Sudeepa Bhattacharyya

An NMR approach to structural proteomics

The influx of genomic sequence information has led to the concept of structural proteomics, the determination of protein structures on a genome-wide scale. Here we describe an approach to structural proteomics of small proteins using NMR spectroscopy. Over 500 small proteins from several organisms were cloned, expressed, purified, and evaluated by NMR. Although there was variability among proteomes, overall 20% of these proteins were found to be readily amenable to NMR structure determination. NMR sample preparation was centralized in one facility, and a distributive approach was used for NMR data collection and analysis. Twelve structures are reported here as part of this approach, which allowed us to infer putative functions for several conserved hypothetical proteins.

Potential of extracellular microRNAs as biomarkers of acetaminophen toxicity in children.

UNLABELLED Developing biomarkers for detecting acetaminophen (APAP) toxicity has been widely investigated. Recent studies of adults with APAP-induced liver injury have reported human serum microRNA-122 (miR-122) as a novel biomarker of APAP-induced liver injury. The goal of this study was to examine extracellular microRNAs (miRNAs) as potential biomarkers for APAP liver injury in children. Global levels of serum and urine miRNAs were examined in three pediatric subgroups: 1) healthy children (n=10), 2) hospitalized children receiving therapeutic doses of APAP (n=10) and 3) children hospitalized for APAP overdose (n=8). Out of 147 miRNAs detected in the APAP overdose group, eight showed significantly increased median levels in serum (miR-122, -375, -423-5p, -30d-5p, -125b-5p, -4732-5p, -204-5p, and -574-3p), compared to the other groups. Analysis of urine samples from the same patients had significantly increased median levels of four miRNAs (miR-375, -940, -9-3p and -302a) compared to the other groups. Importantly, correlation of peak serum APAP protein adduct levels (an indicator of the oxidation of APAP to the reactive metabolite N-acetyl-para-quinone imine) with peak miRNA levels showed that the highest correlation was observed for serum miR-122 (R=0.94; p<0.01) followed by miR-375 (R=0.70; p=0.05). CONCLUSION Our findings demonstrate that miRNAs are increased in children with APAP toxicity and correlate with APAP protein adducts, suggesting a potential role as biomarkers of APAP toxicity.

Serum biomarker profile associated with high bone turnover and BMD in postmenopausal women.

Early diagnosis of the onset of osteoporosis is key to the delivery of effective therapy. Biochemical markers of bone turnover provide a means of evaluating skeletal dynamics that complements static measurements of BMD by DXA. Conventional clinical measurements of bone turnover, primarily the estimation of collagen and its breakdown products in the blood or urine, lack both sensitivity and specificity as a reliable diagnostic tool. As a result, improved tests are needed to augment the use of BMD measurements as the principle diagnostic modality. In this study, the serum proteome of 58 postmenopausal women with high or low/normal bone turnover (training set) was analyzed by surface enhanced laser‐desorption/ionization time‐of‐flight mass spectrometry, and a diagnostic fingerprint was identified using a variety of statistical and machine learning tools. The diagnostic fingerprint was validated in a separate distinct test set, consisting of serum samples from an additional 59 postmenopausal women obtained from the same Mayo cohort, with a gap of 2 yr. Specific protein peaks that discriminate between postmenopausal patients with high or low/normal bone turnover were identified and validated. Multiple supervised learning approaches were able to classify the level of bone turnover in the training set with 80% sensitivity and 100% specificity. In addition, the individual protein peaks were also significantly correlated with BMD measurements in these patients. Four of the major discriminatory peaks in the diagnostic profile were identified as fragments of interalpha‐trypsin‐inhibitor heavy chain H4 precursor (ITIH4), a plasma kallikrein‐sensitive glycoprotein that is a component of the host response system. These data suggest that these serum protein fragments are the serum‐borne reflection of the increased osteoclast activity, leading to the increased bone turnover that is associated with decreasing BMD and presumably an increased risk of fracture. In conjunction with the identification of the individual proteins, this protein fingerprint may provide a novel approach to evaluate high bone turnover states.

Translational biomarkers of acetaminophen-induced acute liver injury

Acetaminophen (APAP) is a commonly used analgesic drug that can cause liver injury, liver necrosis and liver failure. APAP-induced liver injury is associated with glutathione depletion, the formation of APAP protein adducts, the generation of reactive oxygen and nitrogen species and mitochondrial injury. The systems biology omics technologies (transcriptomics, proteomics and metabolomics) have been used to discover potential translational biomarkers of liver injury. The following review provides a summary of the systems biology discovery process, analytical validation of biomarkers and translation of omics biomarkers from the nonclinical to clinical setting in APAP-induced liver injury.

Acylcarnitine Profiles in Acetaminophen Toxicity in the Mouse: Comparison to Toxicity, Metabolism and Hepatocyte Regeneration

High doses of acetaminophen (APAP) result in hepatotoxicity that involves metabolic activation of the parent compound, covalent binding of the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI) to liver proteins, and depletion of hepatic glutathione. Impaired fatty acid β-oxidation has been implicated in previous studies of APAP-induced hepatotoxicity. To better understand relationships between toxicity and fatty acid β-oxidation in the liver in APAP toxicity, metabolomic assays for long chain acylcarnitines were examined in relationship to established markers of liver toxicity, oxidative metabolism, and liver regeneration in a time course study in mice. Male B6C3F1 mice were treated with APAP (200 mg/kg IP) or saline and sacrificed at 1, 2, 4, 8, 24 or 48 h after APAP. At 1 h, hepatic glutathione was depleted and APAP protein adducts were markedly increased. Alanine aminotransferase (ALT) levels were elevated at 4 and 8 h, while proliferating cell nuclear antigen (PCNA) expression, indicative of hepatocyte regeneration, was apparent at 24 h and 48 h. Elevations of palmitoyl, oleoyl and myristoyl carnitine were apparent by 2–4 h, concurrent with the onset of Oil Red O staining in liver sections. By 8 h, acylcarnitine levels were below baseline levels and remained low at 24 and 48 h. A partial least squares (PLS) model suggested a direct association of acylcarnitine accumulation in serum to APAP protein adduct and hepatic glutathione levels in mice. Overall, the kinetics of serum acylcarnitines in APAP toxicity in mice followed a biphasic pattern involving early elevation after the metabolism phases of toxicity and later depletion of acylcarnitines.

Biomarkers that discriminate multiple myeloma patients with or without skeletal involvement detected using SELDI-TOF mass spectrometry and statistical and machine learning tools

Multiple Myeloma (MM) is a severely debilitating neoplastic disease of B cell origin, with the primary source of morbidity and mortality associated with unrestrained bone destruction. Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) was used to screen for potential biomarkers indicative of skeletal involvement in patients with MM. Serum samples from 48 MM patients, 24 with more than three bone lesions and 24 with no evidence of bone lesions were fractionated and analyzed in duplicate using copper ion loaded immobilized metal affinity SELDI chip arrays. The spectra obtained were compiled, normalized, and mass peaks with mass-to-charge ratios (m/z) between 2000 and 20,000 Da identified. Peak information from all fractions was combined together and analyzed using univariate statistics, as well as a linear, partial least squares discriminant analysis (PLS-DA), and a non-linear, random forest (RF), classification algorithm. The PLS-DA model resulted in prediction accuracy between 96–100%, while the RF model was able to achieve a specificity and sensitivity of 87.5% each. Both models as well as multiple comparison adjusted univariate analysis identified a set of four peaks that were the most discriminating between the two groups of patients and hold promise as potential biomarkers for future diagnostic and/or therapeutic purposes.

Proteomic analysis of bone cancer: a review of current and future developments.

The ability of sophisticated proteomic approaches to scrutinize the dynamic nature of protein expression, cellular and subcellular protein distribution, post-translational modifications, and protein–protein interactions has culminated in the identification of many potential new therapeutic targets and an abundance of cancer-related biomarkers. From a proteomics perspective, amongst the most under-studied diseases are bone cancers, such as myeloma, osteosarcoma and breast and prostate cancer bony metastases. This review focuses on the recent advances in proteomic technology that have thrust the skeletal cancer field into this exciting age of proteomics, and highlights the future work that is required to adapt this technology to specifically interrogate the skeletal consequences of malignancy.

Metabolomics evaluation of hydroxyproline as a potential marker of melamine and cyanuric acid nephrotoxicity in male and female Fischer F344 rats

Following kidney failure in domesticated pets in the US and kidney issues requiring hospitalization with some deaths in children in China, investigators determined the cause was adulteration of pet foods and baby formula with melamine. It has since been noted that exposure of rats to melamine and cyanuric acid forms melamine cyanurate crystals in the kidney leading to acute nephrotoxicity. This metabolomics study aimed to identify biomarkers of melamine and cyanuric acid-induced renal injury. Male and female Fischer 344 rats were fed a diet fortified with varying doses of melamine and cyanuric acid for 28 days. Analysis of urinary amino acids showed hydroxyproline was increased in both sexes in a manner consistent with the clinical chemistry and histopathology data; most prominent when total urine output was taken into account. Furthermore, rats with the highest levels of urinary hydroxyproline were the only rats that exhibited fibrosis within the kidney. Clinical chemistry and histopathology indicated male rats were slightly more affected than female rats following dosing with the 120 and 180 ppm formulations; hydroxyproline excretion also supports this finding. Hydroxyproline may be a noninvasive urinary biomarker for detection of acute kidney injury potentially associated with kidney fibrosis.

Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity

Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury.

Oral Glutamine Reduces Radiation Morbidity in Breast Conservation Surgery

This study examined the effect of oral glutamine (Gln) on radiation injury in breast cancer patients undergoing radiation therapy. The radiation injury was evaluated using Radiation Therapy Oncology Group (RTOG) scales. Cosmesis was scored. Blood Gln and glutathione (GSH) levels were determined. Serum protein profiling was determined using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Patients receiving Gln scored significantly better in RTOG score than the patients receiving placebo. Cosmetic scores averaged excellent in the Gln group vs fair to good in the placebo group. Blood Gln and GSH levels were significantly higher in the Gln group vs the placebo group. Serum protein profiling with SELDI-TOF MS identified a novel Gln-responsive protein that showed amino acid similarity with myoglobin. These results suggest that Gln is an effective way to reduce radiation morbidity to breast cancer and is associated with the increased expression of a novel serum protein biomarker.