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1.
J Immunol ; 211(9): 1348-1358, 2023 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-37737664

RESUMO

Cytotoxic lymphocytes eliminate cancer cells through the release of lytic granules, a specialized form of secretory lysosomes. This compartment is part of the pleomorphic endolysosomal system and is distinguished by its highly dynamic Ca2+ signaling machinery. Several transient receptor potential (TRP) calcium channels play essential roles in endolysosomal Ca2+ signaling and ensure the proper function of these organelles. In this study, we examined the role of TRPML1 (TRP cation channel, mucolipin subfamily, member 1) in regulating the homeostasis of secretory lysosomes and their cross-talk with mitochondria in human NK cells. We found that genetic deletion of TRPML1, which localizes to lysosomes in NK cells, led to mitochondrial fragmentation with evidence of collapsed mitochondrial cristae. Consequently, TRPML1-/- NK92 (NK92ML1-/-) displayed loss of mitochondrial membrane potential, increased reactive oxygen species stress, reduced ATP production, and compromised respiratory capacity. Using sensitive organelle-specific probes, we observed that mitochondria in NK92ML1-/- cells exhibited evidence of Ca2+ overload. Moreover, pharmacological activation of the TRPML1 channel in primary NK cells resulted in upregulation of LC3-II, whereas genetic deletion impeded autophagic flux and increased accumulation of dysfunctional mitochondria. Thus, TRPML1 impacts autophagy and clearance of damaged mitochondria. Taken together, these results suggest that an intimate interorganelle communication in NK cells is orchestrated by the lysosomal Ca2+ channel TRPML1.


Assuntos
Canais de Cálcio , Canais de Potencial de Receptor Transitório , Humanos , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo , Mitocôndrias/metabolismo , Lisossomos/metabolismo , Células Matadoras Naturais/metabolismo
2.
J Prosthet Dent ; 126(1): 92.e1-92.e8, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34049698

RESUMO

STATEMENT OF PROBLEM: Rapid osseointegration between implant and bone tissue for early loading of a prosthesis with sufficient primary stability depends on the surface characteristics of the implant. The development and characterization of suitable surface coatings on dental implants is a major challenge. PURPOSE: The purpose of this in vitro study was to evaluate and compare the osteogenic potential and cytotoxicity of unmodified zirconia, acid-etched zirconia, bioactive glass-coated zirconia, and tamarind kernel polysaccharide with hydrophilic acrylic acid (TKP-AA) hydrogel-coated zirconia. MATERIAL AND METHODS: Thirty-six disks each of unmodified zirconia, acid-etched, 45S5 bioactive glass-coated, and TKP-AA hydrogel-coated zirconia were evaluated for osteogenic potential and cytotoxic effect by using human osteoblast Saos-2 cells. The surface topography of the disks and the morphology of the cells grown on these surfaces were examined by scanning electron microscopy (n=3). The cell attachment was evaluated by confocal imaging (n=3). The cytotoxic effect was evaluated by cell viability assay (n=9). Osteoblast maturation was assessed by alkaline phosphatase assay (n=9) and cell mineralization by alizarin red staining (n=9). ANOVA and Bonferroni multiple comparison post hoc tests were used to evaluate the statistical significance of the intergroup differences in these characteristics (α=.05). RESULTS: The surface modifications resulted in distinct changes in the surface morphology of zirconia disks and the growth of Saos-2 cells. Zirconia disks coated with TKP-AA promoted higher proliferation of osteoblasts compared with unmodified disks (P<.001). Similarly, the surface modifications significantly increased the differentiation of mesenchymal stem cells to osteoblasts as compared with uncoated zirconia (P<.001). However, the rate of differentiation to osteoblasts was similar among the surface modifications. Acid-etched and TKP-AA-coated disks promoted mineralization of osteoblasts to the same extent, except bioactive glass coating, which significantly increased the rate of mineralization (P<.001). CONCLUSIONS: Surface modification of zirconia by acid etching and coating with Bioglass or TKP-AA hydrogel resulted in the improved growth and differentiation of osteoblasts. TKP-AA hydrogel coating promoted the proliferation of osteoblasts, whereas Bioglass coating showed better mineralization. TKP-AA hydrogel coating is a promising candidate for improving the osseointegration of dental implants that warrants further investigation.


Assuntos
Implantes Dentários , Osseointegração , Proliferação de Células , Humanos , Osteoblastos , Propriedades de Superfície , Titânio , Zircônio
3.
Reprod Domest Anim ; 55(11): 1619-1628, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32920930

RESUMO

Sperm cells perform precise chemotactic and thermotactic movement which is crucial for fertilization. However, the key molecules involved in detection of different chemical and physical stimuli which guide the sperm during navigation are not well understood. Ca2+ -signalling mediated by ion channels seem to play important role in motility and other fertility parameters. In this work, we explored the endogenous localization pattern of TRPV channels in the mature spermatozoa of avian species. Using sperm from white pekin duck (Anas platyrhynchos) as the representative avian model, we demonstrate that duck sperm endogenously express the thermosensitive channels TRPV1, TRPV2, TRPV3, TRPV4, and highly Ca2+ -selective channels TRPV5 and TRPV6 in specific yet differential locations. All of these TRPV channels are enriched in the sperm tail, indicating their relevance in sperm motility. Interestingly, the TRPV3 and TRPV4 channels are present in the mitochondrial region. Calcium selective TRPV5 channel is exclusively present in sperm tail and is most abundant among the TRPV channels. This is the first report describing the endogenous presence of TRPV2 and TRPV3 channels in the sperm of any species. Using confocal imaging and super-resolution imaging, we demonstrate that though the TRPV channels are evolutionarily closely related, they have distinct localization pattern in the duck sperm, which could impact their role in fertilization.


Assuntos
Patos , Espermatozoides/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Regulação da Expressão Gênica , Masculino , Microscopia Confocal/veterinária , Mitocôndrias , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/citologia , Canais de Cátion TRPV/genética
4.
Biochem Biophys Res Commun ; 512(2): 295-302, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30885439

RESUMO

TRPA1 is a non-selective cation channel originated in invertebrates. The genomic locus containing TRPA1 gene remains highly conserved and retained in all vertebrates. TRPA1 gene is evolutionarily selected, yet maintained as a highly diverged protein. Throughout the vertebrate evolution, the extracellular loops of TRPA1 become most diverged indicating that TRPA1 may be involved in detecting large spectrum and uncertain stimulus which is critical for adaptive benefit. We tested the expression of TRPA1 in mature sperm from different vertebrates. This is the first report demonstrating that TRPA1 is expressed endogenously in mature spermatozoa of multiple species representing entire vertebrate phyla. However, its specific localization within sperm remains species-specific. Accordingly, we report that in rodents TRPA1 expression correlates with different stages of spermatogenesis. We propose that presence of endogenous TRPA1 in testes and in mature sperm provides reproductive benefit.


Assuntos
Evolução Molecular , Espermatogênese/genética , Canal de Cátion TRPA1/genética , Vertebrados/genética , Animais , Humanos , Masculino , Filogenia , Especificidade da Espécie , Espermatogênese/fisiologia , Sintenia , Canal de Cátion TRPA1/química , Canal de Cátion TRPA1/fisiologia , Vertebrados/classificação , Vertebrados/fisiologia
6.
Connect Tissue Res ; 59(sup1): 111-121, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29458266

RESUMO

PURPOSE: With increased life expectancy, disorders in lifestyle and other clinical conditions, and the changes in the connective tissues such as in bone, impose diverse biomedical problems. Cells belong to osteogenic lineages are extremely specific for their surface requirements. Therefore, suitable surfaces are the critical bottle neck for successful bone tissue engineering. This study involves assessment of polysaccharide-based hydrogel which effectively allows growth, differentiation and mineralisation of osteogenic cells even in the absence of osteogenic inducing factors. MATERIALS AND METHODS: Tamarind Kernel Polysaccharide was grafted with acrylic acid at different mole ratio. The critical parameter, surface morphology for bio application was assessed by SEM. MTT assay has been performed with hydrogels on Saos-2 cells. The biocompatibility and adhesion of different cell lines (F-11, Saos-2, Raw 264.7 and MSCs) on hydrogel surface was performed by Phalloidin and DAPI staining. Further the differentiation, mineralization and expression of different osteogenic markers, ALP assay, Alizarin Red staining and q-PCR was performed. RESULTS: The hydrogels show highly porous and interconnected pores. MTT assay demonstrates the hydrogel have no cytotoxicity towards Saos-2 cells and are suitable for proliferation of different lineage of cell lines. ALP, Alizarin red staining and q-PCR assay shows that the hydrogel surface enhances the differentiation, mineralization and expression of different osteogenic genes in Saos-2 cells in the absence of any osteogenic inducing factors. Conclusion Synthesized hydrogel surface triggers signalling events towards osteogenesis even in the absence of added growth factors. We proposed that this material can be used for effective bone tissue engineering in vitro at low cost.


Assuntos
Acrilatos/química , Osso e Ossos/metabolismo , Hidrogéis/química , Osteogênese , Polissacarídeos/química , Tamarindus/química , Engenharia Tecidual , Animais , Osso e Ossos/citologia , Camundongos , Células RAW 264.7 , Ratos
7.
Biochem Biophys Res Commun ; 473(4): 781-788, 2016 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-27003252

RESUMO

Transient Receptor Potential Vanilloid sub-type 4 (TRPV4) is a non-selective cationic channel involved in regulation of temperature, osmolality and different ligand-dependent Ca(2+)-influx. Recently, we have demonstrated that TRPV4 is conserved in all vertebrates. Now we demonstrate that TRPV4 is endogenously expressed in all vertebrate sperm cells ranging from fish to mammals. In human sperm, TRPV4 is present as N-glycosylated protein and its activation induces Ca(2+)-influx. Its expression and localization differs in swim-up and swim-down cells suggesting that TRPV4 is an important determining factor for sperm motility. We demonstrate that pharmacological activation or inhibition of TRPV4 regulates Ca(2+)-wave propagation from head to tail. Such findings may have wide application in male fertility-infertility, contraception and conservation of endangered species as well.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Bufonidae , Células Cultivadas , Patos , Regulação da Expressão Gênica/fisiologia , Humanos , Lagartos , Masculino , Especificidade da Espécie
10.
Biochem Biophys Res Commun ; 456(1): 312-9, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25434996

RESUMO

TRPV4 is involved in several physiological and sensory functions as well as with several diseases and genetic disorders, though the molecular mechanisms for these are unclear. In this work we have analyzed molecular evolution and structure-function relationship of TRPV4 using sequences from different species. TRPV4 has evolved during early vertebrate origin (450million years). Synteny analysis confirms that TRPV4 has coevolved with two enzymes involved in sterol biosynthesis, namely MVK and GLTP. Cholesterol-recognizing motifs are present within highly conserved TM4-Loop4-TM5 region of TRPV4. TRPV4 is present in lipid raft where it co-localizes with Caveolin1 and Filipin. TM4-Loop4-TM5 region as well as Loop4 alone can physically interact with cholesterol, its precursor mevalonate and derivatives such as stigmasterol and aldosterone. Mobility of TRPV4-GFP depends on membrane cholesterol level. Molecular evolution of TRPV4 shared striking parallelism with the cholesterol bio-synthesis pathways at the genetic, molecular and metabolic levels. We conclude that interaction with sterols and cholesterol-dependent membrane dynamics have influence on TRPV4 function. These results may have importance on TRPV4-medaited cellular functions and pathophysiology.


Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Caveolina 1/metabolismo , Biologia Computacional , Bases de Dados de Proteínas , Evolução Molecular , Filipina/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Software
11.
Gen Comp Endocrinol ; 220: 23-32, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25449179

RESUMO

While effects of different steroids on the gene expression and regulation are well established, it is proven that steroids can also exert rapid non-genomic actions in several tissues and cells. In most cases, these non-genomic rapid effects of steroids are actually due to intracellular mobilization of Ca(2+)- and other ions suggesting that Ca(2+) channels are involved in such effects. Transient Receptor Potential (TRP) ion channels or TRPs are the largest group of non-selective and polymodal ion channels which cause Ca(2+)-influx in response to different physical and chemical stimuli. While non-genomic actions of different steroids on different ion channels have been established to some extent, involvement of TRPs in such functions is largely unexplored. In this review, we critically analyze the literature and summarize how different steroids as well as their metabolic precursors and derivatives can exert non-genomic effects by acting on different TRPs qualitatively and/or quantitatively. Such effects have physiological repercussion on systems such as in sperm cells, immune cells, bone cells, neuronal cells and many others. Different TRPs are also endogenously expressed in diverse steroid-producing tissues and thus may have importance in steroid synthesis as well, a process which is tightly controlled by the intracellular Ca(2+) concentrations. Tissue and cell-specific expression of TRP channels are also regulated by different steroids. Understanding of the crosstalk between TRP channels and different steroids may have strong significance in physiological, endocrinological and pharmacological context and in future these compounds can also be used as potential biomedicine.


Assuntos
Cálcio/metabolismo , Esteroides/uso terapêutico , Canais de Potencial de Receptor Transitório/fisiologia , Humanos , Esteroides/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo
12.
Blood Adv ; 7(16): 4492-4504, 2023 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-37327114

RESUMO

The functionality of natural killer (NK) cells is tuned during education and is associated with remodeling of the lysosomal compartment. We hypothesized that genetic variation in killer cell immunoglobulin-like receptor (KIR) and HLA, which is known to influence the functional strength of NK cells, fine-tunes the payload of effector molecules stored in secretory lysosomes. To address this possibility, we performed a high-resolution analysis of KIR and HLA class I genes in 365 blood donors and linked genotypes to granzyme B loading and functional phenotypes. We found that granzyme B levels varied across individuals but were stable over time in each individual and genetically determined by allelic variation in HLA class I genes. A broad mapping of surface receptors and lysosomal effector molecules revealed that DNAM-1 and granzyme B levels served as robust metric of the functional state in NK cells. Variation in granzyme B levels at rest was tightly linked to the lytic hit and downstream killing of major histocompatibility complex-deficient target cells. Together, these data provide insights into how variation in genetically hardwired receptor pairs tunes the releasable granzyme B pool in NK cells, resulting in predictable hierarchies in global NK cell function.


Assuntos
Células Matadoras Naturais , Receptores KIR , Granzimas/genética , Granzimas/metabolismo , Receptores KIR/genética , Receptores KIR/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Genótipo
13.
ACS Omega ; 7(11): 9537-9550, 2022 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-35350319

RESUMO

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.

14.
Front Cell Dev Biol ; 10: 867057, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36211461

RESUMO

Bi-directional crosstalk between Ca2+ signaling and ROS modulates physiological processes as a part of a regulatory circuit including sperm function. The role of transient receptor potential vanilloid 1 (TRPV1) in this regard cannot be undermined. This is the first report demonstrating the Ca2+-sensitive TRPV1 channel to be under-expressed in spermatozoa of subfertile men, idiopathic infertile men, and normozoospermic infertile males with high ROS (idiopathic infertility and unilateral varicocele). To study the effect of TRPV1 in determining the fertility outcome, we compared the expression profile of TRPV1 in spermatozoa of male partners who achieved pregnancy by natural conception (NC+, n = 10), IVF (IVF+, n = 23), or ICSI (ICSI +, n = 9) and their respective counterparts with failed pregnancy NC (n = 7), IVF (n = 23), or ICSI (n = 10), by both immunocytochemistry and flow-cytometry. Reduced expression of TRPV1 in sperm of IVF ± and ICSI ± men with respect to that NC+ men imply its role in mediating successful fertilization. Unsuccessful pregnancy outcome with an underexpression of TRPV1 in sperm of NC-/IVF-/ICSI-men suggests its role in conception and maintenance of pregnancy. Since ROS is regarded as one of the major contributors to sperm dysfunction, the effect of H2O2 +/- TRPV1 modulators (RTX/iRTX) on acrosomal reaction and calcium influx was evaluated to confirm TRPV1 as a redox sensor in human sperm. A significant increment in the percentage of acrosome reacted spermatozoa along with augmented Ca2+-influx was observed after H2O2 treatment, both in the presence or absence of TRPV1 agonist resiniferatoxin (RTX). The effect was attenuated by the TRPV1 antagonist iodoresiniferatoxin (iRTX), indicating the involvement of TRPV1 in mediating H2O2 response. Enhancement of motility and triggering of acrosomal reaction post TRPV1 activation suggested that disruption of these signaling cascades in vivo, possibly due to down-regulation of TRPV1 in these subfertile males. Bioinformatic analysis of the crosstalk between TRPV1 with fertility candidate proteins (reported to influence IVF outcome) revealed cell death and survival, cellular compromise, and embryonic development to be the primary networks affected by anomalous TRPV1 expression. We therefore postulate that TRPV1 can act as a redox sensor, and its expression in spermatozoa may serve as a fertility marker.

15.
ACS Infect Dis ; 7(8): 2068-2072, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-33779133

RESUMO

Antibacterial activity of silver nanoparticles is often associated with toxicity to the host. We here report that noncytotoxic doses of silver nanoparticles coated with zinc oxide, Ag@ZnO, can stimulate proliferation and migration of human keratinocytes, HaCaT, with increased expression of Ki67 and vinculin at the leading edge of wounds. Interestingly, Ag@ZnO stimulates keratinocytes to produce the antimicrobial peptides hBD2 and RNase7, promoting antibacterial activity against both extracellular and intracellular Staphylococcus aureus isolated from wounds. Overall, these results suggest that Ag@ZnO has the potential to significantly improve treatment outcomes in clearing wound infection.


Assuntos
Nanopartículas Metálicas , Óxido de Zinco , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Humanos , Queratinócitos , Proteínas Citotóxicas Formadoras de Poros , Prata
16.
Sci Rep ; 11(1): 19263, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34584119

RESUMO

Urinary tract infection frequently caused by E. coli is one of the most common bacterial infections. Increasing antibiotic resistance jeopardizes successful treatment and alternative treatment strategies are therefore mandatory. Metformin, an oral antidiabetic drug, has been shown to activate macrophages in the protection against certain infecting microorganisms. Since epithelial cells often form the first line of defense, we here investigated the effect on uroepithelial cells during E. coli infection. Metformin upregulated the human antimicrobial peptides cathelicidin LL-37 and RNase7 via modulation of the TRPA1 channel and AMPK pathway. Interestingly, metformin stimulation enriched both LL-37 and TRPA1 in lysosomes. In addition, metformin specifically increased nitric oxide and mitochondrial, but not cytosolic ROS. Moreover, metformin also triggered mRNA expression of the proinflammatory cytokines IL1B, CXCL8 and growth factor GDF15 in human uroepithelial cells. The GDF15 peptide stimulated macrophages increased LL-37 expression, with increased bacterial killing. In conclusion, metformin stimulation strengthened the innate immunity of uroepithelial cells inducing enhanced extracellular and intracellular bacterial killing suggesting a favorable role of metformin in the host defense.


Assuntos
Infecções por Escherichia coli/tratamento farmacológico , Metformina/farmacologia , Infecções Urinárias/tratamento farmacológico , Urotélio/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Linhagem Celular , Citocinas/metabolismo , Reposicionamento de Medicamentos , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Humanos , Imunidade Inata/efeitos dos fármacos , Metformina/uso terapêutico , Ribonucleases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Canal de Cátion TRPA1/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Infecções Urinárias/imunologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/imunologia , Urotélio/imunologia , Urotélio/microbiologia , Catelicidinas
17.
Sci Rep ; 11(1): 3730, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33580126

RESUMO

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.


Assuntos
Osteoblastos/metabolismo , Canais de Cátion TRPM/metabolismo , Engenharia Tecidual/métodos , Animais , Benzamidas/metabolismo , Benzamidas/farmacologia , Células da Medula Óssea/citologia , Osso e Ossos/metabolismo , Diferenciação Celular , Células Cultivadas , Feminino , Hidrogéis/química , Hidrogéis/farmacologia , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Osteogênese , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPM/antagonistas & inibidores , Tiofenos/metabolismo , Tiofenos/farmacologia
18.
ACS Appl Mater Interfaces ; 11(46): 42998-43017, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31664808

RESUMO

Multidrug resistant (MDR) bacteria have emerged as a major clinical challenge. The unavailability of effective antibiotics has necessitated the use of emerging nanoparticles as alternatives. In this work, we have developed carbohydrate-coated bimetallic nanoparticles (Au-AgNP, 30-40 nm diameter) that are nontoxic toward mammalian cells yet highly effective against MDR strains as compared to their monometallic counterparts (Ag-NP, Au-NP). The Au-AgNP is much more effective against Gram-negative MDR Escherichia coli and Enterobacter cloacae when compared to most of the potent antibiotics. We demonstrate that in vivo, Au-AgNP is at least 11000 times more effective than Gentamicin in eliminating MDR Methicillin Resistant Staphylococcus aureus (MRSA) infecting mice skin wounds. Au-AgNP is able to heal and regenerate infected wounds faster and in scar-free manner. In vivo results show that this Au-AgNP is very effective antibacterial agent against MDR strains and does not produce adverse toxicity. We conclude that this bimetallic nanoparticle can be safe in complete skin regeneration in bacteria infected wounds.


Assuntos
Bactérias/crescimento & desenvolvimento , Materiais Revestidos Biocompatíveis , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Ouro , Nanopartículas Metálicas , Prata , Cicatrização/efeitos dos fármacos , Infecção dos Ferimentos , Animais , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Ouro/química , Ouro/farmacologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Prata/química , Prata/farmacologia , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/metabolismo , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/patologia
19.
Biosci Rep ; 39(9)2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31488616

RESUMO

Transient receptor potential channel subfamily A member 1 (TRPA1) is a non-selective cationic channel, identified initially as a cold sensory receptor. TRPA1 responds to diverse exogenous and endogenous stimuli associated with pain and inflammation. However, the information on the role of TRPA1 toward T-cell responses remains scanty. In silico data suggest that TRPA1 can play an important role in the T-cell activation process. In this work, we explored the endogenous expression of TRPA1 and its function in T cells. By reverse transcription polymerase chain reaction (RT-PCR), confocal microscopy and flow cytometry, we demonstrated that TRPA1 is endogenously expressed in primary murine splenic T cells as well as in primary human T cells. TRPA1 is primarily located at the cell surface. TRPA1-specific activator namely allyl isothiocyanate (AITC) increases intracellular calcium ion (Ca2+) levels while two different inhibitors namely A-967079 as well as HC-030031 reduce intracellular Ca2+ levels in T cells; TRPA1 inhibition also reduces TCR-mediated calcium influx. TRPA1 expression was found to be increased during αCD3/αCD28 (TCR) or Concanavalin A (ConA)-driven stimulation in T cells. TRPA1-specific inhibitor treatment prevented induction of cluster of differentiation 25 (CD25), cluster of differentiation 69 (CD69) in ConA/TCR stimulated T cells and secretion of cytokines like tumor necrosis factor (TNF), interferon γ (IFN-γ), and interleukin 2 (IL-2) suggesting that endogenous activity of TRPA1 may be involved in T-cell activation. Collectively these results may have implication in T cell-mediated responses and indicate possible role of TRPA1 in immunological disorders.


Assuntos
Imunidade Celular/genética , Linfócitos T/imunologia , Canal de Cátion TRPA1/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Acetanilidas/farmacologia , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Cálcio/imunologia , Cálcio/metabolismo , Simulação por Computador , Concanavalina A/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade Celular/imunologia , Interferon gama/imunologia , Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Isotiocianatos/farmacologia , Lectinas Tipo C/imunologia , Camundongos , Oximas/farmacologia , Cultura Primária de Células , Purinas/farmacologia , Canal de Cátion TRPA1/agonistas , Canal de Cátion TRPA1/antagonistas & inibidores , Canal de Cátion TRPA1/imunologia , Fator de Necrose Tumoral alfa/imunologia
20.
Macromol Biosci ; 17(3)2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27767291

RESUMO

Bone related problems are increasing as a consequence of increased life expectancy, disorders in life style, and other medical conditions enforcing the need for functional bones prepared in vitro at affordable cost. Lack of suitable surface which promotes growth of both osteogenic and nonosteogenic cells is a major limitation. Here a novel biomaterial is reported that is synthesized from natural polysaccharide, namely, tamarind kernel polysaccharide (TKP), which is grafted with hydrophilic acrylic acid (AA) by radical polymerization. Modification in surface functionality removes unwanted proteins and alters hydrophilic/hydrophobic balance. TKP-AA is suitable for the growth of different nonosteogenic and osteogenic cells. This material is suitable for osteoblasts and promotes in vitro mineralization and differentiation without the addition of exogenous growth factors. TKP-AA can be used for the growth of mesenchymal stem cell-derived osteoblasts. It is suggested that TKP-AA can potentially be used as a scaffold for diverse cell types and particularly for bone tissue engineering at low cost.


Assuntos
Materiais Biocompatíveis/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Osteogênese/efeitos dos fármacos , Polissacarídeos/química , Animais , Materiais Biocompatíveis/administração & dosagem , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Hidrogel de Polietilenoglicol-Dimetacrilato/administração & dosagem , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Osteoblastos/efeitos dos fármacos , Polissacarídeos/administração & dosagem , Ratos , Alicerces Teciduais/química
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