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Abstract: Drosophila melanogaster is a prominent organism in developmental biology research and in studies related to pathophysiological conditions like cancer and Alzheimer’s disease. The fruit fly gut contains several cytochrome P450s (CYP450s), which have central roles in Drosophila development and in the normal physiology of the gut. Since the crystal structures of these proteins have not been deciphered yet, we modeled the structure of 29 different D. melanogaster gut CYP450s using Prime (Schrödinger). The sequences of chosen D. melanogaster gut CYP450s were compared with that of their human counterparts. The common gut (and liver) microsomal CYP450s in humans were chosen for structural comparison to find the homology and identity % of D. melanogaster CYPs with that of their human counterparts. The modeled structures were validated using PROCHECK and the best fit models were used for docking several known human pharmacological agents/drugs to the modeled D. melanogaster gut CYP450s. Based on the binding affinities (ΔG values) of the selected drug molecules with the modeled fly gut CYPs, the plausible differences in metabolism of the prominent drugs in humans and flies were projected. The gut is involved in the absorption of oral drugs/pharmacological agents, and hence, upregulation of intestinal CYP450 and their reactions with endobiotics and xenobiotics is envisaged. The insights gleaned from this work can validate D. melanogaster as a model organism for studying intestinal drug metabolism, particularly in the context of a) toxicology of pharmacological agents to the gut cells and b) how gut P450 metabolites/products can influence gut homeostasis. This work can help establish a platform for further in vitro investigations on how intestinal CYP450 metabolism can influence gut health. The data from this work can be used for further in silico studies and this work can serve as a platform for future in vitro investigations on intestinal CYP450-mediated metabolism of endo- and xeno-biotics in D. melanogaster.

The Z-scheme electron transport chain (ETC) explanation for photosynthesis starts with the serial/sequential transfer of electrons sourced from water molecules bound at Photosystem II via a deterministic array of redox centers (of various stationary/mobile proteins), before \"sinking\" via the reduction of NADP+ bound at flavin-enzyme reductase. Several research groups’ finding that additives (like bicarbonate) enhance the light reaction had divided the research community because it violated the Z-scheme. The untenable aspects of the Z-scheme perception were demonstrated earlier and a murburn bioenergetics (a stochastic/parallel paradigm of ion-radical equilibriums) model was proposed to explain photophosphorylation and Emerson effect. Herein, we further support the murburn model with accurate thermodynamic calculations, which show that the cost of one-electron abstraction from bicarbonate [491 kJ/mol] is lower than water [527 kJ/mol]. Further, copious thioredoxin enables the capture of photoactivated electrons in milieu, which aid in the reduction of nicotinamide nucleotides. The diffusible reactive species (DRS) generated in milieu sponsor phosphorylations and oxygenic reactions. With structural analysis of Photosystems and interacting molecules, we chart out the equations of reactions that explain the loss of labeled O-atom traces in delocalized oxygenesis. Thus, this essay discredits the Z-scheme and explains key outstanding observations in the field.

Aims: We aim to demonstrate why deeming diffusible reactive oxygen species (DROS) as toxic wastes do not afford a comprehensive understanding of cytochrome P450 mediated microsomal xenobiotic metabolism (mXM). Background: Current pharmacokinetic investigations consider reactive oxygen species formed in microsomal reactions as toxic waste products, whereas our works (Manoj et al., 2016) showed that DROS are the reaction mainstay in cytochrome P450 mediated metabolism and that they play significant roles in explaining several unexplained physiologies. Objective: Herein, we strive to detail the thermodynamic and kinetic foundations of murburn precepts of cytochrome P450 mediated drug metabolism. Methodology: Primarily, in silico approaches (using pdb crystal structure files), murburn reaction chemistry logic and thermodynamic calculations to elucidate the new model of CYP-mediated drug metabolism. The theoretical foundations are used to explain experimental observations. Results: We visually elucidate how murburn model better explains- (i) promiscuity of the unique P450-reductase; (ii) prolific activity and inhibitions of CYP3A4; (iii) structure-function correlations of important key CYP2 family isozymes- 2C9, 2D6 and 2E1; and (iv) mutation studies and mechanism-based inactivation of CYPs. Several other miscellaneous aspects of CYP reaction chemistry are also addressed. Conclusion: In the light of our findings that DROS are crucial for explaining reaction outcomes in mXM, approaches for understanding drug-drug interactions and methodologies for lead drug candidates' optimizations should be revisited.

Quinones are found in the lipid membranes of prokaryotes like E. coli and cyanobacteria, and are also abundant in eukaryotic mitochondria and chloroplasts. They are intricately involved in the reaction mechanism of redox phosphorylations. In the Mitchellian chemiosmotic school of thought, membrane-lodged quinones are perceived as highly mobile conveyors of two-electron equivalents from the first leg of Electron Transport Chain (ETC) to the 'second pit-stop' of Cytochrome bc1 or b6f complex (CBC), where they undergo a regenerative 'Q-cycle'. In Manoj's murburn mechanism, the membrane-lodged quinones are perceived as relatively slow-moving one- or two- electron donors/acceptors, enabling charge separation and the CBC resets a one-electron paradigm via 'turbo logic'. Herein, we compare various purviews of the two mechanistic schools with respect to: constraints in mobility, protons' availability, binding of quinones with proteins, structural features of the protein complexes, energetics of reaction, overall reaction logic, etc. From various perspectives, the murburn mechanism appeals as a viable alternative explanation well-rooted in thermodynamics/kinetics and one which lends adequate structure-function correlations for the roles of quinones, lipid membrane and associated proteins.

The inefficiency of cyanide/HCN (CN) binding with heme proteins (under physiological regimes) is demonstrated with an assessment of thermodynamics, kinetics, and inhibition constants. The acute onset of toxicity and CN’s mg/Kg LD50(μM lethal concentration) suggests that the classical hemeFe binding-based inhibition rationale is untenable to account for the toxicity of CN. In vitro mechanistic probing of CN-mediated inhibition of hemeFe reductionist systems was explored as a murburn model for mitochondrial oxidative phosphorylation (mOxPhos). The effect of CN in haloperoxidase catalyzed chlorine moiety transfer to small organics was considered as an analogous probe for phosphate group transfer in mOxPhos. Similarly, inclusion of CN in peroxidase-catalase mediated one-electron oxidation of small organics was used to explore electron transfer outcomes in mOxPhos, leading to water formation. The free energy correlations from a Hammett study and IC50/Hill slopes analyses and comparison with ligands$\left( {\text{CO}}/{{{{\text{H}}_{2}}\text{S}}/{\text{N}_{3}^{\text{-}}}\;}\; \right)$provide insights into the involvement of diffusible radicals and proton-equilibriums, explaining analogous outcomes in mOxPhos chemistry. Further, we demonstrate that superoxide (diffusible reactive oxygen species, DROS) enables in vitro ATP synthesis from ADP+phosphate, and show that this reaction is inhibited by CN. Therefore, practically instantaneous CN ion-radical interactions with DROS in matrix catalytically disrupt mOxPhos, explaining the acute lethal effect of CN.

Background: Inactivating mutations in matrix Gla protein (MGP) lead to Keutel syndrome, a rare disease hallmarked by ectopic calcification of cartilage and vascular tissues. Although MGP acts as a strong inhibitor of arterial elastic lamina calcification (elastocalcinosis), its mode of action is unknown. Two sets of conserved residues undergoing posttranslational modifications—4 glutamic acid residues, which are γ-carboxylated by gamma-glutamyl carboxylase; and 3 serine residues, which are phosphorylated by yet unknown kinase(s)—are thought to be essential for MGP’s function. Methods: We pursued a genetic approach to study the roles of MGP’s conserved residues. First, a transgenic line ( SM22a-GlamutMgp ) expressing a mutant form of MGP, in which the conserved glutamic acid residues were mutated to alanine, was generated. The transgene was introduced to Mgp −/− mice to generate a compound mutant, which produced the mutated MGP only in the vascular tissues. We generated a second mouse model ( Mgp S3mut/S3mut ) to mutate MGP’s conserved serine residues to alanine. The initiation and progression of vascular calcification in these models were analyzed by alizarin red staining, histology, and micro-computed tomography imaging. Results: On a regular diet, the arterial walls in the Mgp −/− ; SM22α-GlamutMgp mice were not calcified. However, on a high phosphorus diet, these mice showed wide-spread arterial calcification. In contrast, Mgp S3mut/S3mut mice on a regular diet recapitulated arterial calcification traits of Mgp −/− mice, although with lesser severity. Conclusions: For the first time, we show here that MGP’s conserved serine residues are indispensable for its antimineralization function in the arterial tissues. Although the conserved glutamic acid residues are not essential for this function on a regular diet, they are needed to prevent phosphate-induced arterial elastocalcinosis.

Complex V or FoF1-ATPase is a multimeric protein found in bioenergetic membranes of cells and organelles like mitochondria/chloroplasts. The popular perception on Complex V deems it as a reversible molecular motor, working bi-directionally (breaking or making ATP) via a conformation-change based chemiosmotic rotary ATP synthesis (CRAS) mechanism, driven by proton-gradients or trans-membrane potential (TMP). In continuance of our pursuits against the CRAS model of cellular bioenergetics, herein we demonstrate the validity of the murburn model based in diffusible reactive (oxygen) species (DRS/DROS). Supported by new in silico derived data (that there are ∼12 adenosine nucleotide binding sites on the F1 bulb and not merely 3 sites, as perceived earlier), available structural information, known experimental observations, and thermodynamic/kinetic considerations (that de-solvation of protons from hydronium ions is facile), we deduce that Complex V serves as a physiological chemostat and a murzyme (enzyme working via murburn scheme, employing DRS). That is- Complex V uses ATP (via consumption at ε or proteins of F1 module) as a Michaelis-Menten substrate to serve as a pH-stat by inletting protons via the c-ring of Fo module. Physiologically, Complex V also functions as a murzyme by presenting ADP/Pi (or their reaction intermediates) on the αβ bulb, thereby enabling greater opportunities for DRS/proton-assisted ATP formation. Thus, the murburn paradigm succeeds the CRAS hypothesis for explaining the role of oxygen in mitochondrial physiologies of oxidative phosphorylation, thermogenesis, TMP and homeostasis. Communicated by Ramaswamy H. Sarma

Background: Semi-arid regions have a large livestock population that grazes on nutrient-depleted natural grasses and crop leftovers, resulting in mineral shortages. The goal of this study was to determine the amounts of iron (Fe), copper (Cu), manganese (Mn) and zinc (Zn) in soil, pasture and sheep serum in semi-arid areas with low rainfall (498-522 mm) in order to construct a soil-plant-animal continuum and recommend solutions to increase trace element availability. Methods: Samples were collected from different parts of the continuum [Soil (n=50), pasture (n=50) and blood samples from sheep (n=50)] from the selected study area and the trace elements were analyzed after digestion using atomic absorption spectrophotometer. Descriptive, correlation and t-test analyses were carried out to categorize the deficiency, their significance and soil-plant-animal continuum. Result: Although most soil samples were found to be below threshold levels, most straws and stovers in the field had marginal levels of these micro-minerals. Most sheep serum samples had levels of different micro-minerals that were below the threshold level, implying that many indigenous sheep are deficient in micro-minerals. Although there was a substantial association between feed, fodder and sheep serum, there was no such correlation between trace mineral levels in sheep serum and soil. A higher percentage of these micro-minerals in sheep faeces implies that these nutrients are truly recycled through faeces and urine in semi-arid rural areas. To address the sub-clinical micro-mineral insufficiency in semi-arid regions, micro-mineral supplementation, particularly Zn, Cu and Mn, is required.

Using in silico docking approaches, we scan the various subunits of Complex V (FoF1ATPase) for putative adenosine nucleotide binding sites. We find that multiple generic ADP/ATP binding sites are present on the alpha-beta binding sites and a conserved ATP binding site is present on the epsilon subunit. These findings support the murburn model of Complex V.

It is unresolved why lactate is transported to the liver for further utilization within the physiological purview of Cori cycle, when muscles have more lactate dehydrogenase (LDH) than liver. We point out that the answer lies in thermodynamics/equilibriums. While the utilization of NADH for the reduction of pyruvate to lactate can be mediated via the classical mechanism, the oxidation of lactate (with/without the uphill reduction of NAD

Drosophila melanogaster is a prominent organism in developmental biology research and in studies related to pathophysiological conditions like cancer and Alzheimer's disease. The fruit fly gut contains several cytochrome P450s (CYP450s), which have central roles in Drosophila development and in the normal physiology of the gut. Since the crystal structures of these proteins have not been deciphered yet, we modeled the structure of 29 different D. melanogaster gut CYP450s using Prime (Schrödinger). The sequences of chosen D. melanogaster gut CYP450s were compared with that of their human counterparts. The common gut (and liver) microsomal CYP450s in humans were chosen for structural comparison to find the homology and identity % of D. melanogaster CYPs with that of their human counterparts. The modeled structures were validated using PROCHECK and the best fit models were used for docking several known human pharmacological agents/drugs to the modeled D. melanogaster gut CYP450s. Based on the binding affinities (ΔG values) of the selected drug molecules with the modeled fly gut CYPs, the plausible differences in metabolism of the prominent drugs in humans and flies were projected. The gut is involved in the absorption of oral drugs/pharmacological agents, and hence, upregulation of intestinal CYP450 and their reactions with endobiotics and xenobiotics is envisaged. The insights gleaned from this work can validate D. melanogaster as a model organism for studying intestinal drug metabolism, particularly in the context of a) toxicology of pharmacological agents to the gut cells and b) how gut P450 metabolites/products can influence gut homeostasis. This work can help establish a platform for further in vitro investigations on how intestinal CYP450 metabolism can influence gut health. The data from this work can be used for further in silico studies and this work can serve as a platform for future in vitro investigations on intestinal CYP450-mediated metabolism of endo- and xeno-biotics in D. melanogaster.

Physiological redox conversion of alpha-hydroxy/keto acids is believed to be reversibly carried out by (de)hydrogenases, employing nicotinamide cofactors. With lactate dehydrogenase (LDH) as example, we point out that while the utilization of NADH for the reduction of pyruvate to lactate (the post-glycolytic reaction) can be mediated via the classical Michaelis-Menten mechanism, the oxidation of lactate to pyruvate (with or without the uphill reduction of NADH) necessitates alternative physiological approaches. This reaction could be more efficiently coupled/catalyzed with/by murzyme activities, which employ diffusible reactive (oxygen) species (DRS/DROS/ROS). Such a scheme would enable the cellular system to tide over the unfavorable energy barriers of the forward reaction (~450 kJ/mol; earlier considered to be ~25 kJ/mole!), and give kinetically viable conversions. Further, the new mechanism does not necessitate any ‘smart decision-making’ by the pertinent redox isozyme(s). For LDH, the new theory explains its multimeric nature, non-variant structure of the isozymes’ active sites and accounts for why lactate is transported to the liver for further utilization within the physiological purview of Cori cycle. The theoretical insights, in silico evidence and analyses of literature herein also enrich our understanding of ‘lactic acidosis’ (in clinical context), Warburg effect and approach for cancer therapy.

In this second half of our treatise on oxygenic photosynthesis, we provide support for the murburn model of the light reaction of photosynthesis and ratify key predictions made in the first part. Molecular docking and visualization of various ligands of quinones/quinols (and their derivatives) with PS II/Cytochrome b6f complexes did not support chartered 2e-transport role of quinols. A broad variety of herbicides did not show any affinity/binding-based rationales for inhibition of photosynthesis. We substantiate the proposal that disubstituted phenolics (perceived as protonophores/uncouplers or affinity-based inhibitors in the classical purview) serve as interfacial modulators of diffusible reactive (oxygen) species or DR(O)S. The DRS-based murburn model is evidenced by the identification of multiple ADP-binding sites on the extra-membraneous projection of protein complexes and structure/distribution of the photo/redox catalysts. With a panoramic comparison of the redox metabolic machinery across diverse organellar/cellular systems, we highlight the ubiquitous one-electron murburn facets (cofactors of porphyrin, flavin, FeS, other metal centers and photo/redox active pigments) that enable a facile harnessing of the utility of DRS. In the summative analyses, it is demonstrated that the murburn model of light reaction explains the structures of membrane supercomplexes recently observed in thylakoids and also accounts for several photodynamic experimental observations and evolutionary considerations. In toto, the work provides a new orientation and impetus to photosynthesis research. Communicated by Ramaswamy H. Sarma.

In this first part of our essay on oxygenic photosynthesis, we address the various aspects of classical and murburn explanations. We had recently pointed out that the classical explanations of- trans-membrane potential (TMP)-based chemiosmotic rotary ATP synthesis, quinone-cycle at cytochrome b6f, chartered electron transport by plastocyanin, and modality of light’s interaction with chloroplast pigments- are untenable. In lieu, we proposed a diffusible reactive (oxygen) species (DROS)-based murburn model. In continuum, herein, we assess the operational viability of the cyclic oxygenesis and electron transport chain (ETC) explanations for oxygenic photosynthesis, in the light of updated structural/mechanistic information. Further, thermodynamically validated murburn equations of photolysis-photophosphorylation (Pl-Pp) are provided, along with the rationale for organelle-homeostasis. We propose that the photo-excitation of various pigments leads to the formation of aquated electrons (eaq) and DROS in milieu, which are stabilized by a pool of redox-active elements within chloroplasts. Subsequently, the ‘eaq+DR(O)S’ pool is utilized stochastically via disordered/parallel reactions at the stacked thylakoid membrane interface. Effective charge separation by ‘photosystem switches’ enables delocalized oxygenesis, NAD(P) reduction and ADP phosphorylation. Heat/TMP/DROS generation is also an outcome of this process. Finally, we compare the murburn and classical models of Pl-Pp and delineate agendas for their ratification/falsification. Communicated by Ramaswamy H. Sarma Contemporary beliefs on oxygenic photosynthesis are critiqued.Murburn model is suggested as an alternative explanation.In the new model, diffusible reactive species are the main protagonists.All pigments are deemed photo-redox active in the new stochastic mechanism.NADPH synthesis occurs via simple electron transfers, not via elaborate ETC.Oxygenesis is delocalized and not just centered at Mn-Complex.Energetics of murburn proposal for photophosphorylation is provided.The proposal ushers in a paradigm shift in photosynthesis research. Contemporary beliefs on oxygenic photosynthesis are critiqued. Murburn model is suggested as an alternative explanation. In the new model, diffusible reactive species are the main protagonists. All pigments are deemed photo-redox active in the new stochastic mechanism. NADPH synthesis occurs via simple electron transfers, not via elaborate ETC. Oxygenesis is delocalized and not just centered at Mn-Complex. Energetics of murburn proposal for photophosphorylation is provided. The proposal ushers in a paradigm shift in photosynthesis research.

Blood hemoglobin (Hb) is the most abundant globular protein in humans, known to transport oxygen. Erythrocytes have ~10-3 M concentration levels of ATP in steady-state and we estimate that this high cannot be formed from 10-4 - 10-7 M levels of precursors via substrate-level phosphorylation of glycolysis. To account for this discrepancy, we propose that Hb serves as a ‘murzyme’ (a redox enzyme working along the principles of murburn concept), catalyzing the synthesis of the major amounts of ATP found in erythrocytes. This proposal is along the lines of our earlier works demonstrating DROS (diffusible reactive oxygen species) mediated ATP-synthesis as a thermodynamically and kinetically viable mechanism for physiological oxidative phosphorylation. We support the new hypothesis for Hb with theoretical arguments, experimental findings of reputed peers and in silico explorations. Using in silico methods, we demonstrate that adenoside nucleotide and 2,3-bisphosphoglycerate (2,3-BPG) binding sites are located suitably on the monomer/tetramer, thereby availing facile access to the superoxide emanating from the heme center. Our proposal explains earlier reported in situ experimental findings/suggestions of 2,3-BPG and ADP binding at the same locus on Hb. The binding energy is in the order of 2,3-BPG > NADH > ATP > ADP > AMP and agrees with earlier reports, potentially explaining the bioenergetic physiology of erythrocytes. Also, the newly discovered site for 2,3-BPG shows lower affinity in fetal Hb (as compared to adults) explaining oxygen transfer from mother to embryo. The findings pose significant implications in routine physiology and pathologies like sickle cell anemia and thalassemia.

Coronavirus disease 2019 (COVID-19) is caused by a Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2), which is a positive-strand RNA virus. The SARS-CoV-2 genome and its association to SAR-CoV-1 vary from ca. 66 to 96% depending on the type of betacoronavirideae family members. With several drugs, viz. chloroquine, hydroxychloroquine, ivermectin, artemisinin, remdesivir, azithromycin considered for clinical trials, there has been an inherent need to find distinctive antiviral mechanisms of these drugs. Curcumin, a natural bioactive molecule has been shown to have therapeutic potential for various diseases, and its effect on COVID-19 is also currently being explored. In this study, we show the binding potential of curcumin targeted to a variety of SARS-CoV-2 proteins, viz. spike glycoproteins (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), spike protein-ACE2 (PDB ID: 6M17) along with nsp10 (PDB ID: 6W4H) and RNA dependent RNA polymerase (PDB ID: 6M71) structures. Furthermore, representative docking complexes were validated using molecular dynamics simulations and mechanistic studies at 100 ns was carried on nucleocapsid and nsp10 proteins with curcumin complexes which resulted in stable and efficient binding energies and correlated with that of docked binding energies of the complexes. Both the docking and simulation studies indicate that curcumin has the potential as an antiviral against COVID-19.

This communication discusses the interactions/outcomes of isoprenoid quinones/quinols (Q/QH2) and membrane-bound cytochromes involved in bioenergetic routines. Particularly, we use qualitative and quantitative arguments to counter the idea that highly deterministic electron relays are triggered within Complex III of mitochondria, resulting from the donation of two electrons by QH2 (Yuly et al., PNAS, 2020). In this regard, we propose that murburn concept explains the role(s) of membrane-embedded Q/QH2 in vital metabolic processes such as mitochondrial oxidative phosphorylation (OxPhos) and chloroplastid/cyanobacterial photophosphorylations (PhotoPhos), without invoking highly fastidious universal free energy landscapes or deterministic electron circuitry.

Herein, we demonstrate why deeming diffusible reactive oxygen species (DROS) as toxic wastes does not afford a comprehensive understanding of cytochrome P450 mediated microsomal xenobiotic metabolism (mXM). Using the recent insights unveiled in mechanistic redox enzymology, we reason out the remaining pieces of the mXM mechanistic chemistry and support our proposals with visual evidence and thermodynamic calculations. Particularly, we elucidate how murburn model better explains- (i) promiscuity of the unique P450-reductase; (ii) prolific activity and inhibitions of CYP3A4; (iii) structure-function correlations of important key CYP2 family isozymes- 2C9, 2D6 and 2E1; and (iv) mutation studies and mechanism-based inactivation of CYPs. In the light of our findings, it is opportune to revamp the methodologies employed for screening potential drug candidate from lead molecules.

Quinones are found in the lipid-membranes of prokaryotes like E. coli and cyanobacteria, and are also abundant in eukaryotic mitochondria and chloroplasts. They are intricately involved in the reaction mechanism of redox phosphorylations. In the Mitchellian chemiosmotic school of thought, membrane-lodged quinones are perceived as highly mobile conveyors of two-electron equivalents from the first leg of Electron Transport Chain (ETC) to the ‘second pit-stop’ of Cytochrome bc1 or b6f complex (CBC), where they undergo a regenerative ‘Q-cycle’. In Manoj’s murburn mechanism, the membrane-lodged quinones are perceived as one- or two- electron donors/acceptors, enabling charge separation and the CBC resets a one-electron paradigm via ‘turbo logic’. Herein, we compare various purviews of the two mechanistic schools with respect to: constraints in mobility, protons’ availability, binding of quinones with proteins, structural features of the protein complexes, energetics of reaction, overall reaction logic, etc. From various perspectives, it is concluded that the chemiosmotic Q-cycle is an untenable hypothesis. We project the murburn proposal as one rooted in thermodynamics/kinetics and which provides tangible structure-function correlations for the roles of quinones, lipid membrane and associated proteins.

Murburn concept is a new redox metabolic paradigm which advocates that several redox enzymes generate/stabilize diffusible reactive (oxygen) species (DRS or DROS) to carry out useful electron/moiety transfer reactions at biological membrane interfaces (Manoj 2020a). Herein, we show that the components and principles of redox reactions within chloroplasts/cyanobacteria share several similarities with soluble and simple extracellular or peroxisomal heme-enzymes that carry out electron/group transfer. We explore the comparison in detail with membrane-embedded and complex systems that catalyze: (i) microsomal xenobiotic metabolism and (ii) mitochondrial oxidative phosphorylation. We point out that the murburn interpretations of catalytic phenomena are consistent through the various reaction systems cited above. Further, we argue that evolutionary constraints and the physiological restrictions of neutral pH ranges discount proton-gradient based explanations for bioenergetic phosphorylations in chloroplasts. Therefore, we propose that the highly packed thylakoid membranes with minute aqueous volumes serve to enhance the lifetimes of oxygen-centered radicals and intermediates. The murburn perspective could also potentially explain protein supercomplexes in chloroplasts, and generation of ATP in mitochondria by photo-activation. Our proposal also highlights the evolutionary significance of lipid membranes and utility of oxygen in diverse life processes.

Medial calcification has been associated with diabetes, chronic kidney disease, and genetic disorders like pseudoxanthoma elasticum. Recently, we showed that genetic reduction of arterial elastin content reduces the severity of medial calcification in matrix Gla protein (MGP)-deficient and Eln haploinsufficient Mgp-/-;Eln+/- mice. This study suggests that there might be a direct effect of elastin amount on medial calcification. We studied this using novel in vitro systems, which are based on elastin or elastin-like polypeptides. We first examined the mineral deposition properties of a transfected pigmented epithelial cell line that expresses elastin and other elastic lamina proteins. When grown in inorganic phosphate-supplemented medium, these cells deposited calcium phosphate minerals, which could be prevented by an N'-terminal peptide of MGP (m3pS) carrying phosphorylated serine residues. We next confirmed these findings using a cell-free elastin-like polypeptide (ELP3) scaffold, where the peptide prevented mineral maturation. Overall, this work describes a novel cell culture model for elastocalcinosis and examines the inhibition of mineral deposition by the m3pS peptide in this and a cell-free elastin-based scaffold. Our study provides strong evidence suggesting the critical functional roles of MGP's phosphorylated serine residues in the prevention of elastin calcification and proposes a possible mechanism of their action.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a respiratory syndrome caused by positive RNA virus resulting in outbreak of corona virus disease 2019 (COVID-19). The SARS-CoV-2 genome and its association to SAR-CoV-1 vary from ca. 66% to 96% depending on the type of betacoronavirdeae family members. With several drugs, viz. chloroquine, hydroxychloroquine, ivermectin, quinidine, artemisinin, remdesivir, azithromycin considered for clinical trials, there has been an inherent need to find distinctive antiviral mechanisms of these drugs. On the other hand, curcumin, a natural bioactive molecule has been shown to have a therapeutic potential for various diseases, but no role of it in COVID-19 has been explored. In this work, we show the binding potential of curcumin targeted to a host of SARS-CoV-2 proteins, viz. spike glycoproteins (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), membrane glycoprotein (PDB ID: 6M17) along with nsp10 (PDB ID: 6W4H) and RNA dependent RNA polymerase (PDB ID: 6M71) structures. Our results indicate that curcumin has potential antiviral protein binding affinity towards SARS-CoV-2 proteins which is comparable with other repurposed drugs that are considered for clinical trials.

Robert Emerson’s original observation (1957) that “oxygenesis occurs even with far-red light excitation of Photosystem I” is incompatible with the extant Kok-Joliot cycle’s foundation that “photolysis occurs only at red-light stimulated Photosystem II harboring MnComplex”. Further, the Z-scheme of electron transfer cannot account for Emerson’s observations of enhanced oxygenesis by simultaneous excitation of the two photosystems with both red and far-red light because serially connected components would surely increase systemic resistance to flow of charges, impeding the overall electron transfer process from water to NADP+. To address such discrepancies, we propose that the photo-excitation of various pigments leads to the formation of aquated electrons (eaq) and diffusible reactive oxygen species (DROS) in milieu, which are stabilized by a pool of redox-active elements within chloroplasts. Subsequently, the ‘eaq+DROS’ pool is utilized and routed via disordered and parallel reactions by the ‘photosystem switches’ for NADP reduction, O2 liberation and ADP phosphorylation. The stochastic ‘murburn’ model is thermodynamically and kinetically favorable and evidenced by the identification of multiple ADP-binding sites on PS II/Cytochrome b6f, and structure/distribution of the concerned proteins, complexes and pigments. The new model also explains the observed synergy in functioning of photosystems and plants’ photosynthetic spectral range of 400-700 nm.

Mitochondrial oxidative phosphorylation (mOxPhos) makes ATP, the energy currency of life. Chemiosmosis, a proton centric mechanism, advocates that Complex V harnesses a transmembrane potential (TMP) for ATP synthesis. This perception of cellular respiration requires oxygen to stay tethered at Complex IV (an association inhibited by cyanide) and diffusible reactive oxygen species (DROS) are considered wasteful and toxic products. With new mechanistic insights on heme and flavin enzymes, an oxygen or DROS centric explanation (called murburn concept) was recently proposed for mOxPhos. In the new mechanism, TMP is not directly harnessed, protons are a rate limiting reactant and DROS within matrix serve as the chemical coupling agents that directly link NADH oxidation with ATP synthesis. Herein, we report multiple ADP binding sites and solvent accessible DROS channels in respiratory proteins, which validate the oxygen or DROS centric power generation (ATP synthesis) system in mOxPhos. Since cyanide's heme binding Kd is high (mM), low doses (uM) of cyanide is lethal because cyanide disrupts DROS dynamics in mOxPhos. The critical study also provides comprehensive arguments against Mitchell's and Boyer's explanations and extensive support for murburn concept based holistic perspectives for mOxPhos.

Youth is wasted on the young. Often money is, too.” Thus, individuals forgo their future for nourishment of present. Consequently, individuals after retirement found themselves stuck in a situation where they had to rely on others for earning themselves a living. This forecast makes investing in retirement benefit plan, essential. Despite of various benefits of financial independence in retirement, the population at Lucknow does not sufficiently engage with retirement planning due to lack of financial literacy. The purpose of this study is to identify factors influencing retirement planning behavior among the working individual in Lucknow. Out of 360 questionnaires issued, only 304 were used for the study. This cross-sectional study was undertaken. The finding of this study explore the factor affecting retirement planning behaviour such as financial planning after retirement, income, attitude, culture, financial literacy and goal clarity will provide the strategies for governments, employers, financial advisers and those planning for retirement to implement it to make it simpler leads to financial independence

The use of TiO2 nanoparticles (NPs) in a wide range of products (specially biomedical applications) in day-to-day life and the notable fact that it is photoactive and generates reactive oxygen species (ROS) create curiousness among researchers to study their effects on different biological systems. Heme-enzymes are known to operate redox reactions in living system in tandem with ROS. Horseradish peroxidase (HRP) being a model heme-enzyme from the peroxidase family was systematically studied for enzymatic changes after interaction with TiO2 NPs under physiologically relevant pH and irradiation conditions. This study gives us an extensive idea of the changes in enzyme kinetics, intrinsic fluorescence, interaction efficiency, and stability of the protein–nanoparticle complex formed. The effect of ROS generated by TiO2 NPs under different irradiation conditions was substantially considered as one of the major factors that influenced the enzyme kinetics apart from protein adsorption on the surface of nanoparticles. The impact of excessive use of TiO2 NPs on the activity of HRP can be discerned from the current study.

The impact of bio-remediation agent nZVI on environment is still inadequately understood, especially on aquatic food web. The study presented here has therefore considered both chemical (CS) and biological (BS) synthetic origins of nZVI and their effects on both algae and daphnia. The study is unique in its attempt to explore the possibility of trophic transfer from algae to its immediate higher niche (daphnia as the model). An equal weightage of the effects of both CS and BS nZVI on algae and daphnia has been explored here; hence it allows us to compare the capping of nZVI on toxicity. To examine the causes of observed lethality- ROS generation, effects on the activity of oxidative enzymes, membrane damage and biouptake of nZVI was analysed. The overall outcome of CS and BS nZVI on lethality was significantly different in algae and daphnia, where daphnia demonstrated relatively higher sensitivity against CS nZVI. Algae demonstrated considerable differences in CS and BS nZVI toxicity only at higher concentration. This study did not show a probable biomagnification and trophic transfer from algae to daphnia under the experimental conditions even at the highest exposure concentration. The study instigates the importance of trophic transfer to understand the possible biomagnification of nZVI among organisms of different trophic levels and eventually the consequences on environment.

Since mid-1970s, the proton-centric proposal of ‘chemiosmosis’ became the acclaimed explanation for aerobic respiration. Recently, significant theoretical and experimental evidence were presented for an oxygen-centric ‘murburn’ mechanism of mitochondrial ATP-synthesis. Herein, we compare the predictive capabilities of the two models with respect to the available information on mitochondrial reaction chemistry and the membrane proteins' structure-function correlations. Next, fundamental queries are addressed on thermodynamics of mitochondrial oxidative phosphorylation (mOxPhos): (1) Can the energy of oxygen reduction be utilized for proton transport? (2) Is the trans-membrane proton differential harness-able as a potential energy capable of doing useful work? and (3) Whether the movement of miniscule amounts of mitochondrial protons could give rise to a potential of ~200 mV and if such an electrical energy could sponsor ATP-synthesis. Further, we explore critically if rotary ATPsynthase activity of Complex V can account for physiological ATP-turnovers. We also answer the question- “What is the role of protons in the oxygen-centric murburn scheme of aerobic respiration?” Finally, it is demonstrated that the murburn reaction model explains the fast kinetics, non-integral stoichiometry and high yield of mOxPhos. Strategies are charted to further demarcate the two explanations' relevance in the cellular physiology of aerobic respiration.

The long-standing explanation for cellular respiration (mitochondrial oxidative phosphorylation, mOxPhos) in textbooks is proton-centric and involves the elements of Rotary ATP synthesis, Chemiosmosis principle, Proton pumps and Electron transport chain (in short, the RCPE model). Addressing certain lacunae in the RCPE model, an alternative scheme based on murburn concept was proposed in 2017 (Manoj, 2017). The new proposal is oxygen-centric in essence, and it advocates constructive roles for diffusible reactive oxygen species (DROS) in electron transfer reactions and ATP-synthesis. By the end of 2018, significant arguments and experimental evidences (in vitro, in situ, and in silico) had accumulated supporting the new mechanism. Herein, the authors compare the predictive capabilities of the two models. Theoretical concepts and expectations are detailed to differentiate the two models, and the correlations are cross-checked with the available data/information. Experimental strategies are further charted to delineate and demarcate the two hypotheses’ relevance in mOxPhos.

Cyanide (CN) toxicity is traditionally understood to result from its binding of hemeFe centers, thereby disrupting mitochondrial cytochrome oxidase function and oxygen utilization by other globin proteins. Recently, a diffusible reactive oxygen species (DROS) mediated reaction mechanism called murburn concept was proposed to explain mitochondrial ATP-synthesis and heat generation. Per this purview, it was theorized that CN ion-radical equilibrium dissipates the catalytically vital DROS into futile cycles, producing water. In the current study, a comparative quantitative assessment of the above two explanations is made for: (i) lethal dosage or concentrations of CN, (ii) thermodynamics and kinetics of the binding/reaction, and (iii) correlation of CN with the binding data and reaction chemistry of H2S/CO. The quantitative findings suggest that the hemeFe binding-based toxicity explanation is untenable. CN also inhibited the experimentalin vitroDROS-mediated coupling of inorganic phosphate with ADP. Further, pH-dependent inhibition profiles of heme enzyme catalyzed oxidation of a phenolic (wherein an -OH group reacts with DROS to form water, quite akin to the murburn model of ATP synthesis) indicated that- (i) multiple competitive reactions in milieu controlled outcomes and (ii) low concentrations of CN cannot disrupt activity via a coordination (binding) of cyanide at the distal hemeFe. Therefore, the μM-level IC50and the acutely lethal effect of CN on cellular respiration could be explained by the deleterious interaction of CN ion-radical equilibrium with DROS in matrix, disrupting mitochondrial ATP synthesis. This work supports the murburn explanation for cellular respiration.

The present study deals with the toxicity assessment of two differently synthesized zero valent iron nanoparticles (nZVI, chemical and biological) as well as Fe2+ ions on Artemia salina at three different initial concentrations of 1, 10, and 100 mg/L of these particles. The assessment was done till 96 h at time intervals of 24 h. EC50 value was calculated to evaluate the 50% mortality of Artemia salina at all exposure time durations. Between chemically and biologically synthesized nZVI nanoparticles, insignificant differences in the level of mortality were demonstrated. At even 24 h, Fe2+ ion imparted complete lethality at the highest exposure concentration (100 mg/L). To understand intracellular oxidative stress because of zero valent iron nanoparticles, ROS estimation, SOD activity, GSH activity, and catalase activity was performed which demonstrated that ionic form of iron is quite lethal at high concentrations as compared with the same concentration of nZVI exposure. Lower concentrations of nZVI were more toxic as compared with the ionic form and was in order of CS-nZVI>BS-nZVI>Fe2+. Cell membrane damage and bio-uptake of nanoparticles were also evaluated for all three concentrations of BS-nZVI, CS-nZVI, and Fe2+ using adult Artemia salina in marine water; both of which supported the observations made in toxicity assessment. This study can be further explored to exploit Artemia salina as a model organism and a biomarker in an nZVI prone aquatic system to detect toxic levels of these nanoparticles. (c) 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1617-1627, 2017.

TiO2 NPs is one of the major components of paints and sunscreens and also acts as a catalyst for waste water treatment. The study demonstrates the toxic effects of TiO2 NPs on an anaerobic bacterium, Macrococcus caseolyticus, isolated from the activated sludge of a waste water treatment facility. The cytotoxicity assessment under UVA, visible light, and dark conditions revealed a dose- and exposure-dependent reduction in viability. The examination of cytotoxicity was performed with membrane permeability assessment through LDH (Lactate dehydrogenase) detection. The formation of exopolymeric substances (EPS) was observed to be dose-dependent, and maximum EPS release was noted under UVA condition. The scanning electron microscopy described the aggregation tendency of biofilm to be more pronounced under UVA condition when treated with TiO2 NPs as compared to the control biofilm. Uptake of TiO2 NPs by the biofilm indicated possible remediation of the NPs in waste water by these organisms.

The biocatalytic growth of metal nanoparticles for the detection of environmentally relevant chemical molecules is a recently exploited strategy. The present work provides a new synthesis methodology for gold nanoparticles (GNPs), which could be enzymatically modulated by horseradish peroxidase (HRP). Gold seeds (∼10 nm) were prepared initially by the reduction of HAuCl4 using a minimal concentration of NaBH4, and the further biocatalyzed GNP formation was assisted by HRP, its substrate, and hydrogen peroxide (H2O2 helps in complete formation of GNPs). Upon HRP inhibition, the enzymatic formation of GNPs was observed to decrease with increasing concentrations of phenylhydrazine (HRP inhibitor) using a simple UV-visible spectrophotometer. The current method provides an in situ enzymatic assay based on the biocatalyzed rate of GNP formation for the rapid, reproducible, and cost-effective detection of phenylhydrazine with a detection limit of 4.02 μM (range: 25–200 μM). The proposed technique was also successfully tested in real samples like drinking, tap, and garden water, emphasizing its usefulness for the direct on-site monitoring of phenylhydrazine, and the concept can be extended for the detection of other inhibitors of peroxidase also.

Gold nanoparticles (GNPs) are widely used for medical purposes, both in diagnostics as well as drug delivery, and hence are prone to release and distribution in the environment. Thus, we have explored the effects of GNPs with two distinct surface capping (citrate and PVP), and three different sizes (16, 27, and 37 nm) at 0.01-, 0.1-, and 1-mg L−1 concentrations on a predominant freshwater alga Scenedesmus obliquus in the sterile freshwater matrix. We have also investigated how an abundant metal ion from freshwater, i.e., Zn2+ ions may modulate the effects of the selected GNPs (40 nm, citrate, and PVP capped). Preliminary toxicity results revealed that gold nanoparticles were highly toxic in comparison to zinc ions alone. A significant modulation in the toxicity of Zn ions was not noticed in the presence of GNPs. In contrast, zinc ions minimized the toxicity produced by GNPs (both CIT-37 and PVP-37), despite its individual toxicity. Approximately, about 42, 33, and 25% toxicity reduction was noted at 0.05-, 0.5-, and 5-mg L−1 Zn ions, respectively, for CIT-37 GNPs, while 31% (0.05 mg L−1), 24% (0.5 mg L−1), and 9% (5 mg L−1) of toxicity reduction were noted for PVP-37 GNPs. Maximum toxicity reduction was seen at 0.05 mg L−1 of Zn ions. Abbott modeling substantiated antagonistic effects offered by Zn2+ ions on GNPs. Stability and sedimentation data revealed that the addition of zinc ions gradually induced the aggregation of NPs and in turn significantly reduced the toxicity of GNPs. Thus, the naturally existing ions like Zn2+ have an ability to modulate the toxicity of GNPs in a real-world environment scenario.

Nanocomposites of polymethyl-methacrylate (PMMA) have gained high attention owing to their extensive applications as antibacterial agents. The antibacterial activities of Ag and TiO2 nanoparticles are well established. This study intended to differentiate the antibacterial activities exhibited by PMMA/TiO2/Ag nanocomposite, towards bacterial consortium and single dominant bacterial isolates from packaged drinking water. A silver nanoparticle dose-dependent decline in cell viability of consortium and individual isolates was compared under UVC and dark conditions to evaluate the antibacterial activity of the nanocomposite. To corroborate with the viability results, oxidative stress & cell permeability was also assessed under similar conditions. Surface characterization of PMMA/TiO2/Ag nanocomposite was performed by FTIR, AFM, and SEM analyses after interaction with the bacteria. The PMMA/TiO2/Ag nanocomposite showed enhanced antibacterial activity against single bacterial isolate compared to the consortium. The outcomes from the study with PMMA/TiO2/Ag nanocomposite necessitate relooking at the test design for assessment of antibacterial effects in real conditions incorporating the impact on the consortium of microorganisms instead of individual strains.

This paper presents a high-speed Vedic multiplier based on the Urdhva Tiryagbhyam sutra of Vedic mathematics that incorporates a novel adder based on Quaternary Signed digit number system. Three operations are inherent in multiplication: partial products generation, partial products reduction and addition. A fast adder architecture therefore greatly enhances the speed of the overall process. A Quaternary logic adder architecture is proposed that works on a hybrid of binary and quaternary number systems. A given binary string is first divided into quaternary digits of 2 bits each followed by parallel addition reducing the carry propagation delay. The design doesn't require a radix conversion module as the sum is directly generated in binary using the novel concept of an adjusting bit. The proposed multiplier design is compared with a Vedic multiplier based on multi voltage or multi value logic [MVL], Vedic Multiplier that incorporates a QSD adder with a conversion module for quaternary to binary conversion, Vedic multiplier that uses Carry Select Adder and a commonly used fast multiplication mechanism such as Booth multiplier. All these designs have been developed using Verilog HDL and synthesized by Synopsys Design Compiler. They have been realized using the open source NAN gate 15nm technology library. The proposal shows a maximum of 88.75% speed improvement with respect to Multi Value logic based 128x128 Vedic multiplier while the minimum is 17.47%.

Supplementary_Information_for_Dose_Response_Mar_2018_revision_Final for Murburn Concept: A Molecular Explanation for Hormetic and Idiosyncratic Dose Responses by Abhinav Parashar, Daniel Andrew Gideon, and Kelath Murali Manoj in Dose-Response

This paper proposes a fast, purely combinational implementation of a divider, based on the Dhwajanka Sutra of Vedic mathematics. Vedic mathematics offers algorithms that are computationally efficient over conventional arithmetic algorithms. Dhwajanka Sutra has been chosen as it is an algorithm efficient for all possible cases, unlike other sutras for division (Nikhilam and Paravartya) which are case specific. The simplicity of the Vedic algorithm implemented in combinational form has reduced computation time significantly The proposed design is compared with existing divider architectures implemented on an FPGA namely Non-restoring Algorithm based Divider, Vedic Divider (Paravartya Algorithm), Vedic Divider (Nikhilam Algorithm), Decimal Divider based on Newton Raphson algorithm, Decimal Divider based on SRT algorithm and Combination divider algorithms. The proposed vedic divider shows a maximum of 273.72% speed improvement while the minimum is 99.95%. The results have been validated using the Xilinx ISE Design Suite 14.7, on the Virtex5 (xc5vlx20T-2ff323) FPGA Target Technology and the design has been implemented in Verilog.