Seeking gender-affirming medical care: A phenomenological inquiry on skillful coping with transgender and non-binary adults in the United States Midwest.

This study sought to understand how transgender and gender non-binary (TGNB) individuals skillfully cope with healthcare services and to explore how childhood experiences impact expectations, habits, and meaning-making when utilizing healthcare services. Using an interpretive phenomenological approach, we sampled 17, White TGNB adults in the United States, ages 19 to 57, using semi-structed interviews about childhood experiences with healthcare utilization and adult experiences seeking genderaffirming healthcare. Analysis identified one main theme-Anticipate the worst in healthcare and be pleasantly surprised-and three subthemes: i) contrast between positive childhood and negative adulthood experiences in medical care; ii) coping practices for the worst; and iii) finding your unicorn doctor and medical staff for pleasant experiences. Results indicate participants experienced a disruption and acquisition of new coping practices in healthcare settings and the cultivation of a radical imagination for a more liberated medical world for TGNB people. Implications for providers and medical offices for empowering TGNB adults are described.

Dual-mode optical sensing of organic vapors and proteins with polydiacetylene (PDA)-embedded electrospun nanofibers.

Optical sensors capable of colorimetric visualization and/or fluorescence detection have shown tremendous potential for field technicians and emergency responders, owing to the portability and low cost of such devices. Polydiacetylene (PDA)-enhanced nanofibers are particularly promising due to high surface area, facile functionalization, simple construction, and the versatility to empower either colorimetric or fluorescence signaling. We demonstrate here a dual-mode optical sensing with electrospun nanofibers embedded with various PDAs. The solvent-dependent fluorescent transition of nanofibers generated a pattern that successfully distinguished four common organic solvents. The colorimetric and fluorescent sensing of biotin-avidin interactions by embedding biotinylated-PCDA monomers into silica-reinforced nanofiber mats were realized for detection of biomolecules. Finally, a PDA-based nanofiber sensor array consisting of three monomers has been fabricated for the determination and identification of organic amine vapors using colorimetry and principal component analysis (PCA). The combination of PCA and the strategy of probing analytes in two different concentration ranges (ppm and ppth) led to successful analysis of all eight amines.

Modified Bose-Einstein and Fermi-Dirac statistics if excitations are localized on an intermediate length scale: applications to non-Debye specific heat.

Disordered systems show deviations from the standard Debye theory of specific heat at low temperatures. These deviations are often attributed to two-level systems of uncertain origin. We find that a source of excess specific heat comes from correlations between quanta of energy if excitations are localized on an intermediate length scale. We use simulations of a simplified Creutz model for a system of Ising-like spins coupled to a thermal bath of Einstein-like oscillators. One feature of this model is that energy is quantized in both the system and its bath, ensuring conservation of energy at every step. Another feature is that the exact entropies of both the system and its bath are known at every step, so that their temperatures can be determined independently. We find that there is a mismatch in canonical temperature between the system and its bath. In addition to the usual finite-size effects in the Bose-Einstein and Fermi-Dirac distributions, if excitations in the heat bath are localized on an intermediate length scale, this mismatch is independent of system size up to at least 10(6) particles. We use a model for correlations between quanta of energy to adjust the statistical distributions and yield a thermodynamically consistent temperature. The model includes a chemical potential for units of energy, as is often used for other types of particles that are quantized and conserved. Experimental evidence for this model comes from its ability to characterize the excess specific heat of imperfect crystals at low temperatures.

Abi3bp is a multifunctional autocrine/paracrine factor that regulates mesenchymal stem cell biology.

Mesenchymal stem cells (MSCs) transplanted into injured myocardium promote repair through paracrine mechanisms. We have previously shown that MSCs over-expressing AKT1 (Akt-MSCs) exhibit enhanced properties for cardiac repair. In this study, we investigated the relevance of Abi3bp toward MSC biology. Abi3bp formed extracellular deposits with expression controlled by Akt1 and ubiquitin-mediated degradation. Abi3bp knockdown/knockout stabilized focal adhesions and promoted stress-fiber formation. Furthermore, MSCs from Abi3bp knockout mice displayed severe deficiencies in osteogenic and adipogenic differentiation. Knockout or stable knockdown of Abi3bp increased MSC and Akt-MSC proliferation, promoting S-phase entry via cyclin-d1, ERK1/2, and Src. Upon Abi3bp binding to integrin-ß1 Src associated with paxillin which inhibited proliferation. In vivo, Abi3bp knockout increased MSC number and proliferation in bone marrow, lung, and liver. In summary, we have identified a novel extracellular matrix protein necessary for the switch from proliferation to differentiation in MSCs.

Effects of myocardial infarction on the distribution and transport of nutrients and oxygen in porcine myocardium.

One of the primary limitations of cell therapy for myocardial infarction is the low survival of transplanted cells, with a loss of up to 80% of cells within 3 days of delivery. The aims of this study were to investigate the distribution of nutrients and oxygen in infarcted myocardium and to quantify how macromolecular transport properties might affect cell survival. Transmural myocardial infarction was created by controlled cryoablation in pigs. At 30 days post-infarction, oxygen and metabolite levels were measured in the peripheral skeletal muscle, normal myocardium, the infarct border zone, and the infarct interior. The diffusion coefficients of fluorescein or FITC-labeled dextran (0.3-70 kD) were measured in these tissues using fluorescence recovery after photobleaching. The vascular density was measured via endogenous alkaline phosphatase staining. To examine the influence of these infarct conditions on cells therapeutically used in vivo, skeletal myoblast survival and differentiation were studied in vitro under the oxygen and glucose concentrations measured in the infarct tissue. Glucose and oxygen concentrations, along with vascular density were significantly reduced in infarct when compared to the uninjured myocardium and infarct border zone, although the degree of decrease differed. The diffusivity of molecules smaller than 40 kD was significantly higher in infarct center and border zone as compared to uninjured heart. Skeletal myoblast differentiation and survival were decreased stepwise from control to hypoxia, starvation, and ischemia conditions. Although oxygen, glucose, and vascular density were significantly reduced in infarcted myocardium, the rate of macromolecular diffusion was significantly increased, suggesting that diffusive transport may not be inhibited in infarct tissue, and thus the supply of nutrients to transplanted cells may be possible. in vitro studies mimicking infarct conditions suggest that increasing nutrients available to transplanted cells may significantly increase their ability to survive in infarct.

FRET detection of proteins using fluorescently doped electrospun nanofibers and pattern recognition.

This paper reports the fabrication of solid-state nanofiber sensor arrays and their use for detection of multiple proteins using principal component analysis (PCA). Four cationic and anionic fluorescently embedded nanofibers are generated by an electrospinning method, yielding unique patterns of fluorescence change upon interaction with protein samples. Five metal and nonmetal containing proteins, i.e., hemoglobin, myoglobin, cytochrome c, BSA, and avidin, have been investigated; and the results show that distinct fluorescent patterns can be formed upon the addition of protein samples to the array of solid nanofiber substrates, allowing their unambiguous identification. The nanofiber films are highly repeatable with a batch-to-batch variation of approximately 5% and demonstrated outstanding reusability with less than a 15% loss of fluorescence intensity signal after 5 regenerations of test cycles. For a more practical visualization, a cluster map was generated using PCA of the change-in-fluorescence (ΔI) composite patterns, demonstrating the potential of the method for diagnostic applications.

Nanofibers doped with dendritic fluorophores for protein detection.

We report a solid-state, nanofiber-based optical sensor for detecting proteins with an anionic fluorescent dendrimer (AFD). The AFD was encapsulated in cellulose acetate (CA) electrospun nanofibers, which were deacetylated to cellulose to generate secondary porous structures that are desirable for enhancing molecular interactions, and thus better signaling. The protein sensing properties of the fibers were characterized by monitoring the fluorescence response of cytochrome c (cyt c), hemoglobin (Hgb), and bovine serum albumin (BSA) as a function of concentration. Effective quenching was observed for the metalloproteins, cyt c and Hgb. The effect was primarily due to energy transfer of the imbedded fluorescent dendrimers to the protein, as both proteins contain heme portions. Electron transfer, caused through the electrostatic effects in the binding of the anionic dendrimer to the positive patches of globular proteins, could be responsible as well. BSA, on the other hand, triggered a "turn-on" response in fluorescence, suggesting the negatively charged BSA reduces the pi-pi stacking of the partially dispersed, negatively charged dendritic fluorophores through repulsion forces, which results in an increase in fluorescence. Stern-Volmer constants (K(sv)) of the electrospun fibers were found to be 3.4 x 10(5) and 1.7 x 10(6) M(-1) for cyt c and Hgb, respectively. The reusability of the nanofibers is excellent: the nanofibers demonstrated less than 15% change of fluorescence intensity signal in a 5-cycle test.

Single-laboratory validation of an NMR method for the determination of aloe vera polysaccharide in pharmaceutical formulations.

This report presents a single-laboratory-validated NMR method for determining the quantity of aloe vera polysaccharide in product formulations. The ratio of signal intensities of the acetyl methyl protons to methyl protons of an internal reference varied linearly with concentration (r2 > 0.99) with a lower LOQ of 0.2 g/100 mL for two commercial aloe polysaccharide standards, Acemannan Hydrogel (AH) and Immuno-10 (I-10). The assay was used to quantify these standards in two nonacetylated polysaccharide matrices, dextrin and arabinogalactan, and in a pharmaceutical product. The concentrations of AH in samples containing the polysaccharide matrices agreed within 7% of values determined on the basis of weight and showed within- and between-run RSD values of < 3.5%. The assay of I-10 in the pharmaceutical product was within 7% of the expected values over a range from 50 to 125% of the targeted I-10 concentration, with a between-run RSD of < 7%. The assay showed no interference from other added polysaccharides or from other components of the pharmaceutical formulation and was independent of the molecular size distribution of the aloe polysaccharide present. The NMR assay can be used to validate aloe polysaccharide contained in a product and to follow any chemical degradation that may occur over time.

SFRP2 regulates cardiomyogenic differentiation by inhibiting a positive transcriptional autofeedback loop of Wnt3a.

Wnts comprise a family of 20 lipid-modified glycoproteins in mammals and play critical roles during embryological development and organogenesis of several organ systems, including the heart. They are required for mesoderm formation and have been implicated in promoting cardiomyogenic differentiation of mammalian embryonic stem cells, but the underlying mechanisms regulating Wnt signaling during cardiomyogenesis remain poorly understood. In this report, we show that in a pluripotent mouse embryonal carcinoma stem cell line, SFRP2 inhibits cardiomyogenic differentiation by regulating Wnt3a transcription. SFRP2 inhibited early stages of cardiomyogenesis, preventing mesoderm specification and maintaining the cells in the undifferentiated state. Using a gain- and loss-of-function approach, we demonstrate that although addition of recombinant SFRP2 decreased Wnt3a transcription and cardiomyogenic differentiation, silencing of Sfrp2 led to enhanced Wnt3a transcription, mesoderm formation, and increased cardiomyogenesis. We show that the inhibitory effects of SFRP2 on Wnt transcription are secondary to interruption of a positive feedback effect of Wnt3a on its own transcription. Wnt3a increased its own transcription via the canonical pathway and TCF4 family of transcription factors, and the inhibitory effects of SFRP2 on Wnt3a transcription were associated with disruption of downstream canonical Wnt signaling. The inhibitory effects of Sfrp2 on Wnt3a expression identify Sfrp2 as a "checkpoint gene," which exerts its control on cardiomyogenesis through regulation of Wnt3a transcription.

An in vitro system to evaluate the effects of ischemia on survival of cells used for cell therapy.

Maintaining cell viability is a major challenge associated with transplanting cells into ischemic myocardium to restore function. A likely contributor to significant cell death during cardiac cell therapy is hypoxia/anoxia. We developed a system that enabled quantification and association of cell survival with oxygen and nutrient values within in vitro constructs. Myoblasts were suspended in 2% collagen gels in 1 cm diameter x 1 cm deep constructs. At 48 +/- 3 h post-seeding, oxygen levels were measured using microelectrodes and gels were snap-frozen. Bioluminescence metabolite imaging and TUNEL staining were performed on cryosections. Oxygen and glucose consumption and lactate production rates were calculated by fitting data to Fick's second law of diffusion with Michaelis-Menten kinetics. Oxygen levels dropped to 0 mmHg and glucose levels dropped from 4.28 to 3.18 mM within the first 2000 mum of construct depth. Cell viability dropped to approximately 40% over that same distance and continued to drop further into the construct. We believe this system provides a reproducible and controllable test bed to compare survival, proliferation, and phenotype of various cell inputs (e.g., myoblasts, mesenchymal stem cells, and cardiac stem cells) and the impact of different treatment regimens on the likelihood of survival of transplanted cells.

Intracardiac transplantation of a mixed population of bone marrow cells improves both regional systolic contractility and diastolic relaxation.

BACKGROUND: Pre-clinical and clinical studies suggest that transplantation of bone marrow-derived stem cells can improve global cardiac function. However, no quantitative assessment of regional systolic contraction and correlation with phenotype has been made. Therefore, we used our model of cryoinfarcted rabbit myocardium for intracardiac transplantation of a mixed population of bone marrow-derived cells and assessed both regional function and myogenic conversion of the cells. METHODS: Nineteen New Zealand white rabbits underwent cryoinjury of the left ventricle. Autologous bone marrow (BM) cells were expanded in vitro. After 2 weeks, either 1 x 10(8) mixed BM-derived progenitor cells (BM group, n = 11) or vehicle (control group, n = 8) were injected into the cryoinjured region. Regional systolic function was measured using micromanometry and sonomicrometry before and 4 weeks after cell injection; cell phenotype was evaluated histologically. RESULTS: All animals in the BM group significantly improved both systolic shortening (0.11 +/- 0.7 vs -0.05 +/- 0.05 mm in the control group, p < 0.05) and regional stroke work when compared with control (9.6 +/- 2.4 vs -1.2 +/- 1.2 mm . mm Hg, p < 0.003). In addition, the BM group had improved global diastolic function, as measured by minimum dP/dt and end-diastolic pressure. On histologic assessment, BM cells differentiated toward a myogenic phenotype. CONCLUSIONS: Transplanting a mixed population of marrow-derived cells that can adopt a myogenic phenotype improves regional contractility and diastolic relaxation after myocardial infarction.

Cell therapy for heart failure--muscle, bone marrow, blood, and cardiac-derived stem cells.

Heart failure (HF) affects a rapidly growing population of patients. Despite improvements in the understanding and therapy of many stages of cardiovascular disease, there has been little progress in treating HF. In the late-stage disease, current options are cardiac transplantation and mechanical support--options that are limited to a small patient collective. The ischemically injured failing heart lacks contractile myocardium, functional vasculature, and electrical integrity, which has made treatment of the underlying injury untenable in the past. Restoring all of these components seems an overwhelming challenge. Yet, the concept of cell therapy--tissue repair by transplantation of stem and progenitor cells--has opened new potential options for patients with heart failure. Skeletal myoblasts, bone marrow, and blood-derived stem cells have all shown considerable myogenic and angiogenic potential in vitro and have rapidly moved from bench to bedside. A number of nonrandomized, non-placebo-controlled safety and feasibility studies have been reported and now double-blinded randomized controlled trials are underway. Despite this rapid clinical pace, the exact mechanisms underlying the functional benefits of different cell types are not well understood. Instead, multiple similar mechanism have been ascribed to virtually every cell type. Thus, while the field is exciting and offers unheralded promise to treat patients with CVD, we must proceed with due diligence and caution. Only a deep understanding of the benefits versus the risks, and the mechanisms involved in cell-mediated cardiac repair, will allow us to design clinically valuable tools and fulfill the potential of this exciting 21st century approach to treating cardiovascular disease.

Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation.

Immature skeletal muscle cells, or myoblasts, have been used in cellular cardiomyoplasty in attempts to regenerate cardiac muscle tissue by injection of cells into damaged myocardium. In some studies, muscle tissue within myoblast implant sites may be morphologically similar to cardiac muscle. We hypothesized that identifiable aspects of the cardiac milieu may contribute to growth and development of implanted myoblasts in vivo. To test this hypothesis, we designed a novel in vitro system to mimic some aspects of the electrical and biochemical environment of native myocardium. This system enabled us to separate the three-dimensional (3-D) electrical and biochemical signals that may be involved in myoblast proliferation and plasticity. Myoblasts were grown on 3-D polyglycolic acid mesh scaffolds under control conditions, in the presence of cardiac-like electrical current fluxes, or in the presence of culture medium that had been conditioned by mature cardiomyocytes. Cardiac-like electrical current fluxes caused increased myoblast number in 3-D culture, as determined by DNA assay. The increase in cell number was due to increased cellular proliferation and not differences in apoptosis, as determined by proliferating cell nuclear antigen and TdT-mediated dUTP nick-end labeling. Cardiomyocyte-conditioned medium also significantly increased myoblast proliferation. Expression of transcription factors governing differentiation along skeletal or cardiac lineages was evaluated by immunoblotting. Although these assays are qualitative, no changes in differentiation state along skeletal or cardiac lineages were observed in response to electrical current fluxes. Furthermore, from these experiments, conditioned medium did not appear to alter the differentiation state of skeletal myoblasts. Hence, cardiac milieu appears to stimulate proliferation but does not affect differentiation of skeletal myoblasts.

Video-assisted thoracoscopic transplantation of myoblasts into the heart.

PURPOSE: Currently, cells are transplanted into injured myocardium either through thoracotomy for open surgical delivery or through catheterization for endoventricular or intracoronary delivery; both methods have limitations. Open surgical delivery limits the potential patient population, whereas catheter-based delivery limits the ability to visualize the injection site and confirm delivery of the cells to the appropriate region. In this study, we examine the feasibility of cell transplantation into myocardium using a minimally invasive thoracoscopic approach. DESCRIPTION: Seven swine underwent thoracoscopic cell transplantation. Using a prototype injection device, approximately 10 million myoblasts were injected into the anterior, lateral, posterior, and apical regions of myocardium. Animals were recovered up to 7 days, and after euthanasia, hearts were explanted for histology. EVALUATION: All seven swine had successful delivery of myoblasts into the defined injection sites, as confirmed by analysis of an operative video, magnetic resonance imaging of iron-oxide-labeled cells, and histologic examination. CONCLUSIONS: Thoracoscopic cellular cardiomyoplasty is feasible and allows the surgeon the benefits of direct visualization of the cell injection while minimizing morbidity associated with open cell delivery.

Comparison of intracardiac cell transplantation: autologous skeletal myoblasts versus bone marrow cells.

BACKGROUND: Multiple cell types are being proposed for cardiac repair, but side-by-side comparisons are lacking. We tested the hypothesis that intracardiac transplantation of autologous bone marrow- or skeletal muscle-derived progenitor cells improve regional heart function to a similar degree. METHODS AND RESULTS: Thirty-nine New Zealand White rabbits underwent cryoinjury of the left ventricle and simultaneous hind limb bone marrow aspiration or soleus muscle biopsy. Both muscle and bone marrow cells were expanded in vitro. After 2 weeks, 10(8) skeletal muscle (SM group) or bone marrow-derived progenitor cells (BM group) were injected into the cryoinjured region (SM: n=12; BM: n=8). Medium alone was injected into the remaining animals (Control: n=16). Regional systolic function was measured using micromanometry and sonomicrometry at baseline, before, and 4 weeks after cell injection. Cell treatment resulted in a similar degree of improvement in a derivative of stroke work in the SM and BM groups (P=0.0026 and P=0.0085 versus Control, respectively). No significant difference was seen between BM and SM groups (P=0.9). On histology, engrafted cells were found in all of the cell treated animals. Injected myoblasts formed myotubes or muscle cells throughout the scar that expressed slow and fast myosin heavy chain. A subset of bone marrow cells differentiated toward a myogenic phenotype, as indicated by expression of desmin and alpha-sarcomeric actin in the engrafted areas. CONCLUSIONS: Transplantation and myogenic differentiation of bone marrow-derived progenitor cells increased regional systolic heart function after myocardial injury to a similar degree as skeletal myoblasts.

Aging, progenitor cell exhaustion, and atherosclerosis.

BACKGROUND: Atherosclerosis is largely attributed to chronic vascular injury, as occurs with excess cholesterol; however, the effect of concomitant vascular aging remains unexplained. We hypothesize that the effect of time in atherosclerosis progression is related to obsolescence of endogenous progenitor cells that normally repair and rejuvenate the arteries. METHODS AND RESULTS: Here we show that chronic treatment with bone marrow-derived progenitor cells from young nonatherosclerotic ApoE-/- mice prevents atherosclerosis progression in ApoE-/- recipients despite persistent hypercholesterolemia. In contrast, treatment with bone marrow cells from older ApoE-/- mice with atherosclerosis is much less effective. Cells with vascular progenitor potential are decreased in the bone marrow of aging ApoE-/- mice, but cells injected from donor mice engraft on recipient arteries in areas at risk for atherosclerotic injury. CONCLUSIONS: Our data indicate that progressive progenitor cell deficits may contribute to the development of atherosclerosis.