Delivery of Therapeutic miRNA via Plasma-Polymerised Nanoparticles Rescues Diabetes-Impaired Endothelial Function.

MicroRNAs (miRNAs) are increasingly recognised as key regulators of the development and progression of many diseases due to their ability to modulate gene expression post-translationally. While this makes them an attractive therapeutic target, clinical application of miRNA therapy remains at an early stage and in part is limited by the lack of effective delivery modalities. Here, we determined the feasibility of delivering miRNA using a new class of plasma-polymerised nanoparticles (PPNs), which we have recently isolated and characterised. We showed that PPN-miRNAs have no significant effect on endothelial cell viability in vitro in either normal media or in the presence of high-glucose conditions. Delivery of a miRNA inhibitor targeting miR-503 suppressed glucose-induced miR-503 upregulation and restored the downstream mRNA expression of CCNE1 and CDC25a in endothelial cells. Subsequently, PPN delivery of miR-503 inhibitors enhanced endothelial angiogenesis, including tubulogenesis and migration, in culture conditions that mimic diabetic ischemia. An intramuscular injection of a PPN-miR-503 inhibitor promoted blood-perfusion recovery in the hindlimb of diabetic mice following surgically induced ischemia, linked with an increase in new blood vessel formation. Together, this study demonstrates the effective use of PPN to deliver therapeutic miRNAs in the context of diabetes.

Overexpression of VIRMA confers vulnerability to breast cancers via the m6A-dependent regulation of unfolded protein response.

Virilizer-like m6A methyltransferase-associated protein (VIRMA) maintains the stability of the m6A writer complex. Although VIRMA is critical for RNA m6A deposition, the impact of aberrant VIRMA expression in human diseases remains unclear. We show that VIRMA is amplified and overexpressed in 15-20% of breast cancers. Of the two known VIRMA isoforms, the nuclear-enriched full-length but not the cytoplasmic-localised N-terminal VIRMA promotes m6A-dependent breast tumourigenesis in vitro and in vivo. Mechanistically, we reveal that VIRMA overexpression upregulates the m6A-modified long non-coding RNA, NEAT1, which contributes to breast cancer cell growth. We also show that VIRMA overexpression enriches m6A on transcripts that regulate the unfolded protein response (UPR) pathway but does not promote their translation to activate the UPR under optimal growth conditions. Under stressful conditions that are often present in tumour microenvironments, VIRMA-overexpressing cells display enhanced UPR and increased susceptibility to death. Our study identifies oncogenic VIRMA overexpression as a vulnerability that may be exploited for cancer therapy.

Cross-Section Observational Study to Assess Antimicrobial Resistance Prevalence among Bovine Respiratory Disease Bacterial Isolates from Commercial US Feedlots.

Antimicrobial resistance (AMR) is a global public health threat that jeopardizes efficacy of antibiotics in veterinary and human medicine. Antibiotics are commonly administered to target the bacterial component of bovine respiratory disease (BRD). The objectives of this study were to obtain a better understanding of antibiotic resistance in BRD-associated bacteria (Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni), investigate the clinical significance of AMR by monitoring clinical outcomes, and determine if regional differences exist in AMR trends. Deep pharyngeal swabs were used to sample beef cattle at initial BRD diagnosis (n = 453) from US feedlots representing three geographic regions. Organisms were identified by bacterial culture and subjected to broth microdilution antimicrobial susceptibility testing. Bacterium prevalence include P. multocida (36.0%), M. haemolytica (32.7%), and H. somni (28.5%). Of the Histophilus isolates, 39.5% were resistant to at least one antimicrobial, compared to 11.7% and 8.8% Pasteurella and Mannheimia, respectively. Non-susceptibility across all organisms was 5.7 X more likely in animals that received metaphylaxis, than those that did not (p < 0.0001; OR 5.7; CI 2.6-12.5). During days on feed 21-40, non-susceptibility of Histophilus was 8.7 X more likely than Mannheimia (p = 0.0002; OR 8.7; CI 2.8 to 27.4) and 6 X more likely than Pasteurella (p = 0.0016; OR 6.0; CI 2.0-18.0).

Characterization of Longitudinal Testosterone, Cortisol, and Musth in Male Asian Elephants (Elephas maximus), Effects of Aging, and Adrenal Responses to Social Changes and Health Events.

The conservation of endangered species and sustainability of managed populations requires considerations to ensure the health and welfare of individuals. Male elephants experience a biological phenomenon called "musth", which is characterized by increased testosterone production, temporal gland secretion and urine dribbling, heightened aggression and sexual behavior, and therefore can pose unique challenges for human safety and animal welfare. This study characterized longitudinal (9 to 22 years) patterns of circulating testosterone and cortisol in relation to musth in four adult Asian elephant bulls spanning ages from 12 to 54 years. Age-related effects on musth activity and adrenal responses to social changes and clinical health events were also examined. All bulls exhibited regular annual musth cycles. Circulating cortisol covaried positively with testosterone and musth, highlighting intrinsic patterns that should be considered when evaluating the impact of social, health, and environmental changes on adrenal glucocorticoid activity. Except for an end-of-life cortisol increase in one bull, there was no clear evidence of chronically elevated cortisol secretion outside of musth in any individual. Testosterone decreased with age in sexually mature bulls, whereas age-related changes in cortisol varied across individuals, with the three older bulls showing the greatest rate of change during musth versus inter-musth periods. In contrast to physiological factors, there was no evidence of social factors, such as addition of a new male and death of male herdmates, impacting adrenal glucocorticoid activity in these bulls in the short term. Changes in cortisol were associated with treatment for Mycobacterium tuberculosis (M. tb) in two bulls, increasing after start of treatment and decreasing with cessation of treatment, but were not clearly associated with activation of disease. This study highlights the importance of longitudinal hormone monitoring to track changes in physiological function and responses to social, health, and environmental change in elephant bulls, which is important for making more informed decisions on how to manage male elephants under varying degrees of human care to ensure welfare and safety.

Supporting Zoo Asian Elephant (Elephas maximus) Welfare and Herd Dynamics with a More Complex and Expanded Habitat.

Ensuring good health and welfare is an increasingly important consideration for conservation of endangered species, whether free-ranging or managed to varying degrees under human care. The welfare-based design of a new habitat for Asian elephants at the Oregon Zoo focused on meeting the elephants' physical, physiological, psychological, and social needs 24 h a day and across life stages. The habitat was designed to encourage activity, promote species-typical behaviors, support changing social dynamics, offer increased opportunities for choice, and provide biologically meaningful challenges. In this 4-year study, we monitored elephant health and welfare indicators throughout the transition and acclimation from the previous habitat to the new habitat. Several welfare indicators obtained through longitudinal hormone analyses, behavior assessments, and GPS measurement of walking distance and space use provided evidence that these goals were achieved. The elephants were more active and walked farther on a daily basis in the new habitat, with an average walking distance of over 15 km per day. A switch from primarily caretaker-delivered food to seeking food on their own indicates that the disbursement of food with less temporal and spatial predictability increased foraging opportunities, which better satisfies appetitive motivations important for psychological well-being. All individuals showed adaptive and normal adrenal responses to change and challenge, with the highest fecal glucocorticoid metabolite (FGM) concentrations and variability during the construction phase, and a return to previous baseline concentrations in the new habitat, suggesting they acclimated well to the new environment. The elephants expressed a diverse range of species-typical behaviors and demonstrated social dynamics of a healthy herd in both habitats with transitions of individuals through life stages. They exhibited more autonomy in choosing whom to associate with socially and also by choosing different aspects of their environment with regular indoor/outdoor access and extensive resource use in the new habitat. Findings indicate that the complexity and flexibility of the new habitat and habitat management has been effective in improving overall welfare by providing meaningful challenges and the opportunity to express appetitive behaviors, by offering choice in environmental conditions, and by providing the space and resource distribution to support evolving herd dynamics and increased social equity for individuals.

Risk and resilience of vulnerable families in Hong Kong under the impact of COVID-19: an ecological resilience perspective.

PURPOSE: Hong Kong has experienced four waves of COVID-19 since the first case was confirmed in January 2020. Several studies have highlighted the psychological impacts of the outbreak in Hong Kong but have largely ignored the protective factors that contribute to resilience among vulnerable families. This study adopted an ecological resilience framework to explore the impact of this epidemic on members of families with youth with a delinquent tendency/mental health concerns and the ecological protective factors for these vulnerable families. METHODS: Random sampling based on a sampling frame provided by one of the largest local social service organizations in Hong Kong led to the recruitment of 407 respondents who were interviewed using a battery of standardized questionnaires. RESULTS: The results showed that 30.6% and 11.5% of respondents reported a moderate and a severe level of psychological distress, respectively, almost double the percentages reported in a previous study conducted in Hong Kong before the COVID-19 outbreak. Around 36.6% of respondents indicated they had encountered financial problems and almost 40% indicated aggravated financial circumstances since the outbreak. Hierarchical regression analysis revealed that financial stress was the strongest predictor of psychological distress. Structural equation modeling indicated that family support, indoor leisure activities and community resources significantly mediated the negative influence of COVID-19-related stressors on psychological distress of family members. CONCLUSION: Family leisure activities, family support, community spirit and mutual help within the context of social-distancing restrictions may need to be promoted to benefit vulnerable families in Hong Kong under the COVID-19 epidemic.

Plasma polymerized nanoparticles effectively deliver dual siRNA and drug therapy in vivo.

Multifunctional nanocarriers (MNCs) promise to improve therapeutic outcomes by combining multiple classes of molecules into a single nanostructure, enhancing active targeting of therapeutic agents and facilitating new combination therapies. However, nanocarrier platforms currently approved for clinical use can still only carry a single therapeutic agent. The complexity and escalating costs associated with the synthesis of more complex MNCs have been major technological roadblocks in the pathway for clinical translation. Here, we show that plasma polymerized nanoparticles (PPNs), synthesised in reactive gas discharges, can bind and effectively deliver multiple therapeutic cargo in a facile and cost-effective process compatible with up scaled commercial production. Delivery of siRNA against vascular endothelial growth factor (siVEGF) at extremely low concentrations (0.04 nM), significantly reduced VEGF expression in hard-to-transfect cells when compared with commercial platforms carrying higher siRNA doses (6.25 nM). PPNs carrying a combination of siVEGF and standard of care Paclitaxel (PPN-Dual) at reduced doses (< 100 µg/kg) synergistically modulated the microenvironment of orthotopic breast tumors in mice, and significantly reduced tumor growth. We propose PPNs as a new nanomaterial for delivery of therapeutics, which can be easily functionalised in any laboratory setting without the need for additional wet-chemistry and purification steps.

Immobilized Macrophage Colony-Stimulating Factor (M-CSF) Regulates the Foreign Body Response to Implanted Materials.

The functionality and durability of implanted biomaterials are often compromised by an exaggerated foreign body reaction (FBR). M1/M2 polarization of macrophages is a critical regulator of scaffold-induced FBR. Macrophage colony-stimulating factor (M-CSF), a hematopoietic growth factor, induces macrophages into an M2-like polarized state, leading to immunoregulation and promoting tissue repair. In the present study, we explored the immunomodulatory effects of surface bound M-CSF on poly-l-lactic acid (PLLA)-induced FBR. M-CSF was immobilized on the surface of PLLA via plasma immersion ion implantation (PIII). M-CSF functionalized PLLA, PLLA-only, and PLLA+PIII were assessed in an IL-1ß luciferase reporter mouse to detect real-time levels of IL-1ß expression, reflecting acute inflammation in vivo. Additionally, these different treated scaffolds were implanted subcutaneously into wild-type mice to explore the effect of M-CSF in polarization of M2-like macrophages (CD68+/CD206+), related cytokines (pro-inflammatory: IL-1ß, TNF and MCP-1; anti-inflammatory: IL-10 and TGF-ß), and angiogenesis (CD31) by immunofluorescent staining. Our data demonstrated that IL-1ß activity in M-CSF functionalized scaffolds was ∼50% reduced compared to PLLA-only at day 1 (p < 0.01) and day 2 (p < 0.05) post-implantation. There were >2.6-fold more CD206+ macrophages in M-CSF functionalized PLLA compared to PLLA-only at day 7 (p < 0.001), along with higher levels of IL-10 at both day 7 (p < 0.05) and day 14 (p < 0.01), and TGF-ß at day 3 (p < 0.05), day 7 (p < 0.05), and day 14 (p < 0.001). Lower levels of pro-inflammatory cytokines were also detected in M-CSF functionalized PLLA in the early phase of the immune response compared to PLLA-only: a ∼58% decrease at day 3 in IL-1ß; a ∼91% decrease at day 3 and a ∼66% decrease at day 7 in TNF; and a ∼60% decrease at day 7 in MCP-1. Moreover, enhanced angiogenesis inside and on/near the scaffold was observed in M-CSF functionalized PLLA compared to PLLA-only at day 3 (p < 0.05) and day 7 (p < 0.05), respectively. Overall, M-CSF functionalized PLLA enhanced CD206+ macrophage polarization and angiogenesis, consistent with lower levels of pro-inflammatory cytokines and higher levels of anti-inflammatory cytokines in early stages of the host response, indicating potential immunoregulatory functions on the local environment.

Bioactive Materials Facilitating Targeted Local Modulation of Inflammation.

Cardiovascular disease is an inflammatory disorder that may benefit from appropriate modulation of inflammation. Systemic treatments lower cardiac events but have serious adverse effects. Localized modulation of inflammation in current standard treatments such as bypass grafting may more effectively treat CAD. The present study investigated a bioactive vascular graft coated with the macrophage polarizing cytokine interleukin-4. These grafts repolarize macrophages to anti-inflammatory phenotypes, leading to modulation of the pro-inflammatory microenvironment and ultimately to a reduction of foreign body encapsulation and inhibition of neointimal hyperplasia development. These resulting functional improvements have significant implications for the next generation of synthetic vascular grafts.

Rapid Endothelialization of Off-the-Shelf Small Diameter Silk Vascular Grafts.

Synthetic vascular grafts for small diameter revascularization are lacking. Clinically available conduits expanded polytetrafluorethylene and Dacron fail acutely due to thrombosis and in the longer term from neointimal hyperplasia. We report the bioengineering of a cell-free, silk-based vascular graft. In vitro we demonstrate strong, elastic silk conduits that support rapid endothelial cell attachment and spreading while simultaneously resisting blood clot and fibrin network formation. In vivo rat studies show complete graft patency at all time points, rapid endothelialization, and stabilization and contraction of neointimal hyperplasia. These studies show the potential of silk as an off-the-shelf small diameter vascular graft.

Integration of induced pluripotent stem cell-derived endothelial cells with polycaprolactone/gelatin-based electrospun scaffolds for enhanced therapeutic angiogenesis.

BACKGROUND: Induced pluripotent stem-cell derived endothelial cells (iPSC-ECs) can be generated from any somatic cell and their iPSC sources possess unlimited self-renewal. Previous demonstration of their proangiogenic activity makes them a promising cell type for treatment of ischemic injury. As with many other stem cell approaches, the low rate of in-vivo survival has been a major limitation to the efficacy of iPSC-ECs to date. In this study, we aimed to increase the in-vivo lifetime of iPSC-ECs by culturing them on electrospun polycaprolactone (PCL)/gelatin scaffolds, before quantifying the subsequent impact on their proangiogenic function. METHODS: iPSC-ECs were isolated and stably transfected with a luciferase reporter to facilitate quantification of cell numbers and non-invasive imaging in-vivo PCL/gelatin scaffolds were engineered using electrospinning to obtain woven meshes of nanofibers. iPSC-ECs were cultured on scaffolds for 7 days. Subsequently, cell growth and function were assessed in vitro followed by implantation in a mouseback subcutaneous model for 7 days. RESULTS: Using a matrix of conditions, we found that scaffold blends with ratios of PCL:gelatin of 70:30 (PG73) spun at high flow rates supported the greatest levels of iPSC-EC growth, retention of phenotype, and function in vitro. Implanting iPSC-ECs seeded on PG73 scaffolds in vivo improved their survival up to 3 days, compared to cells directly injected into control wounds, which were no longer observable within 1 h. Enhanced engraftment improved blood perfusion, observed through non-invasive laser Doppler imaging. Immunohistochemistry revealed a corresponding increase in host angiogenic mechanisms characterized by the enhanced recruitment of macrophages and the elevated expression of proangiogenic cytokines vascular endothelial growth factor and placental growth factor. CONCLUSIONS: Knowledge of these mechanisms combined with a deeper understanding of the scaffold parameters influencing this function provides the groundwork for optimizing future iPSC-EC therapies utilizing engraftment platforms. The development of combined scaffold and iPSC-EC therapies could ultimately improve therapeutic angiogenesis and the treatment of ischemic injury.

Modulation of Retrograde Trafficking of KCa3.1 in a Polarized Epithelium.

In epithelia, the intermediate conductance, Ca2+-activated K+ channel (KCa3.1) is targeted to the basolateral membrane (BLM) where this channel plays numerous roles in absorption and secretion. A growing body of research suggests that the membrane resident population of KCa3.1 may be critical in clinical manifestation of diseases. In this study, we investigated the key molecular components that regulate the degradation of KCa3.1 using a Fisher rat thyroid cell line stably expressing KCa3.1. Using immunoblot, Ussing chamber, and pharmacological approaches, we demonstrated that KCa3.1 is targeted exclusively to the BLM, provided a complete time course of degradation of KCa3.1 and degradation time courses of the channel in the presence of pharmacological inhibitors of ubiquitylation and deubiquitylation to advance our understanding of the retrograde trafficking of KCa3.1. We provide a complete degradation profile of KCa3.1 and that the degradation is via an ubiquitin-dependent pathway. Inhibition of E1 ubiquitin activating enzyme by UBEI-41 crippled the ability of the cells to internalize the channel, shown by the increased BLM surface expression resulting in an increased function of the channel as measured by a DCEBIO sensitive K+ current. Additionally, the involvement of deubiquitylases and degradation by the lysosome were also confirmed by treating the cells with PR-619 or leupeptin/pepstatin, respectively; which significantly decreased the degradation rate of membrane KCa3.1. Additionally, we provided the first evidence that KCa3.1 channels were not deubiquitylated at the BLM. These data further define the retrograde trafficking of KCa3.1, and may provide an avenue for therapeutic approach for treatment of disease.

Evaluation of synthetic vascular grafts in a mouse carotid grafting model.

Current animal models for the evaluation of synthetic grafts are lacking many of the molecular tools and transgenic studies available to other branches of biology. A mouse model of vascular grafting would allow for the study of molecular mechanisms of graft failure, including in the context of clinically relevant disease states. In this study, we comprehensively characterise a sutureless grafting model which facilitates the evaluation of synthetic grafts in the mouse carotid artery. Using conduits electrospun from polycaprolactone (PCL) we show the gradual development of a significant neointima within 28 days, found to be greatest at the anastomoses. Histological analysis showed temporal increases in smooth muscle cell and collagen content within the neointima, demonstrating its maturation. Endothelialisation of the PCL grafts, assessed by scanning electron microscopy (SEM) analysis and CD31 staining, was near complete within 28 days, together replicating two critical aspects of graft performance. To further demonstrate the potential of this mouse model, we used longitudinal non-invasive tracking of bone-marrow mononuclear cells from a transgenic mouse strain with a dual reporter construct encoding both luciferase and green fluorescent protein (GFP). This enabled characterisation of mononuclear cell homing and engraftment to PCL using bioluminescence imaging and histological staining over time (7, 14 and 28 days). We observed peak luminescence at 7 days post-graft implantation that persisted until sacrifice at 28 days. Collectively, we have established and characterised a high-throughput model of grafting that allows for the evaluation of key clinical drivers of graft performance.

Non-invasive tracking of injected bone marrow mononuclear cells to injury and implanted biomaterials.

Biomaterial scaffolds enhancing the engraftment of transplanted bone-marrow mononuclear cells (BM-MNC) have enormous potential for tissue regeneration applications. However, development of appropriate materials is challenging given the precise microenvironments required to support BM-MNC engraftment and function. In this study, we have developed a non-invasive, real-time tracking model of injected BM-MNC engraftment to wounds and implanted biomaterial scaffolds. BM-MNCs, encoded with firefly luciferase and enhanced GFP reporter genes, were tail vein injected into subcutaneously wounded mice. Luciferase-dependent cell bioluminescence curves revealed our injected BM-MNCs homed to and engrafted within subcutaneous wound sites over the course of 21days. Further immunohistochemical characterization showed that these engrafted cells drove functional changes by increasing the number of immune cells present at early time points and remodelling cell phenotypes at later time points. Using this model, we subcutaneously implanted electrospun polycaprolactone (PCL) and PCL/Collagen scaffolds, to determine differences in exogenous BM-MNC response to these materials. Following BM-MNC injection, immunohistochemical analysis revealed a high exogenous BM-MNC density around the periphery of PCL scaffolds consistent with a classical foreign body response. In contrast, transplanted BM-MNCs engrafted throughout PCL/Collagen scaffolds indicating an improved biological response. Importantly, these differences were closely correlated with the real-time bioluminescence curves, with PCL/Collagen scaffolds exhibiting a∼2-fold increase in maximum bioluminescence compared with PCL scaffolds. Collectively, these results demonstrate a new longitudinal cell tracking model that can non-invasively determine transplanted BM-MNC homing and engraftment to biomaterials, providing a valuable tool to inform the design scaffolds that help augment current BM-MNC tissue engineering strategies. STATEMENT OF SIGNIFICANCE: Tracking the dynamic behaviour of transplanted bone-marrow mononuclear cells (BM-MNCs) is a long-standing research goal. Conventional methods involving contrast and tracer agents interfere with cellular function while also yielding false signals. The use of bioluminescence addresses these shortcomings while allowing for real-time non-invasive tracking in vivo. Given the failures of transplanted BM-MNCs to engraft into injured tissue, biomaterial scaffolds capable of attracting and enhancing BM-MNC engraftment at sites of injury are highly sought in numerous tissue engineering applications. To this end, the results from this study demonstrate a new longitudinal tracking model that can non-invasively determine exogenous BM-MNC homing and engraftment to biomaterials, providing a valuable tool to inform the design of scaffolds with implications for countless tissue engineering applications.

Far infrared thermal detectors for laser radiometry using a carbon nanotube array.

We present a description of a 1.5 mm long, vertically aligned carbon nanotube array (VANTA) on a thermopile and separately on a pyroelectric detector. Three VANTA samples, having average lengths of 40 µm, 150 µm, and 1.5 mm were evaluated with respect to reflectance at a laser wavelength of 394 µm(760 GHz), and we found that the reflectance decreases substantially with increasing tube length, ranging from 0.38 to 0.23 to 0.01, respectively. The responsivity of the thermopile by electrical heating (98.4 mA/W) was equal to that by optical heating (98.0 mA/W) within the uncertainty of the measurement. We analyzed the frequency response and temporal response and found a thermal decay period of 500 ms, which is consistent with the specific heat of comparable VANTAs in the literature. The extremely low (0.01) reflectance of the 1.5 mm VANTAs and the fact that the array is readily transferable to the detector's surface is, to our knowledge, unprecedented.

Genome-wide haplotype association mapping in mice identifies a genetic variant in CER1 associated with BMD and fracture in southern Chinese women.

BMD is a heritable trait and risk indicator for osteoporosis. In this study, we used a genome-wide haplotype association mapping (HAM) approach to identify a haplotype block within Cer1 that partitions inbred mice strains into high and low BMD groups. A cohort of 1083 high and low BMD human subjects were studied, and a nonsynonymous SNP (rs3747532) in human CER1 was identified to be associated with increased risk of both low BMD in premenopausal women (OR: 2.2; 95% CI: 1.0-4.6; p < 0.05) and increased risk of vertebral fractures (OR: 1.82, p = 0.025) in the postmenopausal cohort. We also showed that Cer1 is expressed in mouse bone and growth plate by RT-PCR, immunohistochemistry, and in situ hybridization, consistent with polymorphisms potentially influencing BMD. Our successful identification of an association with CER1 in humans together with our mouse study suggests that CER1 may play a role in the development of bone or its metabolism. Our study highlights the use of publicly available databases for rapidly surveying the genome for quantitative trait loci.