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Slane L.C.,550 Engineering Drive | Thelen D.G.,550 Engineering Drive | Thelen D.G.,1513 University Ave | Thelen D.G.,1300 University Ave
Medical Engineering and Physics | Year: 2015

The purpose of this study was to investigate middle-age effects on Achilles displacement patterns under passive stretch and eccentric loading. Healthy young (24.1±1.4 years, n=9) and middle-aged (49.0±3.1 years, n=9) adults were positioned prone and the ankle was cyclically dorsiflexed (0.5Hz, 25° range) during passive stretch and active lengthening. Achilles displacements were tracked in cine ultrasound using 2D speckle tracking. Displacements were found to be non-uniform, with mid and deep portions of the tendon displacing more than superficial portions. However, the degree of non-uniformity was significantly reduced in middle-aged adults, suggesting a potential age-related reduction in inter-fascicle sliding or a shift in loading sharing between plantarflexors. Eccentric loading reduced displacement magnitudes, likely reflecting distal tendon stretch induced via active muscle contractions. Changes in tendon displacement with active loading were greater in middle-aged adults, which could reflect greater tendon compliance. The observed age-related changes in Achilles tendon behavior may have implications for both plantarflexor performance and injury risk. © 2015 IPEM.


Chen B.,University of Wisconsin - Madison | Peng Y.,University of Wisconsin - Madison | Valeja S.G.,University of Wisconsin - Madison | Xiu L.,University of Wisconsin - Madison | And 4 more authors.
Analytical Chemistry | Year: 2016

Recent progress in top-down proteomics has led to a demand for mass spectrometry (MS)-compatible chromatography techniques to separate intact proteins using volatile mobile phases. Conventional hydrophobic interaction chromatography (HIC) provides high-resolution separation of proteins under nondenaturing conditions but requires high concentrations of nonvolatile salts. Herein, we introduce a series of more-hydrophobic HIC materials that can retain proteins using MS-compatible concentrations of ammonium acetate. The new HIC materials appear to function as a hybrid form of conventional HIC and reverse phase chromatography. The function of the salt seems to be preserving protein structure rather than promoting retention. Online HIC-MS is feasible for both qualitative and quantitative analysis. This is demonstrated with standard proteins and a complex cell lysate. The mass spectra of proteins from the online HIC-MS exhibit low charge-state distributions, consistent with those commonly observed in native MS. Furthermore, HIC-MS can chromatographically separate proteoforms differing by minor modifications. Hence, this new HIC-MS combination is promising for top-down proteomics. © 2016 American Chemical Society.


Peng Y.,University of Wisconsin - Madison | Gregorich Z.R.,University of Wisconsin - Madison | Valeja S.G.,University of Wisconsin - Madison | Zhang H.,University of Wisconsin - Madison | And 10 more authors.
Molecular and Cellular Proteomics | Year: 2014

Heart failure (HF) is a leading cause of morbidity and mortality worldwide and is most often precipitated by myocardial infarction. However, the molecular changes driving cardiac dysfunction immediately after myocardial infarction remain poorly understood. Myofilament proteins, responsible for cardiac contraction and relaxation, play critical roles in signal reception and transduction in HF. Post-translational modifications of myofilament proteins afford a mechanism for the beat-to-beat regulation of cardiac function. Thus it is of paramount importance to gain a comprehensive understanding of post-translational modifications of myofilament proteins involved in regulating early molecular events in the post-infarcted myocardium. We have developed a novel liquid chromatography-mass spectrometry-based top-down proteomics strategy to comprehensively assess the modifications of key cardiac proteins in the myofilament subproteome extracted from a minimal amount of myocardial tissue with high reproducibility and throughput. The entire procedure, including tissue homogenization, myofilament extraction, and on-line LC/MS, takes less than three hours. Notably, enabled by this novel top-down proteomics technology, we discovered a concerted significant reduction in the phosphorylation of three crucial cardiac proteins in acutely infarcted swine myocardium: cardiac troponin I and myosin regulatory light chain of the myofilaments and, unexpectedly, enigma homolog isoform 2 (ENH2) of the Z-disc. Furthermore, top-down MS allowed us to comprehensively sequence these proteins and pinpoint their phosphorylation sites. For the first time, we have characterized the sequence of ENH2 and identified it as a phosphoprotein. ENH2 is localized at the Z-disc, which has been increasingly recognized for its role as a nodal point in cardiac signaling. Thus our proteomics discovery opens up new avenues for the investigation of concerted signaling between myofilament and Z-disc in the early molecular events that contribute to cardiac dysfunction and progression to HF. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.


Cao X.-J.,1300 University Ave | Oertel D.,1300 University Ave
Journal of Neurophysiology | Year: 2010

Auditory nerve fibers are the major source of excitation to the three groups of principal cells of the ventral cochlear nucleus (VCN), bushy, T stellate, and octopus cells. Shock-evoked excitatory postsynaptic currents (eEPSCs) in slices from mice showed systematic differences between groups of principal cells, indicating that target cells contribute to determining pre- and postsynaptic properties of synapses from spiral ganglion cells. Bushy cells likely to be small spherical bushy cells receive no more than three, most often two, excitatory inputs; those likely to be globular bushy cells receive at least four, most likely five, inputs. T stellate cells receive 6.5 inputs. Octopus cells receive >60 inputs. The N-methyl-D-aspartate (NMDA) components of eEPSCs were largest in T stellate, smaller in bushy, and smallest in octopus cells, and they were larger in neurons from younger than older mice. The average AMPA conductance of a unitary input is 22 ± 15 nS in both groups of bushy cells, <1.5 nS in octopus cells, and 4.6 ± 3 nS in T stellate cells. Sensitivity to philanthotoxin (PhTX) and rectification in the intracellular presence of spermine indicate that AMPA receptors that mediate eEPSCs in T stellate cells contain more GluR2 subunits than those in bushy and octopus cells. The AMPA components of eEPSCs were briefer in bushy (0.5 ms half-width) than in T stellate and octopus cells (0.8-0.9 ms half-width). Widening of eEPSCs in the presence of cyclothiazide (CTZ) indicates that desensitization shortens eEPSCs. CTZ-insensitive synaptic depression of the AMPA components was greater in bushy and octopus than in T stellate cells. Copyright © 2010 The American Physiological Society.


Smidt E.R.,University of Wisconsin - Madison | Conley S.P.,University of Wisconsin - Madison | Zhu J.,1300 University Ave | Zhu J.,University of Wisconsin - Madison | Arriaga F.J.,University of Wisconsin - Madison
Agronomy Journal | Year: 2016

Until recently, soybean [Glycine max (L.) Merr.] fields were often seeded at a single rate. Advances in GPS and variable rate technology (VRT) are allowing growers to use variable rate planting prescriptions to optimize yields and input costs. This study was conducted to find the key predictors for characterizing soybean seed yield from commonly collected precision agriculture data layers. Research was conducted in 11 unique fields both in 2013 and 2014 in Wisconsin and all 22 site-years were following corn [Zea mays (L.)]. Seeding rate, soil sampling, yield, and soil survey data were gathered from each site for analysis. A statistical procedure used in the determination of key prediction parameters, random forest analysis, was used and identified soil map unit as the most important variable when predicting soybean seed yield for the pooled data sets in both 2013 and 2014. The next most important factors were, in order of importance, soil P, soil organic matter, soil available water supply in the upper 100 cm, soil K, and elevation in 2013 and soil P, elevation, soil K, soil organic matter, and soil available water supply in the upper 150 cm during 2014. Individual field random forest analyses determined elevation was the most important predictor of soybean yield, on average, for 2013 and 2014 followed by organic matter, K, P, and pH in 2013 and pH, K, organic matter, and P in 2014. © 2016 American Society of Agronomy 5585 Guilford Road, Madison, WI 53711 USA.


Maddodi N.,University of Wisconsin - Madison | Bhat K.M.R.,University of Wisconsin - Madison | Bhat K.M.R.,Manipal University India | Devi S.,University of Wisconsin - Madison | And 3 more authors.
Journal of Biological Chemistry | Year: 2010

MAP2 is a neuron-specific microtubule-associated protein that binds and stabilizes dendritic microtubules. Previously, we showed that MAP2 expression is (a) activated in cutaneous primary melanoma and (b) inversely associated with melanoma tumor progression. We also showed that ectopic expression of MAP2 in metastatic melanoma cells inhibits cell growth by inducing mitotic spindle defects and apoptosis. However, molecular mechanisms of regulation of MAP2 gene expression in melanoma are not understood. Here, we show that in melanoma cells MAP2 expression is induced by the demethylating agent 5-aza-2′-cytidine, and MAP2 promoter is progressively methylated during melanoma progression, indicating that epigenetic mechanisms are involved in silencing of MAP2 in melanoma. In support of this, methylation of MAP2 promoter DNA in vitro inhibits its activity. Because MAP2 promoter activity levels in melanoma cell lines also correlated with activating mutation in BRAF, a gene that is highly expressed in neurons, we hypothesized that BRAF signaling is involved in MAP2 expression. We show that hyperactivation of BRAF-MEK signaling activates MAP2 expression in melanoma cells by two independent mechanisms, promoter demethylation or down-regulation of neuronal transcription repressor HES1. Our data suggest that BRAF oncogene levels can regulate melanoma neuronal differentiation and tumor progression. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Gregorich Z.R.,University of Wisconsin - Madison | Chang Y.-H.,University of Wisconsin - Madison | Ge Y.,University of Wisconsin - Madison | Ge Y.,1300 University Ave
Pflugers Archiv European Journal of Physiology | Year: 2014

The pathophysiology of heart failure (HF) is diverse, owing to multiple etiologies and aberrations in a number of cellular processes. Therefore, it is essential to understand how defects in the molecular pathways that mediate cellular responses to internal and external stressors function as a system to drive the HF phenotype. Mass spectrometry (MS)-based proteomics strategies have great potential for advancing our understanding of disease mechanisms at the systems level because proteins are the effector molecules for all cell functions and, thus, are directly responsible for determining cell phenotype. Two MS-based proteomics strategies exist: peptide-based bottom-up and protein-based top-down proteomics-each with its own unique strengths and weaknesses for interrogating the proteome. In this review, we will discuss the advantages and disadvantages of bottom-up and top-down MS for protein identification, quantification, and analysis of post-translational modifications, as well as highlight how both of these strategies have contributed to our understanding of the molecular and cellular mechanisms underlying HF. Additionally, the challenges associated with both proteomics approaches will be discussed and insights will be offered regarding the future of MS-based proteomics in HF research. © 2014 Springer-Verlag.

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