Modulation of calcium-influx by carboxymethyl tamarind­gold nanoparticles promotes biomineralization for tissue regeneration.

Gold nanoparticles (AuNPs) have been reported to modulate bone tissue regeneration and are being extensively utilized in biomedical implementations attributable to their low cytotoxicity, biocompatibility and simplicity of functionalization. Lately, biologically synthesized nanoparticles have acquired popularity because of their environmentally acceptable alternatives for diverse applications. Here we report the green synthesis of AuNPs by taking the biopolymer Carboxymethyl Tamarind (CMT) as a unique reducing as well as a stabilizing agent. The synthesized CMT-AuNPs were analyzed by UV-vis spectrophotometer, DLS, FTIR, XRD, TGA, SEM and TEM. These results suggest that CMT-AuNPs possess an average size of 19.93 ± 8.52 nm and have long-term stability. Further, these CMT-AuNPs promote the proliferation together with the differentiation and mineralization of osteoblast cells in a "dose-dependent" manner. Additionally, CMT-AuNPs are non-toxic to SD rats when applied externally. We suggest that the CMT-AuNPs have the potential to be a suitable and non-toxic agent for differentiation and mineralization of osteoblast cells in vitro and this can be tested in vivo as well.

Menthol causes mitochondrial Ca2+-influx, affects structure-function relationship and cools mitochondria.

Menthol is a small bioactive compound able to cause several physiological changes and has multiple molecular targets. Therefore, cellular response against menthol is complex, and still poorly understood. In this work, we used a human osteosarcoma cell line (Saos-2) and analysed the effect of menthol, especially in terms of cellular, subcellular and molecular aspects. We demonstrate that menthol causes increased mitochondrial Ca2+ in a complex manner, which is mainly contributed by intracellular sources, including ER. Menthol also changes the Ca2+-load of individual mitochondrial particles in different conditions. Menthol increases ER-mito contact points, causes mitochondrial morphological changes, and increases mitochondrial ATP, cardiolipin, mitochondrial ROS and reduces mitochondrial membrane potential (ΔΨm). Menthol also prevents the mitochondrial quality damaged by sub-lethal and lethal doses of CCCP. In addition, menthol lowers the mitochondrial temperature within cell and also serves as a cooling agent for the isolated mitochondria in a cell free system too. Notably, menthol-induced reduction of mitochondrial temperature is observed in diverse types of cells, including neuronal, immune and cancer cells. As the higher mitochondrial temperature is a hallmark of several inflammatory, metabolic, disease and age-related disorders, we propose that menthol can serve as an active anti-aging compound against all these disorders. These findings may have relevance in case of several pharmacological and clinical applications of menthol. SIGNIFICANCE STATEMENT: Menthol is a plant-derived bioactive compound that is widely used for several physiological, behavioural, addictive, and medicinal purposes. It is a well-established "cooling and analgesic agent". However, the exact cellular and sub-cellular responses of menthol is poorly understood. In this work, we have characterized the effects of menthol on mitochondrial metabolism. Menthol regulates mitochondrial Ca2+, ATP, superoxides, cardiolipin, membrane-potential, and ER-mito contact sites. Moreover, the cooling agent menthol also cools down mitochondria and protects mitochondrial damage by certain toxins. These findings may promote use of menthol as a useful supplementary agent for anti-aging, anti-cancer, anti-inflammatory purposes where higher mitochondrial temperature is prevalent.

TRPV4 regulates osteoblast differentiation and mitochondrial function that are relevant for channelopathy.

Different ion channels present in the osteoblast regulate the cellular functions including bio-mineralization, a process that is a highly stochastic event. Cellular events and molecular signaling involved in such process is poorly understood. Here we demonstrate that TRPV4, a mechanosensitive ion channel is endogenously present in an osteoblast cell line (MC3T3-E1) and in primary osteoblasts. Pharmacological activation of TRPV4 enhanced intracellular Ca2+-level, expression of osteoblast-specific genes and caused increased bio-mineralization. TRPV4 activation also affects mitochondrial Ca2+-levels and mitochondrial metabolisms. We further demonstrate that different point mutants of TRPV4 induce different mitochondrial morphology and have different levels of mitochondrial translocation, collectively suggesting that TRPV4-mutation-induced bone disorders and other channelopathies are mostly due to mitochondrial abnormalities. These findings may have broad biomedical implications.

TRPV4 regulates mitochondrial Ca2+-status and physiology in primary murine T cells based on their immunological state.

T cell activation process is critically affected by temperature and intracellular Ca2+-signalling. Yet, the nature and the key molecules involved in such complex Ca2+-signalling is poorly understood. It is mostly assumed that ion channels present in the plasma membrane primarily regulate the cytosolic Ca2+-levels exclusively. TRPV4 is a non-selective Ca2+ channel which can be activated at physiological temperature. TRPV4 is involved in several physiological, pathophysiological process as well as different forms of pain. Here we demonstrate that TRPV4 is endogenously expressed in T cell and is present in the mitochondria of T cells. TRPV4 activation increases mitochondrial Ca2+-levels, and alters mitochondrial temperature as well as specific metabolisms. The TRPV4-dependent increment in the mitochondrial Ca2+ is context-dependent and not just passively due to the increment in the cytosolic Ca2+. Our work also indicates that mitochondrial Ca2+-level correlates positively with a series of essential factors, such as mitochondrial membrane potential, mitochondrial ATP production and negatively correlates with certain factors such as mitochondrial temperature. We propose that TRPV4-mediated mitochondrial Ca2+-signalling and other metabolisms has implications in the immune activation process including immune synapse formation. Our data also endorse the re-evaluation of Ca2+-signalling in T cell, especially in the light of mitochondrial Ca2+-buffering and in higher body temperature, such as in case of fever. Presence of TRPV4 in the mitochondria of T cell is relevant for proper and optimum immune response and may provide evolutionary adaptive benefit. These findings may also have broad implications in different pathophysiological process, neuro-immune cross-talks, and channelopathies involving TRPV4.

Highly water-stable, luminescent, and monodisperse polymer-coated CsPbBr3 nanocrystals for imaging in living cells with better sensitivity.

Recently, CsPbX3 (X= Cl, Br, I) nanocrystals (NCs) have evolved as a potential contender for various optoelectronic applications due to some of their excellent photophysical properties. Their superior non-linear optical properties enable them to take part in bioimaging applications due to their longer penetration depth and less scattering effect in living cells. However, the poor stability of perovskite NCs in aqueous media still remains a great challenge for practical usage. Comparatively stable silica-coated NCs have a tendency to agglomerate among other NCs and transform into bigger particles. Such big particles clog the inside of narrow channels during the uptake and can't effectively reach the targeted cells. To tackle such issues, we introduce a fast and reproducible synthesis process of CsPbBr3 NCs that are coated with different long-chained organic ligands/polymers and compared their photophysical properties. Among them, polyvinylpyrrolidone (PVP) encapsulated NCs are highly luminescent in the green spectral region and showed a maximum photoluminescence quantum yield (PLQY) of up to 84%. The incorporation of n-isopropyl acrylamide (NIPAM) along with PVP further improves the stability of the PVP-coated NCs against heat and moisture. These NCs exhibit higher water stability compared to silica-coated NCs and maintained their emission properties for about one week in DI water. The smaller particle size, uniform size distribution, higher structural stability, and better dispersivity of polymer-coated NCs in the aqueous media enable them to perform as fluorescent probes for live cell imaging in mammalian Chinese Hamster Ovary (CHO-K1) cells. There is no adverse affect in the cells' viability and morphology even after long incubation periods (∼72 hours). The dosage of Pb-ions contained in the polymer-coated NCs is calculated as below 5 µg mL-1, which is suitable for live cell imaging. This work provides insight for expanding the use of these NCs significantly into bioimaging applications with higher sensitivity.

TRPV4 interacts with MFN2 and facilitates endoplasmic reticulum-mitochondrial contact points for Ca2+-buffering.

AIM: Mitochondrial fission-fusion events, distribution, and Ca2+-buffering abilities are relevant for several diseases, yet are poorly understood events. TRPV4 channels are a group of thermosensitive ion channel which regulate cellular and mitochondrial Ca2+-level. The underlying mechanisms of the change in mitochondrial dynamics upon modulation of TRPV4 channel are ill explored. MAIN METHODS: We have used TRPV4 expressing stable cell line CHO-K1-V4 and compared with CHO-K1-Mock as a control cell. We have also used mouse bone marrow derived mesenchymal stem cells and purified mitochondria from mouse brain for the interaction study. KEY FINDINGS: Now we demonstrate that expression and/or pharmacological modulation of TRPV4 regulates mitochondrial morphologies and Ca2+-level. TRPV4 interacts with MFN1/MFN2, the mitochondrial regulatory factors. TRPV4 regulates ER-mito contact points. We used different cellular conditions where cytosolic or ER Ca2+-levels were pharmacologically altered. Analysis of ∼55,000 mitochondrial particles, ∼125,000 ER-mito contact points from ∼900 cells in 10 different cellular conditions suggest that ER-mito contact points are inversely regulated with mitochondrial Ca2+-levels where TRPV4 always elevates mitochondrial Ca2+-levels. These findings link TRPV4 with MFN2-mediated diseases and suggest that different TRPV4-induced channelopathies are likely due to mitochondrial abnormalities.

Function and regulation of thermosensitive ion channel TRPV4 in the immune system.

Transient receptor potential vanilloid sub-type 4 (TRPV4) is a six transmembrane protein that acts as a non-selective Ca2+ channel. Notably, TRPV4 is present in almost all animals, from lower eukaryotes to humans and is expressed in diverse tissue and cell types. Accordingly, TRPV4 is endogenously expressed in several types of immune cells that represent both innate and adaptive immune systems of higher organism. TRPV4 is known to be activated by physiological temperature, suggesting that it acts as a molecular temperature sensor and thus plays a key role in temperature-dependent immune activation. It is also activated by diverse endogenous ligands, lipid metabolites, physical and mechanical stimuli. Both expression and function of TRPV4 in various immune cells, including T cells and macrophages, are also modulated by multiple pro- and anti-inflammatory compounds. The results from several laboratories suggest that TRPV4 is involved in the immune activation, a phenomenon with evolutionary significance. Because of its diverse engagement in the neuronal and immune systems, TRPV4 is a potential therapeutic target for several immune-related disorders.

Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis.

Bone defects, including bone loss due to increased osteoclast activity, have become a global health-related issue. Osteoclasts attach to the bone matrix and resorb the same, playing a vital role in bone remodeling. Ca2+ homeostasis plays a pivotal role in the differentiation and maturation of osteoclasts. In this work, we examined the role of TRPV1, a nonselective cation channel, in osteoclast function and differentiation. We demonstrate that endogenous TRPV1 is functional and causes Ca2+ influx upon activation with pharmacological activators [resiniferatoxin (RTX) and capsaicin] at nanomolar concentration, which enhances the generation of osteoclasts, whereas the TRPV1 inhibitor (5'-IRTX) reduces osteoclast differentiation. Activation of TRPV1 upregulates tartrate-resistant acid phosphatase activity and the expression of cathepsin K and calcitonin receptor genes, whereas TRPV1 inhibition reverses this effect. The slow release of capsaicin or RTX at a nanomolar concentration from a polysaccharide-based hydrogel enhances bone marrow macrophage (BMM) differentiation into osteoclasts whereas release of 5'-IRTX, an inhibitor of TRPV1, prevents macrophage fusion and osteoclast formation. We also characterize several subcellular parameters, including reactive oxygen (ROS) and nitrogen (RNS) species in the cytosol, mitochondrial, and lysosomal profiles in BMMs. ROS were found to be unaltered upon TRPV1 modulation. NO, however, had elevated levels upon RTX-mediated TRPV1 activation. Capsaicin altered mitochondrial membrane potential (ΔΨm) of BMMs but not 5'-IRTX. Channel modulation had no significant impact on cytosolic pH but significantly altered the pH of lysosomes, making these organelles less acidic. Since BMMs are precursors for osteoclasts, our findings of the cellular physiology of these cells may have broad implications in understanding the role of thermosensitive ion channels in bone formation and functions, and the TRPV1 modulator-releasing hydrogel may have application in bone tissue engineering and other biomedical sectors.

Lowest aqueous picomolar fluoride ions and in vivo aluminum toxicity detection by an aluminum(iii) binding chemosensor.

Aluminum toxicity in biological systems is a well-known issue yet remains as a prevalent and unsolvable problem due to the lack of proper molecular tools that can detect free aluminum(iii) or Al(iii) ions in vivo. Herein, we report a water-soluble photo-induced electron transfer (PET)-based turn-ON/OFF fluorometric chemosensor for the dual detection of Al(iii) and fluoride ions in aqueous media with a nanomolar (∼1.7 × 10-9 M) and picomolar (∼2 × 10-12 M, lowest ever detection so far) detection limit, respectively. Fluoride ions in sea water could be detected as well as the recognition of non-contamination in drinking water. In addition, using live-cell microscopy, Al(iii) ions were detected in live biological samples in vivo to aid establishing the aluminum-toxicity effect.

TRPM8 channel inhibitor-encapsulated hydrogel as a tunable surface for bone tissue engineering.

A major limitation in the bio-medical sector is the availability of materials suitable for bone tissue engineering using stem cells and methodology converting the stochastic biological events towards definitive as well as efficient bio-mineralization. We show that osteoblasts and Bone Marrow-derived Mesenchymal Stem Cell Pools (BM-MSCP) express TRPM8, a Ca2+-ion channel critical for bone-mineralization. TRPM8 inhibition triggers up-regulation of key osteogenesis factors; and increases mineralization by osteoblasts. We utilized CMT:HEMA, a carbohydrate polymer-based hydrogel that has nanofiber-like structure suitable for optimum delivery of TRPM8-specific activators or inhibitors. This hydrogel is ideal for proper adhesion, growth, and differentiation of osteoblast cell lines, primary osteoblasts, and BM-MSCP. CMT:HEMA coated with AMTB (TRPM8 inhibitor) induces differentiation of BM-MSCP into osteoblasts and subsequent mineralization in a dose-dependent manner. Prolonged and optimum inhibition of TRPM8 by AMTB released from the gels results in upregulation of osteogenic markers. We propose that AMTB-coated CMT:HEMA can be used as a tunable surface for bone tissue engineering. These findings may have broad implications in different bio-medical sectors.