TY - JOUR T1 - High-affinity binding of celastrol to monomeric α-synuclein mitigates in vitro aggregation. JF - J Biomol Struct Dyn Y1 - 2023 A1 - R, Kavya A1 - Aouti, Snehal A1 - Jos, Sneha A1 - Prasad, Thazhe Kootteri A1 - K N, Kumuda A1 - Unni, Sruthi A1 - Padmanabhan, Balasundaram A1 - Kamariah, Neelagandan A1 - Padavattan, Sivaraman A1 - Mythri, Rajeswara Babu AB -

α-Synuclein (αSyn) aggregation is associated with Parkinson's disease (PD). The region αSyn acts as the nucleation 'master controller' and αSyn as a 'secondary nucleation site'. They drive monomeric αSyn to aggregation. Small molecules targeting these motifs are promising for disease-modifying therapy. Using computational techniques, we screened thirty phytochemicals for αSyn binding. The top three compounds were experimentally validated for their binding affinity. Amongst them, celastrol showed high binding affinity. NMR analysis confirmed stable αSyn-celastrol interactions involving several residues in the N-terminus and NAC regions but not in the C-terminal tail. Importantly, celastrol interacted extensively with the key motifs that drive αSyn aggregation. Thioflavin-T assay indicated that celastrol reduced αSyn aggregation. Thus, celastrol holds promise as a potent drug candidate for PD.Communicated by Ramaswamy H. Sarma.

ER - TY - JOUR T1 - Hair Follicle Grafting Therapy Promotes Re-Emergence of Critical Skin Components in Chronic Nonhealing Wounds JF - JID Innovations Y1 - 2021 A1 - Saha, D ED - Thannimangalath, S AB -

An exploding public health crisis is the exponential growth in the incidence of chronic nonhealing ulcers associated with diseases such as diabetes. Various modalities have been developed to stimulate wound closure that is otherwise recalcitrant to standard clinical treatments. However, these approaches primarily focus on the process of re-epithelialization and are often deficient in regenerating the full spectrum of structures necessary for normal skin function. Autologous hair follicle grafting is a recent therapy to stimulate the closure of such nonhealing wounds, and we observed effects beyond the epidermis to other important components of the dermis. We found that hair follicle grafting facilitated the reappearance of various undifferentiated and differentiated layers of the epidermis with the restoration of epidermal junctions. In addition, other important structures that are critical for cutaneous health and function such as the blood and lymph vasculature, nerve fibers, and sweat gland structures were restored in postgrafted wounds. Interestingly, both immune cells and inflammatory signals were substantially decreased, indicating a reduction in the chronic inflammation that is a hallmark of nonhealing wounds. Our observation that punch wounds created on the postgrafted area likewise healed suggests that this is a self-sustaining long-term therapy for patients with chronic wounds.

VL - 1 IS - 3 ER - TY - JOUR T1 - Histological and Immunohistochemical Examination of Stem Cell Proliferation and Reepithelialization in the Wounded Skin. JF - Bio Protoc Y1 - 2021 A1 - Gund, Rupali A1 - Zirmire, Ravindra A1 - J, Haarshaadri A1 - Kansagara, Gaurav A1 - Jamora, Colin AB -

The skin is the largest organ that protects our body from the external environment and it is constantly exposed to pathogenic insults and injury. Repair of damage to this organ is carried out by a complex process involving three overlapping phases of inflammation, proliferation and remodeling. Histological analysis of wounded skin is a convenient approach to examine broad alterations in tissue architecture and investigate cells in their indigenous microenvironment. In this article we present a protocol for immunohistochemical examination of wounded skin to study mechanisms involved in regulating stem cell activity, which is a vital component in the repair of the damaged tissue. Performing such histological analysis enables the understanding of the spatial relationship between cells that interact in the specialized wound microenvironment. The analytical tools described herein permit the quantitative measurement of the regenerative ability of stem cells adjacent to the wound and the extent of re-epithelialization during wound closure. These protocols can be adapted to investigate numerous cellular processes and cell types within the wounded skin.

VL - 11 IS - 2 ER - TY - JOUR T1 - Highly Responsive Fluorescent Assemblies Allow for Unique, Multiparametric Sensing of the Phospholipid Membrane Environment. JF - Chemistry Y1 - 2019 A1 - Gulyani, Akash A1 - Dey, Nilanjan A1 - Bhattacharya, Santanu KW - Fluorescence Polarization KW - Fluorescent Dyes KW - Lipid Bilayers KW - Phospholipids KW - Pyrenes KW - Spectrometry, Fluorescence KW - Water AB -

Despite decades-long extensive research, probes that provide a comprehensive description of the lipid membrane microenvironment are still lacking. Here, a "smart" pyrene-terpyridine probe for multiparametric sensing of lipid membranes is reported. The complexity of the associated local microenvironment can be described by the distinct features of the probe fluorescence. The self-assembly of the probe molecules in phospholipid bilayers was sensitive to membrane order and phase state. The self-assembled probes showed a unique emission, influenced by dye-dye interactions and excited-state charge transfer. Moreover, this emission was sensitive to interfacial hydration, with very specific changes in emission wavelengths and fluorescence lifetimes upon variation of lipid compositions and properties. In parallel, changes in the lipid order and hydration affected the ground-state interactions in the dye aggregates and, thus, could be measured through ratiometric changes in the excitation and emission readouts. In addition, fluorescence anisotropy measurements provided another way to study the nature of dye aggregates in lipid bilayers. Overall, this report demonstrates how multiple aspects of the membrane microenvironment can be sensed through the unique fluorescence signatures of this "smart" probe in lipid membranes, and it establishes a new paradigm in lipid-membrane sensing.

VL - 25 IS - 6 ER - TY - JOUR T1 - Hippocampal and amygdalar cell-specific translation is similar soon after stress but diverge over time. JF - Hippocampus Y1 - 2018 A1 - Madan, Jesvin S A1 - Gupta, Kanika A1 - Chattarji, Sumantra A1 - Bhattacharya, Aditi AB -

Stress is known to cause contrasting patterns of morphological and physiological plasticity in the hippocampus and amygdala. An obligatory cellular process underlying such neural changes is de novo translation and alterations in protein expression. Yet the nature of the translational response to stress in neurons remains largely unexplored. Even less is known about how glia are affected. Using a click-chemistry-based method to label the de novo proteome in live brain slices, we monitored translation in neurons and astrocytes of the basolateral amygdala (BLA) and dorsal hippocampal area CA3 (dCA3) in rats at different time-points after a single 2-hr exposure to immobilization stress. We observed enhancements in neuronal translation in both brain regions 1 hour after stress. This initial increase persisted in the BLA up to 10 days afterwards. In contrast, dCA3 neuronal translation gradually decreased to below control levels 10 days later. Translation profiles of dCA3 astrocytes followed timelines similar to neurons, but in BLA astrocytes translation peaked 1 day later and remained elevated 10 days later. Together our results demonstrate that stress causes an immediate upregulation of protein synthesis in both amygdalar and hippocampal neurons and astrocytes. However, these two areas eventually exhibit opposite temporal profiles of protein expression well after the end of stress. These findings identify new metrics of stress-induced plasticity at the level of cell-type specific proteomic landscape that may provide important insights into the molecular basis of the divergent temporal effects of stress across brain regions and biological scales.

VL - 28 IS - 6 ER - TY - JOUR T1 - Hierarchies in light sensing and dynamic interactions between ocular and extraocular sensory networks in a flatworm. JF - Sci Adv Y1 - 2017 A1 - Shettigar, Nishan A1 - Joshi, Asawari A1 - Dalmeida, Rimple A1 - Gopalkrishna, Rohini A1 - Chakravarthy, Anirudh A1 - Patnaik, Siddharth A1 - Mathew, Manoj A1 - Palakodeti, Dasaradhi A1 - Gulyani, Akash AB -

Light sensing has independently evolved multiple times under diverse selective pressures but has been examined only in a handful among the millions of light-responsive organisms. Unsurprisingly, mechanistic insights into how differential light processing can cause distinct behavioral outputs are limited. We show how an organism can achieve complex light processing with a simple "eye" while also having independent but mutually interacting light sensing networks. Although planarian flatworms lack wavelength-specific eye photoreceptors, a 25 nm change in light wavelength is sufficient to completely switch their phototactic behavior. Quantitative photoassays, eye-brain confocal imaging, and RNA interference/knockdown studies reveal that flatworms are able to compare small differences in the amounts of light absorbed at the eyes through a single eye opsin and convert them into binary behavioral outputs. Because planarians can fully regenerate, eye-brain injury-regeneration studies showed that this acute light intensity sensing and processing are layered on simple light detection. Unlike intact worms, partially regenerated animals with eyes can sense light but cannot sense finer gradients. Planarians also show a "reflex-like," eye-independent (extraocular/whole-body) response to low ultraviolet A light, apart from the "processive" eye-brain-mediated (ocular) response. Competition experiments between ocular and extraocular sensory systems reveal dynamic interchanging hierarchies. In intact worms, cerebral ocular response can override the reflex-like extraocular response. However, injury-regeneration again offers a time window wherein both responses coexist, but the dominance of the ocular response is reversed. Overall, we demonstrate acute light intensity-based behavioral switching and two evolutionarily distinct but interacting light sensing networks in a regenerating organism.

VL - 3 IS - 7 ER - TY - JOUR T1 - Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis. JF - Biochemistry Y1 - 2017 A1 - Plapp, Bryce V A1 - Savarimuthu, Baskar Raj A1 - Ferraro, Daniel J A1 - Rubach, Jon K A1 - Brown, Eric N A1 - Ramaswamy, S KW - 2,2'-Dipyridyl KW - Adenosine Diphosphate Ribose KW - Alcohol Dehydrogenase KW - Animals KW - Catalytic Domain KW - Crystallography, X-Ray KW - Formamides KW - Horses KW - Kinetics KW - Liver KW - Models, Molecular KW - NAD KW - Phenanthrolines KW - Protein Binding KW - Protein Conformation KW - Water KW - Zinc AB -

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5'-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2'-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme-NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ∼1.3 Å from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.

VL - 56 IS - 28 ER -