Mitochondrial Calcium Transport During Autophagy Initiation.

While it has been shown that Ca2+ dynamics at the ER membrane is essential for the initiation of certain types of autophagy such as starvation-induced autophagy, how mitochondrial Ca2+ transport changes during the first stage of autophagy is not systemically characterized. An investigation of mitochondrial Ca2+ dynamics during autophagy initiation may help us determine the relationship between autophagy and mitochondrial Ca2+ fluxes. Here we examine acute mitochondrial and ER calcium responses to a panel of autophagy inducers in different cell types. Mitochondrial Ca2+ transport and Ca2+ transients at the ER membrane are triggered by different autophagy inducers. The mitophagy-inducer-initiated mitochondrial Ca2+ uptake relies on mitochondrial calcium uniporter and may decelerate the following mitophagy. In neurons derived from a Parkinson's patient, mitophagy-inducer-triggered mitochondrial Ca2+ influx is faster, which may slow the ensuing mitophagy.

Consumption of honey ameliorates lipopolysaccharide-induced intestinal barrier dysfunction via upregulation of tight junction proteins.

PURPOSE: The leaky gut barrier is an important factor leading to various inflammatory gastrointestinal disorders. The nutritional value of honey and variety of its health benefits have long been recognized. This study was undertaken to assess the role of Indian mustard honey in preventing lipopolysaccharide (LPS)-induced intestinal barrier dysfunction using a combination of in vitro and in vivo experimental model systems. METHODS: LPS was used to induce intestinal barrier damage in a trans-well model of Caco-2 cells (1 µg/ml) and in Swiss albino mice (5 mg/kg body weight). Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were used to analyse sugar and phenolic components in honey samples. The Caco-2 cell monolayer integrity was evaluated by transepithelial electrical resistance (TEER) and paracellular permeability assays. The histopathology of intestinal tissue was analysed by haematoxylin and eosin dual staining. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to quantify the transcription of genes. The protein expression was analysed by immunofluorescence, western blot and ELISA-based techniques. RESULTS: The in vitro data showed that honey prevented LPS-induced intestinal barrier dysfunction dose dependently as was measured by TEER and paracellular flux of FITC-dextran dye. Further, the in vivo data showed a prophylactic effect of orally administered honey as it prevented the loss of intestinal barrier integrity and villus structure. The cellular localization and expression of tight junction (TJ) proteins were upregulated along with downregulation of pro-inflammatory cytokines in response to the administration of honey with LPS. CONCLUSIONS: The findings of this study suggest a propitious role of honey in the maintenance of TJ protein integrity, thereby preventing LPS-induced intestinal barrier disintegration.

Zinc Acts Synergistically with Berberine for Enhancing Its Efficacy as an Anti-cancer Agent by Inducing Clusterin-Dependent Apoptosis in HT-29 Colorectal Cancer Cells.

It is now widely accepted that anti-cancer medications are most effective when administered in combination. Zinc is an essential micronutrient whilst berberine is a well-known natural phytochemical, both having multiple molecular mechanisms of action. The present study aimed to determine the combinatorial effect of zinc and berberine on the human adenocarcinoma HT-29 cancer cell line. The anti-proliferative activity of berberine and zinc was determined by cell viability and colony-forming assays. The combination index and drug reduction index values of zinc and berberine co-treatments were estimated by suitable software. Flow cytometry was used to analyse cell cycle distribution and Annexin V/PI staining. The expression of apoptosis and zinc signalling markers were analysed by RT-qPCR and immunoblot analysis. Berberine decreased the viability of colon cancer cells in a dose-dependent manner whilst zinc alone had no significant influence on it. However, zinc and berberine co-treatment resulted in a synergistic anti-cancer action which was demonstrated by G2/M phase arrest of cell growth at a lower dose of berberine. Annexin V assay demonstrated that the synergistic impact of zinc and berberine boosted the number of apoptotic cells. Gene expression analysis at both transcriptional and translational levels showed the upregulation of apoptotic (caspase-3 and caspase-8) and a zinc-sensing receptor (GPR39) gene with concomitant downregulation of anti-apoptotic genes like proliferating cell nuclear antigen (PCNA) and clusterin. Our findings showed that the combination of zinc and berberine has synergistic anti-cancer efficacy and thus could be used as a potential chemopreventive option for colon cancer.

Wheatgrass extract imparts neuroprotective actions against scopolamine-induced amnesia in mice.

The rich and diverse phytoconstituents of wheatgrass have established it as a natural antioxidant and detoxifying agent. The anti-inflammatory potential of wheatgrass has been studied extensively. However, the neuroprotective potential of wheatgrass has not been studied in depth. In this study, we investigated the neuroprotective response of wheatgrass against age-related scopolamine-induced amnesia in mice. Scopolamine is an established anticholinergic drug that demonstrates the behavioural and molecular characteristics of Alzheimer's disease. In the current study, wheatgrass extracts (prepared from 5 and 7 day old plantlets) were administered to scopolamine-induced memory deficit mice. The Morris water maze (MWM) and Y-maze tests demonstrated that wheatgrass treatment improves the behavior and simultaneously enhances the memory of amnesic mice. We further evaluated the expression of neuroinflammation related genes and proteins in the hippocampal region of mice. Wheatgrass significantly upregulated the mRNA and protein expression of neuroprotective markers such as BDNF and CREB in scopolamine-induced mice. Simultaneously, wheatgrass also downregulated the expression of inflammatory markers such as TNF-α and tau genes in these mice. The treatment of scopolamine-induced memory impaired mice with wheatgrass resulted in an elevation in the level of the phosphorylated form of ERK and Akt proteins. Wheatgrass treatment of mice also regulated the phosphorylation of tau protein and simultaneously prevented its aggregation in the hippocampal region of the brain. Overall, this study suggests the therapeutic potential of wheatgrass in the treatment of age-related memory impairment, possibly through the involvement of ERK/Akt-CREB-BDNF pathway and concomitantly ameliorating the tau-related pathogenesis.

Triclosan-induced neuroinflammation develops caspase-independent and TNF-α signaling pathway associated necroptosis in Neuro-2a cells.

Triclosan (TCS) is widely used in cosmetics and healthcare industry as a broad-spectrum antibacterial agent. The lipophilic property and persistent nature of TCS has led to severe health issues. In the present study, we have evaluated the neuroinflammatory effect of TCS on mouse Neuro-2a cells. Initial investigation confirmed a dose-dependent loss in viability and morphology of cells in presence of TCS. The transcription and translation studies confirmed a downregulation in the expression of autophagy markers in Neuro-2a cells. The confocal microscopy study revealed that the abrogated autophagy in TCS-treated cells occurred due to loss in the autophagy flux and prevention in the lipidation of autophagosome bilayer. The fluorescence microscopy also confirmed a loss in the formation of autophagolysosomes in neuronal cells with increasing TCS concentrations. TCS treatment resulted in loss of mitochondrial integrity in cells as evidenced by a decrease in mitochondrial membrane potential in JC-1 staining. Further, the transcriptional and translational studies confirmed the activation of TNF-α signaling pathway in TCS-treated cells thus enhancing the expression of RIPK1, RIPK3 and MLKL proteins and their phosphorylated forms. TCS was also found to increase the tau protein pathogenesis in Neuro-2a cells, which alludes to the development of tau-associated neurodegeneration. Altogether, this study confirms the neuroinflammatory actions of TCS in Neuro-2a cells involving a TNF-α-induced MLKL-mediated signaling.

Wheatgrass inhibits the lipopolysaccharide-stimulated inflammatory effect in RAW 264.7 macrophages.

Inflammation is a multifaceted set of cellular communications generated against foreign infection, toxic influence or autoimmune injury. The present study investigates the anti-inflammatory effect of wheatgrass extract against the harmful impact of lipopolysaccharide (LPS) in macrophage cells, i.e., RAW 264.7 cells. Our results indicate that 5- and 7- days old wheatgrass extracts inhibit the LPS-stimulated production of nitric oxide. Moreover, wheatgrass extract significantly downregulates the mRNA expression of LPS-stimulated various pro-inflammatory markers, tumor necrosis factor-α, interleukin-6, interleukin-1ß, AP-1 and also iNOS-2 and COX-2. Our flow cytometry analyses confirmed that wheatgrass extract prevents the generation of reactive oxygen species in LPS-stimulated RAW 264.7 cells, thus arresting oxidative stress in cells. The immunoblot analyses also confirmed a significant reduction in the expression of inflammatory proteins, namely, iNOS-2 and COX-2, in wheatgrass extract-treated cells, compared to LPS-stimulated condition. The NF-κB transactivation assay further confirmed the inhibitory effect of wheatgrass extracts on the LPS-stimulated expression of NF-κB. Molecular docking based studies showed the plausible binding of two significant wheatgrass constituents, i.e., apigenin and myo-inositol with COX-2 protein, with binding energies of -10.59 kcal/mol and -7.88 kcal/mol, respectively. Based on the above results, wheatgrass may be considered as a potential therapeutic candidate for preventing inflammation.

Black pepper and piperine induce anticancer effects on leukemia cell line.

The black pepper, most commonly used in Indian cuisines for ages, is considered as "king of spices." The present study evaluates the anticancer potential of black pepper and its main constituent, i.e. alkaloid piperine, against human leukemia cell line, K-562 cells. Gas chromatography-mass spectrometry (GC-MS) analysis confirmed the presence of piperine in black pepper extract. The methanolic extract of black pepper (BP-M) and pure piperine (PIP) showed a strong cytotoxic effect against this cell line. Both BP-M and PIP generated apoptotic bodies in K-562 cells and caused nuclear condensation as visualized by fluorescent microscopy, which was further confirmed by flow cytometry analysis. BP-M and PIP also generated reactive oxygen species in K-562 cells as established by flow cytometry. The translation of Bax, caspase-3 and caspase-9 genes was found to be upregulated with subsequent downregulation of Bcl-2 gene. The anti-proliferative effect of both BP-M and PIP was also observed by trypan blue staining and was further confirmed by the downregulated expression of proliferating cell nuclear antigen (PCNA). The molecular docking studies showed the binding of PIP with PCNA and Bcl-2 and supported the in vitro findings. The docking studies also proposed the binding of PIP to ADP binding pocket of Apaf-1 protein. Taken together, these findings signify the anticancer potential of both black pepper and PIP, thus proposing black pepper as a potent nutraceutical for preventing the progression of chronic myeloid leukemia.

Black pepper prevents anemia of inflammation by inhibiting hepcidin over-expression through BMP6-SMAD1/ IL6-STAT3 signaling pathway.

Hepcidin, a circulatory hepatic peptide hormone, is associated with systemic iron homeostasis. Inflammation leads to an increase in hepcidin expression, which dysregulates body iron level. The related disorder, anemia of inflammation, is the second most prevalent anemia-related disorder worldwide. In the present study, we conducted in vitro and in vivo studies to evaluate the effect of black pepper (BP) and its major bioactive alkaloid, piperine, on anemia of inflammation. The initial in vitro study using human hepatocyte cell line, HepG2, confirmed that among different black pepper extracts: methanol (BPME), ethanol (BPEE) and aqueous (BPAE), BPME to be most effective in downregulating transcription of hepcidin gene. Further, BPME and piperine significantly downregulated hepcidin protein expression at 200 µg/ml and 100 µM concentrations, respectively. In the next phase, BPME and piperine were found to significantly attenuate BMP-6 and IL-6 induced hepcidin overexpression by downregulating the increased level of pSMAD1 and pSTAT3 proteins, respectively. For in vivo study, we first subcutaneously injected male BALB/c mice with oil of turpentine, thrice within a period of two weeks, in order to enhance the expression of hepcidin. After that, the intraperitoneal administration of BPME and piperine at 70 and 25 mg/kg body weight, respectively, on alternate days for a period of another two weeks resulted in downregulation of hepcidin overexpression in diseased mice, as confirmed by RT-PCR and immunoblot analysis. The histopathology of liver tissue confirmed increased iron bioavailability in BPME and piperine treated animals. The molecular docking-based interaction studies demonstrated the binding potential of piperine with SMAD1 and STAT3 proteins. The binding patterns supported the proposed inhibition of hepcidin activating proteins. All together, these findings suggest black pepper as a therapeutic candidate for the treatment of anemia of inflammation.

Evaporation induced self-assembly of different shapes and sizes of nanoparticles: A molecular dynamics study.

Molecular dynamics simulations of Lennard-Jones particles have been performed to study the self-assembled structure of nanoparticles (NPs) formed upon evaporation of nanofluid droplets on a heated surface. Different shapes of NPs such as a sphere, cube, triangle, and rod are considered in this work for the nanofluid. The influence of solvent-surface and NP-surface interaction strengths, size, and shape of NPs is analyzed on the structure of the NP deposit formed upon evaporation. The solvophilic substrate leads to the formation of different structures such as the hemispherical clump, monolayer, and ring depending on the size, shape, and interaction between other pairs of atoms. On the other hand, the solvophobic substrate always leads to a clump of NPs. Structural and thermodynamic properties are calculated to characterize the self-assembled structures. The low pair energy and high excess entropy are the characteristics of a ring structure. Furthermore, the mean square displacement of NPs is found to be lower for the ring structure compared to the hemispherical clump structure, and this observation is independent of the shape and size of the NP. The change in arrangement from disorder to order is observed for rod shaped NPs during evaporation.

A coarse-grain molecular dynamics study of oil-water interfaces in the presence of silica nanoparticles and nonionic surfactants.

In this work, we have studied the effect of hydrophilic silica nanoparticles (NPs), in the presence of nonionic surfactants (Triethylene glycol monododecyl ether and Tween 20), on the oil-water (n-octane-water, n-dodecane-water and n-hexadecane-water) interfacial tensions (IFTs) at 300 K, using coarse-grained molecular dynamics simulations based on the MARTINI force field. Simulation results indicate that silica NPs solely do not affect the IFT. However, the silica NPs may or may not increase the IFT of oil-water containing nonionic surfactant, depending on the tendency of the surfactant to adsorb on the surface of NPs. The adsorption occurs due to the formation of hydrogen bonds, and adsorption increases with a decrease in pH, as seen in experimental studies. In this work, we found that the oil-water IFT increases with an increasing amount of adsorption of the surfactant on NPs. At a fixed amount of adsorption of the surfactant on NPs, the IFT behavior is indifferent to the change in concentration of NPs. However, the IFT decreases with an increase in surfactant concentration. We present a detailed analysis of the density profile and intrinsic width of the interface. The IFT behavior is found to correlate extremely well with the intrinsic width of the interface. The current study provides an explanation for the increase in IFT observed in a recent experiment [N. R. Biswal et al., J. Phys. Chem. B 120, 7265-7274 (2016)] for various types of NPs and nonionic surfactant systems.