A comprehensive diagnostic approach combining phylogenetic disease bracketing and CT imaging reveals osteomyelitis in a Tyrannosaurus rex.

Traditional palaeontological techniques of disease characterisation are limited to the analysis of osseous fossils, requiring several lines of evidence to support diagnoses. This study presents a novel stepwise concept for comprehensive diagnosis of pathologies in fossils by computed tomography imaging for morphological assessment combined with likelihood estimation based on systematic phylogenetic disease bracketing. This approach was applied to characterise pathologies of the left fibula and fused caudal vertebrae of the non-avian dinosaur Tyrannosaurus rex. Initial morphological assessment narrowed the differential diagnosis to neoplasia or infection. Subsequent data review from phylogenetically closely related species at the clade level revealed neoplasia rates as low as 3.1% and 1.8%, while infectious-disease rates were 32.0% and 53.9% in extant dinosaurs (birds) and non-avian reptiles, respectively. Furthermore, the survey of literature revealed that within the phylogenetic disease bracket the oldest case of bone infection (osteomyelitis) was identified in the mandible of a 275-million-year-old captorhinid eureptile Labidosaurus. These findings demonstrate low probability of a neoplastic aetiology of the examined pathologies in the Tyrannosaurus rex and in turn, suggest that they correspond to multiple foci of osteomyelitis.

Eye Movements in Frogs and Salamanders-Testing the Palatal Buccal Pump Hypothesis.

In frogs and salamanders, movements of the eyeballs in association with an open palate have often been proposed to play a functional role in lung breathing. In this "palatal buccal pump," the eyeballs are elevated during the lowering of the buccal floor to suck air in through the nares, and the eyeballs are lowered during elevation of the buccal floor to help press air into the lungs. Here, we used X-Ray Reconstruction of Moving Morphology to investigate eye movements during lung breathing and feeding in bullfrogs and axolotls. Our data do not show eye movements that would be in accordance with the palatal buccal pump. On the contrary, there is a small passive elevation of the eyeballs when the buccal floor is raised. Inward drawing of the eyeballs occurs only during body motion and for prey transport in bullfrogs, but this was not observed in axolotls. Each eye movement in bullfrogs has a vertical, a mediolateral, and an anteroposterior component. Considering the surprisingly weak posterior motion component of the eyeballs, their main role in prey transport might be fixing the prey by pressing it against the buccal floor. The retraction of the buccal floor would then contribute to the posterior push of the prey. Because our study provides no evidence for a palatal buccal pump in frogs and salamanders, there is also no experimental support for the idea of a palatal buccal pump in extinct temnospondyl amphibians, in contrast to earlier suggestions.

Movimientos oculares en ranas y salamandras: prueba de la hipótesis de la bomba bucal palatina (Eye movements in frogs and salamanders ­ testing the palatal buccal pump hypothesis) En ranas y salamandras, los movimientos oculares asociados con el paladar abierto a menudo se ha propuesto que desempeñan un papel funcional en la respiración pulmonar. En esta "bomba bucal palatina", los globos oculares se elevan durante la bajada del piso bucal para inhalar por las narinas, y los globos oculares se bajan durante la elevación del piso bucal para ayudar a presionar el aire hacia los pulmones. Aquí utilizamos la Reconstrucción de Rayos X de la Morfología en Movimiento para investigar los movimientos oculares durante la respiración pulmonar y la alimentación en ranas mugidoras y ajolotes. Nuestros datos no muestran movimientos oculares que estarían de acuerdo con la bomba bucal palatina. Por el contrario, hay una pequeña elevación pasiva de los globos oculares cuando se eleva el suelo bucal. La retracción interna de los globos oculares ocurre solo durante el movimiento del cuerpo y para el transporte de presas en las ranas mugidoras, pero esto no se observó en los ajolotes. Cada movimiento ocular en las ranas mugidoras tiene un componente vertical, mediolateral y anteroposterior. Considerando el componente de movimiento posterior sorprendentemente pequeño de los globos oculares, su función principal en el transporte de presas podría ser la fijación de la presa presionándola contra el suelo bucal. La retracción del suelo bucal contribuiría entonces al empuje posterior de la presa. Debido a que nuestro estudio no proporciona evidencia de una bomba bucal palatina en ranas y salamandras, tampoco hay apoyo experimental para la idea de una bomba bucal palatina en anfibios temnospóndilos extintos, en contraste con sugerencias anteriores. translated to Spanish by Y.E. Jimenez (yordano_jimenez@brown.edu).

Chemometrical analysis of proteomics data obtained from three cell types treated with multi-walled carbon nanotubes and TiO2 nanobelts$.

Applications of nanomaterials in biomedical, industrial, and consumer goods areas are expanding rapidly because of their unique physicochemical properties. Hazard assessment of nanosubstances is necessary for the protection of human and ecological health. We studied the proteomics patterns of three cell lines: co-culture of Caco-2 and HT29-MTX cells, primary small airway epithelial cells, and THP-1macrophage-like cells. The cells were exposed at 10 µg and 100 µg concentrations for 3 and 24 hours to multi-walled carbon nanotubes and TiO2 nanobelts (TiO2-NB). The data were analysed with the hierarchical clustering method and principal components analysis. In all cases, time of exposure is the most important factor in separation and clustering of proteomics patterns. Furthermore, the sets of proteins, which are specific for long (24 hours) exposure, are identified.

Congenital malformations of the vertebral column in ancient amphibians.

Temnospondyls, the largest group of Palaeozoic and Mesozoic amphibians, primitively possess rhachitomous vertebrae with multipartite centra (consisting of one horse-shoe-shaped inter- and paired pleurocentra). In a group of temnospondyls, the stereospondyls, the intercentra became pronounced and disc-like, whereas the pleurocentra were reduced. We report the presence of congenital vertebral malformations (hemi, wedge and block vertebrae) in Permian and Triassic temnospondyls, showing that defects of formation and segmentation in the tetrapod vertebral column represent a fundamental failure of somitogenesis that can be followed throughout tetrapod evolution. This is irrespective of the type of affected vertebra, that is, rhachitomous or stereospondylous, and all components of the vertebra can be involved (intercentrum, pleurocentrum and neural arch), either together or independently on their own. This is the oldest known occurrence of wedge vertebra and congenital block vertebra described in fossil tetrapods. The frequency of vertebral congenital malformations in amphibians appears unchanged from the Holocene.

Differential protein expression during aging in ventricular myocardium of Fischer 344 x Brown Norway hybrid rats.

The aging heart undergoes well characterized structural changes associated with functional decline, though the underlying mechanisms are not understood. The aim of this study was to determine to what extent ventricular myocardial protein expression was altered with age and which proteins underwent protein nitration. Fischer 344 x Brown Norway F1 hybrid (FBN) rats of four age groups were used, 4, 12, 24, and 34 months. Differential protein expression was determined by 2-DE and proteins were identified by peptide mass fingerprinting. Altered protein nitration with age was assessed by immunoblotting. Over 1000 protein spots per sample were detected, and 255 were found to be differentially expressed when all aged groups were compared to young rats (4 months) (p0.05). A strong positive correlation between differential protein expression and increasing age (p=0.03, R(2)=0.997) indicated a progressive, rather than abrupt, change with age. Of 46 differentially expressed proteins identified, seventeen have roles in apoptosis, ten in hypertrophy, seven in fibrosis, and three in diastolic dysfunction, aging-associated processes previously reported in both human and FBN rat heart. Protein expression alterations detected here could have beneficial effects on cardiac function; thus, our data indicate a largely adaptive change in protein expression during aging. In contrast, differential protein nitration increased abruptly, rather than progressively, at 24 months of age. Altogether, the results suggest that differential myocardial protein expression occurs in a progressive manner during aging, and that a proteomic-based approach is an effective method for the identification of potential therapeutic targets to mitigate aging-related myocardial dysfunction.

Investigation of protein expression in magnetic field-treated human glioma cells.

We previously reported phenotypic changes in human breast cancer cells following low-level magnetic field (MF) exposure. Here proteomic methods were used to investigate the biochemical effect of MF exposure in SF767 human glioma cells. Protein alterations were studied after exposure to 1.2 microTesla (microT) MF [12 milliGauss (mG), 60 Hertz (Hz)] +/- epidermal growth factor (EGF). SF767 cells were exposed for 3 h to sham conditions (<0.2 microT ambient field strength) or 1.2 microT MF (+/-EGF; 10 ng/ml). Solubilized protein fractions (sham; 1.2 microT; sham + EGF; 1.2 microT + EGF) were loaded for electrophoresis by 2D-PAGE and stained using a colloidal Coomassie blue technique to resolve and characterize the proteins. Protein patterns were compared across groups via Student's t-test using PDQUEST software. Cell profiles revealed significant alterations in the spot density of a subset of treated cells. Automated spot excision and processing was performed prior to peptide mass fingerprinting proteins of interest. Fifty-seven proteins from the detectable pool were identified and/or found to differ significantly across treatment groups. The mean abundance of 10 identified proteins was altered following 1.2 microT exposure. In the presence of EGF six proteins were altered after low magnetic field treatment by increasing (4) or decreasing (2) in abundance. The results suggest that the analysis of differentially expressed proteins in SF767 cells may be useful as biomarkers for biological changes caused by exposure to magnetic fields.

Protein expression changes in the nucleus accumbens and amygdala of inbred alcohol-preferring rats given either continuous or scheduled access to ethanol.

Chronic ethanol (EtOH) drinking produces neuronal alterations within the limbic system. To investigate changes in protein expression levels associated with EtOH drinking, inbred alcohol-preferring (iP) rats were given one of three EtOH access conditions in their home-cages: continuous ethanol (CE: 24h/day, 7days/week access to EtOH), multiple scheduled access (MSA: four 1-h sessions during the dark cycle/day, 5 days/week) to EtOH, or remained EtOH-naïve. Both MSA and CE groups consumed between 6 and 6.5g of EtOH/kg/day after the 3rd week of access. On the first day of EtOH access for the seventh week, access was terminated at the end of the fourth MSA session for MSA rats and the corresponding time point (2300h) for CE rats. Ten h later, the rats were decapitated, brains extracted, the nucleus accumbens (NAcc) and amygdala (AMYG) microdissected, and protein isolated for 2-dimensional gel electrophoretic analyses. In the NAcc, MSA altered expression levels for 12 of the 14 identified proteins, compared with controls, with six of these proteins altered by CE access, as well. In the AMYG, CE access changed expression levels for 22 of the 27 identified proteins, compared with controls, with 8 of these proteins altered by MSA, as well. The proteins could be grouped into functional categories of chaperones, cytoskeleton, intracellular communication, membrane transport, metabolism, energy production, or neurotransmission. Overall, it appears that EtOH drinking and the conditions under which EtOH is consumed, differentially affect protein expression levels between the NAcc and AMYG. This may reflect differences in neuroanatomical and/or functional characteristics associated with EtOH self-administration and possibly withdrawal, between these two brain structures.

Effect of JP-8 jet fuel exposure on protein expression in human keratinocyte cells in culture.

Dermal exposure to jet fuel is a significant occupational hazard. Previous studies have investigated its absorption and disposition in skin, and the systemic biochemical and immunotoxicological sequelae to exposure. Despite studies of JP-8 jet fuel components in murine, porcine or human keratinocyte cell cultures, proteomic analysis of JP-8 exposure has not been investigated. This study was conducted to examine the effect of JP-8 administration on the human epidermal keratinocyte (HEK) proteome. Using a two-dimensional electrophoretic approach combined with mass spectrometric-based protein identification, we analyzed protein expression in HEK exposed to 0.1% JP-8 in culture medium for 24 h. JP-8 exposure resulted in significant expression differences (p<0.02) in 35 of the 929 proteins matched and analyzed. Approximately, a third of these alterations were increased in protein expression, two-thirds declined with JP-8 exposure. Peptide mass fingerprint identification of effected proteins revealed a variety of functional implications. In general, altered proteins involved endocytotic/exocytotic mechanisms and their cytoskeletal components, cell stress, and those involved in vesicular function.

Changes in gene and protein expression in magnetic field-treated human glioma cells.

Because few cancer studies have examined protein profiles and genetic regulation from a single carcinogen exposure, the objective of this study was to determine genetic change via microarray and to evaluate whether that change was a precursor to cellular protein changes. In separate but experimentally identical studies, human glioma SF767 cells were exposed for 3 h to 60-Hz magnetic fields (sham or 1.2 muT). Microarray results suggested that magnetic field treatment resulted in the up-regulation of 5 genes, whereas 25 genes were down-regulated. The mean abundance of 10 identified proteins was altered following 1.2 muT exposure relative to sham (3 increase, 7 decrease). These studies suggest a limited but complicated response in the glioma cells to the magnetic field treatment.

Proteomics: applications and opportunities in preclinical drug development.

Advances in DNA sequencing and the near-term availability of whole genome sequences for several pharmaceutically relevant organisms promise to dramatically alter the breadth and scale of high-throughput proteomic studies. The substantial amount of literature is available in the public domain, demonstrate the potential of proteomics in the preclinical phases of pharmaceutical development. Over the next few years, it is anticipated that functional genomics and proteomics will have major impacts on the clinical phases of drug development. Expected benefits are earlier proof-of-concept studies in man and increased efficiency of clinical trials through the availability of biologically relevant markers for drug efficacy and safety.

Toxicity of chemical mixtures: proteomic analysis of persisting liver and kidney protein alterations induced by repeated exposure of rats to JP-8 jet fuel vapor.

Male Sprague-Dawley rats were exposed by whole body inhalation to 1000 mg/m3 +/- 10% JP-8 jet fuel vapor or room air control conditions for 6 h/day, 5 days/week for six consecutive weeks. Following a rest period of 82 days rats were sacrificed, and liver and kidney tissues examined by proteomic methods for both total protein abundance and protein charge modification. Kidney and lung samples were solubilized and separated via large scale, high resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Through the use of peptide mass fingerprinting, confirmed by sequence tag analysis, three altered proteins were identified and quantified. Numerical, but not significantly different increases were found in total abundance of lamin A (NCBI Accession No. 1346413) in the liver, and of 10-formyltetrahydrofolate dehydrogenase (10-FTHF DH, #1346044) and glutathione-S-transferase (GST; #2393724) in the kidneys of vapor-exposed subjects. Protein charge modification index (CMI) analysis indicated significant alterations (P < 0.001) in expressed lamin A and 10-FTHF DH. These persisting changes in liver and kidney proteins are discussed in terms of possible alterations in the functional capacity of exposed subjects.

Proteomic analysis of the renal effects of simulated occupational jet fuel exposure.

We analyzed protein expression in the cytosolic fraction prepared from whole kidneys in male Swiss-Webster mice exposed 1 h/day for five days to aerosolized JP-8 jet fuel at a concentration of 1000 mg/m3, simulating military occupational exposure. Kidney cytosol samples were solubilized and separated via large-scale, high-resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant changes in soluble kidney proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass finger-printing and related to ultrastructural abnormalities, altered protein processing, metabolic effects, and paradoxical stress protein/detoxification system responses. These results demonstrate a significant but comparatively moderate JP-8 effect on protein expression in the kidney and provide novel molecular evidence of JP-8 nephrotoxicity. Human risk is suggested by these data but conclusive assessment awaits a noninvasive search for biomarkers in JP-8 exposed humans.

Proteomic analysis of simulated occupational jet fuel exposure in the lung.

We analyzed protein expression in the cytosolic fraction prepared from whole lung tissue in male Swiss-Webster mice exposed 1 h/day for seven days to aerosolized JP-8 jet fuel at concentrations of 1000 and 2500 mg/m3, simulating military occupational exposure. Lung cytosol samples were solubilized and separated via large scale, high resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant quantitative and qualitative changes in tissue cytosol proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass fingerprinting, confirmed by sequence tag analysis, and related to impaired protein synthetic machinery, toxic/metabolic stress and detoxification systems, ultrastructural damage, and functional responses to CO2 handling, acid-base homeostasis and fluid secretion. These results demonstrate a significant but comparatively moderate JP-8 effect on protein expression and corroborate previous morphological and biochemical evidence. Further molecular marker development and mechanistic inferences from these observations await proteomic analysis of whole tissue homogenates and other cell compartment, i.e., mitochondria, microsomes, and nuclei of lung and other targets.

Alterations in rabbit kidney protein expression following lead exposure as analyzed by two-dimensional gel electrophoresis.

It was recently reported that low blood lead levels impaired kidney function in men. To develop a set of molecular markers of renal lead exposure and effect, we investigated changes in renal protein expression while approximating occupational lead exposure at subchronic, low blood levels. Lead was administered to male Dutch Belted rabbits as a lead acetate solution adjusted weekly to achieve and maintain the target blood lead levels of 0, 20, 40, and 80 microg/dL for 15 weeks. Lead exposure did not affect kidney or body weights. The effect of increasing blood lead on protein expression was evaluated in rabbit kidney by large-scale two-dimensional electrophoresis (2-DE). Significant quantitative changes (p < 0.05) occurred in a dose-related manner in 12 proteins at 20 microg/dL exposure, 25 at 40 microg/dL, and 102 at 80 microg/dL. At a higher level of significance (p < 0.001), 40 microg/dL blood lead resulted in one protein alteration and 80 microg/dL affected 14 proteins. A set of quantitatively altered charge variants was tentatively identified as glutathione-S-transferase (GST), based on similar observations in rodents subjected to short-term, very high lead exposure. The significance of the protein alterations observed as markers of toxicity awaits their conclusive identification. Investigation of the kidney 2-DE profile in lead-exposed rabbit may be useful in understanding the mechanism of lead nephrotoxicity in humans.

Regional protein alterations in rat kidneys induced by lead exposure.

Lead is a potent neuro- and nephrotoxin in humans and a renal carcinogen in rats. Previous studies have detected lead-induced increases in the activities of specific detoxification enzymes in distinct kidney cell types preceding irreversible renal damage. While preferential susceptibility of the highly vascularized cortex to the effects of lead is clear, lead effects on the medullary region have remained unexplored. The present study was undertaken to investigate the extent to which regional renal protein expression differs and to determine which, if any, regionally distinct protein markers indicative of lead's renotoxic mechanism might be detected in kidney cortical and medullary cytosols. We examined protein expression in these two functionally and anatomically distinct regions, and identified several proteins that are differentially expressed in those regions and were significantly altered by lead. Kidney cytosols from rats injected with lead acetate (114 mg/kg, three consecutive daily injections) were separated by two-dimensional electrophoresis. Lead exposure significantly (P<0.001) altered the abundance (either or) of 76 proteins in the cortex and only 13 in the medulla. Eleven of the proteins altered in the protein patterns were conclusively identified either by matrix-assisted laser desorption/ionization mass spectrometry/electrospray ionization-mass spectrometry (MALDI-MS/ESI-MS) analysis of peptide digests, immunological methods, or by gel matching. Several of the cortical proteins altered by lead were unchanged in the medulla while others underwent similar but lesser alterations. These observations reflect the complexity of lead's nephrotoxicity and endorse the application of proteomics in mechanistic studies as well as biomarker development in a variety of toxicologic paradigms.

Differential expression of cytosolic proteins in the rat kidney cortex and medulla: preliminary proteomics.

The rodent kidney is a target of many xenobiotics and is typified by regionally specific structure and function. This renders distinct regions of the kidney differentially susceptible to toxic exposure and effect. To characterize these differences at the proteome level, protein patterns from male rat kidney cortex and medulla cytosols were examined by two-dimensional electrophoresis (2-DE) and image analysis and prominent proteins identified immunologically or by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and electrospray/ionization-tandem mass spectrometry (ESI-MS/MS) sequence tag identification. An average of 727 protein spots were resolved and matched to the cortex cytosol reference pattern, and 716 in the medulla. Of this total, 127 proteins were found to differ in abundance (86 higher in cortex; 41 higher in medulla) (P < 0.001). Of those proteins that were detectable in both cortex and medulla, the abundance of 97 differed significantly while 30 proteins were found to be unique to one region or the other (26 in cortex, 4 in medulla). Twenty protein spots were identified and their regional differences are discussed. These results both confirm and expand our understanding of the molecular heterogeneity characterizing structurally and functionally distinct regions of the kidney and serve as a useful foundation for future nephrotoxicologic studies.

Glutathione S-transferases: two-dimensional electrophoretic protein markers of lead exposure.

Glutathione S-transferases (GST) are a family of detoxification isoenzymes that catalyze the conjugation of xenobiotics and their metabolites with reduced glutathione. Lead exposure in rats is known to induce GST isoenzymes in the liver and kidney. These changes in expression have potential use as biomarkers of lead exposure. Because two-dimensional electrophoresis (2-DE) enables one to analyze both protein abundance changes and chemical changes in protein structure, 2-DE was used to determine the effect of in vivo lead exposure on GST isoform expression in rat kidney cytosols. Male Sprague-Dawley rats were exposed to inorganic lead, and proteins were separated by conventional ISO-DALT and NEPHGE-DALT techniques and blotted for immunological identification. Lead exposure caused detectable inductions in both GSTP1 and GSTM1 and quantifiable charge modification in GSTP1. These preliminary data confirm the utility of 2-D electrophoretic GST analysis as indicative of lead exposure and toxicity and support its use for further elaboration of lead's effects on renal protein expression.

Two-dimensional electrophoresis of precision-cut testis slices: toxicologic application.

Advances in tissue slice technology and a recent novel application of this technique to reproductive toxicology using bovine testis have demonstrated the remarkable utility of this approach. The objective of the present study was to combine this in vitro toxicity test system with large-scale two-dimensional polyacrylamide gel electrophoresis (2-DE) to detect and study alterations in testicular-slice protein patterns as molecular correlates of 1,3,5-trinitrobenzene (TNB) and 1,3-dinitrobenzene (DNB) toxicity. Previous studies have shown that testicular slices remain viable for > 24 h and, as measured by protein synthesis inhibition, TNB causes dose-related injury. Tissue-slices were prepared from bovine testicles incubated for 2, 4 or 6 h and exposed to either 100 microM, 500 microM or 1 mM DNB or TNB in the incubation medium. Slices were collected, solubilized, and separated by large scale 2-DE. Resulting protein patterns were then examined by image analysis, which revealed coefficients of variation in protein spot abundance comparable to patterns from fresh rodent tissue samples. Furthermore, specific protein alterations indicated dose-related inductions and declines in protein abundance, some progressive over time. The results of this investigation demonstrate the potential toxicologic utility of combining in vitro tissue-slice technology with high-resolution 2-DE protein mapping. The consolidation of these methods offers a novel approach for toxicity screening and testing, reduces experimental cost, and reduces the use of laboratory animals.

Two-dimensional electrophoretic analysis of myocardial proteins from lead-exposed rabbits.

Despite reported adverse effects, the cardiovascular toxicity of lead remains controversial. The purpose of the present study was to determine if low-level subchronic exposure of rabbits to lead would produce detectable, concentration-dependent changes in myocardial proteins. Lead was administered to male Dutch Belted rabbits as a lead acetate solution, adjusted weekly to achieve and maintain the target blood lead levels of 0, 20, 40, and 80 microg/dL for 15 weeks. Lead exposures did not affect heart or body weights. Myocardial concentrations of lead at sacrifice were 58+/-25, 69+/-23, 102+/-62, and 105+/-37 ng/g. Of 808 individual proteins resolved by two-dimensional electrophoresis (2-DE) in ventricular homogenates, 162 had coefficients of variation < 20%. A number of proteins were tentatively identified based on coordinate positions homologous to other established 2-DE patterns. Despite variable expression of some protein spots, none of the protein abundances analyzed were found to be significantly altered (P < 0.001) by the lead exposures studied. Therefore results show no detectable effect of a low-body burden of lead on major myocardial proteins of the rabbit.