scholarly journals Three-Dimensional (3D) Printed Silver Nanoparticles/Alginate/Nanocrystalline Cellulose Hydrogels: Study of the Antimicrobial and Cytotoxicity Efficacy

Nanomaterials ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 844 ◽  
Author(s):  
Carlo Bergonzi ◽  
Giulia Remaggi ◽  
Claudia Graiff ◽  
Laura Bergamonti ◽  
Marianna Potenza ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Morphological Characterization ◽  
Nanocrystalline Cellulose ◽  
In Vitro Tests ◽  
Mechanical Resistance ◽  
Natural Polymers ◽  
Targeted Analysis

Here, a formulation of silver nanoparticles (AgNPs) and two natural polymers such as alginate (ALG) and nanocrystalline cellulose (CNC) was developed for the 3D printing of scaffolds with large surface area, improved mechanical resistance and sustained capabilities to promote antimicrobial and cytotoxic effects. Mechanical resistance, water content, morphological characterization and silver distribution of the scaffolds were provided. As for applications, a comparable antimicrobial potency against S. aureus and P. aeruginosa was demonstrated by in vitro tests as function of the AgNP concentration in the scaffold (Minimal Inhibitory Concentration value: 10 mg/mL). By reusing the 3D system the antimicrobial efficacy was demonstrated over at least three applications. The cytotoxicity effects caused by administration of AgNPs to hepatocellular carcinoma (HepG2) cell culture through ALG and ALG/CNC scaffold were discussed as a function of time and dose. Finally, the liquid chromatography-mass spectrometry (LC-MS) technique was used for targeted analysis of pro-apoptotic initiation and executioner caspases, anti-apoptotic and proliferative proteins and the hepatocyte growth factor, and provided insights about molecular mechanisms involved in cell death induction.

scholarly journals Antibacterial Effect of Silver Nanoparticles on Klebsiella spp

2020 ◽  
Vol 1 (2) ◽  
pp. 8-15
Author(s):  
Gislanne Stéphanne Estevam da Silva ◽  
Rivaldo Leon Bezerra Cabral ◽  
Nathalie de Sena Pereira ◽  
José Heriberto Oliveira do Nascimento ◽  
Dany G kramer
Keyword(s):  
Silver Nanoparticles ◽  
Bacterial Resistance ◽  
Brain Heart Infusion ◽  
Trisodium Citrate ◽  
Antimicrobial Activities ◽  
Positive Control ◽  
Negative Control ◽  
In Vitro Tests ◽  
Klebsiella Spp

Silver nanoparticles (AgNP) can be incorporated into medical devices, such as tissues, to circumvent bacterial resistance such as Klebsiella spp, which can lead to skin and mucosal infections. Thus, the aim of the present study was to synthesize silver nanoparticles for later incorporation into cotton fabrics and in vitro tests against Klebsiella spp. The AgNP colloidal solution was synthesized (AgNO3 - 0.1 mM, 100 mM trisodium citrate, polyvinylpyrrolidone - 0.24 g, H2OH2) and then impregnated into the cotton fabric pretreated with poly diallyl dimethylammonium chloride (PDDA) of 100/500 tissue, shaken for 30 minutes). The material produced was analyzed by the FTIR; DLS and reflectance spectroscopy. The tests of the antimicrobial activities were by the microdilution technique against Klebsiella spp, in tubes containing Brain Heart Infusion (BHI), with the solution of silver (1); Tissue containing AgNP - 4 mm (2); Negative control (3) and positive control - ceftriaxone (4). Regarding MIC, the inhibitory activity occurred of the dilutions between 1/2 and 1/16. The AgNP particles had an average size of 24.75 nm. As synthesized AgNPs demonstrate the excellent antimicrobial activity against Klebsiella spp, with special emphasis on applications in nanotechnology and nanomedicine, targeting multiresistant antibiotic bacteria.

scholarly journals Pleiotropic effects of FGFR1 on cell proliferation, survival, and migration in a 3D mammary epithelial cell model

2005 ◽  
Vol 171 (4) ◽  
pp. 663-673 ◽  
Author(s):  
Wa Xian ◽  
Kathryn L. Schwertfeger ◽  
Tracy Vargo-Gogola ◽  
Jeffrey M. Rosen
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cell ◽  
Mammary Epithelial Cell ◽  
Molecular Mechanisms ◽  
Cell Model ◽  
Smooth Muscle Actin ◽  
Three Dimensional ◽  
Mammary Epithelial ◽  
Matrix Metalloproteinase 3

Members of the fibroblast growth factor (FGF) family and the FGF receptors (FGFRs) have been implicated in mediating various aspects of mammary gland development and transformation. To elucidate the molecular mechanisms of FGFR1 action in a context that mimics polarized epithelial cells, we have developed an in vitro three-dimensional HC11 mouse mammary epithelial cell culture model expressing a drug-inducible FGFR1 (iFGFR1). Using this conditional model, iFGFR1 activation in these growth-arrested and polarized mammary acini initially led to reinitiation of cell proliferation, increased survival of luminal cells, and loss of cell polarity, resulting in the disruption of acinar structures characterized by the absence of an empty lumen. iFGFR1 activation also resulted in a gain of invasive properties and the induction of matrix metalloproteinase 3 (MMP-3), causing the cleavage of E-cadherin and increased expression of smooth muscle actin and vimentin. The addition of a pan MMP inhibitor abolished these phenotypes but did not prevent the effects of iFGFR1 on cell proliferation or survival.

scholarly journals Vinculin regulates directionality and cell polarity in two- and three-dimensional matrix and three-dimensional microtrack migration

2016 ◽  
Vol 27 (9) ◽  
pp. 1431-1441 ◽  
Author(s):  
Aniqua Rahman ◽  
Shawn P. Carey ◽  
Casey M. Kraning-Rush ◽  
Zachary E. Goldblatt ◽  
Francois Bordeleau ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Focal Adhesion ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Focal Adhesions ◽  
Three Dimensions ◽  
Collagen Matrices ◽  
Migration Direction ◽  
Unidirectional Motion

During metastasis, cells can use proteolytic activity to form tube-like “microtracks” within the extracellular matrix (ECM). Using these microtracks, cells can migrate unimpeded through the stroma. To investigate the molecular mechanisms of microtrack migration, we developed an in vitro three-dimensional (3D) micromolded collagen platform. When in microtracks, cells tend to migrate unidirectionally. Because focal adhesions are the primary mechanism by which cells interact with the ECM, we examined the roles of several focal adhesion molecules in driving unidirectional motion. Vinculin knockdown results in the repeated reversal of migration direction compared with control cells. Tracking the position of the Golgi centroid relative to the position of the nucleus centroid reveals that vinculin knockdown disrupts cell polarity in microtracks. Vinculin also directs migration on two-dimensional (2D) substrates and in 3D uniform collagen matrices, as indicated by reduced speed, shorter net displacement, and decreased directionality in vinculin-deficient cells. In addition, vinculin is necessary for focal adhesion kinase (FAK) activation in three dimensions, as vinculin knockdown results in reduced FAK activation in both 3D uniform collagen matrices and microtracks but not on 2D substrates, and, accordingly, FAK inhibition halts cell migration in 3D microtracks. Together these data indicate that vinculin plays a key role in polarization during migration.

scholarly journals Intercellular coupling between peripheral circadian oscillators by TGF-β signaling

2021 ◽  
Vol 7 (30) ◽  
pp. eabg5174
Author(s):  
Anna-Marie Finger ◽  
Sebastian Jäschke ◽  
Marta del Olmo ◽  
Robert Hurwitz ◽  
Adrián E. Granada ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Clock Genes ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Transforming Growth Factor Β ◽  
Molecular Clocks ◽  
Peripheral Oscillators ◽  
Circadian Oscillators

Coupling between cell-autonomous circadian oscillators is crucial to prevent desynchronization of cellular networks and disruption of circadian tissue functions. While neuronal oscillators within the mammalian central clock, the suprachiasmatic nucleus, couple intercellularly, coupling among peripheral oscillators is controversial and the molecular mechanisms are unknown. Using two- and three-dimensional mammalian culture models in vitro (mainly human U-2 OS cells) and ex vivo, we show that peripheral oscillators couple via paracrine pathways. We identify transforming growth factor–β (TGF-β) as peripheral coupling factor that mediates paracrine phase adjustment of molecular clocks through transcriptional regulation of core-clock genes. Disruption of TGF-β signaling causes desynchronization of oscillator networks resulting in reduced amplitude and increased sensitivity toward external zeitgebers. Our findings reveal an unknown mechanism for peripheral clock synchrony with implications for rhythmic organ functions and circadian health.

scholarly journals Two clusters of surface-exposed amino acid residues enable high-affinity binding of retinal degeneration-3 (RD3) protein to retinal guanylyl cyclase

2020 ◽  
Vol 295 (31) ◽  
pp. 10781-10793 ◽  
Author(s):  
Igor V. Peshenko ◽  
Alexander M. Dizhoor
Keyword(s):  
Retinal Degeneration ◽  
Guanylyl Cyclase ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Hek293 Cells ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Retinal Guanylyl Cyclase ◽  
Functional Interface

Retinal degeneration-3 (RD3) protein protects photoreceptors from degeneration by preventing retinal guanylyl cyclase (RetGC) activation via calcium-sensing guanylyl cyclase–activating proteins (GCAP), and RD3 truncation causes severe congenital blindness in humans and other animals. The three-dimensional structure of RD3 has recently been established, but the molecular mechanisms of its inhibitory binding to RetGC remain unclear. Here, we report the results of probing 133 surface-exposed residues in RD3 by single substitutions and deletions to identify side chains that are critical for the inhibitory binding of RD3 to RetGC. We tested the effects of these substitutions and deletions in vitro by reconstituting purified RD3 variants with GCAP1-activated human RetGC1. Although the vast majority of the surface-exposed residues tolerated substitutions without loss of RD3's inhibitory activity, substitutions in two distinct narrow clusters located on the opposite sides of the molecule effectively suppressed RD3 binding to the cyclase. The first surface-exposed cluster included residues adjacent to Leu63 in the loop connecting helices 1 and 2. The second cluster surrounded Arg101 on a surface of helix 3. Single substitutions in those two clusters drastically, i.e. up to 245-fold, reduced the IC50 for the cyclase inhibition. Inactivation of the two binding sites completely disabled binding of RD3 to RetGC1 in living HEK293 cells. In contrast, deletion of 49 C-terminal residues did not affect the apparent affinity of RD3 for RetGC. Our findings identify the functional interface on RD3 required for its inhibitory binding to RetGC, a process essential for protecting photoreceptors from degeneration.

scholarly journals Direct SARS-CoV-2 infection of the human inner ear may underlie COVID-19-associated audiovestibular dysfunction

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Minjin Jeong ◽  
Karen E. Ocwieja ◽  
Dongjun Han ◽  
P. Ashley Wackym ◽  
Yichen Zhang ◽  
...  
Keyword(s):  
Inner Ear ◽  
Hair Cells ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
In Vitro Models ◽  
Cellular Models ◽  
Induced Pluripotent ◽  
Human Inner Ear

Abstract Background COVID-19 is a pandemic respiratory and vascular disease caused by SARS-CoV-2 virus. There is a growing number of sensory deficits associated with COVID-19 and molecular mechanisms underlying these deficits are incompletely understood. Methods We report a series of ten COVID-19 patients with audiovestibular symptoms such as hearing loss, vestibular dysfunction and tinnitus. To investigate the causal relationship between SARS-CoV-2 and audiovestibular dysfunction, we examine human inner ear tissue, human inner ear in vitro cellular models, and mouse inner ear tissue. Results We demonstrate that adult human inner ear tissue co-expresses the angiotensin-converting enzyme 2 (ACE2) receptor for SARS-CoV-2 virus, and the transmembrane protease serine 2 (TMPRSS2) and FURIN cofactors required for virus entry. Furthermore, hair cells and Schwann cells in explanted human vestibular tissue can be infected by SARS-CoV-2, as demonstrated by confocal microscopy. We establish three human induced pluripotent stem cell (hiPSC)-derived in vitro models of the inner ear for infection: two-dimensional otic prosensory cells (OPCs) and Schwann cell precursors (SCPs), and three-dimensional inner ear organoids. Both OPCs and SCPs express ACE2, TMPRSS2, and FURIN, with lower ACE2 and FURIN expression in SCPs. OPCs are permissive to SARS-CoV-2 infection; lower infection rates exist in isogenic SCPs. The inner ear organoids show that hair cells express ACE2 and are targets for SARS-CoV-2. Conclusions Our results provide mechanistic explanations of audiovestibular dysfunction in COVID-19 patients and introduce hiPSC-derived systems for studying infectious human otologic disease.

scholarly journals Cortical Spheroid Model for Studying the Effects of Ischemic Brain Injury

2021 ◽  
Author(s):  
Rachel M McLaughlin ◽  
Amanda Laguna ◽  
Ilayda Top ◽  
Christien Hernadez ◽  
Liane L Livi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Cost Effective ◽  
Glucose Deprivation ◽  
In Vitro Models ◽  
3D Architecture ◽  
A Cell ◽  
Antioxidant Agent

Stroke is a devastating neurological disorder and a leading cause of death and long-term disability. Despite many decades of research, there are still very few therapeutic options for patients suffering from stroke or its consequences. This is partially due to the limitations of current research models, including traditional in vitro models which lack the three-dimensional (3D) architecture and cellular make-up of the in vivo brain. 3D spheroids derived from primary postnatal rat cortex provide an in vivo-relevant model containing a similar cellular composition to the native cortex and a cell-synthesized extracellular matrix. These spheroids are cost-effective, highly reproducible, and can be produced in a high-throughput manner, making this model an ideal candidate for screening potential therapeutics. To study the cellular and molecular mechanisms of stroke in this model, spheroids were deprived of glucose, oxygen, or both oxygen and glucose for 24 hours. Both oxygen and oxygen-glucose deprived spheroids demonstrated many of the hallmarks of stroke, including a decrease in metabolism, an increase in neural dysfunction, and an increase in reactive astrocytes. Pretreatment of spheroids with the antioxidant agent N-acetylcysteine (NAC) mitigated the decrease in ATP seen after 24 hours of oxygen-glucose deprivation. Together, these results show the utility of our 3D cortical spheroid model for studying ischemic injury and its potential for screening stroke therapeutics.

Molecular Mechanisms of Mutations in Factor XIII A-subunit Deficiency: In vitro Expression in COS-cells Demonstrates Intracellular Degradation of the Mutant Proteins

1997 ◽  
Vol 77 (06) ◽  
pp. 1068-1072 ◽  
Author(s):  
Aarno Palotie ◽  
Hanna Mikkola ◽  
Laszlo Muszbek ◽  
Gizela Haramura ◽  
Eija Hämäläinen ◽  
...  
Keyword(s):  
Factor Xiii ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Subunit Gene ◽  
Three Dimensional Model ◽  
Mutant Proteins ◽  
Cos Cells ◽  
Intracellular Degradation ◽  
A Subunit

SummaryFactor XIII deficiency is an autosomal recessive bleeding disorder that is largely caused by various mutations in FXIII A-subunit gene. Characteristically, the patients lack both A-subunit activity and antigen in the circulation. Here we have analysed the consequences of four mis-sense mutations (Met242→Thr, Arg252→Ile, Arg326→Gln, Leu498 to Pro) and one stop mutation (Arg661→Stop) in the FXIII A-subunit gene by expression in COS-cells. After transient transfection each mutant cDNA expressed mRNA at an equal level to the wild type FXIII. However, the mutant polypeptides accumulated in the cells in significantly reduced quantities and demonstrated only very low enzymatic activity. Analysis of immunoprecipitated metabolically labelled polypeptides demonstrated remarkable instability and intracellular degradation of all mutant FXIII proteins. These results verify the deleterious nature of the individual amino acid changes and confirm that protein instability and susceptibility to proteolysis are consequences of the mutations, as predicted from the three-dimensional model of crystallised FXIII A-subunit.

scholarly journals Genetic regulation of murine pituitary development

2015 ◽  
Vol 54 (2) ◽  
pp. R55-R73 ◽  
Author(s):  
Karine Rizzoti
Keyword(s):  
Endocrine Cells ◽  
Molecular Mechanisms ◽  
Genetic Regulation ◽  
Three Dimensional ◽  
Significant Progress ◽  
Pituitary Development ◽  
Tumour Formation ◽  
Ventral Diencephalon

Significant progress has been made recently in unravelling the embryonic events leading to pituitary morphogenesis, bothin vivoandin vitro. This includes dissection of the molecular mechanisms controlling patterning of the ventral diencephalon that regulate formation of the pituitary anlagen or Rathke's pouch. There is also a better characterisation of processes that underlie maintenance of pituitary progenitors, specification of endocrine lineages and the three-dimensional organisation of newly differentiated endocrine cells. Furthermore, a population of adult pituitary stem cells (SCs), originating from embryonic progenitors, have been described and shown to have not only regenerative potential, but also the capacity to induce tumour formation. Finally, the successful recapitulationin vitroof embryonic events leading to generation of endocrine cells from embryonic SCs, and their subsequent transplantation, represents exciting advances towards the use of regenerative medicine to treat endocrine deficits. In this review, an up-to-date description of pituitary morphogenesis will be provided and discussed with particular reference to pituitary SC studies.

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