Effects of in utero exposure to Δ-9-tetrahydrocannabinol on cardiac extracellular matrix expression and vascular transcriptome in rhesus macaques.

Delta-9-tetrahydrocannabinol (THC), the psychoactive component of cannabis, remains a schedule I substance, thus safety data regarding the effects on the cardiovascular and prenatal health are limited. Importantly, there is evidence showing prenatal cannabis exposure can negatively impact fetal organ development, including the cardiovascular system. THC can cross the placenta and bind to cannabinoid receptors expressed in the developing fetus, including on endothelial cells. To understand the impact of prenatal THC exposure on the fetal cardiovascular system, we used our rhesus macaque model of prenatal daily edible THC consumption. Before conception, animals were acclimated to THC (2.5 mg/7 kg/day, equivalent to a heavy medical cannabis dose) and maintained on this dose daily throughout pregnancy. Fetal tissue samples were collected at gestational day 155 (full term is 168 days). Our model showed that in utero THC exposure was associated with a decreased heart weight-to-body weight ratio in offspring, warranting further mechanistic investigation. Histological examination of the fetal cardiac and vascular tissues did not reveal any significant effect of THC exposure on the maturity of collagen within the fetal heart or the aorta. Total collagen III expression and elastin production and organization were unchanged. However, bulk RNA-sequencing of vascular cells in the umbilical vein, umbilical artery, and fetal aorta demonstrated that THC alters the fetal vascular transcriptome and is associated with upregulated expression of genes involved in carbohydrate metabolism and inflammation. The long-term consequences of these findings are unknown but suggest that prenatal THC exposure may affect cardiovascular development in offspring.NEW & NOTEWORTHY Prenatal cannabis use is increasing and despite the public health relevance, there is limited safety data regarding its impact on offspring cardiovascular health outcomes. We used a translational, nonhuman primate model of daily edible Δ-9-tetrahydrocannabinol (THC) consumption during pregnancy to assess its effects on the fetal cardiovascular system. THC-exposed fetal vascular tissues displayed upregulation of genes involved in cellular metabolism and inflammation, suggesting that prenatal THC exposure may impact fetal vascular tissues.

Oligo cyc-DEP: On-chip cyclic immunofluorescence profiling of cell-derived nanoparticles.

We present a follow-on technique for the cyclic-immunofluorescence profiling of suspension particles isolated using dielectrophoresis. The original lab-on-chip technique ("cyc-DEP" [cyclic immunofluorescent imaging on dielectrophoretic chip]) was designed for the multiplex surveillance of circulating biomarkers. Nanoparticles were collected from low-volume liquid biopsies using microfluidic dielectrophoretic chip technology. Subsequent rounds of cyclic immunofluorescent labeling and quenching were imaged and quantified with a custom algorithm to detect multiple proteins. While cyc-DEP improved assay multiplicity, long runtimes threatened its clinical adoption. Here, we modify the original cyc-DEP platform to reduce assay runtimes. Nanoparticles were formulated from human prostate adenocarcinoma cells and collected using dielectrophoresis. Three proteins were labeled on-chip with a mixture of short oligonucleotide-conjugated antibodies. The sample was then incubated with complementary fluorophore-conjugated oligonucleotides, which were dehybridized using an ethylene carbonate buffer after each round of imaging. Oligonucleotide removal exhibited an average quenching efficiency of 98 ± 3% (n = 12 quenching events), matching the original cyc-DEP platform. The presented "oligo cyc-DEP" platform achieved clinically relevant sample-to-answer times, reducing the duration for three rounds of cyclic immunolabeling from approximately 20 to 6.5 h-a 67% decrease attributed to rapid fluorophore removal and the consolidated co-incubation of antibodies.

cyc-DEP: Cyclic immunofluorescence profiling of particles collected using dielectrophoresis.

Cancer is a highly heterogenous disease that requires precise detection tools and active surveillance methods. Liquid biopsy assays provide an agnostic way to follow the complex trajectory of cancer, providing better patient stratification tools for optimized treatment. Here, we present the development of a low-volume liquid biopsy assay called cyc-DEP (cyclic immunofluorescent imaging on dielectrophoretic chip) to profile biomarkers collected on a dielectrophoretic microfluidic chip platform. To enable on-chip cyclic imaging, we optimized a fluorophore quenching method and sequential rounds of on-chip staining with fluorescently conjugated primary antibodies. cyc-DEP allows for the quantification of a multiplex array of proteins using 25 µl of a patient plasma sample. We utilized nanoparticles from a prostate adenocarcinoma (LNCaP) cell line and a panel of six target proteins to develop our proof-of-concept technique. We then used cyc-DEP to quantify blood plasma levels of target proteins from healthy individuals, low-grade and high-grade prostate cancer patients (n = 3 each) in order to demonstrate that our platform is suitable for liquid biopsy analysis in its present form. To ensure accurate quantification of signal intensities and comparisons between different samples, we incorporated a signal intensity normalization method (fluorescent beads) and a custom signal intensity quantification algorithm that account for the distribution of signal across hundreds of collection regions on each chip. Our technique enabled a threefold improvement in multiplicity for detecting proteins associated with fluid samples, opening doors for early detection, and active surveillance through quantification of a multiplex array of biomarkers from low-volume liquid biopsies.

Conformational and topological correlations in non-frustated triblock copolymers with homopolymers.

The phase behavior of non-frustrated ABC block copolymers polymers, modeling poly(isoprene-b-styrene-b-ethylene oxide) (ISO), is studied using dissipative particle dynamic (DPD) simulations. The phase diagram showed a wide composition range for the alternating gyroid morphology, which can be transformed to a chiral metamaterial. A quantitative analysis of topology was developed, that correlates the location of a block relative to the interface with the block's end-to-end distance. This analysis showed that the A-blocks stretched as they were located deeper in the A-rich region. To further expand the stability of the alternating gyroid phase, A-selective homopolymers of different lengths were co-assembled with the ABC copolymer at several compositions. Topological analysis showed that homopolymers with lengths shorter than or equal to the A-block length filled the middle of the networks, decreasing packing frustration and stabilizing them, while longer homopolymers stretched across the network but allowed for the formation of stable, novel morphologies. Adding homopolymers to triblock copolymer melts increases tunability of the network, offering greater control over the final stable phase and bridging two separate regions in the phase diagram.

Does volunteering decrease burnout? Healthcare professional and student perspectives on burnout and volunteering.

Background: Burnout among healthcare providers is a significant crisis in our healthcare system, especially in the context of the COVID-19 pandemic. The aim of this study was to understand what motivates healthcare workers and students to volunteer in their community as well as examine how volunteering relates to burnout. These findings can help health organizations better meet the needs of healthcare workers, as well as provide insights for non-profits that rely on volunteer professionals. Methods: Healthcare providers (N = 8), graduate healthcare students (N = 10), and undergraduate students (N = 14) who volunteered at community health fairs completed the OLBI burnout assessment and an individual semi-structured interview to characterize their attitudes toward volunteering and its relationship with burnout. Interviews were recorded, transcribed, and analyzed using a phenomenological approach, comparing themes across levels of burnout among providers and students. Results: Participants described that feeling burnt out decreased one's likelihood to volunteer, but also that volunteering prevented burnout. The OLBI scores showed that 79.2 and 20.8% of students were low and moderately burnt out respectively, and 87.5 and 12.5% of health professionals were low and moderately burnt out, respectively. Students volunteered for professional development while healthcare professionals cited a desire for a change in their day-to-day work as a reason to volunteer. Both students and health professionals often volunteered because they wanted to make a difference, it made them feel good, and/or they felt a responsibility to volunteer. COVID-19 had a wide range of effects on burnout and motivations to volunteer. Conclusion: Volunteering may be useful for preventing burnout among healthcare workers and students, but may not be helpful for those already experiencing burnout. Interview responses and the fact that none of the volunteers had high burnout levels according to their OLBI scores suggest those who choose to volunteer may be less burnt out. Healthcare organizations and schools can encourage volunteering by emphasizing the difference healthcare students and professionals can make through volunteering in the community. Increasing convenience and emphasizing professional development can help recruit and retain healthcare student volunteers. Highlighting the chance to diversify their scope of practice may help recruit and retain healthcare professional volunteers.

Pharmacologic targeting of coagulation factors XII and XI by monoclonal antibodies reduces thrombosis in nitinol stents under flow.

BACKGROUND: Cardiovascular implantable devices, such as vascular stents, are critical for the treatment of cardiovascular diseases. However, their success is dependent on robust and often long-term antithrombotic therapies. Yet, the current standard-of-care therapies often pose significant bleeding risks to patients. Coagulation factor (F)XI and FXII have emerged as potentially safe and efficacious targets to safely reduce pathologic thrombin generation in medical devices. OBJECTIVES: To study the efficacy of monoclonal antibody-targeting FXII and FXI of the contact pathway in preventing vascular device-related thrombosis. METHODS: The effects of inhibition of FXII and FXI using function-blocking monoclonal antibodies were examined in a nonhuman primate model of nitinol stent-related thrombosis under arterial and venous flow conditions. RESULTS: We found that function-blocking antibodies of FXII and FXI reduced markers of stent-induced thrombosis in vitro and ex vivo. However, FXI inhibition resulted in more effective mitigation of thrombosis markers under varied flow conditions. CONCLUSION: This work provides further support for the translation of contact pathway of coagulation inhibitors for their adjunctive clinical use with cardiovascular devices.

Development of a novel Guinea Pig model producing transgenerational endothelial transcriptional changes driven by maternal food restriction and a second metabolic insult of high fat diet.

Developmental programming of chronic adverse cardiovascular health outcomes has been studied both using numerous human populations and an array of animal models. However, the mechanisms that produce transgenerational effects have been difficult to study due to a lack of developmentally relevant models. As such, how increased disease risk is carried to the second generation has been poorly studied. We hypothesized that the endothelium which mediates many acute and chronic vascular inflammatory responses is a key player in these effects, and epidemiological studies implicate transgenerational nutritional effects on endothelial health. To study the mutigenerational effects of maternal undernutrition on offspring endothelial health, we developed a model of transgenerational nutritional stress in guinea pigs, a translationally relevant precocial species with a relatively short lifespan. First- and second-generation offspring were subjected to a high fat diet in adolescence to exacerbate negative cardiovascular health. To assess transcriptional changes, we performed bulk RNA-sequencing in carotid artery endothelial cells, with groups stratified as prenatal control or food restricted, and postnatal control or high fat diet. We detected statistically significant gene alterations for each dietary permutation, some of which were unique to treatments and other transcriptional signatures shared by multiple or all conditions. These findings highlight a core group of genes altered by high fat diet that is shared by all cohorts and a divergence of transgenerational effects between the prenatal ad libitum and dietary restriction groups. This study establishes the groundwork for this model to be used to better understand the interplay of prenatal stress and genetic reprogramming.

In Silico Screening and Testing of FDA-Approved Small Molecules to Block SARS-CoV-2 Entry to the Host Cell by Inhibiting Spike Protein Cleavage.

The COVID-19 pandemic began in 2019, but it is still active. The development of an effective vaccine reduced the number of deaths; however, a treatment is still needed. Here, we aimed to inhibit viral entry to the host cell by inhibiting spike (S) protein cleavage by several proteases. We developed a computational pipeline to repurpose FDA-approved drugs to inhibit protease activity and thus prevent S protein cleavage. We tested some of our drug candidates and demonstrated a decrease in protease activity. We believe our pipeline will be beneficial in identifying a drug regimen for COVID-19 patients.

Hemocompatibility of micropatterned biomaterial surfaces is dependent on topographical feature size.

Small-diameter synthetic vascular grafts that have improved hemocompatibility and patency remain an unmet clinical need due to thrombosis. A surface modification that has potential to attenuate these failure mechanisms while promoting an endothelial layer is the micropatterning of luminal surfaces. Anisotropic features have been shown to downregulate smooth muscle cell proliferation, direct endothelial migration, and attenuate platelet adhesion and activation. However, the effect of micropatterning feature size and orientation relative to whole blood flow has yet to be investigated within a systematic study. In this work, hemocompatibility of micropattern grating sizes of 2, 5, and 10 µm were investigated. The thrombogenicity of the micropattern surface modifications were characterized by quantifying FXIIa activity, fibrin formation, and static platelet adhesion in vitro. Additionally, dynamic platelet attachment and end-point fibrin formation were quantified using an established, flowing whole blood ex vivo non-human primate shunt model without antiplatelet or anticoagulant therapies. We observed a higher trend in platelet attachment and significantly increased fibrin formation for larger features. We then investigated the orientation of 2 µm gratings relative to whole blood flow and found no significant differences between the various orientations for platelet attachment, rate of linear platelet attachment, or end-point fibrin formation. MicroCT analysis of micropatterned grafts was utilized to quantify luminal patency. This work is a significant step in the development of novel synthetic biomaterials with improved understanding of hemocompatibility for use in cardiovascular applications.

Molecular modelling of the FOXO4-TP53 interaction to design senolytic peptides for the elimination of senescent cancer cells.

BACKGROUND: Senescent cells accumulate in tissues over time as part of the natural ageing process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to develop a senolytic that acts not only on ageing cells but also senescent cancer cells to prevent cancer recurrence or progression. METHODS: We used molecular modelling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cells both in cell culture and in vivo. FINDINGS: Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it disrupts FOXO4-TP53 foci, activates TP53 mediated apoptosis and preferentially binds FOXO4 compared to TP53. Next, we show that intratumoural delivery of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis and a survival advantage in mouse models of melanoma. Finally, we show that repeated systemic delivery of ES2 to older mice results in reduced senescent cell numbers in the liver with minimal toxicity. INTERPRETATION: Taken together, our results reveal that peptides can be generated to specifically target and eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. FUNDING: This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health & Science University.