stability profile, the in vitro profile of the new series is essentially identical to that observed with the original one. A prototypical compound from the new series demonstrates potentiation in an in vivo model of infection.

Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy.

Whole-cell assays were implemented to search for efflux pump inhibitors (EPIs) of the three multidrug resistance efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN) that contribute to fluoroquinolone resistance in clinical isolates of Pseudomonas aeruginosa. Secondary assays were developed to identify lead compounds with exquisite activities as inhibitors. A broad-spectrum EPI which is active against all three known Mex efflux pumps from P. aeruginosa and their close Escherichia coli efflux pump homolog (AcrAB-TolC) was discovered. When this compound, MC-207,110, was used, the intrinsic resistance of P. aeruginosa to fluoroquinolones was decreased significantly (eightfold for levofloxacin). Acquired resistance due to the overexpression of efflux pumps was also decreased (32- to 64-fold reduction in the MIC of levofloxacin). Similarly, 32- to 64-fold reductions in MICs in the presence of MC-207,110 were observed for strains with overexpressed efflux pumps and various target mutations that confer resistance to levofloxacin (e.g., gyrA and parC). We also compared the frequencies of emergence of levofloxacin-resistant variants in the wild-type strain at four times the MIC of levofloxacin (1 microg/ml) when it was used either alone or in combination with EPI. In the case of levofloxacin alone, the frequency was approximately 10(-7) CFU/ml. In contrast, with an EPI, the frequency was below the level of detection (<10(-11)). In summary, we have demonstrated that inhibition of efflux pumps (i) decreased the level of intrinsic resistance significantly, (ii) reversed acquired resistance, and (iii) resulted in a decreased frequency of emergence of P. aeruginosa strains that are highly resistant to fluoroquinolones.

Transformants of Metarhizium anisopliae sf. anisopliae overexpressing chitinase from Metarhizium anisopliae sf. acridum show early induction of native chitinase but are not altered in pathogenicity to Manduca sexta.

Extracellular chitinase activity has been implicated in the pathogenesis of several fungal infections. Following induction with chitin, the insect pathogens Metarhizium anisopliae sf. acridum ARSEF strain 324 and Metarhizium anisopliae sf. anisopliae ARSEF strain 2575 secrete 44-kDa basic and acidic isoforms of endochitinase, respectively. The gene from strain 324 (Chit1) was cloned and inserted into the genome of strain 2575 under the control of Aspergillus regulatory elements such that transgenic 2575 (2575-Chit(+)) expressed CHIT1 in a noninducing medium (i.e., not containing chitin). Isoelectric focusing followed by a zymogram technique revealed that neither wild-type 2575 nor 2575-Chit(+) produced significant amounts of the native 2575 acidic chitinase in a noninducing medium. However, in a chitin-containing medium, 2575-Chit(+) produced the native chitinase earlier than strain 2575, soon after secretion of CHIT1. We hypothesize that this is due to the production of soluble inducers following chitin hydrolysis by CHIT1 and that M. anisopliae uses enzymes expressed at low levels to sense the nature of the polymeric nutrient present in the immediate environment. However, the chitinase overproducers did not show altered virulence to caterpillars (Manduca sexta) compared to the wild-type fungus, suggesting that wild-type levels of chitinase are not limiting for cuticle penetration.

In vitro studies of the impact of transfusion on the detection of alloantibodies after autoadsorption.

BACKGROUND: In a patient with warm autoantibodies who has recently received a transfusion, it is not recommended to perform adsorptions using autologous RBCs to detect alloantibodies. Although not scientifically documented, this position is based on the theory that transfused RBCs in the patient's circulation would be capable of adsorbing alloantibodies that may be present. This in vitro study was designed to determine what percentage of transfused RBCs might completely remove alloantibodies in vivo. STUDY DESIGN AND METHODS: Selected D, E, K, Fy(a), and Jk(a) antibodies were adsorbed with mixtures of antigen-positive and antigen-negative RBCs to determine the lowest concentration of antigen-positive RBCs capable of removing all alloantibody reactivity. The percentage of antigen-positive RBCs in each mixture was determined by flow cytometry. RESULTS: Small amounts of antigen-positive RBCs (2-6%, as determined by flow cytometry) completely removed anti-D, -E, and -Fy(a) reactivity. Reactivity of two examples of anti-K was removed by 11 percent and 17 percent of K+ RBCs, respectively. Anti-Jk(a) reactivity was completely removed by 4 to 5 percent Jk(a+) RBCs using a PEG adsorption; the endpoint (>11%) was estimated, but complete adsorption with ZZAP-treated RBCs was not performed. CONCLUSION: Small amounts of antigen-positive RBCs are generally capable of removing all alloantibody reactivity. Thus, waiting for 3 months after transfusion before performing autologous adsorptions is a prudent policy.

Cloning, expression, and substrate specificity of a fungal chymotrypsin. Evidence for lateral gene transfer from an actinomycete bacterium.

Unlike trypsins, chymotrypsins have not until now been found in fungi. Expressed sequence tag analysis of the deuteromycete Metarhizium anisopliae identified two trypsins (family S1) and a novel chymotrypsin (CHY1). CHY1 resembles actinomycete (bacterial) chymotrypsins (family S2) rather than other eukaryote enzymes (family S1) in being synthesized as a precursor species (374 amino acids, pI/MW: 5.07/38,279) containing a large N-terminal fragment (186 amino acids). Chy1 was expressed in Pichia pastoris yielding an enzyme with a chymotrypsin specificity for branched aliphatic and aromatic C-terminal amino acids. This is predictable as key catalytic residues determining the specificity of Streptomyces griseus chymotrypsins are conserved with CHY1. Mature (secreted) CHY1 (pI/MW: 8.29/18,499) shows closest overall amino acid identity to S. griseus protease C (55%) and clustered with other secreted bacterial S2 chymotrypsins that diverged widely from animal and endocellular bacterial enzymes in phylogenetic trees of the chymotrypsin superfamily. Conversely, actinomycete chymotrypsins are much more closely related to fungal proteases than to other eubacterial sequences. Complete genomes of yeast, gram eubacteria, archaebacteria, and mitochondria do not contain paralogous genes. Expressed sequence tag data bases from other fungi also lack chymotrypsin homologs. In light of this patchy distribution, we conclude that chy1 probably arose by lateral gene transfer from an actinomycete bacterium.

Lack of host specialization in Aspergillus flavus.

Aspergillus spp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans for their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains of A. flavus did not affect nonwounded bean leaves, corn kernels, or insects at 22 degrees C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton balls, is produced by most A. flavus isolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. All A. flavus strains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast to A. flavus, strains of A. nidulans and A. fumigatus could not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate that A. flavus has parasitic attributes that are lacking in A. fumigatus and A. nidulans but that individual strains of A. flavus are not specialized to particular hosts.