ABSTRACT
Titanium and titanium alloys are widely used in medical devices and implants; thus, the biocompatibility of these metals is of great importance. In this study, glioblastoma astrocytoma cellular responses to Ti65-Zr18-Nb16-Mo1 (Ti65M, metastable medium-entropy alloy), Ti-13Nb-7Sn-4Mo (TNSM, titanium alloy), and commercially pure titanium (CP-Ti) were studied. Several physical parameters (crystal phase structure, surface roughness and hardness) of the titanium alloys were measured, and the correlation with the cellular viability was investigated. Finally, the relative protein expression in cellular proliferation pathways was measured and compared with mRNA expression assessed with quantitative real-time reverse transcription polymerase chain reaction assay (qRT-PCR).
Subject(s)
Alloys , Titanium , Alloys/chemistry , Titanium/chemistry , Osteoblasts/metabolism , Metals/metabolism , Hardness , Materials Testing , Biocompatible Materials/chemistryABSTRACT
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) technology is a powerful method for genetic modification (and regulation) that is of great current interest. The development of new, economical methods of detecting and extracting Cas9 (and/or dCas9) from transfected cells is thus an important advance. In this work, we employed molecular imprinting, using two peptides from the Cas9 protein, to make magnetic peptide-imprinted chitosan nanoparticles. dCas9 was encoded in a plasmid which was then transfected into human embryonic kidney (HEK293T) cells. The expression of dCas9 protein was measured by using total protein kits. Finally, the imprinted nanoparticles were used to extract dCas9 from transfected cell homogenates.
Subject(s)
CRISPR-Associated Protein 9/isolation & purification , Chitosan/chemistry , Molecular Imprinting , Nanoparticles/chemistry , Peptides/chemistry , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Epitopes/isolation & purification , Gene Editing , HEK293 Cells , Humans , Magnets/chemistry , TransfectionABSTRACT
Molecularly imprinted polymer (MIP)-based electrochemical sensors for the protein α-synuclein (a marker for Parkinson's disease) were developed using a peptide epitope from the protein. MIPs doped with various concentrations and species of transition metal dichalcogenides (TMDs) to enhance conductivity were electropolymerized with and without template molecules. The current during the electropolymerization was compared with that associated with the electrochemical response (at 0.24~0.29 V vs. ref. electrode) to target peptide molecules in the finished sensor. We found that this relationship can aid in the rational design of conductive MIPs for the recognition of biomarkers in biological fluids. The sensing range and limit of detection of TMD-doped imprinted poly(AN-co-MSAN)-coated electrodes were 0.001-100 pg/mL and 0.5 fg/mL (SNR = 3), respectively. To show the potential applicability of the MIP electrochemical sensor, cell culture medium from PD patient-specific midbrain organoids generated from induced pluripotent stem cells was analyzed. α-Synuclein levels were found to be significantly reduced in the organoids from PD patients, compared to those generated from age-matched controls. The relative standard deviation and recovery are less than 5% and 95-115%, respectively. Preparation of TMD-doped α-synuclein (SNCA) peptide-imprinted poly(AN-co-MSAN)-coated electrodes.
Subject(s)
Disulfides/chemistry , Molecularly Imprinted Polymers/chemistry , Molybdenum/chemistry , Sulfides/chemistry , Tungsten Compounds/chemistry , alpha-Synuclein/analysis , Electrochemical Techniques/methods , Humans , Limit of Detection , Mesencephalon/chemistry , Organoids/chemistry , Parkinson Disease/diagnosis , Peptide Fragments/chemistry , alpha-Synuclein/chemistryABSTRACT
Oyster shells are rich in calcium, and thus, the potential use of waste shells is in the production of calcium phosphate (CaP) minerals for osteopathic biomedical applications, such as scaffolds for bone regeneration. Implanted scaffolds should stimulate the differentiation of induced pluripotent stem cells (iPSCs) into osteoblasts. In this study, oyster shells were used to produce nano-grade hydroxyapatite (HA) powder by the liquid-phase precipitation. Then, biphasic CaP (BCP) bioceramics with two different phase ratios were obtained by the foaming of HA nanopowders and sintering by two different two-stage heat treatment processes. The different sintering conditions yielded differences in structure and morphology of the BCPs, as determined by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Brunauer-Emmett-Teller (BET) surface area analysis. We then set out to determine which of these materials were most biocompatible, by co-culturing with iPSCs and examining the gene expression in molecular pathways involved in self-renewal and differentiation of iPSCs. We found that sintering for a shorter time at higher temperatures gave higher expression levels of markers for proliferation and (early) differentiation of the osteoblast. The differences in biocompatibility may be related to a more hierarchical pore structure (micropores within macropores) obtained with briefer, high-temperature sintering.
Subject(s)
Animal Shells/chemistry , Hydroxyapatites/chemistry , Induced Pluripotent Stem Cells/metabolism , Animal Shells/metabolism , Animals , Biocompatible Materials/chemistry , Bone Regeneration/physiology , Calcium Phosphates/chemistry , Cell Adhesion/physiology , Cell Differentiation/drug effects , Ceramics/chemistry , Humans , Hydroxyapatites/chemical synthesis , Hydroxyapatites/metabolism , Hydroxyapatites/pharmacology , Induced Pluripotent Stem Cells/drug effects , Osteoblasts/cytology , Osteogenesis/drug effects , Osteogenesis/physiology , Ostreidae/metabolism , Porosity/drug effects , Tissue Engineering/methods , Tissue Scaffolds/chemistryABSTRACT
Porous silicon is of current interest for cardiac tissue engineering applications. While porous silicon is considered to be a biocompatible material, it is important to assess whether post-etching surface treatments can further improve biocompatibility and perhaps modify cellular behavior in desirable ways. In this work, porous silicon was formed by electrochemically etching with hydrofluoric acid, and was then treated with oxygen plasma or supercritical carbon dioxide (scCO2). These processes yielded porous silicon with a thickness of around 4 µm. The different post-etch treatments gave surfaces that differed greatly in hydrophilicity: oxygen plasma-treated porous silicon had a highly hydrophilic surface, while scCO2 gave a more hydrophobic surface. The viabilities of H9c2 cardiomyocytes grown on etched surfaces with and without these two post-etch treatments was examined; viability was found to be highest on porous silicon treated with scCO2. Most significantly, the expression of some key genes in the angiogenesis pathway was strongly elevated in cells grown on the scCO2-treated porous silicon, compared to cells grown on the untreated or plasma-treated porous silicon. In addition, the expression of several apoptosis genes were suppressed, relative to the untreated or plasma-treated surfaces.
Subject(s)
Biocompatible Materials/chemistry , Carbon Dioxide/chemistry , Myocytes, Cardiac , Silicon/chemistry , Bioengineering , Cell Survival , Porosity , Spectrum Analysis , Surface PropertiesABSTRACT
This work reports on a modularized electrochemical method for the determination of the hormones cortisol, progesterone, testosterone and 17ß-estradiol in urine. These hormones were employed as templates when generating molecular imprints from aniline and metanilic acid by electropolymerization on the surface of screen-printed electrodes. The electrically conductive imprint was characterized by SEM, AFM and cyclic voltammetry. A four-channel system was then established to enable simultaneous determination of the hormones by cyclic voltammetry. The detection limits for cortisol, progesterone, testosterone and 17ß-estradiol are as low as 2, 2.5, 10 and 9 ag·mL-1 (for S/N = 3). Graphical abstract A four-channel system was established to enable simultaneous determination of 4 steroid hormones by cyclic voltammetry and by using moleculalry imprinted polymers.
Subject(s)
Electrochemical Techniques/methods , Estradiol/urine , Hydrocortisone/urine , Polymers/chemistry , Progesterone/urine , Testosterone/urine , Aniline Compounds/chemistry , Electrochemical Techniques/instrumentation , Electrodes , Equipment Design , Humans , Limit of Detection , Molecular Imprinting , Polymerization , Polymers/chemical synthesis , Sulfanilic Acids/chemistryABSTRACT
In human adrenarche during childhood, the secretion of dehydroepiandrosterone (DHEA) from the adrenal gland increases due to its increased synthesis and/or decreased metabolism. DHEA is synthesized by 17α-hydroxylase/17,20-lyase, and is metabolized by 3ß-hydroxysteroid dehydrogenase type 2 (3ßHSD2). In this study, the inhibition of purified human 3ßHSD2 by the adrenal steroids, androstenedione, cortisone, and cortisol, was investigated and related to changes in secondary enzyme structure. Solubilized, purified 3ßHSD2 was inhibited competitively by androstenedione with high affinity, by cortisone at lower affinity, and by cortisol only at very high, nonphysiologic levels. When purified 3ßHSD2 was bound to lipid vesicles, the competitive Ki values for androstenedione and cortisone were slightly decreased, and the Ki value of cortisol was decreased 2.5-fold, although still at a nonphysiologic level. The circular dichroism spectrum that measured 3ßHSD2 secondary structure was significantly altered by the binding of cortisol, but not by androstenedione and cortisone. Our import studies show that 3ßHSD2 binds in the intermitochondrial space as a membrane-associated protein. Androstenedione inhibits purified 3ßHSD2 at physiologic levels, but similar actions for cortisol and cortisone are not supported. In summary, our results have clarified the mechanisms for limiting the metabolism of DHEA during human adrenarche.
Subject(s)
17-Hydroxysteroid Dehydrogenases/antagonists & inhibitors , Adrenarche/drug effects , Adrenarche/physiology , Androstenedione/pharmacology , Enzyme Inhibitors/pharmacology , Feedback, Physiological/drug effects , 17-Hydroxysteroid Dehydrogenases/chemistry , 17-Hydroxysteroid Dehydrogenases/metabolism , Adrenal Glands/drug effects , Adrenal Glands/metabolism , Adrenarche/metabolism , Androstenedione/metabolism , Cell Line , Cortisone/metabolism , Cortisone/pharmacology , Enzyme Inhibitors/metabolism , Humans , Hydrocortisone/metabolism , Hydrocortisone/pharmacology , Liposomes/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Protein Conformation , Protein Transport/drug effects , SolubilityABSTRACT
Incorporation of superparamagnetic nanoparticles into molecularly imprinted polymers (MIPs) is useful for both bioseparations and for concentration and sensing of biomedically relevant target molecules in physiological fluids, through the application of a magnetic field. In this study, we combined the separation and concentration of a target (phenylalanine) in urine, using magnetic molecularly imprinted polymeric composite nanoparticles, with optical sensing, to improve assay sensitivity. This target is important as a catecholamine precursor, and as an important amino acid constituent of proteins. Poly(ethylene-co-vinyl alcohol)s were imprinted with target molecules, and showed a high imprinting effectiveness (target binding compared with binding to non-imprinted polymer particles.) Fluorescence spectrophotometry was used to measure binding of the target, and also binding of possible interfering compounds. These measurements suggest that functional groups on phenylalanine dominate the selectivity of the synthesized MIPs. Finally, the composite nanoparticles were used to separate and sense the target molecule in urine by Raman scattering microscopy.
Subject(s)
Biosensing Techniques/instrumentation , Magnetite Nanoparticles/chemistry , Phenylalanine/urine , Polyvinyls/chemistry , Catecholamines/chemistry , Humans , Nanocomposites/chemistry , Particle Size , Polymers/chemistry , Spectrometry, FluorescenceABSTRACT
Microcontact imprinting of cells often involves the deposition of a polymer solution onto a monolayer cell stamp, followed by solvent evaporation. Thus, the concentration of the polymer may play an important role in the final morphology and efficacy of the imprinted film. In this work, various concentrations of poly(ethylene-co-vinyl alcohol) (EVAL) were dissolved in dimethyl sulfoxide (DMSO) for the microcontact imprinting of algae on an electrode. Scanning electron microscopy and fluorescence spectrometry were used to characterize the surface morphology and recognition capacity of algae to the algae-imprinted cavities. The readsorption of algae onto algae-imprinted EVAL thin films was quantified to obtain the EVAL concentration that maximized algal binding. Finally, the power and current density of an algal biofuel cell with the algae-imprinted EVAL-coated electrode were measured and found to be approximately double those of such a cell with a Pt/indium tin oxide (ITO)/poly(ethylene terephthalate) (PET) electrode.
Subject(s)
Biofuels , Chlamydomonas reinhardtii/chemistry , Dimethyl Sulfoxide/chemistry , Polyvinyls/chemistry , Chlamydomonas reinhardtii/ultrastructureABSTRACT
An MXene is a novel two-dimensional transition metal carbide or nitride, with a typical formula of Mn+1XnTx (M = transition metals, X = carbon or nitrogen, and T = functional groups). MXenes have found wide application in biomedicine and biosensing, owing to their high biocompatibility, abundant reactive surface groups, good conductivity, and photothermal properties. Applications include photo- and electrochemical sensors, energy storage, and electronics. This review will highlight recent applications of MXene and MXene-derived materials in drug delivery, tissue engineering, antimicrobial activity, and biosensors (optical and electrochemical). We further elaborate on recent developments in utilizing MXenes for photothermal cancer therapy, and we explore multimodal treatments, including the integration of chemotherapeutic agents or magnetic nanoparticles for enhanced therapeutic efficacy. The high surface area and reactivity of MXenes provide an interface to respond to the changes in the environment, allowing MXene-based drug carriers to respond to changes in pH, reactive oxygen species (ROS), and electrical signals for controlled release applications. Furthermore, the conductivity of MXene enables it to provide electrical stimulation for cultured cells and endows it with photocatalytic capabilities that can be used in antibiotic applications. Wearable and in situ sensors incorporating MXenes are also included. Major challenges and future development directions of MXenes in biomedical applications are also discussed. The remarkable properties of MXenes will undoubtedly lead to their increasing use in the applications discussed here, as well as many others.
Subject(s)
Anti-Bacterial Agents , Carbon , Nitrites , Transition Elements , Combined Modality Therapy , Drug CarriersABSTRACT
Programmed death ligand 1 (PD-L1) has been shown to suppress the anti-tumor immune response of some lung cancer patients, and thus PD-L1 expression may be a valuable predictor of the efficacy of anti-PD-1/PD-L1 monoclonal therapy in such patients. In this work, a sandwich approach to fluorescence resonance energy transfer (FRET) was used with green-emitting Yb3+/Ho3+-doped upconversion nanoparticles (UCNPs) and a rhodamine-conjugated conductive polymer as donor and acceptor, respectively. Yb3+/Ho3+-doped UCNPs were synthesized and then coated with poly(ethylene-co-vinyl alcohol), pEVAL, imprinted with PD-L1 peptide. Epitope-imprinted composite nanoparticles were characterized by dynamic light scattering, superconducting quantum interference magnetometry, and atomic force microscopy. Poly(triphenylamine rhodamine-3-acetic acid-co-3,4-ethoxylenedioxythiophene)s copolymers (p(TPAR-co-EDOT)) were imprinted with various epitopes of PD-L1 by in situ electrochemical polymerization. The epitope-imprinted polymer-coated electrodes were then characterized by scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. Finally, the sandwich sensing of various PD-L1 concentrations with peptide-imprinted p(TPAR-co-EDOT)-coated substrate and UCNP-containing magnetic peptide-imprinted pEVAL nanoparticles by FRET was conducted to measure the concentration of PD-L1 in A549 lung cancer cell lysate.
Subject(s)
Biosensing Techniques , Lung Neoplasms , Nanoparticles , Humans , Fluorescence Resonance Energy Transfer , Polymers/chemistry , B7-H1 Antigen , Nanoparticles/chemistry , Peptides , Rhodamines , EpitopesABSTRACT
The inner mitochondrial membrane protein 3ß-hydroxysteroid dehydrogenase 2 (3ßHSD2) synthesizes progesterone and androstenedione through its dehydrogenase and isomerase activities. This bifunctionality requires 3ßHSD2 to undergo a conformational change. Given its proximity to the proton pump, we hypothesized that pH influences 3ßHSD2 conformation and thus activity. Circular dichroism (CD) showed that between pH 7.4 and 4.5, 3ßHSD2 retained its primarily α-helical character with a decrease in α-helical content at lower pH values, whereas the ß-sheet content remained unchanged throughout. Titrating the pH back to 7.4 restored the original conformation within 25 min. Metabolic conversion assays indicated peak 3ßHSD2 activity at pH 4.5 with ~2-fold more progesterone synthesized at pH 4.5 than at pH 3.5 and 7.4. Increasing the 3ßHSD2 concentration from 1 to 40 µg resulted in a 7-fold increase in progesterone at pH 4.5, but no change at pH 7.4. Incubation with guanidinum hydrochloride (GdmHCl) showed a three-step cooperative unfolding of 3ßHSD2 from pH 7.4 to 4.5, possibly due to the native state unfolding to the intermediate ion core state. With further decreases in pH, increasing concentrations of GdmHCl led to rapid two-step unfolding that may represent complete loss of structure. Between pH 4 and 5, the two intermediate states appeared stable. Stopped-flow kinetics showed slower unfolding at around pH 4, where the protein is in a pseudostable state. Based on our data, we conclude that at pH 4-5, 3ßHSD2 takes on a molten globule conformation that promotes the dual functionality of the enzyme.
Subject(s)
Mitochondria/enzymology , Mitochondrial Membranes/enzymology , Progesterone Reductase/chemistry , Progesterone Reductase/metabolism , Animals , Cell Line , Humans , Hydrogen-Ion Concentration , Kinetics , Mice , Mitochondria/chemistry , Mitochondria/genetics , Mitochondrial Membranes/chemistry , Progesterone Reductase/genetics , Protein Conformation , Protein FoldingABSTRACT
Lipid-coated microbubbles and emulsions are of interest as possible ultrasound-mediated drug delivery vehicles and for their interesting behaviors and fundamental properties. We and others have noted that bubbles coated with the long chain saturated phospholipid distearoylphosphatidylcholine (DSPC) rapidly shrink to a quasistable size when repeatedly insonated with short ultrasound pulses; such stability may adversely affect the bubble's subsequent ability to deliver its pharmacological cargo. Bubbles coated with the unsaturated lipid dioleoylphosphatidylcholine (DOPC) did not show stability but did undergo an abrupt change from rapid initial shrinkage to a slow persistent shrinkage, leading ultimately to dissolution or dispersion. As DOPC and DSPC differ not only in chain saturation but also phase behavior, we performed additional studies using dimyristoyl PC (DMPC) as a coat lipid and controlled the solution temperature to study bubble behavior on exposure to repeated ultrasound pulses for the same coat, in both fluid and gel phases. We find, first, that essentially all bubbles show an initially rapid shrinkage, in which gas loss exceeds the limit imposed by gas diffusion into the surrounding medium; this rapid shrinkage may be evidence of nanoscopic bubble fragmentation. Second, upon reaching a fraction of their initial size, bubbles begin a slower shrinkage with a shrinkage rate that depends on the resting phase state of the coat lipid: fluid DMPC monolayers give a more rapid shrinkage than gel phase. DOPC-coated bubbles showed no temperature-dependent responses in the same temperature range. The results are especially interesting in that bubble compression during the pulse is likely to adiabatically heat the bubble and fluidize the coat, regardless of its initial phase state; thus, some structural feature of the resting coat, such as defect lines in the gel phase, may be important in the subsequent response to the ~3 µs ultrasound pulse.
Subject(s)
Dimyristoylphosphatidylcholine/chemistry , Temperature , Ultrasonics , Fluorocarbons/chemistry , Phase Transition , PressureABSTRACT
Insulin may help to control blood glucose levels in diabetes; however, the long-term release of insulin is important for therapy. In this work, four guide RNAs (gRNA) for factors that promote specification and maturation of insulin-producing cells were synthesized: pancreatic and duodenal homeobox 1 (PDX1), protoendocrine factor (neurogenin 3, NGN3), NK6 homeobox 1 (NKX6.1), and musculoaponeurotic fibrosarcoma oncogene family A (MAFA). These gRNAs were used to form ribonucleoproteins (RNPs) with tracRNA and dCas9-VPR, and were then immobilized on magnetic peptide-imprinted chitosan nanoparticles, which enhanced transfection. The production and release of insulin from transfected cells were then measured using ELISA and staining with anti-insulin antibodies. The expression of the genes was evaluated using qRT-PCR; this was also used to investigate the cascade of additional transcriptional regulators. The magnitude and duration of insulin production were evaluated for single and repeated transfections (using different transfection schedules) to identify the most promising protocol.
Subject(s)
Insulin-Secreting Cells , Transcription Factors , Transcription Factors/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Insulin-Secreting Cells/metabolism , Insulin/metabolismABSTRACT
A CRISPRa transcription activation system was used to upregulate insulin expression in HEK293T cells. To increase the delivery of the targeted CRISPR/dCas9a, magnetic chitosan nanoparticles, imprinted with a peptide from the Cas9 protein, were developed, characterized, and then bound to dCas9a that was complexed with a guide RNA (gRNA). The adsorption of dCas9 proteins conjugated with activators (SunTag, VPR, and p300) to the nanoparticles was monitored using both ELISA kits and Cas9 staining. Finally, the nanoparticles were used to deliver dCas9a that was complexed with a synthetic gRNA into HEK293T cells to activate their insulin gene expression. Delivery and gene expression were examined using quantitative real-time polymerase chain reaction (qRT-PCR) and staining of insulin. Finally, the long-term release of insulin and the cellular pathway related to stimulation by glucose were also investigated.
ABSTRACT
In the adrenals, testes, and ovaries, 3ß-hydroxysteroid dehydrogenase type 2 (3ßHSD2) catalyzes the conversion of pregnenolone to progesterone and dehydroepiandrostenedione to androstenedione. Alterations in this pathway can have deleterious effects, including sexual development impairment, spontaneous abortion, and breast cancer. 3ßHSD2, synthesized in the cytosol, is imported into the inner mitochondrial membrane (IMM) by translocases. Steroidogenesis requires that 3ßHSD2 acts as both a dehydrogenase and isomerase. To achieve this dual functionality, 3ßHSD2 must undergo a conformational change; however, what triggers that change remains unknown. We propose that 3ßHSD2 associates with IMM or outer mitochondrial membrane translocases facing the intermembrane space (IMS) and that this interaction promotes the conformational change needed for full activity. Fractionation assays demonstrate that 3ßHSD2 associated with the IMM but did not integrate into the membrane. Through mass spectrometry and Western blotting of mitochondrial complexes and density gradient ultracentrifugation, we show that that 3ßHSD2 formed a transient association with the translocases Tim50 and Tom22 and with Tim23. This association occurred primarily through the interaction of Tim50 with the N terminus of 3ßHSD2 and contributed to enzymatic activity. Tim50 knockdown inhibited catalysis of dehydroepiandrostenedione to androstenedione and pregnenolone to progesterone. Although Tim50 knockdown decreased 3ßHSD2 expression, restoration of expression via proteasome and protease inhibition did not rescue activity. In addition, protein fingerprinting and CD spectroscopy reveal the flexibility of 3ßHSD2, a necessary characteristic for forming multiple associations. In summary, Tim50 regulates 3ßHSD2 expression and activity, representing a new role for translocases in steroidogenesis.
Subject(s)
3-Hydroxysteroid Dehydrogenases/biosynthesis , Adrenal Glands/metabolism , Androstenedione/biosynthesis , Dehydroepiandrosterone/metabolism , Gene Expression Regulation, Enzymologic/physiology , Gonads/metabolism , Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , 3-Hydroxysteroid Dehydrogenases/genetics , Androstenedione/genetics , Animals , Cell Line , Dehydroepiandrosterone/genetics , Female , Gene Knockdown Techniques , Humans , Male , Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/biosynthesis , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/genetics , Protein Structure, Tertiary , SwineABSTRACT
The impact of compartmental expression of steroidogenic enzymes and of changes in flux through delta5 and delta4 metabolism on sex steroid synthesis was investigated by rebuilding pathways using recombinant enzyme expression by infection of insect cells with recombinant baculovirus constructs. Human cytochromes 17alpha-hydroxylase/17,20-lyase (P450c17) and aromatase (P450arom), always coexpressed with their redox partner NADPH-P450 oxidoreductase (CPR) and 3beta-hydroxysteroid dehydrogenase/delta5-4 isomerase (3betaHSD; types 1 or 2), were compartmentally expressed in different cell populations or coexpressed together with pregnenolone (100 nM) as substrate. Estrone was compared among cell compartments expressing different enzyme combinations or in cells coexpressing all enzymes (experiment 1). Additionally, P450c17, 3betaHSD, and CPR were all coexpressed, and androstenedione was measured in cells with different 3betaHSD expression levels or activity using an inhibitor, trilostane (experiment 2). Steroids were measured by immunoassay and mass spectrometry. In experiment 1, partitioning of P450c17, P450arom, and 3betaHSD markedly decreased estrone synthesis in comparison to cells coexpressing enzymes in different combinations. However, partitioning P450arom with 3betaHSD from P450c17 in different cell populations resulted in more estrone than either of the other two-cell compartment models. In experiment 2 (cells coexpressing P450c17, 3betaHSD, and CPR), androstenedione secretion was (paradoxically) higher at lower levels of 3betaHSD, and partial inhibition of 3betaHSD by trilostane also increased androstenedione when 3betaHSD expression was high. We conclude 1) that tissue or cell-specific, partitioned expression of sex steroid synthesizing enzymes limits rather than maximizes estrogen synthesis and 2) that limiting metabolism by 3betaHSD can paradoxically promote androgen synthesis when 3betaHSD expression is high by promoting delta5-steroid flux.
Subject(s)
Androgens/biosynthesis , Cytochrome P-450 Enzyme System/metabolism , Estrogens/biosynthesis , Gene Expression Regulation, Enzymologic/physiology , 17-alpha-Hydroxyprogesterone/metabolism , Animals , Cell Line , Cytochrome P-450 Enzyme System/genetics , Female , Granulosa Cells/metabolism , Humans , Insecta , Protein Isoforms , Recombinant Proteins , Substrate SpecificityABSTRACT
Fc epsilonRI on mast cells form a synapse when presented with mobile, bilayer-incorporated Ag. In this study, we show that receptor reorganization within the contacting mast cell membrane is markedly different upon binding of mobile and immobilized ligands. Rat basophilic leukemia mast cells primed with fluorescent anti-DNP IgE were engaged by surfaces presenting either bilayer-incorporated, monovalent DNP-lipid (mobile ligand), or chemically cross-linked, multivalent DNP (immobilized ligand). Total internal reflection fluorescence imaging and electron microscopy methods were used to visualize receptor reorganization at the contact site. The spatial relationships of Fc epsilonRI to other cellular components at the synapse, such as actin, cholesterol, and linker for activation of T cells, were also analyzed. Stimulation of mast cells with immobilized polyvalent ligand resulted in typical levels of degranulation. Remarkably, degranulation also followed interaction of mast cells, with bilayers presenting mobile, monovalent ligand. Receptors engaged with mobile ligand coalesce into large, cholesterol-rich clusters that occupy the central portion of the contacting membrane. These data indicate that Fc epsilonRI cross-linking is not an obligatory step in triggering mast cell signaling and suggest that dense populations of mobile receptors are capable of initiating low-level degranulation upon ligand recognition.
Subject(s)
Cell Communication/immunology , Cell Membrane/immunology , Cell Membrane/metabolism , Immunological Synapses/metabolism , Mast Cells/immunology , Receptors, IgE/metabolism , Actins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line, Tumor , Cell Membrane/ultrastructure , Cholesterol/metabolism , Cross-Linking Reagents/metabolism , Immunoglobulin E/metabolism , Immunological Synapses/ultrastructure , Ligands , Lipid Bilayers/chemistry , Lipid Bilayers/immunology , Lipid Bilayers/metabolism , Mast Cells/metabolism , Mast Cells/ultrastructure , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Protein Binding/immunology , Rats , Receptors, IgE/chemistry , Receptors, IgE/ultrastructure , Surface PropertiesABSTRACT
Fractures of the talar neck comprise almost 50% of fractures of the talus and may result in significant long-term morbidity. It is of paramount importance to ensure anatomic reduction of the fracture not only for fracture healing but also for minimizing future posttraumatic arthritic sequelae. In addition to conventional radiographs and computed tomography scans, the Canale view has proven to be beneficial, especially when evaluating for varus displacement. This study investigated whether the original method of performing the Canale view could be modified for improved evaluation for varus displacement. Simulated talar neck fractures were created in 6 cadaveric specimens. These were placed into varying amounts of varus displacement; the Canale view was performed with progressive degrees of eversion, from 0° to 25°, resulting in 108 total views. Blinded evaluation was performed, and a ranking system was used to determine the most beneficial degree(s) of eversion for evaluating varus malalignment. Multiple statistical analyses were performed. A significant difference was seen between the high and low range of values of eversion. A significantly lower ranking was achieved with 10° of eversion. As opposed to a single view taken at 15° of eversion, a range of angles may be most beneficial in evaluating varus displacement in talar neck fractures.
Subject(s)
Fractures, Bone/diagnostic imaging , Patient Positioning/methods , Talus/diagnostic imaging , Aged , Aged, 80 and over , Cadaver , Female , Humans , Male , Middle Aged , Talus/injuries , Tomography, X-Ray ComputedABSTRACT
The level of C-reactive protein (CRP) in serum is frequently used to evaluate risk of coronary heart disease, and its concentration is related to cardiovascular disease, fibrosis and inflammation, cancer, and viral infections. In this work, three novel peptides, never previously used as imprinted templates, were selected, synthesized, and employed for epitope imprinting. Various imprinting concentrations of the template and various ratios of aniline (AN) to m-aminobenzenesulfonic acid (MSAN) were used in electropolymerization to form molecularly imprinted polymers (MIPs). The imprinting template and functional monomer concentrations were optimized to maximize the electrochemical response to target peptides. The surface morphologies of peptide- and non-imprinting poly(AN-co-MSAN) were observed using a scanning electron microscope (SEM) and an atomic force microscope (AFM). Moreover, the effect of doping of MIPs with a very small percentage of an MXene (e.g. Ti2C at 0.1 wt% in the preparation solution) on the electrochemical response was also studied. Ti2C doping dramatically increased sensing range from 0.1 to 100 fg/mL to 10000 fg/mL, and electrochemical responses were amplified by a factor of approximately 1.3 within the sensing range. Finally, commercially available serum was diluted and then measured using the MXene-doped PIP-coated electrodes to estimate the accuracy compared with ELISA results.