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.     

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.     

Alterations in Cortical Sensorimotor Connectivity following Complete Cervical Spinal Cord Injury: A Prospective Resting-State fMRI Study

Functional magnetic resonance imaging (fMRI) studies have demonstrated alterations during task-induced brain activation in spinal cord injury (SCI) patients. The interruption to structural integrity of the spinal cord and the resultant disrupted flow of bidirectional communication between the brain and the spinal cord might contribute to the observed dynamic reorganization (neural plasticity). However, the effect of SCI on brain resting-state connectivity patterns remains unclear. We undertook a prospective resting-state fMRI (rs-fMRI) study to explore changes to cortical activation patterns following SCI. With institutional review board approval, rs-fMRI data was obtained in eleven patients with complete cervical SCI (>2 years post injury) and nine age-matched controls. The data was processed using the Analysis of Functional Neuroimages software. Region of interest (ROI) based analysis was performed to study changes in the sensorimotor network using pre- and post-central gyri as seed regions. Two-sampled t-test was carried out to check for significant differences between the two groups. SCI patients showed decreased functional connectivity in motor and sensory cortical regions when compared to controls. The decrease was noted in ipsilateral, contralateral, and interhemispheric regions for left and right precentral ROIs. Additionally, the left postcentral ROI demonstrated increased connectivity with the thalamus bilaterally in SCI patients. Our results suggest that cortical activation patterns in the sensorimotor network undergo dynamic reorganization following SCI. The presence of these changes in chronic spinal cord injury patients is suggestive of the inherent neural plasticity within the central nervous system.     

Biomass production,nutrient cycling,and carbon fixation by Salicornia brachiata Roxb.: A promising halophyte for coastal saline soil rehabilitation

In order to increase our understanding of the interaction of soil-halophyte (Salicornia brachiata) relations and phytoremediation, we investigated the aboveground biomass, carbon fixation, and nutrient composition (N, P, K, Na, Ca, and Mg) of S. brachiata using six sampling sites with varying characteristics over one growing season in intertidal marshes. Simultaneously, soil characteristics and nutrient concentrations were also estimated. There was a significant variation in soil characteristics and nutrient contents spatially (except pH) as well as temporally. Nutrient contents in aboveground biomass of S. brachiata were also significantly differed spatially (except C and Cl) as well as temporally. Aboveground biomass of S. brachiata ranged from 2.51 to 6.07 t/ha at maturity and it was positively correlated with soil electrical conductivity and available Na, whereas negatively with soil pH. The K/Na ratio in plant was below one, showing tolerance to salinity. The aboveground C fixation values ranged from 0.77 to 1.93 C t/ha at all six sampling sites. This study provides new understandings into nutrient cycling—C fixation potential of highly salt-tolerant halophyte S. brachiata growing on intertidal soils of India. S. brachiata have a potential for amelioration of the salinity due to higher Na bioaccumulation factor.     

Compositional and Solvent Engineering in Dion–Jacobson 2D Perovskites Boosts Solar Cell Efficiency and Stability

Hybrid halide 2D perovskites deserve special attention because they exhibit superior environmental stability compared with their 3D analogs. The closer interlayer distance discovered in 2D Dion–Jacobson (DJ) type of halide perovskites relative to 2D Ruddlesden–Popper (RP) perovskites implies better carrier charge transport and superior performance in solar cells. Here, the structure and properties of 2D DJ perovskites employing 3‐(aminomethyl)piperidinium (3AMP²⁺) as the spacing cation and a mixture of methylammonium (MA⁺) and formamidinium (FA⁺) cations in the perovskite cages are presented. Using single‐crystal X‐ray crystallography, it is found that the mixed‐cation (3AMP)(MA_0.75FA_0.25)₃Pb₄I₁₃ perovskite has a narrower bandgap, less distorted inorganic framework, and larger Pb? I? Pb angles than the single‐cation (3AMP)(MA)₃Pb₄I₁₃. Furthermore, the (3AMP)(MA_0.75FA_0.25)₃Pb₄I₁₃ films made by a solvent‐engineering method with a small amount of hydriodic acid have a much better film morphology and crystalline quality and more preferred perpendicular orientation. As a result, the (3AMP)(MA_0.75FA_0.25)₃Pb₄I₁₃‐based solar cells exhibit a champion power conversion efficiency of 12.04% with a high fill factor of 81.04% and a 50% average efficiency improvement compared to the pristine (3AMP)(MA)₃Pb₄I₁₃ cells. Most importantly, the 2D DJ 3AMP‐based perovskite films and devices show better air and light stability than the 2D RP butylammonium‐based perovskites and their 3D analogs.