The temporomandibular joint disc of Asian elephant (Elephas maximus) and African elephant (Loxodonta africana)

The temporomandibular joint (TMJ) is a synovial articulation between the mandibular head of the condylar process of the mandible and the mandibular fossa of the squamous temporal bone. Extensions of the fibrous capsule into the joint space form a biconcave disc that functions as an articulating surface and divides the joint into dorsal and ventral compartments. The TMJ disc plays a crucial role in normal functioning of the joint, and differences in cranial morphology, mastication patterns, and diet are reflected in the material and biochemical properties of the disc. The purpose of the present case study was to compare the regional histologic differences between two elephant genera and quantify the biochemical and biomechanical properties of the African elephant disc. This study provides a unique insight into the elephant TMJ disc and also provides a comparison between the African and the Asian elephant genera. The results demonstrate several remarkable findings. First, structure–function relationships exist within the elephant TMJ disc. Second, regional variations exist in the elephant TMJ disc, and these are likely to correlate with its functional requirement. Additionally, it is apparent that some properties of the disc vary with the specific anatomy, diet requirement, and jaw motion. Finally, in comparison with the TMJ disc of other species, it is clear that, although the elephant disc is unique, it has properties that transcend and are preserved among the species.     

MapQuant: open-source software for large-scale protein quantification

Whole-cell protein quantification using MS has proven to be a challenging task. Detection efficiency varies significantly from peptide to peptide, molecular identities are not evident a priori, and peptides are dispersed unevenly throughout the multidimensional data space. To overcome these challenges we developed an open-source software package, MapQuant, to quantify comprehensively organic species detected in large MS datasets. MapQuant treats an LC/MS experiment as an image and utilizes standard image processing techniques to perform noise filtering, watershed segmentation, peak finding, peak fitting, peak clustering, charge-state determination and carbon-content estimation. MapQuant reports abundance values that respond linearly with the amount of sample analyzed on both low- and high-resolution instruments (over a 1000-fold dynamic range). Background noise added to a sample, either as a medium-complexity peptide mixture or as a high-complexity trypsinized proteome, exerts negligible effects on the abundance values reported by MapQuant and with coefficients of variance comparable to other methods. Finally, MapQuant's ability to define accurate mass and retention time features of isotopic clusters on a high-resolution mass spectrometer can increase protein sequence coverage by assigning sequence identities to observed isotopic clusters without corresponding MS/MS data.     

Dynamic Acclimation of Photosynthesis Increases Plant Fitness in Changing Environments

Plants growing in different environments develop with different photosynthetic capacities—developmental acclimation of photosynthesis. It is also possible for fully developed leaves to change their photosynthetic capacity—dynamic acclimation. The importance of acclimation has not previously been demonstrated. Here, we show that developmental and dynamic acclimation are distinct processes. Furthermore, we demonstrate that dynamic acclimation plays an important role in increasing the fitness of plants in natural environments. Plants of Arabidopsis (Arabidopsis thaliana) were grown at low light and then transferred to high light for up to 9 d. This resulted in an increase in photosynthetic capacity of approximately 40%. A microarray analysis showed that transfer to high light resulted in a substantial but transient increase in expression of a gene, At1g61800, encoding a glucose-6-phosphate/phosphate translocator GPT2. Plants where this gene was disrupted were unable to undergo dynamic acclimation. They were, however, still able to acclimate developmentally. When grown under controlled conditions, fitness, measured as seed output and germination, was identical, regardless of GPT2 expression. Under naturally variable conditions, however, fitness was substantially reduced in plants lacking the ability to acclimate. Seed production was halved in gpt2− plants, relative to wild type, and germination of the seed produced substantially less. Dynamic acclimation of photosynthesis is thus shown to play a crucial and previously unrecognized role in determining the fitness of plants growing in changing environments.It has long been recognized that when plants are grown under a particular set of conditions they adjust their photosynthetic capacity to match those conditions (for review, see Walters, 2005). For example, early work from Bjorkman and Holmgren (1963) showed that plants of Solidago virgaurea had different photosynthetic capacities when grown either in sun or shade. In spite of its long history, however, neither the mechanism nor the significance of this response is understood (Walters, 2005). Work from Murchie and Horton (1997) showed that there is substantial variation between species in their ability to acclimate, with plants from semishaded habitats having the greatest variation in photosynthetic capacity, suggesting that there is both a benefit and cost of acclimation. Neither benefit nor cost has been demonstrated.Photosynthetic acclimation can be observed at levels ranging from whole-plant morphology to the detailed stoichiometry of the photosynthetic apparatus (Boardman, 1977; Walters, 2005). Plants grown at low light tend to invest more in leaves than in roots and to have thinner leaves. They have more chlorophyll-containing light-harvesting proteins relative to light-using enzymes involved in electron transport and metabolism, meaning that photosynthesis saturates with light at a lower irradiance. Plants can also adjust the relative proportions of the different photosystems to suit the light quality they experience (Chow et al., 1990; Walters and Horton, 1995a, 1995b).Most studies that have examined the acclimation of plants have done so by making measurements on material that has experienced only one set of conditions—e.g. either high or low light. Differences between plants therefore reflect the conditions experienced as the leaves develop, with leaf morphology and composition being optimized for the conditions seen. Plants do not, however, exist in static environments. Even for a plant growing in an unshaded location, the light incident on a leaf can vary by an order of magnitude from second to second, day to day, and week to week depending on the weather conditions. This variation will typically be accompanied by variation in the temperature, which will also impact on metabolic capacity.When plants are exposed to light at irradiances that are above saturating for photosynthesis, which may result from increases in light or from environmental conditions (e.g. cold, drought) restricting metabolism, they are liable to suffer from stress (Demmig-Adams and Adams, 1992). Specifically, excess light can give rise to reactive oxygen species (Asada, 2006). The damaging effects of this can be limited by investing in antioxidant systems; however, these are metabolically expensive with, for example, substantial amounts of individual antioxidants such as ascorbic acid, being found in the chloroplast (Asada, 2006). It seems likely therefore that the ability of a plant to minimize stress, by adjusting photosynthetic capacity to suit as well as possible the prevailing conditions, will benefit the plant and increase overall fitness.In this study, we have investigated photosynthetic acclimation of the model plant Arabidopsis (Arabidopsis thaliana). Starting with a microarray analysis, we have identified a gene that is essential for acclimation to increases in irradiance. We further show that the ability to acclimate to changes in light has a major role in determining fitness under naturally variable light conditions.     

Use of staurosporine, an actin-modifying agent, to enhance fibrochondrocyte matrix gene expression and synthesis

Modulation of the actin cytoskeleton in chondrocytes has been used to prevent or reverse dedifferentiation and to enhance protein synthesis. We have hypothesized that an actin-modifying agent, staurosporine, could be used with fibrochondrocytes to increase the gene expression and synthesis of critical fibrocartilage proteins. A range of concentrations (0.1–100 nM) was applied to fibrochondrocytes in monolayer and evaluated after 24 h and after 4 days. High-dose staurosporine treatment (10–100 nM) increased cartilage oligomeric matrix protein 60– to 500-fold and aggrecan gene expression two-fold. This effective range of staurosporine was then applied to scaffoldless tissue-engineered fibrochondrocyte constructs for 4 weeks. Whereas glycosaminoglycan synthesis was not affected, collagen content doubled, from 27.6 ± 8.8 μg in the untreated constructs to 55.2 ± 12.2 μg per construct with 100 nM treatment. When analyzed for specific collagens, the 10-nM group showed a significant increase in collagen type I content, whereas collagen type II was unaffected. A concomitant dose-dependent reduction was noted in construct contraction, reflecting the actin-disrupting action of staurosporine. Thus, staurosporine increases the gene expression for important matrix proteins and can be used to enhance matrix production and reduce contraction in tissue-engineered fibrocartilage constructs. The authors gratefully acknowledge NIAMS R01 AR 47839–2 for funding this work, and the Hertz Foundation for their support of G. Hoben.     

Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector

Traditional methods of transgene delivery in livestock are inefficient. Recently, human immunodeficiency virus (HIV-1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. We now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector. We used this vector to deliver a green fluorescent protein expressing transgene; 31% of injected/transferred eggs resulted in a transgenic founder animal and 95% of founder animals displayed green fluorescence. This compares favourably with results using HIV-1 based vectors, and is substantially more efficient than the standard pronuclear microinjection method, indicating that lentiviral transgene delivery may be a general tool with which to efficiently generate transgenic mammals.     

Sequence-based linkage analysis

The rapid decrease in the cost of DNA sequencing will enable its use for novel applications. Here, we investigate the use of DNA sequencing for simultaneous discovery and genotyping of polymorphisms in family linkage studies. In the proposed approach, short contiguous segments of genomic DNA, regularly spaced across the genome, are resequenced in each pedigree member, and all sequence polymorphisms discovered within a pedigree are used as genetic markers. We use computer simulations consistent with observed human sequence diversity to show that segments of 500-1,000 base pairs, spaced at intervals of 1-2 Mb across the genome, provide linkage information that equals or exceeds that of traditional marker-based approaches. We validate these results experimentally by implementing the sequence-based linkage approach for chromosome 19 in CEPH pedigrees.     

A 3.9-centimorgan-resolution human single-nucleotide polymorphism linkage map and screening set

Recent advances in technologies for high-throughout single-nucleotide polymorphism (SNP)-based genotyping have improved efficiency and cost so that it is now becoming reasonable to consider the use of SNPs for genomewide linkage analysis. However, a suitable screening set of SNPs and a corresponding linkage map have yet to be described. The SNP maps described here fill this void and provide a resource for fast genome scanning for disease genes. We have evaluated 6,297 SNPs in a diversity panel composed of European Americans, African Americans, and Asians. The markers were assessed for assay robustness, suitable allele frequencies, and informativeness of multi-SNP clusters. Individuals from 56 Centre d'Etude du Polymorphisme Humain pedigrees, with >770 potentially informative meioses altogether, were genotyped with a subset of 2,988 SNPs, for map construction. Extensive genotyping-error analysis was performed, and the resulting SNP linkage map has an average map resolution of 3.9 cM, with map positions containing either a single SNP or several tightly linked SNPs. The order of markers on this map compares favorably with several other linkage and physical maps. We compared map distances between the SNP linkage map and the interpolated SNP linkage map constructed by the deCode Genetics group. We also evaluated cM/Mb distance ratios in females and males, along each chromosome, showing broadly defined regions of increased and decreased rates of recombination. Evaluations indicate that this SNP screening set is more informative than the Marshfield Clinic's commonly used microsatellite-based screening set.     

Continuous high-titer HIV-1 vector production

Human immunodeficiency virus type 1 (HIV-1)-based vectors are currently made by transient transfection, or using packaging cell lines in which expression of HIV-1 Gag and Pol proteins is induced. Continuous vector production by cells in which HIV-1 Gag-Pol is stably expressed would allow rapid and reproducible generation of large vector batches. However, attempts to make stable HIV-1 packaging cells by transfection of plasmids encoding HIV-1 Gag-Pol have resulted in cells which secrete only low levels of p24 antigen (20-80 ng/ml), possibly because of the cytotoxicity of HIV-1 protease. Infection of cells with HIV-1 can result in stable virus production; cell clones that produce up to 1,000 ng/ml secreted p24 antigen have been described. Here we report that expression of HIV-1 Gag-Pol by a murine leukemia virus (MLV) vector allows constitutive, long-term, high-level (up to 850 ng/ml p24) expression of HIV-1 Gag. Stable packaging cells were constructed using codon-optimized HIV-1 Gag-Pol and envelope proteins of gammaretroviruses; these producer cells could make up to 10(7) 293T infectious units (i.u.)/ml (20 293T i.u./cell/day) for at least three months in culture.     

Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease resulting in the selective death of motor neurons in the brain and spinal cord. Some familial cases of ALS are caused by dominant mutations in the gene encoding superoxide dismutase (SOD1). The emergence of interfering RNA (RNAi) for specific gene silencing could be therapeutically beneficial for the treatment of such dominantly inherited diseases. We generated a lentiviral vector to mediate expression of RNAi molecules specifically targeting the human SOD1 gene (SOD1). Injection of this vector into various muscle groups of mice engineered to overexpress a mutated form of human SOD1 (SOD1(G93A)) resulted in an efficient and specific reduction of SOD1 expression and improved survival of vulnerable motor neurons in the brainstem and spinal cord. Furthermore, SOD1 silencing mediated an improved motor performance in these animals, resulting in a considerable delay in the onset of ALS symptoms by more than 100% and an extension in survival by nearly 80% of their normal life span. These data are the first to show a substantial extension of survival in an animal model of a fatal, dominantly inherited neurodegenerative condition using RNAi and provide the highest therapeutic efficacy observed in this field to date.     

Growth factor impact on articular cartilage subpopulations

We have examined the effects of growth factor stimulation on superficial and growth zone chondrocyte populations. Zonal articular chondrocytes from 8-month-old Spanish goat distal femurs were plated in monolayer cultures and stimulated by using insulin-like growth factor I (IGF-I), basic fibroblast growth factor (bFGF), and transforming growth factor-β1 (TGF-β1). Gene expression for collagen I and II, aggrecan, and superficial zone protein were evaluated every week for 3 weeks. Finally, proteoglycan and collagen deposition were measured for each experimental group. Major differences existed in the behavior of superficial and growth zone chondrocytes, the most apparent being the higher capacity for protein synthesis by the growth zone population. Variations also existed regarding growth factor treatment. TGF-β1 had the greatest effect on proliferation over 8 days. With respect to differentiation, IGF-I increased average collagen II gene expression in the growth zone populations in comparison with growth zone controls. IGF-I increased aggrecan gene expression for the same groups. Superficial zone populations exhibited lower collagen II, collagen I, and aggrecan gene expression than the growth zone populations under all conditions. However, superficial zone protein expression was dramatically elevated in superficial zone populations by TGF-β1. Collagen I expression showed a general increase under all conditions compared with initial values. Combined biosynthesis results showed that the superficial populations secreted little to no collagen, especially collagen II, in comparison with their growth zone counterparts. Glycosaminoglycan production was also much lower than for the growth zone groups. TGF-β1 and IGF-I increased collagen II production in the growth zone populations. TGF-β1 increased glycosaminoglycan secretions in the superficial zone populations and in the growth zone populations, whereas IGF-I produced an increase in glycosaminoglycan secretion only in the growth zone populations. Thus, growth factors elicit different proliferation, gene expression, and biosynthesis responses from zonal chondrocyte subpopulations.