Similar Substrates Research Articles

Touchscreen tasks in mice to demonstrate differences between hippocampal and striatal functions

In mammals, hippocampal and striatal regions are engaged in separable cognitive processes usually assessed through species-specific paradigms. To reconcile cognitive testing among species, translational advantages of the touchscreen-based automated method have been recently promoted. However, it remains undetermined whether similar neural substrates would be involved in such behavioral tasks both in humans and rodents. To address this question, the effects of hippocampal or dorso-striatal fiber-sparing lesions were first assessed in mice through a battery of tasks (experiment A) comprising the acquisition of two touchscreen paradigms, the Paired Associates Learning (dPAL) and Visuo-Motor Conditional Learning (VMCL) tasks, and a more classical T-maze alternation task. Additionally, we sought to determine whether post-acquisition hippocampal lesions would alter memory retrieval in the dPAL task (experiment B). Pre-training lesions of dorsal striatum caused major impairments in all paradigms. In contrast, pre-training hippocampal lesions disrupted the performance of animals trained in the T-maze assay, but spared the acquisition in touchscreen tasks. Nonetheless, post-training hippocampal lesions severely impacted the recall of the previously learned dPAL task. Altogether, our data show that, after having demonstrated their potential in genetically modified mice, touchscreens also reveal perfectly adapted to taxing functional implications of brain structures in mice by means of lesion approaches. Unlike its human counterpart requiring an intact hippocampus, the acquisition of the dPAL task requires the integrity of the dorsal striatum in mice. The hippocampus only later intervenes, when acquired information needs to be retrieved. Touchscreen assays may therefore be suited to study striatal- or hippocampal-dependent forms of learnings in mice.

Bacterial community responses to a gradient of alkaline mountaintop mine drainage in Central Appalachian streams.

Microbial community composition and diversity change along chemical gradients, leading to the expectation that microbial community information might provide new gradient characterizations. Here we examine stream bacteria composition and diversity along a strong chemical gradient in Central Appalachian streams. Coal mining in the region generates alkaline mine drainage (AlkMD), causing dramatic increases in conductivity, alkalinity, sulfate and metals sufficient to degrade stream macrobiota communities throughout the ecoregion. In this study, we examined the relationship between water and biofilm chemistry and biofilm bacteria taxonomic composition in streams where active and reclaimed surface coal mines occupied 0-96% of watershed surface area. We incubated wood veneers in each stream site for 4 months to develop biofilms on similar substrates. We sampled water chemistry at the time of deployment and collection, and after 1 month. Following incubation, we collected biofilms for microbial and chemical characterization. Microbial composition was determined by pyrosequencing 16S rRNA amplicons. Biofilm subsamples were analyzed by inductively coupled plasma mass spectrometry to determine metal concentrations. Our results show that microbial community composition differed significantly between AlkMD-exposed and AlkMD-unexposed sites, and that compositional dissimilarity increased with AlkMD loading. Diversity was not correlated with pH or extent of upstream mining, but instead correlated with biofilm concentrations of Cd, Mn, Zn and Ni. Within mined sites, the extent of upstream mining was negatively correlated with taxonomic richness. Despite major compositional shifts, functional capacity predicted with PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) correlated with mining in only 3 of 43 level-2 KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthology groups.

Establishment of a dipeptidyl peptidases (DPP) 8/9 expressing cell model for evaluating the selectivity of DPP4 inhibitors

Dipeptidyl peptidases (DPPs) 8 and 9 are homologous, cytoplasmic postproline-cutting enzymes, which have similar enzymatic activity and preferred substrates as DPP4. DPP4 is a well-known target for treating diabetes mellitus. With the increased concern of non-selectivity and toxicities caused by DPP4 inhibitors, it is essential to establish new ex vivo system to investigate DPP4 inhibitors' effect on DPP8 and DPP9. Here we reported a newly established cell model system by cloning and transfecting human DPP8/9 genes into HEK 293 cells. We then used this model to evaluate the clinically applied DPP4 inhibitors' effect on DPP8/9, by direct enzymatic activity assay. Given the difference of cellular locations between DPP4 and DPP8/9, we also evaluated the influence of these drugs on intracellular DPP8/9 activity and cell viability by extracellular treatment with different inhibitors. Direct enzymatic activity assay revealed significant and concentration-dependent inhibition effect of vildagliptin, saxagliptin on DPP8/9. Extracellular incubation of DPP8/9 over expressed cells with sitagliptin, vildagliptin, saxagliptin, alogliptin and linagliptin, showed only mild inhibition on DPP8/9. Moreover, all of these drugs showed no significant influence on cell viability. Our results demonstrated that the DPP8/9 over-expressing cell model system is a very useful and promising system for investigating the selectivity and associated toxicity of DPP4 inhibitors on DPP8/9.

A new genus and species of the family Microdispidae (Acari: Prostigmata) associated with Oryctes nasicornis (Coleoptera: Scarabaeidae) and redescription of the monotypic genus Vietodispus Mahunka, 1975

BackgroundMites of the family Microdispidae (Acari: Prostigmata: Heterostigmata) are fungivorous, usually living in soil, litter, decaying plant materials, and similar substrates, and adult females have phoretic associations with arthropods, particularly insects. The family Microdispidae is poorly studied, and because of the vague descriptions of most of the genera and species, the taxonomy of this family is in a state of confusion. Previous to this study, more than 109 microdispid species in 17 genera have been described worldwide.ResultsIn this study, a new genus, Neomicrodispus gen. nov. (Acari: Microdispidae) (type species Neomicrodispus iranicus sp. nov.) is described and illustrated based on phoretic females recovered from Oryctes nasicornis (Coleoptera: Scarabaeidae), which were collected from the forests in northern Iran. Also, the monotypic genus Vietodispus Mahunka, 1975 (type species Vietodispus calcaratus) is redescribed based on its holotype lent from the Hungarian Natural History Museum. The representatives of the family Microdispidae associated with arthropods are reviewed.ConclusionsThere are now 18 genera of this family worldwide that are primarily defined by reductions in their morphology. Because of poor description of some genera and species, synonymies or decreasing in systematic ranks may be expected. A review of arthropod associations, provided in this paper, showed that these mites are primarily associated with ants and beetles, where a rich source of various kinds of fungi particles is easily obtained.

Diversity Within the O-linked Protein Glycosylation Systems of Acinetobacter Species

The opportunistic human pathogen Acinetobacter baumannii is a concern to health care systems worldwide because of its persistence in clinical settings and the growing frequency of multiple drug resistant infections. To combat this threat, it is necessary to understand factors associated with disease and environmental persistence of A. baumannii. Recently, it was shown that a single biosynthetic pathway was responsible for the generation of capsule polysaccharide and O-linked protein glycosylation. Because of the requirement of these carbohydrates for virulence and the non-template driven nature of glycan biogenesis we investigated the composition, diversity, and properties of the Acinetobacter glycoproteome. Utilizing global and targeted mass spectrometry methods, we examined 15 strains and found extensive glycan diversity in the O-linked glycoproteome of Acinetobacter. Comparison of the 26 glycoproteins identified revealed that different A. baumannii strains target similar protein substrates, both in characteristics of the sites of O-glycosylation and protein identity. Surprisingly, glycan micro-heterogeneity was also observed within nearly all isolates examined demonstrating glycan heterogeneity is a widespread phenomena in Acinetobacter O-linked glycosylation. By comparing the 11 main glycoforms and over 20 alternative glycoforms characterized within the 15 strains, trends within the glycan utilized for O-linked glycosylation could be observed. These trends reveal Acinetobacter O-linked glycosylation favors short (three to five residue) glycans with limited branching containing negatively charged sugars such as GlcNAc3NAcA4OAc or legionaminic/pseudaminic acid derivatives. These observations suggest that although highly diverse, the capsule/O-linked glycan biosynthetic pathways generate glycans with similar characteristics across all A. baumannii.

The role of post-translational modifications in fine-tuning BLM helicase function during DNA repair.

RecQ-like helicases are a highly conserved family of proteins which are critical for preserving genome integrity. Genome instability is considered a hallmark of cancer and mutations within three of the five human RECQ genes cause hereditary syndromes that are associated with cancer predisposition. The human RecQ-like helicase BLM has a central role in DNA damage signaling, repair, replication, and telomere maintenance. BLM and its budding yeast orthologue Sgs1 unwind double-stranded DNA intermediates. Intriguingly, BLM functions in both a pro- and anti-recombinogenic manner upon replicative damage, acting on similar substrates. Thus, BLM activity must be intricately controlled to prevent illegitimate recombination events that could have detrimental effects on genome integrity. In recent years it has become evident that post-translational modifications (PTMs) of BLM allow a fine-tuning of its function. To date, BLM phosphorylation, ubiquitination, and SUMOylation have been identified, in turn regulating its subcellular localization, protein-protein interactions, and protein stability. In this review, we will discuss the cellular context of when and how these different modifications of BLM occur. We will reflect on the current model of how PTMs control BLM function during DNA damage repair and compare this to what is known about post-translational regulation of the budding yeast orthologue Sgs1. Finally, we will provide an outlook toward future research, in particular to dissect the cross-talk between the individual PTMs on BLM.

Formation and characterization of porous silicon–samarium/gadolinium nanocomposites: effect of substrate oxidation and biosynthesis process

Samarium and gadolinium nanoparticles synthesized by bioreduction process have been incorporated into nanostructured porous silicon template to form a nanocomposite. The structural and optical properties of PS–Gd and PS–Sm nanocomposites have been studied through TEM, SEM and UV–Vis spectroscopy. Extent of infiltration has been verified through reflectance interference Fourier transform spectroscopy as a function of substrate oxidation conditions. The substrates oxidized at 600 °C showed the maximum infiltration and the corresponding change of optical thickness due to nanoparticles. Such biodegradable nanocomposites in the form of particles can have potential applications in localized drug delivery and enhancement of the image contrast and optoelectronic devices. The results here reported open an energy-cheap procedure to take advantages of small rare earth nanoparticles and produced nanocomposites with their immersion in SiO2 substrates, with the perspective to be replied in other similar substrates under controlled conditions.

Correlation of neurological soft signs and neurocognitive performance in first episode psychosis

Neurological soft signs and neurocognitive impairments are commonly observed in first episode psychosis but the correlation of these factors remains controversial. Here, we evaluated 30 patients with remitted first episode psychosis and 30 healthy controls for the presence and severity of neurological soft signs (using the Neurological Evaluation Scale - NES) and for neurocognitive impairments (using seven subtests of the Cambridge Neuropsychological Test Automated Battery - CANTAB). NES score was higher in patients compared to controls. Neurocognitive impairment was evident in patients in the following domains: working memory, spatial recognition memory, attention set shifting, planning and inhibition. The NES revealed significant correlations with spatial working memory performance and Intra-Extra Dimensional Set Shifting (as a component of executive function). These correlations were observed both in patients and in controls. Planning and inhibition showed correlation with the total NES score and the sequencing of complex motor acts in both groups. In addition, spatial span and spatial recognition memory showed significant correlation with total NES score and the sequencing of complex motor acts in controls. The correlation between sequencing of complex motor acts and specific domains of neurocognitive tasks suggests that similar neuroanatomical substrates might be implicated in these processes.

Fragmentation of Bicyclic γ-Silyloxy-β-hydroxy-α-diazolactones as an Approach to Ynolides

Medium-sized ynolides were prepared by the Lewis acid-mediated fragmentation of bicyclic γ-silyloxy-β-hydroxy-α-diazolactones in which the Cβ–Cγ bond is the ring fusion bond. Although these lactone fragmentation substrates reacted somewhat less efficiently than their carbocyclic counterparts, the fragmentation provided 11-membered ynolides in up to 84% yield. Unlike prior fragmentations of similar substrates, elevated temperatures were required to obtain optimum yields of the ynolide products. The ynolides reported herein have ring sizes of 10 or 11, which are the smallest reported to date.

N-Cadherin-Mediated Cell Signaling in Nucleus Pulposus Cells of the Intervertebral Disc

Introduction Immature nucleus pulposus (NP) cells of the intervertebral disc (IVD) are large, vacuolated cells that form cell clusters with strong cell-cell interactions.1 With maturation and aging, NP cells lose their ability to form these cell clusters, with aging-associated changes in NP cell phenotype, morphology, and proteoglycan synthesis that may contribute to IVD degeneration.2-4 Therefore it is important to understand the mechanisms governing immature NP cell cluster behavior toward the goal of revealing factors that can promote immature, healthy NP cell phenotypes. Prior work has shown healthy, immature NP cells preferentially bind laminin proteins, exhibit potential for cell cluster formation, and maintain matrix production when cultured upon soft, laminin-containing substrates.2 N-cadherin has been identified as a cell-cell adhesion marker that is present in immature NP cells, but disappears with age.5-6 The goal of this study was to determine if N-cadherin was an important molecule in regulating cell-cell interactions in immature NP cell cluster formation and to test for a regulatory role in maintaining an immature NP phenotype for IVD cells in vitro. Materials and Methods Porcine nucleus pulposus (NP) cells were isolated from IVD of immature pigs (3-6 mos).2 Laminin-rich substrates were constructed from basement membrane extract (BME, 13.7 mg/mL, Trevigen) to generate soft (300 Pa) or stiff (BME-coated glass, > 2,900 Pa) substrates; similar substrates were constructed of “soft” and “stiff” type I collagen (col 1, 4 mg/mL, Sigma). NP cells were plated (65,000 cells/cm2) on the substrates with the following treatment conditions for up to 4 days: N-cadherin (40 μg/mL) blocking antibody, E-cadherin (20 μg/mL) blocking antibody or no treatment control. NP cells were stained for polymerized F-actin (phalloidin, Invitrogen), anti-N-cadherin (Abcam), or anti-E-cadherin (Abcam) antibodies with propidium iodide nuclei counterstain. Changes in matrix production were analyzed in collected media and recovered cells via biochemical assays for sGAG (DMMB assay) and DNA (picogreen). Additionally, gene expression for immature NP markers, N-cadherin, Brachyury-T, aggrecan and type I collagen, were analyzed via quantitative real-time PCR. Results NP cells cultured upon soft BME substrates maintained their rounded morphology at all times with formation of 3D cell clusters ( Fig. 1A ). On stiff BME and all col 1 substrates, NP cells spread out and attached with formation of numerous actin stress fibers. Higher N-cadherin expression was observed on soft BME compared with all other substrates; E-cadherin expression was absent in porcine NP cells on all substrates. Additionally, significantly higher matrix production was observed on soft BME substrates compared with soft col 1 ( Fig. 1B ), and higher gene expression for NP markers was observed on soft BME substrates compared with all other substrates ( Fig. 1C ). NP cell cluster formation, proteoglycan synthesis and NP marker gene expression on soft BME substrates was influenced by N-cadherin blocking antibody but not E-cadherin blocking antibody ( Fig. 1A -C). [Figure: see text] Conclusion Soft BME substrates promote NP cell clustering in vitro with an associated expression of N-cadherin. In NP cells, it appears that N-cadherin-mediated signaling helps promote matrix production and elevated expression for multiple molecular markers of an immature NP phenotype. Treatment with an N-cadherin blocking antibody resulted in loss of all features of the immature NP cell on soft BME substrates. These findings establish the importance of N-cadherin in mediating immature NP cell cluster formation and maintenance of the immature phenotype. Ongoing studies aim to understand the downstream signaling mechanisms governed by N-cadherin-mediated signaling that promote the immature NP phenotype. Acknowledgments This study was supported with funds from NIH R01EB002263, R01AR047442, R01AR057410, T32GM008555, North Carolina Biotechnology Center, and with the NSF Graduate Research Fellowship. Disclosure of Interest None declared References Trout JJ, Buckwalter JA, Moore KC, Landas SK. Ultrastructure of the human intervertebral disc. I. Changes in notochordal cells with age. Tissue Cell 1982;14(2):359–369 Gilchrist CL, Darling EM, Chen J, Setton LA. Extracellular matrix ligand and stiffness modulate immature nucleus pulposus cell-cell interactions. PLoS ONE 2011;6(11):e27170 Boos N, Weissbach S, Rohrbach H, Weiler C, Spratt KF, Nerlich AG. Classification of age-related changes in lumbar intervertebral discs: 2002 Volvo Award in basic science. Spine 2002;27(23):2631–2644 Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine 1995;20(11):1307–1314 Minogue BM, Richardson SM, Zeef LA, Freemont AJ, Hoyland JA. Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes. Arthritis Res Ther 2010;12(1):R22 Minogue BM, Richardson SM, Zeef LA, Freemont AJ, Hoyland JA. Characterization of the human nucleus pulposus cell phenotype and evaluation of novel marker gene expression to define adult stem cell differentiation. Arthritis Rheum 2010;62(12):3695–3705

Assessing use and suitability of scanning electron microscopy in the analysis of micro remains in dental calculus

Dental calculus is increasingly recognized as a major reservoir of dietary information. Palaeodietary studies using plant and animal micro remains (e.g. phytoliths, pollen, sponge spicules, and starch grains) trapped in calculus have the potential to revise our knowledge of the dietary role of plants in past populations. The conventional methods used to isolate and identify these micro remains rely on removing them from their microenvironment in the calculus, thus the microenvironment that traps and preserves micro remains is not understood. By using scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM–EDX) on modern chimpanzee calculus from the Taï Forest, Côte d'Ivoire, and human calculus from the Chalcolithic site of Camino del Molino, Spain, we present the first reported observations on characteristics of the matrix setting that are conducive to the survival of starch in dental calculus. We also assess the potential for SEM–EDX to detect starch and differentiate it from structurally and molecularly similar substrates. We demonstrate that SEM–EDX may offer a non-destructive technique for studying micro remains in certain contexts. Finally, we compare traditional optical analytical techniques (OM) with less invasive electron microscopy. The results indicate that SEM–EDX and OM are both effective for observing micro remains in calculus, but differ in their analytical resolution to identify different micro remains, and we therefore recommend a sequential use of both techniques.

In SilicoPrediction ofEscherichia coliProteins Targeting the Host Cell Nucleus, with Special Reference to Their Role in Colon Cancer Etiology

The potential role of Escherichia coli in the development of colorectal carcinoma (CRC) has been investigated in many studies. Although the exact mechanism is not clear, chronic inflammation caused by E. coli and other related events are suggested as possible causes behind E. coli-induced colon cancer. It has been found that CRC cells, but not normal cells, are colonized by an intracellular form of E. coli. We predicted nuclear targeting of bacterial proteins in the host cell through computational tools nuclear localization signal (NLS) mapper and balanced subcellular localization predictor (BaCeILo). During intracellular E. coli residence, such targeting is highly likely and may have a possible role in colon cancer etiology. We observed that several gene expression-associated proteins of E. coli can migrate to the host nucleus during intracellular infections. This situation provides an opportunity for competitive interaction of host and pathogen proteins with similar cellular substrates, thereby increasing the chances of development of colon cancer. Moreover, the results indicated that proteins localized in the membrane of E. coli mostly act as secretary proteins in host cells. No exact correlation was observed between NLS prediction and nuclear localization prediction by BaCeILo. This is partly because of a number of reasons, including that only 30% of nuclear proteins carry NLS and that proteins <40 kDa molecular weight can passively target the host nucleus. This study concludes that detection of gene expression-specific E. coli proteins and their targeting of the nucleus may have a profound impact on CRC etiology.

Secondary Forest Succession in a Post-Agricultural Landscape in the Hudson Valley, New York

Quercus spp. (oaks) were the most abundant trees in the pre-colonial forests of the Hudson Valley. Over the last century, secondary forests have replaced many of the agricultural fields that were established after these forests were cut. Mohonk Preserve's Foothills, New Paltz, NY, includes secondary forest stands ranging in age from about 30 to 100 y. Stands on similar substrates and soils differ not only in age, but also in the factors that influenced forest development at and since field abandonment such as seed sources and herbivory. This study analyzed the composition of 8 stands of various ages to evaluate the possible importance of site-specific differences in these factors. Oaks dominate only one stand, and Acer saccharum (Sugar Maple) is the major sapling species in all well-drained stands. We attribute differences in stand composition, particularly the lack of oaks and the prevalence of Sugar Maple, mainly to differences in drainage, seed sources, and extent of deer browse.

Effect of base steels on mechanical behavior of adhesive joints with dissimilar steel substrates

The hybrid steel materials (e.g., advanced high strength steels combined with mild steels) have been increasingly applied in vehicle components to achieve the compromise among mechanical performance, lightweight and material cost. A significant issue related to the bonding of dissimilar steel materials is that the differences in substrate physical properties often lead to bond separation at strength levels far less than the bond strength established by the adhesive manufacturer for the joint with similar substrates. This research studied several important factors influencing the strengths of adhesive-bonded lap shear (LS) and coach-peel (CP) joints. Four types of steel substrates were used to fabricate the unbalanced adhesive joints (dissimilar steels or same steels with different thicknesses). The quasi-static tensile tests of the unbalanced joints were performed. A finite-element model was proposed to characterize the fracture behavior of joints. The effects of substrate properties such as bending stiffness and load-carrying capability of substrates on the joint strength (peak force) and fracture modes were investigated. It was observed that the interfacial fracture is prone to occur on the substrate with the relatively weak yield strength. A cohesive fracture can be produced when the load-carrying capabilities of the two substrates are similar. In order to optimize the load-carrying capability of the unbalanced joint, a design guideline was put forward.

Plant and Animal Glycolate Oxidases Have a Common Eukaryotic Ancestor and Convergently Duplicated to Evolve Long-Chain 2-Hydroxy Acid Oxidases

Glycolate oxidase (GOX) is a crucial enzyme of plant photorespiration. The encoding gene is thought to have originated from endosymbiotic gene transfer between the eukaryotic host and the cyanobacterial endosymbiont at the base of plantae. However, animals also possess GOX activities. Plant and animal GOX belong to the gene family of (L)-2-hydroxyacid-oxidases ((L)-2-HAOX). We find that all (L)-2-HAOX proteins in animals and archaeplastida go back to one ancestral eukaryotic sequence; the sole exceptions are green algae of the chlorophyta lineage. Chlorophyta replaced the ancestral eukaryotic (L)-2-HAOX with a bacterial ortholog, a lactate oxidase that may have been obtained through the primary endosymbiosis at the base of plantae; independent losses of this gene may explain its absence in other algal lineages (glaucophyta, rhodophyta, and charophyta). We also show that in addition to GOX, plants possess (L)-2-HAOX proteins with different specificities for medium- and long-chain hydroxyacids (lHAOX), likely involved in fatty acid and protein catabolism. Vertebrates possess lHAOX proteins acting on similar substrates as plant lHAOX; however, the existence of GOX and lHAOX subfamilies in both plants and animals is not due to shared ancestry but is the result of convergent evolution in the two most complex eukaryotic lineages. On the basis of targeting sequences and predicted substrate specificities, we conclude that the biological role of plantae (L)-2-HAOX in photorespiration evolved by co-opting an existing peroxisomal protein.

ChemInform Abstract: The Tricky Task of Nitrate/Nitrite Antiport

AbstractReview: 11 refs.

Two-stage conversion of crude glycerol to energy using dark fermentation linked with microbial fuel cell or microbial electrolysis cell

Crude glycerol is a main byproduct of the biodiesel industry, and the beneficial use of waste glycerol has been a major challenge. This study characterises the conversion of crude glycerol into bioenergy such as H2 and electricity using a two-stage process linking dark fermentation with a microbial fuel cell (MFC) or microbial electrolysis cell (MEC). The results showed that fermentation achieved a maximum H2 rate of 332 mL/L and a yield of 0.55 mol H2/mol glycerol, accompanied by 20% of organic removal. Fed with the raw fermentation products with an initial COD of 7610 mg/L, a two-chamber MFC produced 92 mW/m(2) in power density and removed 50% of COD. The Columbic efficiency was 14%. When fed with 50% diluted fermentation product, a similar power output (90m W/m(2)) and COD removal (49%) were obtained, but the CE doubled to 27%. Similar substrates were used to produce H2 in two-chamber MECs, and the diluted influent had a higher performance, with the highest yield at 106 mL H2/g COD and a CE of 24%. These results demonstrate that dark fermentation linked with MFC/MEC can be a feasible option for conversion of waste glycerol into bioenergy.

Computation and Experiment Reveal That the Ring-Rearrangement Metathesis of Himbert Cycloadducts Can Be Subject to Kinetic or Thermodynamic Control

Unusual observations in the ring-rearrangement metathesis of Himbert arene/allene cycloadducts to form fused polycylic lactams led to a more in-depth experimental study that yielded conflicting results. Differences in reactivity within related systems and unexpected changes in diastereoselectivity among other similar substrates were not readily explained on the basis of the experimental results. Computational investigations demonstrated substrate-dependent changes in reaction pathways (ring-opening metathesis/ring-closing metathesis [ROM/RCM] cascade vs ring-closing metathesis/ring-opening metathesis [RCM/ROM] cascade). Furthermore, some reactions were judged to be under thermodynamic control and others under kinetic control. The greater understanding of the most likely reaction pathways and their energetics provides a reasonable explanation for the previously irreconcilable results.

Role of Material-Driven Fibronectin Fibrillogenesis in Protein Remodeling

Protein remodeling at the cell–material interface is an important phenomenon that should be incorporated into the design of advanced biomaterials for tissue engineering. In this work, we address the relationship between fibronectin (FN) activity at the material interface and remodeling, including proteolytic cascades. To do so, we studied FN adsorption on two chemically similar substrates, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA), which resulted in different distribution and conformation of the protein at the material interface: FN organized spontaneously upon adsorption on PEA into physiological-like fibrils, through a process called material-driven FN fibrillogenesis. The amount of adsorbed FN and its conformation were investigated in two different coating concentrations (2 and 20 μg/mL). Since FN activity at the material interface determines the initial cellular response, we followed the formation of focal adhesions (vinculin) and subsequent cell signaling by focal adhesion kinase (FAK) expression and its phosphorylation (pFAK). More detailed studies were performed to get further insights into integrin binding by crosslinking and extraction followed by immunofluorescence, as well as protein and gene expression for α5 and αv. To correlate cell adhesion with matrix degradation, gene expression and activity (zymography) of matrix metalloproteinases (MMPs) were measured. Overall, we demonstrated that the material-driven FN fibrillogenesis triggers proteolytic activity: MMP activity was higher on the material-driven FN fibrils, as a compensatory mechanism to the inability of cells to reorganize this FN network.

Structure and Function of Cytochrome P450S in Insect Adaptation to Natural and Synthetic Toxins: Insights Gained from Molecular Modeling

Over evolutionary time, insect herbivores have adapted to the presence of natural toxins and more recently to synthetic insecticides in or on the plants they consume. Biochemical analyses and molecular modeling of the cytochrome P450 monooxygenases (P450s) that metabolize these compounds have provided insight into the many variations affecting their catalytic activity. Phylogenetically distinct P450s may metabolize similar substrates, and phylogenetically similar P450s may metabolize different substrates; as well, some P450s process broad arrays of both phytochemicals and synthetic insecticides, while closely related P450s are restricted to a narrow range of phytochemicals. Mapped on the predicted three-dimensional structures of insect P450s developed from available mammalian P450 crystal structures, differences in multiple regions of the insect proteins reveal the evolutionary processes occurring as P450 genes have duplicated and diverged. Analyses of site-directed mutants in select lepidopteran and dipteran P450s demonstrate that slight changes in the catalytic site, the putative product release channel, and the proximal surface (interacting with electron transfer partners such as cytochrome P450 reductase and cytochrome b5) yield pronounced activity differences. Additionally, changes in the catalytic site and in the linker region preceding the proline-hinge influence P450 folding. With predicted structures available for many mammalian P450s involved in metabolism of xenobiotics, it is possible to record allelic variation relative to catalytically important regions in the overall P450 structure and to predict functionally critical differences. Together with information on the relative levels of allelic variant transcripts, comprehensive characterization of the mechanisms that modulate metabolism of natural and synthetic xenobiotics in insects can yield insights into plant-insect coevolution and into novel approaches for chemical pest management.