Arundhuti Sen

Evidence of altered phosphatidylcholine metabolism in Alzheimer's disease

Abberant lipid metabolism is implicated in Alzheimers disease (AD) pathophysiology, but the connections between AD and lipid metabolic pathways are not fully understood. To investigate plasma lipids in AD, a multiplatform screen (n = 35 by liquid chromatography-mass spectrometry and n = 35 by nuclear magnetic resonance) was developed, which enabled the comprehensive analysis of plasma from 3 groups (individuals with AD, individuals with mild cognitive impairment (MCI), and age-matched controls). This screen identified 3 phosphatidylcholine (PC) molecules that were significantly diminished in AD cases. In a subsequent validation study (n = 141), PC variation in a bigger sample set was investigated, and the same 3 PCs were found to be significantly lower in AD patients: PC 16:0/20:5 (p < 0.001), 16:0/22:6 (p < 0.05), and 18:0/22:6 (p < 0.01). A receiver operating characteristic (ROC) analysis of the PCs, combined with apolipoprotein E (ApoE) data, produced an area under the curve predictive value of 0.828. Confirmatory investigations into the background biochemistry indiciated no significant change in plasma levels of 3 additional PCs of similar structure, total choline containing compounds or total plasma omega fatty acids, adding to the evidence that specific PCs play a role in AD pathology.

A [2+2] cross-photodimerisation of photostable olefins via a three-component cocrystal solid solution.

A ditopic hydrogen-bond-donor template in the form of resorcinol facilitates a [2+2] cross-photodimerisation of 4-Cl-stilbazole and 4-Me-stilbazole in a rare cocrystal solid solution. A photoreaction does not proceed with the olefins individually or as a solid solution composed solely of the two olefins.

Extension and Limits of the Network of Coupled Motions Correlated to Hydride Transfer in Dihydrofolate Reductase

Enzyme catalysis has been studied extensively, but the role of enzyme dynamics in the catalyzed chemical conversion is still an enigma. The enzyme dihydrofolate reductase (DHFR) is often used as a model system to assess a network of coupled motions across the protein that may affect the catalyzed chemical transformation. Molecular dynamics simulations, quantum mechanical/molecular mechanical studies, and bioinformatics studies have suggested the presence of a “global dynamic network” of residues in DHFR. Earlier studies of two DHFR distal mutants, G121V and M42W, indicated that these residues affect the chemical step synergistically. While this finding was in accordance with the concept of a network of functional motions across the protein, two residues do not constitute a network. To better define the extent and limits of the proposed network, the current work studied two remote residues predicted to be part of the same network: W133 and F125. The effect of mutations in these residues on the nature of the chemical step was examined via measurements of the temperature-dependence of the intrinsic kinetic isotope effects (KIEs) and other kinetic parameters, and double mutants were used to tie the findings to G121 and M42. The findings indicate that residue F125, which was implicated by both calculations and bioinformatic methods, is a part of the same global dynamic network as G121 and M42, while W133, implicated only by bioinformatics, is not. These findings extend our understanding of the proposed network and the relations between functional and genomic couplings. Delineating that network illuminates the need to consider remote residues and protein structural dynamics in the rational design of drugs and of biomimetic catalysts.

Triple Isotopic Labeling and Kinetic Isotope Effects: Exposing H-Transfer Steps in Enzymatic Systems

Kinetic isotope effect (KIE) studies can provide insight into the mechanism and kinetics of specific chemical steps in complex catalytic cascades. Recent results from hydrogen KIE measurements have examined correlations between enzyme dynamics and catalytic function, leading to a surge of studies in this area. Unfortunately, most enzymatic H-transfer reactions are not rate limiting, and the observed KIEs do not reliably reflect the intrinsic KIEs on the chemical step of interest. Given their importance to understanding the chemical step under study, accurate determination of the intrinsic KIE from the observed data is essential. In 1975, Northrop developed an elegant method to assess intrinsic KIEs from their observed values [Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions. Biochemistry 14, 2644-2651]. The Northrop method involves KIE measurements using all three hydrogen isotopes, where one of them serves as the reference isotope. This method has been successfully used with different combinations of observed KIEs over the years, but criteria for a rational choice of reference isotope have never before been experimentally determined. Here we compare different reference isotopes (and hence distinct experimental designs) using the reduction of dihydrofolate and dihydrobiopterin by two dissimilar enzymes as model reactions. A number of isotopic labeling patterns have been applied to facilitate the comparative study of reference isotopes. The results demonstrate the versatility of the Northrop method and that such experiments are limited only by synthetic techniques, availability of starting materials, and the experimental error associated with the use of distinct combinations of isotopologues.

Analysis of polar urinary metabolites for metabolic phenotyping using supercritical fluid chromatography and mass spectrometry

Highlights • An SFC method has been developed for the analysis of polar urinary metabolites.• 12 stationary phases, 9 modifier additives and 3 temperatures were evaluated.• DIOL and 2-PIC columns provide highest peak capacity and overall resolution.• Ammonium formate, ammonium hydroxide and water considerably improved separation.• Alkylamine additives should be strongly considered for polar SFC-UV analysis.

Metabolic Phenotype of the Healthy Rodent Model Using In-Vial Extraction of Dried Serum, Urine, and Cerebrospinal Fluid Spots

High-throughput multiplatform metabolomics experiments are becoming an integral part of clinical and systems biology research. Such methods call for the adoption of robust sample storage and transport formats for small volumes of biofluids. One such format is the dried biofluid spot, which combines small volume requirements with easy portability. Here, we describe ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics of dried rodent serum, urine, and cerebrospinal fluid spots. An in-vial extraction and UHPLC-MS analysis method was first developed and validated by fingerprinting two test fluids, rat serum and RPMI cell nutrient medium. Data for these extracts were compared in terms of (i) peak area measurements of selected features to assess reproducibility and (ii) total fingerprint variation after data pretreatment. Results showed that percentage peak area variation was found to range between 1.4 and 9.4% relative standard deviation (RSD) for a representative set of molecular features. Upon application of the method to spots bearing serum, urine or cerebrospinal fluid (CSF) from healthy rats and mice, a total of 1,182 and 2,309 reproducible molecular features were obtained in positive and negative ionization modes, respectively, of which 610 (positive) and 991 (negative) were found in both rats and mice. Feature matching was used to detect similarities and differences between biofluids, with the biggest overlap found between fingerprints obtained in urine and CSF. Our results thus demonstrate the potential of such direct fingerprinting of dried biofluid spots as a viable alternative to the use of small (10-15 μL) volumes of neat biofluids in animal studies.

Quantum tunnelling in enzyme-catalyzed reactions

Historically, the study of enzymatic catalysis has been characterised by multidisciplinary approaches to the investigation of a range of issues: structural features relevant to catalysis, substrate binding and product-release patterns, the role of functional residues (e.g. general bases or acids)etc...

Lipidomics comparing DCD and DBD liver allografts uncovers lysophospholipids elevated in recipients undergoing early allograft dysfunction

Finding specific biomarkers of liver damage in clinical evaluations could increase the pool of available organs for transplantation. Lipids are key regulators in cell necrosis and hence this study hypothesised that lipid levels could be altered in organs suffering severe ischemia. Matched pre- and post-transplant biopsies from donation after circulatory death (DCD, n = 36, mean warm ischemia time = 21min) and donation after brain death (DBD, n = 76, warm ischemia time = none) were collected. Lipidomic discovery and multivariate analysis (MVA) were applied. Afterwards, univariate analysis and clinical associations were conducted for selected lipids differentiating between these two groups. MVA grouped DCD vs. DBD (p = 6.20 × 10−12) and 12 phospholipids were selected for intact lipid measurements. Two lysophosphatidylcholines, LysoPC (16:0) and LysoPC (18:0), showed higher levels in DCD at pre-transplantation (q < 0.01). Lysophosphatidylcholines were associated with aspartate aminotransferase (AST) 14-day post-transplantation (q < 0.05) and were more abundant in recipients undergoing early allograft dysfunction (EAD) (p < 0.05). A receiver-operating characteristics (ROC) curve combining both lipid levels predicted EAD with 82% accuracy. These findings suggest that LysoPC (16:0) and LysoPC (18:0) might have a role in signalling liver tissue damage due to warm ischemia before transplantation.

Self-assembled enzymatic monolayer directly bound to a gold surface: activity and molecular recognition force spectroscopy studies.

Escherichia coli dihydrofolate reductase (ecDHFR) has one surface cysteine, C152, located opposite and distal to the active site. Here, we show that the enzyme spontaneously assembles on an ultraflat gold surface as a homogeneous, covalently bound monolayer. Surprisingly, the activity of the gold-immobilized ecDHFR as measured by radiographic analysis was found to be similar to that of the free enzyme in solution. Molecular recognition force spectroscopy was used to study the dissociation forces involved in the rupture of AFM probe-tethered methotrexate (MTX, a tight-binding inhibitor of DHFR) from the gold-immobilized enzyme. Treatment of the ecDHFR monolayer with free MTX diminished the interaction of the functionalized tip with the surface, suggesting that the interaction was indeed active-site specific. These findings demonstrate the viability of a simple and direct enzymatic surface-functionalization without the use of spacers, thus, opening the door to further applications in the area of biomacromolecular force spectroscopy.

Quantum effects in enzyme kinetics

Historically, the study of enzymatic catalysis has been characterised by multidisciplinary approaches to the investigation of a range of issues: structural features relevant to catalysis, substrate binding and product-release patterns, the role of functional residues (e.g. general bases or acids)etc...