RAB7 deficiency impairs pulmonary artery endothelial function and promotes pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in the development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health, and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from patients with PAH. Endothelial haploinsufficiency of RAB7 caused spontaneous pulmonary hypertension (PH) in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA-Seq, and RAB7-silenced ECs showed impaired angiogenesis and expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, suggesting inhibition of autophagy at the predegradation level. Furthermore, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in rats with chronic hypoxia/SU5416. In conclusion, we demonstrate for the first time to our knowledge the fundamental impairment of EC function by loss of RAB7, causing PH, and show RAB7 activation to be a potential therapeutic strategy in a preclinical model of PH.

Hyperoxic exposure alters intracellular bioenergetics distribution in human pulmonary cells.

AIMS: Pulmonary oxygen toxicity is caused by exposure to a high fraction of inspired oxygen, which damages multiple cell types within the lung. The cellular basis for pulmonary oxygen toxicity includes mitochondrial dysfunction. The aim of this study was to identify the effects of hyperoxic exposure on mitochondrial bioenergetic and dynamic functions in pulmonary cells. MAIN METHODS: Mitochondrial respiration, inner membrane potential, dynamics (including motility), and distribution of mitochondrial bioenergetic capacity in two intracellular regions were quantified using cultured human lung microvascular endothelial cells, human pulmonary artery endothelial cells and A549 cells. Hyperoxic (95 % O2) exposures lasted 24, 48 and 72 h, durations relevant to mechanical ventilation in intensive care settings. KEY FINDINGS: Mitochondrial motility was altered following all hyperoxic exposures utilized in experiments. Inhomogeneities in inner membrane potential and respiration parameters were present in each cell type following hyperoxia. The partitioning of ATP-linked respiration was also hyperoxia-duration and cell type dependent. Hyperoxic exposure lasting 48 h or longer provoked the largest alterations in mitochondrial motility and the greatest decreases in ATP-linked respiration, with a suggestion of decreases in respiration complex protein levels. SIGNIFICANCE: Hyperoxic exposures of different durations produce intracellular inhomogeneities in mitochondrial dynamics and bioenergetics in pulmonary cells. Oxygen therapy is utilized commonly in clinical care and can induce undesirable decrements in bioenergy function needed to maintain pulmonary cell function and viability. There may be adjunctive or prophylactic measures that can be employed during hyperoxic exposures to prevent the mitochondrial dysfunction that signals the presence of oxygen toxcity.

RAB7 deficiency impairs pulmonary artery endothelial function and promotes pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from PAH patients. Endothelial haploinsufficiency of RAB7 caused spontaneous PH in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA sequencing and RAB7 silenced ECs showed impaired angiogenesis, expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, which suggests inhibition of autophagy at the pre-degradation level. Further, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in chronic hypoxia/SU5416 rats. In conclusion, we demonstrate here for the first time the fundamental impairment of EC function by loss of RAB7 that leads to PH and show RAB7 activation as a potential therapeutic strategy in a preclinical model of PH.

Mitochondrial dynamics involves molecular and mechanical events in motility, fusion and fission.

Mitochondria are cell organelles that play pivotal roles in maintaining cell survival, cellular metabolic homeostasis, and cell death. Mitochondria are highly dynamic entities which undergo fusion and fission, and have been shown to be very motile in vivo in neurons and in vitro in multiple cell lines. Fusion and fission are essential for maintaining mitochondrial homeostasis through control of morphology, content exchange, inheritance of mitochondria, maintenance of mitochondrial DNA, and removal of damaged mitochondria by autophagy. Mitochondrial motility occurs through mechanical and molecular mechanisms which translocate mitochondria to sites of high energy demand. Motility also plays an important role in intracellular signaling. Here, we review key features that mediate mitochondrial dynamics and explore methods to advance the study of mitochondrial motility as well as mitochondrial dynamics-related diseases and mitochondrial-targeted therapeutics.

Hyperbaric oxygen alters intracellular bioenergetics distribution in human dermal fibroblasts.

AIMS: Hyperbaric oxygen therapy (HBOT), used to promote wound healing, has limited efficacy in many clinical conditions. Wound healing exerts bioenergetic demands on cells that can exceed their intrinsic bioenergetic capacity to proliferate and migrate. The aim of this investigation was to quantify the effects of HBOT on mitochondrial dynamics and bioenergetics functions in cells relevant to wound healing. MAIN METHODS: High-resolution respirometry and fluorescence microscopy were used to quantify mitochondrial respiration, intermembrane potential, dynamics, including motility, and the intracellular distribution of mitochondrial bioenergetic capacity partitioned into perinuclear and cell peripheral regions in cultured human dermal fibroblasts. Cells were subjected to a range of gas mixtures and hyperbaric pressures, including conditions utilized in clinical care. KEY FINDINGS: Motility was reduced immediately following all HBOT exposures utilized in experiments. Inhomogeneities in intermembrane potential and respiration parameters were produced by different HBOT conditions. The partitioning of ATP-linked respiration was also HBOT-condition dependent. Application of HBOT at common clinical pressure and oxygen conditions resulted in the largest immediate decrement in mitochondrial motility and reductions in ATP-linked respiration in both the cell periphery and perinuclear zones. Aberrations in motility and respiration were also present 6 h after exposure. SIGNIFICANCE: HBOT produces intracellular distinctions and inhomogeneities in mitochondrial dynamics and bioenergetics. HBOT as is commonly applied in clinical medicine induced undesirable and persistent alterations in bioenergy function needed to support cell migration and/or proliferation. There may be alternative HBOT parameters that more effectively engender maintenance and adequacy of intracellular bioenergy supply to promote wound healing.

Difficulty in Advancing Flexible Epidural Catheters When Establishing Labor Analgesia: An Observational Open-Label Randomized Trial.

BACKGROUND: While flexible epidural catheters reduce the risk of paresthesia and intravascular cannulation, they may be more challenging to advance beyond the tip of a Tuohy needle. This may increase placement time, number of attempts, and possibly complications when establishing labor analgesia. This study investigated the ability to advance flexible epidural catheters through different epidural needles from 2 commonly used, commercially available, epidural kits. METHODS: We hypothesized that the multiorifice wire-reinforced polyamide nylon blend epidural catheters will have a higher rate of successful first attempt insertion than the single-end hole wire-reinforced polyurethane catheters for the establishment of labor analgesia. The primary outcome was a difference in proportions of failure to advance the epidural catheter between the 2 epidural kits and was tested by a χ2 test. Two-hundred forty epidural kits were collected (n = 120/group) for 240 laboring patients requesting epidural analgesia in this open-label clinical trial from November 2018 to September 2019. Two-week time intervals were randomized for the exclusive use of 1 of the 2 kits in this study, where all patients received labor analgesia through either the flexible epidural catheter "A" or the flexible epidural catheter "B." Engineering properties of the equipment used were then determined. RESULTS: Flexible epidural catheter "A," the single-end hole wire-reinforced polyurethane catheter, did not advance at the first attempt in 15% (n = 18 of 120) of the parturients compared to 0.8% (n = 1 of 120) of the catheter "B," the multiorifice wire-reinforced polyamide nylon blend epidural catheter (P < .0001). Twenty-five additional epidural needle manipulations were recorded in the laboring patients who received catheter "A," while 1 epidural needle manipulation was recorded in the parturients who received catheter "B" (P < .0001). Bending stiffness of the epidural catheters used from kit "B" was twice the bending stiffness of the catheters used from kit "A" (bending stiffness catheters "A" 0.64 ± 0.04 N·mm2 versus bending stiffness catheters "B" 1.28 ± 0.20 N·mm2, P = .0038), and the angle formed by the needle and the epidural catheter from kit "A" was less acute than the angle formed from kit "B" (kit "A" 14.17 ± 1.72° versus kit "B" 21.83 ± 1.33°, P = .0036), with a mean difference of 7.66° between the 2 kits' angles. CONCLUSIONS: The incidence of an inability to advance single-end hole wire-reinforced polyurethane catheter was higher compared to the use of multiorifice wire-reinforced polyamide nylon blend epidural catheter. Variation of morphological features of epidural needles and catheters may play a critical role in determining the successful establishment of labor epidural analgesia.

Biophysical Considerations in the Rational Design and Cellular Targeting of Flexible Polymeric Nanoparticles.

How nanoparticle (NP) mechanical properties impact multivalent ligand-receptor-mediated binding to cell surfaces, the avidity, propensity for internalization, and effects due to crowding remains unknown or unquantified. Through computational analyses, the effects of NP composition from soft, deformable NPs to rigid spheres, effect of tethers, the crowding of NPs at the membrane surface, and the cell membrane properties such as cytoskeletal interactions are addressed. Analyses of binding mechanisms of three distinct NPs that differ in type and rigidity (core-corona flexible NP, rigid NP, and rigid-tethered NP) but are otherwise similar in size and ligand surface density are reported; moreover, for the case of flexible NP, NP stiffness is tuned by varying the internal crosslinking density. Biophysical modeling of NP binding to membranes together with thermodynamic analysis powered by free energy calculations is employed, and it is shown that efficient cellular targeting and uptake of NP functionalized with targeting ligand molecules can be shaped by factors including NP flexibility and crowding, receptor-ligand binding avidity, state of the membrane cytoskeleton, and curvature inducing proteins. Rational design principles that confer tension, membrane excess area, and cytoskeletal sensing properties to the NP which can be exploited for cell-specific targeting of NP are uncovered.

In vitro comparison of hydroxocobalamin (B12a) and the mitochondrial directed therapy by a succinate prodrug in a cellular model of cyanide poisoning.

The objective of this study was to compare the use of hydroxocobalamin (B12a) and a succinate prodrug to evaluate for improvement in mitochondrial function in an in vitro model of cyanide poisoning. Peripheral blood mononuclear cells (PBMC) and human aortic smooth muscle cells (HASMC) incubated with 50 mM of sodium cyanide (CN) for five minutes serving as the CN group compared to controls. We investigated the following: (1) Mitochondrial respiration; (2) Superoxide and mitochondrial membrane potential with microscopy; (3) Citrate synthase protein expression. All experiments were performed with a cell concentration of 2-3 × 106 cells/ml for both PBMC and HASMC. There were four conditions: (1) Control (no exposure); (2) Cyanide (exposure only); (3) B12a (cyanide exposure followed by B12a treatment); (4) NV118 (cyanide followed by NV118 treatment). In this study the key findings include: (1) Improvement in key mitochondrial respiratory states with the succinate prodrug (NV118) but not B12a; (2) Attenuation of superoxide production with treatment of NV118 but not with B12a treatment; (3) The changes in respiration were not secondary to increased mitochondrial content as measured by citrate synthase; (4) The use of easily accessible human blood cells showed similar mitochondrial response to both cyanide and treatment to HASMC. The use of a succinate prodrug to circumvent partial CIV inhibition by cyanide with clear reversal of cellular respiration and superoxide production that was not attributed to changes in mitochondrial content not seen by the use of B12a.

Multiscale modeling of protein membrane interactions for nanoparticle targeting in drug delivery.

Nanoparticle (NP)-based imaging and drug delivery systems for systemic (e.g. intravenous) therapeutic and diagnostic applications are inherently a complex integration of biology and engineering. A broad range of length and time scales are essential to hydrodynamic and microscopic molecular interactions mediating NP (drug nanocarriers, imaging agents) motion in blood flow, cell binding/uptake, and tissue accumulation. A computational model of time-dependent tissue delivery, providing in silico prediction of organ-specific accumulation of NPs, can be leveraged in NP design and clinical applications. In this article, we provide the current state-of-the-art and future outlook for the development of predictive models for NP transport, targeting, and distribution through the integration of new computational schemes rooted in statistical mechanics and transport. The resulting multiscale model will comprehensively incorporate: (i) hydrodynamic interactions in the vascular scales relevant to NP margination; (ii) physical and mechanical forces defining cellular and tissue architecture and epitope accessibility mediating NP adhesion; and (iii) subcellular and paracellular interactions including molecular-level targeting impacting NP uptake.

Ex vivo use of cell-permeable succinate prodrug attenuates mitochondrial dysfunction in blood cells obtained from carbon monoxide-poisoned individuals.

The purpose of this study was to evaluate a new pharmacological strategy using a first-generation succinate prodrug, NV118, in peripheral blood mononuclear cells (PBMCs) obtained from subjects with carbon monoxide (CO) poisoning and healthy controls. We obtained human blood cells from subjects with CO poisoning and healthy control subjects. Intact PBMCs from subjects in the CO and Control group were analyzed with high-resolution respirometry measured in pmol O2 per second per 10-6 PBMCs. In addition to obtaining baseline respiration, NV118 (100 µM) was injected, and the same parameters of respiration were obtained for comparison in PBMCs. We measured mitochondrial dynamics with microscopy with the same conditions. We enrolled 37 patients (17 in the CO group and 20 in the Control group for comparison) in the study. PMBCs obtained from subjects in the CO group had overall significantly lower respiration compared with the Control group (P < 0.0001). There was a significant increase in respiration with NV118, specifically with an increase in maximum respiration and respiration from complex II and complex IV (P < 0.0001). The mitochondria in PBMCs demonstrated an overall increase in net movement compared with the Control group. Our results of this study suggest that the therapeutic compound, NV118, increases respiration at complex II and IV as well as restoration of mitochondrial movement in PBMCs obtained from subjects with CO poisoning. Mitochondrial-directed therapy offers a potential future strategy with further exploration in vivo.

Prophylaxis of mitochondrial dysfunction caused by cellular decompression from hyperbaric exposure.

Mitochondrial dysfunction occurring in response to cellular perturbations can include altered mitochondrial motility and bioenergetic function having intracellular heterogeneity. Exogenous mitochondrial directed therapy may correct these dysfunctions. Using in vitro approaches, we find that cell perturbations induced by rapid decompression from hyperbaric conditions with specific gas exposures has differential effects on mitochondrial motility, inner membrane potential, cellular respiration, reactive oxygen species production, impaired maintenance of cell shape and altered intracellular distribution of bioenergetic capacity in perinuclear and cell peripheral domains. Addition of a first-generation cell-permeable succinate prodrug to support mitochondrial function has positive overall effects in blunting the resultant bioenergy responses. Our results with this model of perturbed cell function induced by rapid decompression indicate that alterations in bioenergetic state are partitioned within the cell, as directly assessed by a combination of mitochondrial respiration and dynamics measurements. Reductions in the observed level of dysfunction produced can be achieved with application of the cell-permeable succinate prodrug.

Nanoparticle transport phenomena in confined flows.

Nanoparticles submerged in confined flow fields occur in several technological applications involving heat and mass transfer in nanoscale systems. Describing the transport with nanoparticles in confined flows poses additional challenges due to the coupling between the thermal effects and fluid forces. Here, we focus on the relevant literature related to Brownian motion, hydrodynamic interactions and transport associated with nanoparticles in confined flows. We review the literature on the several techniques that are based on the principles of non-equilibrium statistical mechanics and computational fluid dynamics in order to simultaneously preserve the fluctuation-dissipation relationship and the prevailing hydrodynamic correlations. Through a review of select examples, we discuss the treatments of the temporal dynamics from the colloidal scales to the molecular scales pertaining to nanoscale fluid dynamics and heat transfer. As evident from this review, there, indeed has been little progress made in regard to the accurate modeling of heat transport in nanofluids flowing in confined geometries such as tubes. Therefore the associated mechanisms with such processes remain unexplained. This review has revealed that the information available in open literature on the transport properties of nanofluids is often contradictory and confusing. It has been very difficult to draw definitive conclusions. The quality of work reported on this topic is non-uniform. A significant portion of this review pertains to the treatment of the fluid dynamic aspects of the nanoparticle transport problem. By simultaneously treating the energy transport in ways discussed in this review as related to momentum transport, the ultimate goal of understanding nanoscale heat transport in confined flows may be achieved.

Cross-linker-Modulated Nanogel Flexibility Correlates with Tunable Targeting to a Sterically Impeded Endothelial Marker.

Deformability of injectable nanocarriers impacts rheological behavior, drug loading, and affinity target adhesion. Here, we present atomic force microscopy (AFM) and spectroscopy measurements of nanocarrier Young's moduli, tune the moduli of deformable nanocarriers with cross-linkers, and demonstrate vascular targeting behavior that correlates with Young's modulus. Homobifunctional cross-linkers were introduced into lysozyme-dextran nanogels (NGs). Single particle-scale AFM measurements determined NG moduli varying from ∼50-150 kPa for unmodified NGs or NGs with a short hydrophilic cross-linker (2,2'-(ethylenedioxy)bis(ethylamine), EOD) to ∼350 kPa for NGs modified with a longer hydrophilic cross-linker (4,9-dioxa-1,12-dodecanediamine, DODD) to ∼10 MPa for NGs modified with a longer hydrophobic cross-linker (1,12-diaminododecane, DAD). Cross-linked NGs were conjugated to antibodies for plasmalemma vesicle associated protein (PLVAP), a caveolar endothelial marker that cannot be accessed by rigid particles larger than ∼100 nm. In previous work, 150 nm NGs effectively targeted PLVAP, where rigid particles of similar diameter did not. EOD-modified NGs targeted PLVAP less effectively than unmodified NGs, but more effectively than DODD or DAD modified NGs, which both yielded low levels of targeting, resembling results previously obtained with polystyrene particles. Cross-linked NGs were also conjugated to antibodies against intracellular adhesion molecule-1 (ICAM-1), an endothelial marker accessible to large rigid particles. Cross-linked NGs and unmodified NGs targeted uniformly to ICAM-1. We thus demonstrate cross-linker modification of NGs, AFM determination of NG mechanical properties varying with cross-linker, and tuning of specific sterically constrained vascular targeting behavior in correlation with cross-linker-modified NG mechanical properties.

Nanofluid Dynamics of Flexible Polymeric Nanoparticles Under Wall Confinement.

Describing the hydrodynamics of nanoparticles in fluid media poses interesting challenges due to the coupling between the Brownian and hydrodynamic forces at the nanoscale. We focus on multiscale formulations of Brownian motion and hydrodynamic interactions (HI) of a single flexible polymeric nanoparticle in confining flows using the Brownian Dynamics method. The nanoparticle is modeled as a self-avoiding freely jointed polymer chain that is subject to Brownian forces, hydrodynamics forces, and repulsive interactions with the confining wall. To accommodate the effect of the wall, the hydrodynamic lift due to the wall is included in the mobility of a bead of the polymer chain which depends on its proximity to the wall. Using the example of a flexible polymeric nanoparticle, we illustrate temporal dynamics pertaining to the colloidal scale as well as the nanoscale.

Stiffness can mediate balance between hydrodynamic forces and avidity to impact the targeting of flexible polymeric nanoparticles in flow.

We report computational investigations of deformable polymeric nanoparticles (NPs) under colloidal suspension flow and adhesive environment. We employ a coarse-grained model for the polymeric NP and perform Brownian dynamics (BD) simulations with hydrodynamic interactions and in the presence of wall-confinement, particulate margination, and wall-adhesion for obtaining NP microstructure, shape, and anisotropic and inhomogeneous transport properties for different NP stiffness. These microscopic properties are utilized in solving the Fokker-Planck equation to obtain the spatial distribution of NP subject to shear, margination due to colloidal microparticles, and confinement due to a vessel wall. Comparing our computational results for the amount of NP margination to the near-wall adhesion regime with those of our binding experiments in cell culture under shear, we found quantitative agreement on shear-enhanced binding, the effect of particulate volume fraction, and the effect of NP stiffness. For the experimentally realized polymeric NP, our model predicts that the shear and volume fraction mediated enhancement in targeting has a hydrodynamic transport origin and is not due to a multivalent binding effect. However, for ultrasoft polymeric NPs, our model predicts a substantial increase in targeting due to multivalent binding. Our results are also in general agreement with experiments of tissue targeting measurements in vivo in mice, however, one needs to exercise caution in extending the modeling treatment to in vivo conditions owing to model approximations. The reported combined computational approach and results are expected to enable fine-tuning of design and optimization of flexible NP in targeted drug delivery applications.

Mitochondrial networking in human blood cells with application in acute care illnesses.

Mitochondria are dynamic organelles that adapt in response to environmental stresses or mutations. Dynamic processes involving mitochondria include their locomotion within cells and fusion and fission events in which mitochondrial join together or split apart. Various imaging strategies have been utilized to track mitochondrial dynamics. One common limitation of most of the methods available is that the time required to perform the technique and analyze the results prohibits application to clinical diagnosis and therapy. We recently demonstrated "whole-cell" mitochondrial analysis in a two-dimensional fashion with fluorescence microscopy. Our developed technique allows evaluation of whole-cell mitochondrial networking, including assessment of mitochondrial motility and rates of fission and fusion events using human blood cells (peripheral blood mononuclear cells (PBMCs)) on a clinically relevant timescale. We demonstrate this methodology in a cohort of healthy subjects as well as a cohort of hospitalized subjects having sepsis, an acute care illness. As there is increasing use of human blood cells as a proxy of organ mitochondrial function with respiration in various disease states, the addition of mitochondrial dynamics will now allow for more thorough clinical evaluation of mitochondrial networking in human disease with potential exploration of therapeutics.

Alterations in Mitochondrial Function in Blood Cells Obtained From Patients With Sepsis Presenting to an Emergency Department.

OBJECTIVE: Mitochondrial dysfunction has been implicated as a key cellular event leading to organ dysfunction in sepsis. Our objective is to measure changes in mitochondrial bioenergetics in subjects with early presentation of sepsis to provide insight into the incompletely understood pathophysiology of the dysregulated host response in sepsis. DESIGN: Prospective observational study. SETTING: Single site tertiary academic emergency department. SUBJECTS: We enrolled a total of 48 subjects in the study, 10 with sepsis or septic shock, 10 with infection without sepsis, 14 older and 14 younger healthy controls. INTERVENTIONS: Peripheral blood mononuclear cells were measured with high-resolution respirometry (OROBOROS O2K). MEASUREMENTS AND MAIN RESULTS: The median age in patients with sepsis, infection only, older control and younger controls were 63, 34, 61, and 29 years old, respectively. In the Sepsis group, the median 1st 24-h SOFA score was 8, and the initial median lactate was 4.2 mmol/dL, compared with 1.1 in the Infection Group. The 30-day mortality of the sepsis/septic shock group was 50%, with a median length of stay of 7-days. The Sepsis Group had significantly lower routine and Max respiration when compared with the other groups as well as uncoupled Complex I respiration. There was also a significant decrease in ATP-linked respiration along with the Spare Reserve Capacity in the Sepsis Group when compared with the other group. There were no age-related differences in respiration between the Older and Younger control group. CONCLUSIONS: Bedside measurement of mitochondrial respiration can be minimally invasive and performed in a timely manner. Mitochondrial dysfunction, detected by decreased oxygen consumption utilized for energy production and depleted cellular bioenergetics reserve.

Acute decompression following simulated dive conditions alters mitochondrial respiration and motility.

While barotrauma, decompression sickness, and drowning-related injuries are common morbidities associated with diving and decompression from depth, it remains unclear what impact rapid decompression has on mitochondrial function. In vitro diving simulation was performed with human dermal fibroblast cells subjected to control, air, nitrogen, and oxygen dive conditions. With the exception of the gas mixture, all other related variables, including absolute pressure exposure, dive and decompression rates, and temperature, were held constant. High-resolution respirometry was used to examine key respiratory states. Mitochondrial dynamic function, including net movement, number, and rates of fusion/fission events, was obtained from fluorescence microscopy imaging. Effects of the dive conditions on cell cytoskeleton were assessed by imaging both actin and microtubules. Maximum respiration was lower in fibroblasts in the air group than in the control and nitrogen groups. The oxygen group had overall lower respiration when compared with all other groups. All groups demonstrated lower mitochondrial motility when compared with the control group. Rates of fusion and fission events were the same between all groups. There were visible differences in cell morphology consistent with the actin staining; however, there were no appreciable changes to the microtubules. This is the first study to directly assess mitochondrial respiration and dynamics in a cell model of decompression. Both hyperbaric oxygen and air dive conditions produce deleterious effects on overall mitochondrial health in fibroblasts.

Flexible Nanoparticles Reach Sterically Obscured Endothelial Targets Inaccessible to Rigid Nanoparticles.

Molecular targeting of nanoparticle drug carriers promises maximized therapeutic impact to sites of disease or injury with minimized systemic effects. Precise targeting demands addressing to subcellular features. Caveolae, invaginations in cell membranes implicated in transcytosis and inflammatory signaling, are appealing subcellular targets. Caveolar geometry has been reported to impose a ≈50 nm size cutoff on nanocarrier access to plasmalemma vesicle associated protein (PLVAP), a marker found in caveolae in the lungs. The use of deformable nanocarriers to overcome that size cutoff is explored in this study. Lysozyme-dextran nanogels (NGs) are synthesized with ≈150 or ≈300 nm mean diameter. Atomic force microscopy indicates the NGs deform on complementary surfaces. Quartz crystal microbalance data indicate that NGs form softer monolayers (≈60 kPa) than polystyrene particles (≈8 MPa). NGs deform during flow through microfluidic channels, and modeling of NG extrusion through porous filters yields sieving diameters less than 25 nm for NGs with 150 and 300 nm hydrodynamic diameters. NGs of 150 and 300 nm diameter target PLVAP in mouse lungs while counterpart rigid polystyrene particles do not. The data in this study indicate a role for mechanical deformability in targeting large high-payload drug-delivery vehicles to sterically obscured targets like PLVAP.

Translational Application of Measuring Mitochondrial Functions in Blood Cells Obtained from Patients with Acute Poisoning.

It is conservatively estimated that 5,000 deaths per year and 20,000 injuries in the USA are due to poisonings caused by chemical exposures (e.g., carbon monoxide, cyanide, hydrogen sulfide, phosphides) that are cellular inhibitors. These chemical agents result in mitochondrial inhibition resulting in cardiac arrest and/or shock. These cellular inhibitors have multi-organ effects, but cardiovascular collapse is the primary cause of death marked by hypotension, lactic acidosis, and cardiac arrest. The mitochondria play a central role in cellular metabolism where oxygen consumption through the electron transport system is tightly coupled to ATP production and regulated by metabolic demands. There has been increasing use of human blood cells such as peripheral blood mononuclear cells and platelets, as surrogate markers of mitochondrial function in organs due to acute care illnesses. We demonstrate the clinical applicability of measuring mitochondrial bioenergetic and dynamic function in blood cells obtained from patients with acute poisoning using carbon monoxide poisoning as an illustration of our technique. Our methods have potential application to guide therapy and gauge severity of disease in poisoning related to cellular inhibitors of public health concern.