On the regeneration of fish scales: structure and mechanical behavior.

Fish scales serve as a dermal armor that provides protection from physical injury. Owing to a number of outstanding properties, fish scales are inspiring new concepts for layered engineered materials and next-generation flexible armors. Although past efforts have primarily focused on the structure and mechanical behavior of ontogenetic scales, the structure-property relationships of regenerated scales have received limited attention. In the present study, common carp (Cyprinus carpio) acquired from the wild were held live in an aquatic laboratory at 10°C and 20°C. Ontogenetic scales were extracted from the fish for analysis, as well as regenerated scales after approximately 1 year of development and growth. Their microstructure was characterized using microscopy and Raman spectroscopy, and the mechanical properties were evaluated in uniaxial tension to failure under hydrated conditions. The strength, strain to fracture and toughness of the regenerated scales were significantly lower than those of ontogenetic scales from the same fish, regardless of the water temperature. Scales that regenerated at 20°C exhibited significantly higher strength, strain to fracture and toughness than those regenerated at 10°C. The regenerated scales exhibited a highly mineralized outer layer, but no distinct limiting layer or external elasmodine; they also possessed a significantly lower number of plies in the basal layer than the ontogenetic scales. The results suggest that a mineralized layer develops preferentially during scale regeneration with the topology needed for protection, prior to the development of other qualities.

Contributions of intermolecular bonding and lubrication to the mechanical behavior of a natural armor.

Among many dermal armors, fish scales have become a source of inspiration in the pursuit of "next-generation" structural materials. Although fish scales function in a hydrated environment, the role of water and intermolecular hydrogen bonding to their unique structural behavior has not been elucidated. Water molecules reside within and adjacent to the interpeptide locations of the collagen fibrils of the elasmodine and provide lubrication to the protein molecules during deformation. We evaluated the contributions of this lubrication and the intermolecular bonding to the mechanical behavior of elasmodine scales from the Black Carp (Mylopharyngodon piceus). Scales were exposed to polar solvents, followed by axial loading to failure and the deformation mechanisms were characterized via optical mechanics. Displacement of intermolecular water molecules by liquid polar solvents caused significant (p ≤ 0.05) increases in stiffness, strength and toughness of the scales. Removal of this lubrication decreased the capacity for non-linear deformation and toughness, which results from the increased resistance to fibril rotations and sliding caused by molecular friction. The intermolecular lubrication is a key component of the "protecto-flexibility" of scales and these natural armors as a system; it can serve as an important component of biomimetic-driven designs for flexible armor systems. STATEMENT OF SIGNIFICANCE: The natural armor of fish has become a topic of substantial scientific interest. Hydration is important to these materials as water molecules reside within the interpeptide locations of the collagen fibrils of the elasmodine and provide lubrication to the protein molecules during deformation. We explored the opportunity for tuning the mechanical behavior of scales as a model for next-generation engineering materials by adjusting the extent of hydrogen bonding with polar solvents and the corresponding interpeptide molecular lubrication. Removal of this lubrication decreased the capacity for non-linear deformation and toughness due to an increase in resistance to fibril rotations and sliding as imparted by molecular friction. We show that intermolecular lubrication is a key component of the "protecto-flexibility" of natural armors and it is an essential element of biomimetic approaches to develop flexible armor systems.

Non-canonical Wnt signalling regulates scarring in biliary disease via the planar cell polarity receptors.

The number of patients diagnosed with chronic bile duct disease is increasing and in most cases these diseases result in chronic ductular scarring, necessitating liver transplantation. The formation of ductular scaring affects liver function; however, scar-generating portal fibroblasts also provide important instructive signals to promote the proliferation and differentiation of biliary epithelial cells. Therefore, understanding whether we can reduce scar formation while maintaining a pro-regenerative microenvironment will be essential in developing treatments for biliary disease. Here, we describe how regenerating biliary epithelial cells express Wnt-Planar Cell Polarity signalling components following bile duct injury and promote the formation of ductular scars by upregulating pro-fibrogenic cytokines and positively regulating collagen-deposition. Inhibiting the production of Wnt-ligands reduces the amount of scar formed around the bile duct, without reducing the development of the pro-regenerative microenvironment required for ductular regeneration, demonstrating that scarring and regeneration can be uncoupled in adult biliary disease and regeneration.

A non-canonical mismatch repair pathway in prokaryotes.

Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS-MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS-MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature.

Cervical Vertebral Lesions in Equine Stenotic Myelopathy.

Skeletal lesions in the articular processes of cervical vertebrae C2 to C7 were compared between Thoroughbred horses with cervical stenotic myelopathy (17 males, 2 females; age, 6-50 months) and controls (6 males, 3 females; age, 9-67 months). Lesions identified by magnetic resonance imaging occurred with an increased frequency and severity in diseased horses and were not limited to sites of spinal cord compression. Lesions involved both the articular cartilage and trabecular bone and were further characterized using micro-computed tomography and histopathology. The most common histologic lesions included osteochondrosis, osseous cyst-like structures, fibrous tissue replacement of trabecular bone, retained cartilage matrix spicules, and osteosclerosis. Osseous cyst-like structures were interpreted to be true bone cysts given they were a closed cavity with a cellular lining that separated the cyst from surrounding bone. This is the first report of bone cysts in the cervical articular processes of horses with cervical stenotic myelopathy. The morphology and distribution of the lesions provide additional support for the previously proposed pathogenesis that developmental abnormalities with likely secondary biomechanical influences on the cervical spine contribute to equine cervical stenotic myelopathy.

Whole genome analysis using microarrays.

The development of microarray technology has allowed the genomes of mycobacteria to be directly compared to identify DNA regions that differ between strains due to deletion, insertion, or sequence divergence. The use of microarrays in comparative genomics has proved to be a valuable tool for comparing both mycobacterial species and strains. We describe here the methodology for comparing two mycobacterial DNA samples by microarray hybridization, from labeling and slide preparation, to DNA microarray analysis options. Further developments in microarray design and methodology promise to ensure that microarrays remain an important resource for comparative genomic studies in the future.

Natural methods of protein stabilization: thermostable biocatalysts.

Enzymes that are naturally found in thermophilic and hyperthermophilic organisms are being used as robust biocatalysts in the fine chemical and pharmaceutical industries. They have important use in these industries due to their increased stability which is often required during commercial reaction conditions. The approach used in these studies is to learn how nature has managed to stabilize these proteins using a detailed knowledge of their biochemical properties and three-dimensional structures. This is illustrated with several different classes of enzyme that have been studied at Exeter. These include alcohol dehydrogenase, aminoacylase, pyroglutamyl carboxypeptidase, gamma-lactamase, dehalogenase and lysophospholipase.

Inactivation of polyketide synthase and related genes results in the loss of complex lipids in Mycobacterium tuberculosis H37Rv.

AIMS: Phthiocerol dimycocerosate (PDIM) waxes and other lipids are necessary for successful Mycobacterium tuberculosis infection, although the exact role of PDIM in host-pathogen interactions remains unclear. In this study, we investigated the contribution of tesA, drrB, pks6 and pks11 genes in complex lipid biosynthesis in M. tuberculosis. METHODS AND RESULTS: Four mutants were selected from M. tuberculosis H37Rv transposon mutant library. The transposon insertion sites were confirmed to be within the M. tuberculosis open reading frames for tesA (a probable thioesterase), drrB (predicted ABC transporter), pks11 (putative chalcone synthase) and pks6 (polyketide synthase). The first three of these transposon mutants were unable to generate PDIM and the fourth lacked novel polar lipids. CONCLUSIONS: Mycobacterium tuberculosis can be cultivated in vitro without the involvement of certain lipid synthesis genes, which may be necessary for in vivo pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of transposon mutants is a new functional genomic approach for the eventual definition of the mycobacterial 'lipidome'.

Trans-activation of the maize transposable element, Ds, in Brassica napus.

A two-component transposable element system consisting of a stabilized Activator ( Ac(st)) and a chimeric Dissociation ( Ds) element has been introduced into the genome of Brassica napus. Analyses performed on F2 progeny derived from crosses between Ac(st)- and Ds-bearing parents confirm that Ac transposase catalyzes the somatic excision of the Ds element in both embryonic and non-embryonic tissues of this important crop species. The data further reveal that the vast majority of plants containing both Ac(st) and Ds exhibit Ds excision. However, the level of excision is low and germinal Ds excision events are not observed. We estimate that germinal excision of Ds occurs at a frequency of < 0.2%. RT-PCR analysis of the Ac(st) transcript in somatically active seedlings reveals that introns III and IV are highly misprocessed. The pattern of transcript processing is very similar to that observed in germinally inactive but somatically active Arabidopsis seedlings. We suggest that Ds excision activity in B. napus is highly dependent on the efficiency of Ac(st) transcript processing.

Fas-acting memory.

Genetic and behavioral analysis points to a role for fasciclin II in controlling odor memory and alcohol sensitivity in Drosophila.

What can we teach Drosophila? What can they teach us?

A number of single gene mutations dramatically reduce the ability of fruit flies to learn or to remember. Cloning of the affected genes implicated the adenylyl cyclase second-messenger system as key in learning and memory. The expression patterns of these genes, in combination with other data, indicates that brain structures called mushroom bodies are crucial for olfactory learning. However, the mushroom bodies are not dedicated solely to olfactory processing; they also mediate higher cognitive functions in the fly, such as visual context generalization. Molecular genetic manipulations, coupled with behavioral studies of the fly, will identify rudimentary neural circuits that underly multisensory learning and perhaps also the circuits that mediate more-complex brain functions, such as attention.

A "no-hybrids" screen for functional antagonizers of human p53 transactivator function: dominant negativity in fission yeast.

We have developed a functional "no-hybrids" screen in the fission yeast Schizosaccharomyces pombe based on the transcription transactivator activity of human p53. The screen can be used to identify antagonizers and modulators of p53 activity. Expression of functional full-length human p53 is conditionally lethal to the screen reporter strains. Co-expression of specific inhibitory proteins promotes cell survival and growth. We have validated the "no-hybrids" system by (a) successful modeling of human wild-type p53 interaction with SV40 large T antigen, Mdm2 and a panel of tumor-derived human p53 mutants, (b) demonstrating the screening system's efficiency through identification of a dominant negative fragment of p53 itself in a library screen context and (c) using Drosophila p53 to demonstrate that the system can detect evolutionarily distant p53 homologues based on their transactivator activity. The "no-hybrids" screen will be of utility in searches for p53 function-modulators of both cellular and viral origin.

Flies, genes, and learning.

Flies can learn. For the past 25 years, researchers have isolated mutants, engineered mutants with transgenes, and tested likely suspect mutants from other screens for learning ability. There have been notable surprises-conventional second messenger systems co-opted for intricate associative learning tasks, two entirely separate forms of long-term memory, a cell-adhesion molecule that is necessary for short-term memory. The most recent surprise is the mechanistic kinship revealed between learning and addictive drug response behaviors in flies. The flow of new insight is likely to quicken with the completion of the fly genome and the arrival of more selective methods of gene expression.

The epidermal and dermal changes associated with microdermabrasion.

BACKGROUND: Microdermabrasion has become a popular method of skin rejuvenation for treating dyschromia, fine wrinkles, and mild scarring. OBJECTIVE: To analyze the onset and extent of the dermatologic changes associated with microdermabrasion. METHODS: Ten volunteers, ages 31-62 years, underwent a series of six aluminum oxide microdermabrasion facial treatments 7-10 days apart. Skin biopsy specimens were obtained prior to the study, after three treatments, and after six treatments. RESULTS: Compared to the controls, the treated areas demonstrated the following histologic changes: thickening of the epidermis and dermis, flattening of the rete pegs, vascular ectasia and perivascular inflammation, and hyalinization of the papillary dermis with newly deposited collagen and elastic fibers. CONCLUSION: This study suggests that microdermabrasion produces clinical improvement by a mechanism resembling a reparative process at the dermal and epidermal levels.

The amnesiac gene product is expressed in two neurons in the Drosophila brain that are critical for memory.

Mutations in the amnesiac gene in Drosophila affect both memory retention and ethanol sensitivity. The predicted amnesiac gene product, AMN, is an apparent preproneuropeptide, and previous studies suggest that it stimulates cAMP synthesis. Here we show that, unlike other learning-related Drosophila proteins, AMN is not preferentially expressed in mushroom bodies. Instead, it is strongly expressed in two large neurons that project over all the lobes of the mushroom bodies, a finding that suggests a modulatory role for AMN in memory formation. Genetically engineered blockade of vesicle recycling in these cells abbreviates memory as in the amnesiac mutant. Moreover, restoration of amn gene expression to these cells reestablishes normal olfactory memory in an amn deletion background. These results indicate that AMN neuropeptide release onto the mushroom bodies is critical for normal olfactory memory.

Synthesis and properties of 2-(naphthosultamyl)methyl-carbapenems with potent anti-MRSA activity: discovery of L-786,392.

A series of 1beta-methyl-2-(naphthosultamyl)methyl-carbapenems bearing dicationic groups on the naphthosultamyl moiety was prepared and evaluated for activity against resistant gram-positive bacteria. Based on a combination of excellent in vitro antibacterial activity, acceptable mouse acute toxicity, and a desirable fragmentation pattern on beta-lactam ring opening, the analog 2g (L-786,392) was selected for extended evaluation.

Bond strengths of conventional and simplified bonding systems.

PURPOSE: To compare the shear bond strengths of composite to dentin using conventional (three-component) and simplified (two-component) adhesive systems. MATERIALS AND METHODS: 100 bovine teeth were mounted in phenolic rings and ground to 600-grit to obtain 90 flat facial dentin surfaces and 10 flat facial enamel surfaces. The dentin specimens were assigned to nine treatment groups of 10 teeth each. Three groups were assigned to conventional, three-component bonding systems: All-Bond 2, OptiBond FL, and Scotchbond Multi-Purpose Plus. Six groups were assigned to simplified, two-component bonding systems: Clearfil Liner Bond 2, Fuji Bond LC, One-Step, OptiBond Solo, Prime & Bond 2.1, and Tenure Quik with Fluoride. The enamel specimens were used as the control group with Scotchbond Multi-Purpose Plus Adhesive. Each ground surface was first conditioned according to the manufacturers' directions. After rinsing, the surface of each specimen was left visibly moist prior to application of the bonding system. Each bonding system was applied according to its manufacturer's directions. The corresponding composite restorative materials were applied in 4.4 mm diameter molds to the adhesive surface and light-cured from four directions. The completed specimens were stored in water 48 hours before testing. Shear bond strengths were measured using an Instron universal testing machine. Data were subjected to one-way ANOVA and Tukey's multiple comparison test. RESULTS: Mean shear bond strengths of the conventional systems ranged from 16.3 to 20.6 MPa. Mean shear bond strengths of the simplified systems ranged from 14.7 to 17.4 MPa. The mean shear bond strength of the control (enamel bonding) was 21.4 MPa. The mean shear bond strengths of the conventional and simplified systems were not significantly different from each other or from the control system.

Defining the minimal requirements for papilloma viral E6-mediated inhibition of human p53 activity in fission yeast.

The majority of human anogenital carcinomas show evidence of papillomavirus infection. To facilitate viral replication, viruses disable key cellular responses which would otherwise precipitate cell suicide. An obligate factor in one such response is the p53 tumour suppressor protein. p53 gene mutation is an infrequent event in anogenital cancer, apparently due to the action of HPV E6 protein, which inhibits wild-type p53 function by stimulating the degradation of p53 protein. p53 is required for the apoptotic response that is triggered in untransformed cells following inappropriate cell-cycling. E6 directed inhibition of p53 function thus facilitates the survival of transformed cells. We have developed a genetically tractable model that reports E6 protein-mediated human p53 inactivation in the fission yeast Schizosaccharomyces pombe. Functional dissection of the requirements for E6 directed inhibition in this system reveal an absolute requirement for the presence of both E6 protein and the human E3 ubiquitin ligase, E6-AP. Using a defined set of E6 mutants we show that degradation of p53 protein rather than E6/p53 association is likely required for E6-mediated inhibition. This S. pombe based system represents a candidate screen for novel antiviral agents that act by disrupting the E6/E6-AP/p53 interaction.

Inhibitors of the bacterial cell wall biosynthesis enzyme MurD.

A series of transition-state analog inhibitors of the D-glutamic acid-adding enzyme (MurD) of bacterial peptidoglycan biosynthesis has been synthesized and evaluated for inhibition of the E. coli enzyme.