Pankaj K. Choudhary

Early dysregulation of the mitochondrial proteome in a mouse model of Alzheimer's disease

Mitochondrial structural and functional alterations appear to play to an important role in the pathogenesis of Alzheimers disease (AD). In the present study, we used a quantitative comparative proteomic profiling approach to analyze changes in the mitochondrial proteome in AD. A triple transgenic mouse model of AD (3xTg-AD) which harbors mutations in three human transgenes, APP(Swe), PS1(M146V) and Tau(P301L), was used in these experiments. Quantitative differences in the mitochondrial proteome between the cerebral cortices of 6-month-old male 3xTg-AD and non-transgenic mice were determined by using two-dimensional difference gel electrophoresis (2D-DIGE) and tandem mass spectrometry. We identified 23 different proteins whose expression levels differed significantly between triple transgenic and non-transgenic mitochondria. Both down-regulated and up-regulated mitochondrial proteins were observed in transgenic AD cortices. Proteins which were dysregulated in 3xTg-AD cortices functioned in a wide variety of metabolic pathways, including the citric acid cycle, oxidative phosphorylation, pyruvate metabolism, glycolysis, oxidative stress, fatty acid oxidation, ketone body metabolism, ion transport, apoptosis, and mitochondrial protein synthesis. These alterations in the mitochondrial proteome of the cerebral cortices of triple transgenic AD mice occurred before the development of significant amyloid plaque and neurofibrillary tangles, indicating that mitochondrial dysregulation is an early event in AD.

Krüppel-like Factor 4 activates HBG gene expression in primary erythroid cells

The SP1/Krüppel‐like Factor (SP1/KLF) family of transcription factors plays a role in diverse cellular processes, including proliferation, differentiation and control of gene transcription. The discovery of KLF1 (EKLF), a key regulator of HBB (β‐globin) gene expression, expanded our understanding of the role of KLFs in erythropoiesis. In this study, we investigated a mechanism of HBG (γ‐globin) regulation by KLF4. siRNA‐mediated gene silencing and enforced expression of KLF4 in K562 cells substantiated the ability of KLF4 to positively regulate endogenous HBG gene transcription. The physiological significance of this finding was confirmed in primary erythroid cells, where KLF4 knockdown at day 11 significantly attenuated HBG mRNA levels and enforced expression at day 28 stimulated the silenced HBG genes. In vitro binding characterization using the γ‐CACCC and β‐CACCC probes demonstrated KLF4 preferentially binds the endogenous γ‐CACCC, while CREB binding protein (CREBBP) binding was not selective. Co‐immunoprecipitation studies confirmed protein‐protein interaction between KLF4 and CREBBP. Furthermore, sequential chromatin immunoprecipitation assays showed co‐localization of both factors in the γ‐CACCC region. Subsequent luciferase reporter studies demonstrated that KLF4 trans‐activated HBG promoter activity and that CREBBP enforced expression resulted in gene repression. Our data supports a model of antagonistic interaction of KLF4/CREBBP trans‐factors in HBG regulation.

Pharmaco-Proteomic Study of Hydroxyurea-Induced Modifications in the Sickle Red Blood Cell Membrane Proteome

Hydroxyurea (HU) is an effective oral drug for the management of homozygous sickle cell anemia (SS) in part because it increases fetal hemoglobin (HbF) levels within sickle red blood cells (RBCs) and thus reduces sickling. However, results from the Multicenter Study of HU suggested that clinical symptoms often improved before a significant increase in HbF levels occurred. This indicated that HU may be acting through the modification of additional cellular mechanisms that are yet to be identified. Hence, in this study, we focused on the analysis of the sickle RBC membrane proteome +/− HU treatment. 2D-DIGE (Two Dimensional Difference In-Gel Electrophoresis) technology and tandem mass spectrometry has been used to determine quantitative differences between sickle cell membrane proteins in the presence and absence of a clinically relevant concentration of HU. In vitro protein profiling of 13 sickle RBC membrane samples +/− 50 μM HU identified 10 statistically significant protein spots. Of these, the most remarkable class of proteins to show a statistically significant increase was the anti-oxidant enzymes—catalase, thioredoxin peroxidase and biliver-din reductase and the chaperonin containing TCP1 complex assisting in the folding of RBC cytoskeletal proteins. Interestingly, catalase immunoblots showed an increase in the acidic forms of the enzyme within sickle RBC membranes on incubation with 50 μM HU. We further identified this modification in catalase to be phosphorylation and demonstrated that HU exposed SS RBC membranes showed a 2-fold increase in tyrosine phosphorylation of catalase as compared to counterparts not exposed to HU. These results present an attractive model for HU-induced post-translational modification and potential activation of catalase in mature sickle RBCs. These findings also identify protein targets of HU other than fetal hemoglobin and enhance the understanding of the drug mechanism.

Measuring Agreement in Method Comparison Studies — A Review

Assessment of agreement between two or more methods of measurement is of considerable importance in many areas. In particular, in medicine, new methods or devices that are cheaper, easier to use, or less invasive, are routinely developed. Agreement between a new method and a traditional reference or gold standard must be evaluated before the new one is put into practice. Various methodologies have been proposed for this purpose in recent years. We review the literature focussing on the assessment of agreement between two methods, and on the selection of the best when several methods are compared with a reference. A real data set is analyzed to illustrate the various approaches.

Protein profiling of sickle cell versus control RBC core membrane skeletons by ICAT technology and tandem mass spectrometry

A proteomic approach using a cleavable ICAT reagent and nano-LC ESI tandem mass spectrometry was used to perform protein profiling of core RBC membrane skeleton proteins between sickle cell patients (SS) and controls (AA), and determine the efficacy of this technology. The data was validated through Peptide/Protein Prophet and protein ratios were calculated through ASAPratio. Through an ANOVA test, it was determined that there is no significant difference in the mean ratios from control populations (AA1/AA2) and sickle cell versus control populations (AA/SS). The mean ratios were not significantly different from 1.0 in either comparison for the core skeleton proteins (α spectrin, β spectrin, band 4.1 and actin). On the natural-log scale, the variation (standard deviation) of the method was determined to be 14.1% and the variation contributed by the samples was 13.8% which together give a total variation of 19.7% in the ratios.

In Vivo Pharmaco-Proteomic Analysis of Hydroxyurea Induced Changes in the Sickle Red Blood Cell Membrane Proteome

Hydroxyurea (HU) is an effective drug for the treatment of sickle cell disease (SCD). The main clinical benefit of HU is thought to derive from its capacity to increase fetal hemoglobin (HbF) production. However, other effects leading to clinical benefit, such as improved blood rheology, have been suggested. In order to understand HU-induced changes at the proteomic level, we profiled sickle RBC membranes from of HU-treated and untreated patients. Our previous in vitro profiling studies on sickle RBC membranes identified a significant increase in predominantly anti-oxidant enzymes, protein repair and degradation components and a few RBC cytoskeletal proteins. In the present study, using 2D-DIGE (Two-Dimensional Difference In-Gel Electrophoresis) and tandem mass spectrometry, we detected 32 different proteins that significantly changed in abundance in the HU treatment group. The proteins that significantly increased in abundance were mostly membrane skeletal components involved in the regulation of RBC shape and flexibility, and those showing a significant decrease were components of the protein repair and degradation machinery. RBC palmitoylated membrane protein 55 (p55) is significantly increased in abundance at low (in vitro) and high (in vivo) concentrations of HU. Palmitoylated p55 may be an important target of HU-dependent regulation of the sickle RBC membrane, consistent with our earlier in vitro studies.

Characterization of Cement Particles Found in Peri-implantitis-Affected Human Biopsy Specimens.

PURPOSE Peri-implantitis is a disease characterized by soft tissue inflammation and continued loss of supporting bone, which can result in implant failure. Peri-implantitis is a multifactorial disease, and one of its triggering factors may be the presence of excess cement in the soft tissues surrounding an implant. This descriptive study evaluated the composition of foreign particles from 36 human biopsy specimens with 19 specimens selected for analysis. The biopsy specimens were obtained from soft tissues affected by peri-implantitis around cement-retained implant crowns and compared with the elemental composition of commercial luting cement. MATERIALS AND METHODS Nineteen biopsy specimens were chosen for the comparison, and five test cements (TempBond, Telio, Premier Implant Cement, Intermediate Restorative Material, and Relyx) were analyzed using scanning electron microscopy equipped with energy dispersive x-ray spectroscopy. This enabled the identification of the chemical composition of foreign particles embedded in the tissue specimens and the composition of the five cements. Statistical analysis was conducted using classification trees to pair the particles present in each specimen with the known cements. RESULTS The particles in each biopsy specimen could be associated with one of the commercial cements with a level of probability ranging between .79 and 1. TempBond particles were found in one biopsy specimen, Telio particles in seven, Premier Implant Cement particles in four, Relyx particles in four, and Intermediate Restorative Material particles in three. CONCLUSION Particles found in human soft tissue biopsy specimens around implants affected by peri-implant disease were associated with five commercially available dental cements.

Measuring agreement in method comparison studies with heteroscedastic measurements

We propose a methodology for evaluation of agreement between two methods of measuring a continuous variable whose variability changes with magnitude. This problem routinely arises in method comparison studies that are common in health-related disciplines. Assuming replicated measurements, we first model the data using a heteroscedastic mixed-effects model, wherein a suitably defined true measurement serves as the variance covariate. Fitting this model poses some computational difficulties as the likelihood function is not available in a closed form. We deal with this issue by suggesting four estimation methods to obtain approximate maximum likelihood estimates. Two of these methods are based on numerical approximation of the likelihood, and the other two are based on approximation of the model. Next, we extend the existing agreement evaluation methodology designed for homoscedastic data to work under the proposed heteroscedastic model. This methodology can be used with any scalar measure of agreement. Simulations show that the suggested inference procedures generally work well for moderately large samples. They are illustrated by analyzing a data set of cholesterol measurements.

Bayesian and Frequentist Methodologies for Analyzing Method Comparison Studies With Multiple Methods

Evaluation of agreement among multiple methods of clinical measurement is a topic of considerable interest in health sciences. As in an analysis of variance comparing more than two treatment means, when more than two measurement methods are compared, performing multiple comparisons and ranking pairs of methods on the basis of their extent of agreement are of primary concern. This article develops frequentist and Bayesian methodologies for this purpose. In particular, simultaneous confidence bounds and simultaneous credible bounds are developed for multiple comparisons. Moreover, two approaches are described for ranking method pairs—one based on simultaneous bounds and the other based on posterior probabilities of possible orderings. The proposed methodologies can be used with any scalar measure of agreement. Their small-sample performance is evaluated using simulation. Extension of the basic methodologies to incorporate covariates is illustrated using a blood pressure dataset.

Visual scoring of eggshell patterns has poor repeatability

AbstractEggshell pattern scoring, a method to quantify the degree of surface maculation, can potentially be a quick, inexpensive and reliable method to obtain information on eggshell appearance and spot patterns. The key pigment responsible for red-brownish hues, protoporphyrin IX, is often localized as spots, either on the surface or in distinct layers within the eggshell. Heritable pigment spotting has been linked to factors such as breeding performance and eggshell strength. In this study, we investigated whether pigment scoring of eggshell patterns is repeatable within and between observers, by testing observers under standardised conditions, using the eggshells of two commonly studied passerines, Great Tits (Parus major) and Blue Tits (Cyanistes caeruleus). We found that repeatability of eggshell scores was poor, both within and between observers for both the species. We, therefore, encourage future studies to use alternative methods for quantifying spot patterns, such as digital image analysis, a technique which has already been used extensively.ZusammenfassungOptische Kategorisierung von Eierschalenmustern besitzt nur geringe Wiederholpräzision Die Kategorisierung von Eierschalenmustern, eine Methode zur Quantifizierung der Intensität der Oberflächenfleckung, kann eine schnelle, kostengünstige und zuverlässige Methode darstellen, um Informationen über Eierschalenerscheinung und Fleckenverteilung zu erlangen. Das Pigment, das hauptsächlich für die rot-braunen Farbtöne verantwortlich ist, das Protoporphyrin IX, kommt oft lokalisiert in Flecken entweder direkt an der Oberfläche oder in eng-begrenzten Schichten innerhalb der Eierschale vor. Erbliche, pigmentbedingte Fleckung ist mit Faktoren wie Bruterfolg und Schalenstärke in Verbindung gebracht worden. In der vorliegenden Studie untersuchen wir unter standardisierten Bedingungen am Beispiel der Eierschalen zweier beliebter Studienobjekte unter den Singvögeln, der Kohlmeise (Parus major) und der Blaumeise (Cyanistes caeruleus), inwiefern die optische Kategorisierung von Eierschalenmustern wiederholbar ist, sowohl durch ein und denselben Beobachter, als auch zwischen verschiedenen Beobachtern. Unsere Untersuchung zeigt für beide Meisenarten, dass die Wiederholbarkeit der Kategorisierung von Eierschalen beschränkt ist, sowohl im Vergleich wiederholter Kategorisierungen durch denselben Beobachter, als auch im Vergleich zwischen Beobachtern. Wir empfehlen daher in zukünftigen Untersuchungen alternative Methoden zur Quantifizierung von Fleckenmustern zu verwenden, zum Beispiel die digitale Bildanalyse, eine schon heute weit verbreitete Technik.