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Journal of Dental Research Jul 2019The temporomandibular joint (TMJ) disc nutrient environment profoundly affects cell energy metabolism, proliferation, and biosynthesis. Due to technical challenges of in...
The temporomandibular joint (TMJ) disc nutrient environment profoundly affects cell energy metabolism, proliferation, and biosynthesis. Due to technical challenges of in vivo measurements, the human TMJ disc extracellular nutrient environment under load, which depends on metabolic rates, solute diffusion, and disc morphometry, remains unknown. Therefore, the study objective was to predict the TMJ disc nutrient environment under loading conditions using combined experimental and computational modeling approaches. Specifically, glucose consumption and lactate production rates of porcine TMJ discs were measured under varying tissue culture conditions ( = 40 discs), and mechanical strain-dependent glucose and lactate diffusivities were measured using a custom diffusion chamber ( = 6 discs). TMJ anatomy and loading area were obtained from magnetic resonance imaging of healthy human volunteers ( = 11, male, 30 ± 9 y). Using experimentally determined nutrient metabolic rates, solute diffusivities, TMJ anatomy, and loading areas, subject-specific finite element (FE) models were developed to predict the 3-dimensional nutrient profiles in unloaded and loaded TMJ discs (unloaded, 0% strain, 20% strain). From the FE models, glucose, lactate, and oxygen concentration ranges for unloaded healthy human TMJ discs were 0.6 to 4.0 mM, 0.9 to 5.0 mM, and 0% to 6%, respectively, with steep gradients in the anterior and posterior bands. Sustained mechanical loading significantly reduced nutrient levels ( < 0.001), with a critical zone in which cells may die representing approximately 13.5% of the total disc volume. In conclusion, this study experimentally determined TMJ disc metabolic rates, solute diffusivities, and disc morphometry, and through subject-specific FE modeling, revealed critical interactions between mechanical loading and nutrient supply and metabolism for the in vivo human TMJ disc. The results suggest that TMJ disc homeostasis may be vulnerable to pathological loading (e.g., clenching, bruxism), which impedes nutrient supply. Given difficulties associated with direct in vivo measurements, this study provides a new approach to systematically investigate homeostatic and degenerative mechanisms associated with the TMJ disc.
Topics: Adult; Animals; Biomechanical Phenomena; Diffusion; Energy Metabolism; Glucose; Humans; Lactic Acid; Male; Nutrients; Oxygen; Stress, Mechanical; Swine; Temporomandibular Joint Disc; Young Adult
PubMed: 31126205
DOI: 10.1177/0022034519851044 -
The Journal of Chemical Physics Aug 2023Most biological processes in living cells rely on interactions between proteins. Live-cell compatible approaches that can quantify to what extent a given protein... (Review)
Review
Most biological processes in living cells rely on interactions between proteins. Live-cell compatible approaches that can quantify to what extent a given protein participates in homo- and hetero-oligomeric complexes of different size and subunit composition are therefore critical to advance our understanding of how cellular physiology is governed by these molecular interactions. Biomolecular complex formation changes the diffusion coefficient of constituent proteins, and these changes can be measured using fluorescence microscopy-based approaches, such as single-molecule tracking, fluorescence correlation spectroscopy, and fluorescence recovery after photobleaching. In this review, we focus on the use of single-molecule tracking to identify, resolve, and quantify the presence of freely-diffusing proteins and protein complexes in living cells. We compare and contrast different data analysis methods that are currently employed in the field and discuss experimental designs that can aid the interpretation of the obtained results. Comparisons of diffusion rates for different proteins and protein complexes in intracellular aqueous environments reported in the recent literature reveal a clear and systematic deviation from the Stokes-Einstein diffusion theory. While a complete and quantitative theoretical explanation of why such deviations manifest is missing, the available data suggest the possibility of weighing freely-diffusing proteins and protein complexes in living cells by measuring their diffusion coefficients. Mapping individual diffusive states to protein complexes of defined molecular weight, subunit stoichiometry, and structure promises to provide key new insights into how protein-protein interactions regulate protein conformational, translational, and rotational dynamics, and ultimately protein function.
Topics: Single Molecule Imaging; Diffusion; Microscopy, Fluorescence; Photobleaching; Protein Conformation
PubMed: 37589409
DOI: 10.1063/5.0155638 -
Respiratory Physiology & Neurobiology Jul 2017Roughton and Forster (RF) proposed to split the lung diffusing capacity into two contributions describing first, diffusion to red blood cells (RBC), and second, capture...
Roughton and Forster (RF) proposed to split the lung diffusing capacity into two contributions describing first, diffusion to red blood cells (RBC), and second, capture by diffusion from the RBC surface and reaction with haemoglobin. Solving the diffusion-reaction equations for simplified capillary-RBC structures, we investigate the RF interpretation. This reveals first that the conventional extrapolation to zero pressure of 1/DLCO on PO is not a correct measure of the diffusive component. Consequently the capillary volumes deduced from this extrapolation are erroneous. Secondly, capture mechanisms are different for CO and NO: while DLCO characterizes "volume absorption" in the RBC and is correlated with hematocrit, DLNO quantifies "surface absorption" and provide information about the morphology of the space between the alveolar surface and the RBC surfaces. In conclusion, the RF approach may lead to erroneous physiological interpretations of DLCO; nevertheless, the measurement of DLCO and DLNO bring different types of information that give the potential for a better understanding of respiratory diseases.
Topics: Capillaries; Carbon Monoxide; Diffusion; Erythrocytes; Humans; Lung; Models, Cardiovascular; Nitric Oxide; Pulmonary Diffusing Capacity
PubMed: 28049017
DOI: 10.1016/j.resp.2016.12.014 -
Redox Biology Apr 2022Hydrogen peroxide is a major redox signaling molecule underlying a novel paradigm of cell function and communication. A role for HO as an intercellular signaling...
Hydrogen peroxide is a major redox signaling molecule underlying a novel paradigm of cell function and communication. A role for HO as an intercellular signaling molecule and neuromodulator in the brain has become increasingly apparent, with evidence showing this biological oxidant to regulate neuronal polarity, connectivity, synaptic transmission and tuning of neuronal networks. This notion is supported by its ability to diffuse in the extracellular space, from source of production to target. It is, thus, crucial to understand extracellular HO concentration dynamics in the living brain and the factors which shape its diffusion pattern and half-life. To address this issue, we have used a novel microsensor to measure HO concentration dynamics in the brain extracellular matrix both in an ex vivo model using rodent brain slices and in vivo. We found that exogenously applied HO is removed from the extracellular space with an average half-life of t = 2.2 s in vivo. We determined the in vivo effective diffusion coefficient of HO to be D* = 2.5 × 10 cm s. This allows it to diffuse over 100 μm in the extracellular space within its half-life. Considering this, we can tentatively place HO within the class of volume neurotransmitters, connecting all cell types within the complex network of brain tissue, regardless of whether they are physically connected. These quantitative details of HO diffusion and half-life in the brain allow us to interpret the physiology of the redox signal and lay the pavement to then address dysregulation in redox homeostasis associated with disease processes.
Topics: Brain; Diffusion; Hydrogen Peroxide; Oxidation-Reduction; Signal Transduction
PubMed: 35101799
DOI: 10.1016/j.redox.2022.102250 -
Nature Communications Nov 2022Control over vesicle size during nanoscale liposome synthesis is critical for defining the pharmaceutical properties of liposomal nanomedicines. Microfluidic...
Control over vesicle size during nanoscale liposome synthesis is critical for defining the pharmaceutical properties of liposomal nanomedicines. Microfluidic technologies capable of size-tunable liposome generation have been widely explored, but scaling these microfluidic platforms for high production throughput without sacrificing size control has proven challenging. Here we describe a microfluidic-enabled process in which highly vortical flow is established around an axisymmetric stream of solvated lipids, simultaneously focusing the lipids while inducing rapid convective and diffusive mixing through application of the vortical flow field. By adjusting the individual buffer and lipid flow rates within the system, the microfluidic vortex focusing technique is capable of generating liposomes with precisely controlled size and low size variance, and may be operated up to the laminar flow limit for high throughput vesicle production. The reliable formation of liposomes as small as 27 nm and mass production rates over 20 g/h is demonstrated, offering a path toward production-scale liposome synthesis using a single continuous-flow vortex focusing device.
Topics: Liposomes; Microfluidics; Diffusion; Lipids
PubMed: 36384946
DOI: 10.1038/s41467-022-34750-3 -
Cell Reports Apr 2017Previous autonomous pattern-formation models often assumed complex molecular and cellular networks. This theoretical study, however, shows that a system composed of one...
Previous autonomous pattern-formation models often assumed complex molecular and cellular networks. This theoretical study, however, shows that a system composed of one substrate with multisite phosphorylation and a pair of kinase and phosphatase can generate autonomous spatial information, including complex stripe patterns. All (de-)phosphorylation reactions are described with a generic Michaelis-Menten scheme, and all species freely diffuse without pre-existing gradients. Computational simulation upon >23,000,000 randomly generated parameter sets revealed the design motifs of cyclic reaction and enzyme sequestration by slow-diffusing substrates. These motifs constitute short-range positive and long-range negative feedback loops to induce Turing instability. The width and height of spatial patterns can be controlled independently by distinct reaction-diffusion processes. Therefore, multisite reversible post-translational modification can be a ubiquitous source for various patterns without requiring other complex regulations such as autocatalytic regulation of enzymes and is applicable to molecular mechanisms for inducing subcellular localization of proteins driven by post-translational modifications.
Topics: Diffusion; Enzymes; Kinesics; Models, Biological; Protein Processing, Post-Translational
PubMed: 28445735
DOI: 10.1016/j.celrep.2017.03.081 -
Magnetic Resonance in Medicine Sep 2020To investigate diffusion-time dependency of diffusional kurtosis in the mouse brain using pulsed-gradient spin-echo (PGSE) and oscillating-gradient spin-echo (OGSE)...
PURPOSE
To investigate diffusion-time dependency of diffusional kurtosis in the mouse brain using pulsed-gradient spin-echo (PGSE) and oscillating-gradient spin-echo (OGSE) sequences.
METHODS
3D PGSE and OGSE kurtosis tensor data were acquired from ex vivo brains of adult, cuprizone-treated, and age-matched control mice with diffusion-time (t ) ~ 20 ms and frequency (f) = 70 Hz, respectively. Further, 2D acquisitions were performed at multiple times/frequencies ranging from f = 140 Hz to t = 30 ms with b-values up to 4000 s/mm . Monte Carlo simulations were used to investigate the coupled effects of varying restriction size and permeability on time/frequency-dependence of kurtosis with both diffusion-encoding schemes. Simulations and experiments were further performed to investigate the effect of varying number of cycles in OGSE waveforms.
RESULTS
Kurtosis and diffusivity maps exhibited significant region-specific changes with diffusion time/frequency across both gray and white matter areas. PGSE- and OGSE-based kurtosis maps showed reversed contrast between gray matter regions in the cerebellar and cerebral cortex. Localized time/frequency-dependent changes in kurtosis tensor metrics were found in the splenium of the corpus callosum in cuprizone-treated mouse brains, corresponding to regional demyelination seen with histological assessment. Monte Carlo simulations showed that kurtosis estimates with pulsed- and oscillating-gradient waveforms differ in their sensitivity to exchange. Both simulations and experiments showed dependence of kurtosis on number of cycles in OGSE waveforms for non-zero permeability.
CONCLUSION
The results show significant time/frequency-dependency of diffusional kurtosis in the mouse brain, which can provide sensitivity to probe intrinsic cellular heterogeneity and pathological alterations in gray and white matter.
Topics: Animals; Brain; Corpus Callosum; Diffusion; Diffusion Magnetic Resonance Imaging; Mice; White Matter
PubMed: 32022313
DOI: 10.1002/mrm.28189 -
Lab on a Chip Sep 2019Hydrogels allow for controlling the diffusion rate and amount of solute according to the hydrogel network and thus have found many applications in drug delivery,...
Hydrogels allow for controlling the diffusion rate and amount of solute according to the hydrogel network and thus have found many applications in drug delivery, biomaterials, toxicology, and tissue engineering. This paper describes a 3D-printed microfluidic chip for the straightforward partitioning of hydrogel barriers between microchannels. We use a previously-reported 3-channel architecture whereby the middle channel is filled with a hydrogel - acting like a porous barrier for diffusive transport - and the two side channels act as sink and source; the middle channel communicates with the side channels via orthogonal, small capillary channels that are also responsible for partitioning the hydrogel during filling. Our 3D-printed microfluidic chip is simple to fabricate by stereolithography (SL), inexpensive, reproducible, and convenient, so it is more adequate for transport studies than a microchip fabricated by photolithographic procedures. The chip was fabricated in a resin made of poly(ethylene glycol) diacrylate (PEG-DA) (MW = 258) (PEG-DA-258). The SL process allowed us to print high aspect ratio (37 : 1) capillary channels (27 μm-width and 1 mm-height) and enable the trapping of liquid-phase hydrogels in the hydrogel barrier middle channel. We studied the permeability of hydrogel barriers made of PEG-DA (MW = 700) (PEG-DA-700, 10% polymer content by wt. in water) - as a model of photopolymerizable barriers - and agarose (MW = 120 000, 2% polymer content by wt. in water) - as a model of thermally-gelled barriers. We measured the diffusion of fluorescein, 10k-dextran-Alexa 680 and BSA-Texas Red through these barriers. Fluorescein diffusion was observed through both 10% PEG-DA-700 and 2% agarose barriers while 10k-dextran-Alexa 680 and BSA-Texas Red diffused appreciably only through the 2% agarose hydrogel barrier. Our microfluidic chip facilitates the tuning of such barriers simply by altering the hydrogel materials. The straightforward trapping of selective barriers in 3D-printed microchannels should find wide applicability in drug delivery, tissue engineering, cell separation, and organ-on-a-chip platforms.
Topics: Diffusion; Hydrogels; Microfluidic Analytical Techniques; Polyethylene Glycols; Printing, Three-Dimensional
PubMed: 31502633
DOI: 10.1039/c9lc00535h -
Biophysical Journal Sep 2021Antibody-based therapeutics are the fastest-growing drug class on the market, used to treat aggressive forms of cancer, chronic autoimmune conditions, and numerous other...
Antibody-based therapeutics are the fastest-growing drug class on the market, used to treat aggressive forms of cancer, chronic autoimmune conditions, and numerous other disease states. Although the specificity, affinity, and versatility of therapeutic antibodies can provide an advantage over traditional small-molecule drugs, their development and optimization can be much more challenging and time-consuming. This is, in part, because the ideal formulation buffer systems used for in vitro characterization inadequately reflect the crowded biological environments (serum, endosomal lumen, etc.) that these drugs experience once administered to a patient. Such environments can perturb the binding of antibodies to their antigens and receptors, as well as homo- and hetero-aggregation, thereby altering therapeutic effect and disposition in ways that are incompletely understood. Although excluded volume effects are classically thought to favor binding, weak interactions with co-solutes in crowded conditions can inhibit binding. The second virial coefficient (B) parameter quantifies such weak interactions and can be determined by a variety of techniques in dilute solution, but analogous methods in complex biological fluids are not well established. Here, we demonstrate that fluorescence correlation spectroscopy is able to measure diffusive B-values directly in undiluted serum. Apparent second virial coefficient (B) measurements of antibodies in serum reveal that changes in the balance between attractive and repulsive interactions can dramatically impact global nonideality. Furthermore, our findings suggest that the approach of isolating specific components and completing independent cross-term virial coefficient measurements may not be an effective approach to characterizing nonideality in serum. The approach presented here could enrich our understanding of the effects of biological environments on proteins in general and advance the development of therapeutic antibodies and other protein-based therapeutics.
Topics: Diffusion; Humans; Proteins; Solutions
PubMed: 34384764
DOI: 10.1016/j.bpj.2021.08.007 -
ACS Nano Jan 2021The permeability of hydrogels for the selective transport of molecular penetrants (drugs, toxins, reactants, .) is a central property in the design of soft functional...
The permeability of hydrogels for the selective transport of molecular penetrants (drugs, toxins, reactants, .) is a central property in the design of soft functional materials, for instance in biomedical, pharmaceutical, and nanocatalysis applications. However, the permeation of dense and hydrated polymer membranes is a complex multifaceted molecular-level phenomenon, and our understanding of the underlying physicochemical principles is still very limited. Here, we uncover the molecular principles of permeability and selectivity in hydrogel permeation. We combine the solution-diffusion model for permeability with comprehensive atomistic simulations of molecules of various shapes and polarities in a responsive hydrogel in different hydration states. We find in particular that dense collapsed states are extremely selective, owing to a delicate balance between the partitioning and diffusivity of the penetrants. These properties are sensitively tuned by the penetrant size, shape, and chemistry, leading to vast cancellation effects, which nontrivially contribute to the permeability. The gained insights enable us to formulate semiempirical rules to quantify and extrapolate the permeability categorized by classes of molecules. They can be used as approximate guiding ("rule-of-thumb") principles to optimize penetrant or membrane physicochemical properties for a desired permeability and membrane functionality.
Topics: Diffusion; Hydrogels; Permeability; Polymers
PubMed: 33382598
DOI: 10.1021/acsnano.0c06319