Role of riboflavin in the maintenance of cellular homeostasis.

Riboflavin, also known as vitamin B2, has to be sourced externally by the mammalian cells from food rich in vitamin B2, such as milk and milk products, eggs, lean meat, salmon, almonds, and spinach. The Recommended Dietary Allowance for men and women is 1.3 mg and 1.1 mg daily, respectively. Once in the intestinal lumen, it is absorbed into the gut epithelial cells (brush border) through riboflavin transporters (hRFT). There are 3 known riboflavin transporters: hRFT1, hRFT2, and hRFT3. The specific function and anatomical distribution of each transporter is unclear. However, it is believed that hRFT3 helps translocate riboflavin from the lumen into the gut epithelium, and hRFT1 and hRFT2 help transport it to the RBCs. Once inside the RBC, free riboflavin is sequentially metabolised by flavokinase and FAD synthetase to generate FMN and FAD. FAD acts as a coenzyme for GR and helps transfer electrons from NADPH to oxidised glutathione disulfide (GSSG), thus generating reduced glutathione (GSH). GSH helps maintain redox homeostasis by reducing biomolecules such as sulfhydryl groups. Superoxide dismutase converts superoxide ions into oxygen and hydrogen peroxide (H2O2). H2O2 is neutralised into H2O and O2 by catalase and glutathione peroxidase (GPx). GPx, in turn, is dependent on GSH for this neutralisation. Therefore, riboflavin is a crucial member of this molecular network that maintains a cell’s balance between oxidants and antioxidants.</p

Impaired riboflavin signalling and its role in the pathogenesis of malaria.

Riboflavin deficiency will lead to reduced levels of free riboflavin in the RBCs. This will result in low levels of FMN and FAD. Since GR is dependent on FAD for reducing GSSG into GSH, low levels of FAD will inhibit GR activity and, hence, lead to reduced levels of GSH in the cell. GSH is a crucial antioxidant; low GSH levels will increase free radicals and ROS, such as superoxide ions, H2O2, and nitric oxide. Three main hypotheses were proposed to explain how riboflavin deficiency can lead to low parasitaemia: haemolysis, inhibition of reticulocytosis, and oxidative stress. Sulfhydryl groups form an essential part of the erythrocyte membrane. Low levels of antioxidants in a cell lead to an increase in oxidised membrane sulfhydryl groups, negatively impacting membrane integrity. An increase in ROS levels has also been shown to increase nonspecific cation conductance of the plasma membrane, thus affecting the osmotic balance of the cell. High levels of ROS have also been shown to alter the expression patterns of several housekeeping genes, such as FOX3A, NF kappa B, and AP-2. These proteins act as transcription factors for other genes, such as ICAM1 and TNF alpha which are essential for generating new RBCs (reticulocytosis). ROS have also been shown to interfere with erythropoietic stem cell (ESC) cell division. Therefore, increased levels of ROS and free radicals in ESCc will affect reticulocytosis. Similarly, free radicals and ROS can promote programmed cell death or apoptosis through redox modification of proteins, carbohydrates, lipids, and nucleic acids. These mechanisms can trigger cell death either in synergy or in isolation. While cell death (loss of RBCs) under normal conditions can lead to anaemia, loss of infected RBCs can lead to reduced parasitaemia and protection against malaria. However, it is unclear if the protective effect shown by riboflavin deficiency outweighs the significant haemoglobin or RBC loss associated with riboflavin deficiency-induced anaemia.</p

Novel Green Biomimetic Approach for Preparation of Highly Stable Au-ZnO Heterojunctions with Enhanced Photocatalytic Activity

A simple, ecofriendly, and biomimetic approach using cumin seeds extract (CSE) was developed for the formation of Au-ZnO Schottky contact without employing any chemical capping agents or stabilizers. The various unique phytoconstituents available in cumin seeds extract synergistically convert Au³⁺ ions into Au⁰ on the surface of ZnO, as each phytoconstituent is unique in context to its molecular structure and properties. The as-prepared biogenic Au-ZnO hybrid composites were examined using various spectroscopic and microscopic techniques. The TEM investigation and XRD patterns clearly depict the well dispersed AuNPs with the size range 10–15 nm and face centered cubic lattice on wurtzite ZnO nanostructures. The optical study of the nanocomposites showed two absorption bands: one intense band around 390 nm, which corresponds to ZnO, and a second broad band approximately around 540 nm, which corresponds to Au. The photocatalytic efficacy of Au-ZnO nanocatalysts was investigated by observing the mineralization of an aqueous solution of methylene blue (MB) dye under a 200 W tungsten filament lamp as visible light source. The apparent rate constants were also calculated for degradation processes, and it has been observed that 1 and 3 wt % Au-ZnO nanocomposites respectively have 2.27 and 3.2 times higher photoactivity, compared to pure ZnO. This enhanced photoactivity of biogenic Au-ZnO composite materials was resultant from formation of stable and effective Schottky contact between Au metal and ZnO surfaces

Schematic diagram showing protective action of MEL against ATR immunotoxicity.

<p>ATR treatment activates death receptor (FasL, Fas, FADD, Caspase-8) and mitochondrial (E2F-1, PUMA, Bax) apoptosis (Caspase-3 and cleaved PARP1) signals. In addition, ATR induces ER stress (ATF-6α, XBP-1s, CREB-2, GADD153) signals. MEL inhibits the Fas and mitochondrial apoptosis as well as ER stress. ATR treatment also impairs autophagy by suppressing BECN-1 and upregulating LC3B-II and p62 proteins; whereas MEL restores autophagy by reversing this dysregulation. Dotted line arrows indicate known connecting pathways that were not a part of the present study. Line arrows indicate stimulatory effect and sign T indicates inhibitory effect on the expression of corresponding proteins. Scissor symbol indicates the cleavage of target proteins.</p

ATR-induced Fas mediated apoptosis was inhibited by MEL.

<p>(A) Representative immunoblots of splenocyte lysates from CON, ATR and MEL+ATR mice. FasL, Fas, FADD, Caspase-8 and β-actin proteins were visualized by chemiluminiscence. Corresponding histograms show (B) FasL, Fas and (C) Caspase-8 (CF/FL; p18/p57 and p12/p57) expressions as mean densities normalized by β-actin density. (D) Representative immunoblots of Caspase-3, PARP1 and β-actin. Corresponding histogram (E) shows caspase-3 (CF/FL, p17/p32) and PARP1 (CF/FL, p89/p116) mean densities normalized by β-actin. Data are presented as mean ± SEM of 3 independent experiments (*<i>P</i><0.05, **<i>P</i><0.01 versus CON; ^#<i>P</i><0.05 versus ATR). FL, full length; CF, cleaved fragments.</p

ATR-induced dysregulation of autophagy in splenocytes was ameliorated by MEL.

<p>(A) Representative immunoblots of autophagy markers BECN-1, LC3B (I and II), p62 and loading control β-actin in CON, ATR and MEL+ATR groups. Histograms show (B) BECN-1, (C) LC3B-II and (D) p62 mean densities normalized by β-actin. Data are presented as mean ± SEM of 3 experiments (**<i>P</i><0.01 versus CON; ^#<i>P</i><0.05, ^##<i>P</i><0.01 versus ATR).</p

ATR-induced ER stress response in splenocytes was ameliorated by MEL.

<p>(A) Representative immunoblot showing Calpain1 cleavage and histogram showing ratio of active to inactive Calpain1 (CF/FL, p76/p80) mean density normalized by β-actin. (B) Representative immunoblots of ATF6α, XBP-1, CREB-2, GADD153 and β-actin. Histograms show mean densities of (C) ATF-6α (CF, 70), (D) XBP-1s/XBP-1u ratio (56/32), and (E) PERK proteins (CREB-2 and GADD153) normalized by β-actin density. Data are expressed as mean ± SEM of 3 independent experiments (*<i>P</i><0.05, **<i>P</i><0.01 versus CON; ^#<i>P</i><0.05, ^##<i>P</i><0.01 versus ATR).</p

Exploring the effect of surfactants on the interactions of manganese dioxide nanoparticles with biomolecules

Interactions of manganese dioxide nanoparticles (MnO2 NPs) with vital biomolecules namely deoxyribonucleic acid (DNA) and serum albumin (BSA) have been studied in association with different surfactants by using fluorescence (steady state, synchronous and 3D), UV-visible, resonance light scattering (RLS), dynamic light scattering (DLS), and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The esterase activity of serum albumin was tested in associations with MnO2 NPs and surfactants. The antioxidant potential of prepared NPs was also evaluated (DPPH method). Gel electrophoresis was carried out to analyze the effect of MnO2 NPs and surfactants on DNA. Presence of CTAB, Tween 20, DTAB and Tween 80 enhanced nanoparticle-protein binding. Tween 20 based nanoparticle systems showed long-term stability and biocompatibility. The quenching of BSA fluorescence emission in presence of MnO2 NPs alone and along with Tween 20 revealed stronger association of nanoparticles with proteins. Enhancement in the esterase activity (BSA) was observed in the presence of Tween 20. Furthermore, radical scavenging activity showed highest antioxidant potential in presence of Tween 20. The enthalpy and entropy assessment for protein-NPs association showed the predominance of Vander Waals interactions and hydrogen bonding. The synchronous fluorescence analysis highlighted the involvement of tryptophan (Trp) in the MnO2 NPs-protein interactions. The study evaluates the influence of surfactant on the associations of MnO2 NPs with the essential biomolecules. The findings can be crucially utilized in designing biocompatible MnO2 formulations for long term applications. Communicated by Ramaswamy H. Sarma Influence of conventional surfactants on MnO2 NPs- protein interactions. Tween 20 causes no harm to DNA and protein structures and delivers long-term biocompatibility. Static mode of quenching is observed for MnO2 NPs/Tween 20-protein systems. The thermodynamic analysis signifies the dominance of Vander Waals interactions and hydrogen bonding amongst MnO2 NPs/Tween 20 and BSA systems. The findings will aid in designing stable and biocompatible MnO2 NPs for future therapeutic application.</p

Fibrotic Remodeling of the Extracellular Matrix through a Novel (Engineered, Dual-Function) Antibody Reactive to a Cryptic Epitope on the N-Terminal 30 kDa Fragment of Fibronectin

<div><p>Fibrosis is characterized by excessive accumulation of scar tissue as a result of exaggerated deposition of extracellular matrix (ECM), leading to tissue contraction and impaired function of the organ. Fibronectin (Fn) is an essential component of the ECM, and plays an important role in fibrosis. One such fibrotic pathology is that of proliferative vitreoretinopathy (PVR), a sight-threatening complication which develops as a consequence of failure of surgical repair of retinal detachment. Such patients often require repeated surgeries for retinal re-attachment; therefore, a preventive measure for PVR is of utmost importance. The contractile membranes formed in PVR, are composed of various cell types including the retinal pigment epithelial cells (RPE); fibronectin is an important constituent of the ECM surrounding these cells. Together with the vitreous, fibronectin creates microenvironments in which RPE cells proliferate. We have successfully developed a dual-action, fully human, fibronectin-specific single chain variable fragment antibody (scFv) termed Fn52RGDS, which acts in two ways: i) binds to cryptic sites in fibronectin, and thereby prevents its self polymerization/fibrillogenesis, and ii) interacts with the cell surface receptors, ie., integrins (through an attached “RGD” sequence tag), and thereby blocks the downstream cell signaling events. We demonstrate the ability of this antibody to effectively reduce some of the hallmark features of fibrosis - migration, adhesion, fibronectin polymerization, matrix metalloprotease (MMP) expression, as well as reduction of collagen gel contraction (a model of fibrotic tissue remodeling). The data suggests that the antibody can be used as a rational, novel anti-fibrotic candidate.</p></div

Changes in cell viability and proliferation in the presence of Fn52 and Fn52RGDS.

<p>Panel A: D407 RPE cell viability assessed by the MTT assay. The optical density of the wells containing cells seeded in the presence of patient vitreous/SRF was assigned as 100%. Panel B: RPE cell proliferation was assessed by the BrdU assay. PV (assigned as 100%) represents conditioned media obtained from cells grown in the presence of patient vitreous (and subretinal fluid). In both the assays, the control scFv O27 was also used; the effect of the scFv antibodies was evaluated, using a range of concentrations. Each bar represents mean±SEM (standard error of the mean) calculated from five and three separate experiments respectively.</p