Metabolic profiling of human saliva before and after induced physiological stress by ultra-high performance liquid chromatography–ion mobility–mass spectrometry

A method has been developed for metabolite profiling of the salivary metabolome based on protein precipitation and ultra-high performance liquid chromatography coupled with ion mobility-mass spectrometry (UHPLC–IM–MS). The developed method requires 0.5 mL of human saliva, which is easily obtainable by passive drool. Standard protocols have been established for the collection, storage and pre-treatment of saliva. The use of UHPLC allows rapid global metabolic profiling for biomarker discovery with a cycle time of 15 min. Mass spectrometry imparts the ability to analyse a diverse number of species reproducibly over a wide dynamic range, which is essential for profiling of biofluids. The combination of UHPLC with IM–MS provides an added dimension enabling complex metabolic samples to be separated on the basis of retention time, ion mobility and mass-to-charge ratio in a single chromatographic run. The developed method has been applied to targeted metabolite identification and untargeted metabolite profiling of saliva samples collected before and after exercise-induced physiological stress. δ-Valerolactam has been identified as a potential biomarker on the basis of retention time, MS/MS spectrum and ion mobility drift time.     

Comparative analysis of chromosome 2A molecular organization in diploid and hexaploid wheat

Molecular Biology Reports - Diploid A genome wheat species harbor immense genetic variability which has been targeted and proven useful in wheat improvement. Development and deployment of...     

Palmitate-derivatized human IL-2: a potential anticancer immunotherapeutic of low systemic toxicity

Purpose and experimental design

Recombinant human IL-2 (rhIL-2) is a potent cytokine and FDA-approved anticancer drug. However, its clinical use has been limited by severe toxicity, associated primarily with systemic administration with excess protein distributing freely throughout the body. We hypothesized that rhIL-2 in alternate forms permitting more restricted localization may exert stronger antitumor efficacy and less toxicity. Here, we have tested the utility of palmitate-derivatized rhIL-2. rhIL-2 was reacted with N-hydroxysuccinimide palmitate ester. The resultant lipidated rhIL-2 (pIL-2), when mixed with cells, could spontaneously transfer from solution to cell surfaces. Next, anticancer efficacy of pIL-2 was assessed in two modalities. For adoptive T cell therapy, antitumor cytotoxic T cells (CTLs) were protein transferred (“painted”) with pIL-2 and injected into mice bearing lymphoma. For in situ therapy, pIL-2 was injected intratumorally into mice bearing melanoma. Tumor growth and IL-2-associated toxicity were determined.

Results

In the lymphoma model, painting of the antitumor CTLs with pIL-2 markedly increased their viability and titer. In the melanoma model, intratumoral injection of pIL-2, but not rhIL-2, increased the number of activated CD8⁺ T cells (IFN-γ⁺) in the spleen, reduced lung metastasis and prolonged the survival of treated mice. Moreover, while repeated intratumoral injection of rhIL-2 at an excessively high dose (10 injections of 10,000 IU/mouse) caused marked vascular leakage syndrome, the same regimen using pIL-2 caused no detectable toxicity.

Conclusions

Transferring spontaneously from solution to cell surfaces, pIL-2 may bypass the current limitations of rhIL-2 and, thus, serve as a more effective and tolerable anticancer drug.     

158 A molecular dynamics simulation-based prediction of deleterious angiogenin mutations causing amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons leading to paralysis and death between 3–5?years of diagnosis. Through whole genome association studies, several single nucleotide polymorphisms (SNPs) encoding missense mutations in angiogenin (ANG) protein have been identified as one of the primary factors causing ALS. Structural studies of ANG show that catalytic triad comprising His13, Lys40, and His114 residues imparts ribonucleolytic activity while nuclear localization signal residues ³¹RRR³³ are responsible for nuclear translocation activity. Loss of either ribonucleolytic activity or nuclear translocation activity or both of these functions due to mutations cause ALS. However, the mechanisms of loss-of-functions of ANG mutants are not completely understood. Here, we present a cohesive and comprehensive picture of functional loss mechanisms of all known ALS-associated ANG mutants by extensive molecular dynamics (MD) simulations (Padhi, Kumar, Vasaikar, Jayaram, & Gomes, 2012 Padhi, A. K., Kumar, H., Vasaikar, S. V., Jayaram, B. and Gomes, J. 2012. Mechanisms of loss of functions of human angiogenin variants implicated in amyotrophic lateral sclerosis. PLoS One, 7(2): e32479[PubMed] , [Google Scholar]; AK, 2013 Padhi, A.K., Jayaram, B., & Gomes, J. (accepted for publication). Prediction of functional loss of human angiogenin mutants associated with ALS by molecular dynamics simulations. Scientific Reports (NPG).  [Google Scholar]). Our studies show that conformational switching of catalytic residue His114 is responsible for the loss of ribonucleolytic activity while reduction in solvent-accessible surface area (SASA) of ³¹RRR³³ as a result of local folding is responsible for the loss of nuclear translocation activity (Padhi et al., 2012 Padhi, A. K., Kumar, H., Vasaikar, S. V., Jayaram, B. and Gomes, J. 2012. Mechanisms of loss of functions of human angiogenin variants implicated in amyotrophic lateral sclerosis. PLoS One, 7(2): e32479[PubMed] , [Google Scholar]; AK, 2013 Padhi, A.K., Jayaram, B., & Gomes, J. (accepted for publication). Prediction of functional loss of human angiogenin mutants associated with ALS by molecular dynamics simulations. Scientific Reports (NPG).  [Google Scholar]). Our prediction of loss-of-functions of 17 ANG mutants correlated positively with the reported experimental results. We have subsequently developed a fast molecular dynamics method based on certain global attributes / dynamic markers that can be used to determine whether a mutation is deleterious or benign. To make our method accessible to researchers and clinicians, we created a web server-based tool, ANGDelMut, freely available at http://bioschool.iitd.ernet.in/research.htm, where a user can submit new mutations to ascertain whether they cause ALS. We hope that our method will benefit the community at large and will pave the way for the development of a successful therapy for patients suffering from ALS.     

Extracting Curvature Preferences of Lipids Assembled in Flat Bilayers Shows Possible Kinetic Windows for Genesis of Bilayer Asymmetry and Domain Formation in Biological Membranes

Studies on the assembly of pure lipid components allow mechanistic insights toward understanding the structural and functional aspects of biological membranes. Molecular dynamic (MD) simulations on membrane systems provide molecular details on membrane dynamics that are difficult to obtain experimentally. A large number of MD studies have remained somewhat disconnected from a key concept of amphipathic assembly resulting in membrane structures—shape parameters of lipid molecules in those structures in aqueous environments. This is because most of the MD studies have been done on flat lipid membranes. With the above in view, we analyzed MD simulations of 26 pure lipid patches as a function of (1) lipid type(s) and (2) time of MD simulations along with 35–40 ns trajectories of five pure lipids. We report, for the first time, extraction of curvature preferences of lipids from MD simulations done on flat bilayers. Our results may lead to mechanistic insights into the possible origins of bilayer asymmetries and domain formation in biological membranes.     

Kinetic Enrichment of 34S during Proteobacterial Thiosulfate Oxidation and the Conserved Role of SoxB in S-S Bond Breaking

During chemolithoautotrophic thiosulfate oxidation, the phylogenetically diverged proteobacteria Paracoccus pantotrophus, Tetrathiobacter kashmirensis, and Thiomicrospira crunogena rendered steady enrichment of ³⁴S in the end product sulfate, with overall fractionation ranging between −4.6‰ and +5.8‰. The fractionation kinetics of T. crunogena was essentially similar to that of P. pantotrophus, albeit the former had a slightly higher magnitude and rate of ³⁴S enrichment. In the case of T. kashmirensis, the only significant departure of its fractionation curve from that of P. pantotrophus was observed during the first 36 h of thiosulfate-dependent growth, in the course of which tetrathionate intermediate formation is completed and sulfate production starts. The almost-identical ³⁴S enrichment rates observed during the peak sulfate-producing stage of all three processes indicated the potential involvement of identical S-S bond-breaking enzymes. Concurrent proteomic analyses detected the hydrolase SoxB (which is known to cleave terminal sulfone groups from SoxYZ-bound cysteine S-thiosulfonates, as well as cysteine S-sulfonates, in P. pantotrophus) in the actively sulfate-producing cells of all three species. The inducible expression of soxB during tetrathionate oxidation, as well as the second leg of thiosulfate oxidation, by T. kashmirensis is significant because the current Sox pathway does not accommodate tetrathionate as one of its substrates. Notably, however, no other Sox protein except SoxB could be detected upon matrix-assisted laser desorption ionization mass spectrometry analysis of all such T. kashmirensis proteins as appeared to be thiosulfate inducible in 2-dimensional gel electrophoresis. Instead, several other redox proteins were found to be at least 2-fold overexpressed during thiosulfate- or tetrathionate-dependent growth, thereby indicating that there is more to tetrathionate oxidation than SoxB alone.     

Differential sensitivity of spinach and amaranthus to enhanced UV-B at varying soil nutrient levels: association with gas exchange, UV-B-absorbing compounds and membrane damage

The metabolic reasons associated with differential sensitivity of C₃ and C₄ plant species to enhanced UV-B under varying soil nutrient levels are not well understood. In the present study, spinach (Spinacia oleracea L. var All Green), a C₃ and amaranthus (Amaranthus tricolor L. var Pusa Badi Chaulai), a C₄ plant were subjected to enhanced UV-B (280–315 nm; 7.2 kJ m^?2 day^?1) over ambient under varying soil nutrient levels. The nutrient amendments were recommended Nitrogen (N), Phosphorus (P), Potassium (K), 1.5× recommended NPK, 1.5× recommended N and 1.5× recommended K. Enhanced UV-B negatively affected both the species at all nutrient levels, but the reductions varied with nutrient concentration and combinations. Reductions in photosynthetic rate, stomatal conductance and chlorophyll content were significantly more in spinach compared with amaranthus. The reduction in photosynthetic rate was maximum at 1.5× recommended K and minimum in 1.5× NPK amended plants. The oxidative damage to membranes measured in terms of malondialdehyde content was significantly higher in spinach compared with amaranthus. Enhanced UV-B reduced SOD activity in both the plants except in amaranthus at 1.5× recommended K. POX activity increased under enhanced UV-B at all nutrient levels in amaranthus, but only at 1.5× K in spinach. Amaranthus had significantly higher UV-B-absorbing compounds than spinach even under UV-B stress. Lowest reductions in yield and total biomass under enhanced UV-B compared with ambient were observed in amaranthus grown at 1.5× recommended NPK. Enhanced UV-B did not significantly change the nitrogen use efficiency in amaranthus at all NPK levels, but reduced in spinach except at 1.5× K. These findings suggest that the differential sensitivity of the test species under enhanced UV-B at varying nutrient levels is due to varying antioxidative and UV-B screening capacity, and their ability to utilize nutrients. Amaranthus tolerated enhanced UV-B stress more than spinach at all nutrient levels and 1.5× recommended NPK lowered the sensitivity maximally to enhanced UV-B with respect to photosynthesis, biomass and yield. PCA score has also confirmed the lower sensitivity of amaranthus compared with spinach with respect to the measured physiological and biochemical parameters.     

Within and between Population Variation in Epidermal Club Cell Investment in a Freshwater Prey Fish: A Cautionary Tale for Evolutionary Ecologists

Many prey fishes possess large club cells in their epidermis. The role of these cells has garnered considerable attention from evolutionary ecologists. These cells likely form part of the innate immune system of fishes, however, they also have an alarm function, releasing chemical cues that serve to warn nearby conspecifics of danger. Experiments aimed at understanding the selection pressures leading to the evolution of these cells have been hampered by a surprisingly large intraspecific variation in epidermal club cell (ECC) investment. The goal of our current work was to explore the magnitude and nature of this variation in ECC investment. In a field survey, we documented large differences in ECC investment both within and between several populations of minnows. We then tested whether we could experimentally reduce variation in mean ECC number by raising fish under standard laboratory conditions for 4 weeks. Fish from different populations responded very differently to being held under standard laboratory conditions; some populations showed an increase in ECC investment while others remained unchanged. More importantly, we found some evidence that we could reduce within population variation in ECC investment through time, but could not reduce among-population variation in mean ECC investment. Given the large variation we observed in wild fish and our limited ability to converge mean cell number by holding the fish under standard conditions, we caution that future studies may be hard pressed to find subtle effects of various experimental manipulations; this will make elucidating the selection pressures leading to the evolution of the cells challenging.