Robust Heart Rate Variability Measurement from Facial Videos.

Remote Photoplethysmography (rPPG) is a contactless method that enables the detection of various physiological signals from facial videos. rPPG utilizes a digital camera to detect subtle changes in skin color to measure vital signs such as heart rate variability (HRV), an important biomarker related to the autonomous nervous system. This paper presents a novel contactless HRV extraction algorithm, WaveHRV, based on the Wavelet Scattering Transform technique, followed by adaptive bandpass filtering and inter-beat-interval (IBI) analysis. Furthermore, a novel method is introduced to preprocess noisy contact-based PPG signals. WaveHRV is bench-marked against existing algorithms and public datasets. Our results show that WaveHRV is promising and achieves the lowest mean absolute error (MAE) of 10.5 ms and 6.15 ms for RMSSD and SDNN on the UBFCrPPG dataset.

Dynamic alterations of H3K4me3 and H3K27me3 at ADAM17 and Jagged-1 gene promoters cause an inflammatory switch of endothelial cells.

Histone protein modifications control the inflammatory state of many immune cells. However, how dynamic alteration in histone methylation causes endothelial inflammation and apoptosis is not clearly understood. To examine this, we explored two contrasting histone methylations; an activating histone H3 lysine 4 trimethylation (H3K4me3) and a repressive histone H3 lysine 27 trimethylation (H3K27me3) in endothelial cells (EC) undergoing inflammation. Through computer-aided reconstruction and 3D printing of the human coronary artery, we developed a unique model where EC were exposed to a pattern of oscillatory/disturbed flow as similar to in vivo conditions. Upon induction of endothelial inflammation, we detected a significant rise in H3K4me3 caused by an increase in the expression of SET1/COMPASS family of H3K4 methyltransferases, including MLL1, MLL2, and SET1B. In contrast, EC undergoing inflammation exhibited truncated H3K27me3 level engendered by EZH2 cytosolic translocation through threonine 367 phosphorylation and an increase in the expression of histone demethylating enzyme JMJD3 and UTX. Additionally, many SET1/COMPASS family of proteins, including MLL1 (C), MLL2, and WDR5, were associated with either UTX or JMJD3 or both and such association was elevated in EC upon exposure to inflammatory stimuli. Dynamic enrichment of H3K4me3 and loss of H3K27me3 at Notch-associated gene promoters caused ADAM17 and Jagged-1 derepression and abrupt Notch activation. Conversely, either reducing H3K4me3 or increasing H3K27me3 in EC undergoing inflammation attenuated Notch activation, endothelial inflammation, and apoptosis. Together, these findings indicate that dynamic chromatin modifications may cause an inflammatory and apoptotic switch of EC and that epigenetic reprogramming can potentially improve outcomes in endothelial inflammation-associated cardiovascular diseases.

A new roadmap for social medicine curriculum design based on mixed methods student and faculty evaluations of the preclinical curriculum.

BACKGROUND: To support the development of social medicine curricula that empower medical school graduates to redress health inequities, we conducted a mixed methods student and faculty evaluation of an expanded and innovative preclinical social medicine curriculum. METHODS: We implemented a longitudinal, interactive preclinical social medicine curriculum that was closely integrated with foundational science teaching then conducted a survey-based mixed methods student and faculty curriculum evaluation. Based on these results, we propose a novel conceptual roadmap for social medicine curriculum design. RESULTS: Student and faculty evaluations of an expanded and innovative longitudinal preclinical social medicine curriculum were strongly favorable. Both student and faculty respondents indicated a particular desire for deeper coverage of race and poverty among other social medicine domains. Qualitative student evaluations highlighted the importance of faculty champions to social medicine teaching as well as the educational impact of stories that exemplify the practical impact of the social determinants of health on specific patient experiences. Qualitative faculty evaluations pointed to the challenges of curriculum integration and the need for faculty career development in social medicine teaching. CONCLUSIONS: Based on mixed methods student and faculty curriculum evaluation data, we propose a novel conceptual roadmap for the design of social medicine curricula at other institutions.

The design and implementation of a longitudinal social medicine curriculum at the University of Vermont's Larner College of Medicine.

BACKGROUND: Despite an abundant literature advocating that social determinants of health (SDH) be taught during undergraduate medical education, there are few detailed descriptions of how to design and implement longitudinal core curricula that is delivered to all students and accomplishes this goal. METHODS: In this paper, we describe the design and implementation of a social medicine curriculum at the University of Vermont's Larner College of Medicine (UVM Larner). Using Kern's principles, we designed a longitudinal curriculum that extends through both preclinical and clinical training for all students and focused on integrating SDH material directly into basic science and clinical training. RESULTS: We successfully developed and implemented two primary tools, a "Social Medicine Theme of the Week" (SMTW) in preclinical training, and SDH rounds in the clinical setting to deliver SDH content to all learners at UVM Larner. CONCLUSIONS: Extensive student-faculty partnerships, robust needs assessment, and focusing on longitudinal and integrated SDH content delivery to all students were key features that contributed to successful design and implementation.

Characterization of Button Loci that Promote Homologous Chromosome Pairing and Cell-Type-Specific Interchromosomal Gene Regulation.

Homologous chromosomes colocalize to regulate gene expression in processes including genomic imprinting, X-inactivation, and transvection. In Drosophila, homologous chromosomes pair throughout development, promoting transvection. The "button" model of pairing proposes that specific regions along chromosomes pair with high affinity. Here, we identify buttons interspersed across the fly genome that pair with their homologous sequences, even when relocated to multiple positions in the genome. A majority of transgenes that span a full topologically associating domain (TAD) function as buttons, but not all buttons contain TADs. Additionally, buttons are enriched for insulator protein clusters. Fragments of buttons do not pair, suggesting that combinations of elements within a button are required for pairing. Pairing is necessary but not sufficient for transvection. Additionally, pairing and transvection are stronger in some cell types than in others, suggesting that pairing strength regulates transvection efficiency between cell types. Thus, buttons pair homologous chromosomes to facilitate cell-type-specific interchromosomal gene regulation.

Early Immunomodulatory Effects of Implanted Human Perivascular Stromal Cells During Bone Formation.

Human perivascular stem/stromal cells (PSC) are a multipotent mesodermal progenitor cell population defined by their perivascular residence. PSC are most commonly derived from subcutaneous adipose tissue, and recent studies have demonstrated the high potential for clinical translation of this fluorescence-activated cell sorting-derived cell population for bone tissue engineering. Specifically, purified PSC induce greater bone formation than unpurified stroma taken from the same patient sample. In this study, we examined the differences in early innate immune response to human PSC or unpurified stroma (stromal vascular fraction [SVF]) during the in vivo process of bone formation. Briefly, SVF or PSC from the same patient sample were implanted intramuscularly in the hindlimb of severe combined immunodeficient (SCID) mice using an osteoinductive demineralized bone matrix carrier. Histological examination of early inflammatory infiltrates was examined by hematoxylin and eosin and immunohistochemical staining (Ly-6G, F4/80). Results showed significantly greater neutrophilic and macrophage infiltrates within and around SVF in comparison to PSC-laden implants. Differences in early postoperative inflammation among SVF-laden implants were associated with reduced osteogenic differentiation and bone formation. Similar findings were recapitulated with PSC implantation in immunocompetent mice. Exaggerated postoperative inflammation was associated with increased IL-1α, IL-1ß, IFN-γ, and TNF-α gene expression among SVF samples, and conversely increased IL-6 and IL-10 expression among PSC samples. These data document a robust immunomodulatory effect of implanted PSC, and an inverse correlation between host inflammatory cell infiltration and stromal progenitor cell-mediated ossification.

Vertebral Implantation of NELL-1 Enhances Bone Formation in an Osteoporotic Sheep Model.

BACKGROUND: Vertebral compression fractures related to osteoporosis greatly afflict the aging population. One of the most commonly used therapy today is balloon kyphoplasty. However, this treatment is far from ideal and is associated with significant side effects. NELL-1, an osteoinductive factor that possesses both pro-osteogenic and anti-osteoclastic properties, is a promising candidate for an alternative to current treatment modalities. This study utilizes the pro-osteogenic properties of recombinant human NELL-1 (rhNELL-1) in lumbar spine vertebral defect model in osteoporotic sheep. METHODS: Osteoporosis was induced through ovariectomy, dietary depletion of calcium and vitamin D, and steroid administration. After osteoporotic induction, lumbar vertebral body defect creation was performed. Sheep were randomly implanted with the control vehicle, comprised of hyaluronic acid (HA) with hydroxyapatite-coated ß-tricalcium phosphate (ß-TCP), or the treatment material of rhNELL-1 protein lyophilized onto ß-TCP mixed with HA. Analysis of lumbar spine defect healing was performed by radiographic, histologic, and computer-simulated biomechanical testing. RESULTS: rhNELL-1 treatment significantly increased lumbar spine bone formation, as determined by bone mineral density, % bone volume, and mean cortical width as assessed by micro-computed tomography. Histological analysis revealed a significant increase in bone area and osteoblast number and decrease in osteoclast number around the implant site. Computer-simulated biomechanical analysis of trabecular bone demonstrated that rhNELL-1-treatment resulted in a significantly more stress-resistant composition. CONCLUSION: Our findings suggest rhNELL-1-based vertebral implantation successfully improved cortical and cancellous bone regeneration in the lumbar spine of osteoporotic sheep. rhNELL-1-based bone graft substitutes represent a potential new local therapy.

NELL-1 in the treatment of osteoporotic bone loss.

NELL-1 is a secreted, osteoinductive protein whose expression rheostatically controls skeletal ossification. Overexpression of NELL-1 results in craniosynostosis in humans and mice, whereas lack of Nell-1 expression is associated with skeletal undermineralization. Here we show that Nell-1-haploinsufficient mice have normal skeletal development but undergo age-related osteoporosis, characterized by a reduction in osteoblast:osteoclast (OB:OC) ratio and increased bone fragility. Recombinant NELL-1 binds to integrin ß1 and consequently induces Wnt/ß-catenin signalling, associated with increased OB differentiation and inhibition of OC-directed bone resorption. Systemic delivery of NELL-1 to mice with gonadectomy-induced osteoporosis results in improved bone mineral density. When extended to a large animal model, local delivery of NELL-1 to osteoporotic sheep spine leads to significant increase in bone formation. Altogether, these findings suggest that NELL-1 deficiency plays a role in osteoporosis and demonstrate the potential utility of NELL-1 as a combination anabolic/antiosteoclastic therapeutic for bone loss.

Lentiviral delivery of PPARγ shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture.

INTRODUCTION: Skeletal aging is associated not only with alterations in osteoblast (OB) and osteoclast (OC) number and activity within the basic metabolic unit, but also with increased marrow adiposity. Peroxisome proliferator-activated receptor gamma (PPARγ) is commonly considered the master transcriptional regulator of adipogenesis, however, it has known roles in osteoblast and osteoclast function as well. Here, we designed a lentiviral delivery system for PPARγ shRNA, and examined its effects in vitro on bone marrow stromal cells (BMSC) and in a mouse intramedullary injection model. METHODS: PPARγ shRNA was delivered by a replication-deficient lentiviral vector, after in vitro testing to confirm purity, concentration, and efficacy for Pparg transcript reduction. Next, control green fluorescent protein lentivirus or PPARγ shRNA expressing lentivirus were delivered by intramedullary injection into the femoral bone marrow of male SCID mice. Analyses included daily monitoring of animal health, and postmortem analysis at 4 weeks. Postmortem analyses included high resolution microcomputed tomography (microCT) reconstructions and analysis, routine histology and histomorphometric analysis, quantitative real time polymerase chain reaction analysis of Pparg transcript levels, and immunohistochemical analysis for markers of adipocytes (PPARγ, fatty acid binding protein 4 [FABP4]), osteoblasts (alkaline phosphatase [ALP], osteocalcin [OCN]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP], Cathepsin K). RESULTS: In vitro, PPARγ shRNA delivery significantly reduced Pparg expression in mouse BMSC, accompanied by a significant reduction in lipid droplet accumulation. In vivo, a near total reduction in mature marrow adipocytes was observed at 4 weeks postinjection. This was accompanied by significant reductions in adipocyte-specific markers. Parameters of trabecular bone were significantly increased by both microCT and histomorphometric analysis. By immunohistochemical staining and semi-quantification, a significant increase in OCN+osteoblasts and decrease in TRAP+multinucleated osteoclasts was observed with PPARγ shRNA treatment. DISCUSSION: These findings suggest that acute loss of PPARγ in the bone marrow compartment has a significant role beyond anti-adipose effects. Specifically, we found pro-osteoblastogenic, anti-osteoclastic effects after PPARγ shRNA treatment, resulting in improved trabecular bone architecture. Future studies will examine the isolated and direct effects of PPARγ shRNA on OB and OC cell types, and it may help determine whether PPARγ antagonists are potential therapeutic agents for osteoporotic bone loss.

Human perivascular stem cells show enhanced osteogenesis and vasculogenesis with Nel-like molecule I protein.

An ideal mesenchymal stem cell (MSC) source for bone tissue engineering has yet to be identified. Such an MSC population would be easily harvested in abundance, with minimal morbidity and with high purity. Our laboratories have identified perivascular stem cells (PSCs) as a candidate cell source. PSCs are readily isolatable through fluorescent-activated cell sorting from adipose tissue and have been previously shown to be indistinguishable from MSCs in the phenotype and differentiation potential. PSCs consist of two distinct cell populations: (1) pericytes (CD146+, CD34-, and CD45-), which surround capillaries and microvessels, and (2) adventitial cells (CD146-, CD34+, and CD45-), found within the tunica adventitia of large arteries and veins. We previously demonstrated the osteogenic potential of pericytes by examining pericytes derived from the human fetal pancreas, and illustrated their in vivo trophic and angiogenic effects. In the present study, we used an intramuscular ectopic bone model to develop the translational potential of our original findings using PSCs (as a combination of pericytes and adventitial cells) from human white adipose tissue. We evaluated human PSC (hPSC)-mediated bone formation and vascularization in vivo. We also examined the effects of hPSCs when combined with the novel craniosynostosis-associated protein, Nel-like molecule I (NELL-1). Implants consisting of the demineralized bone matrix putty combined with NELL-1 (3 µg/µL), hPSC (2.5×10(5) cells), or hPSC+NELL-1, were inserted in the bicep femoris of SCID mice. Bone growth was evaluated using microcomputed tomography, histology, and immunohistochemistry over 4 weeks. Results demonstrated the osteogenic potential of hPSCs and the additive effect of hPSC+NELL-1 on bone formation and vasculogenesis. Comparable osteogenesis was observed with NELL-1 as compared to the more commonly used bone morphogenetic protein-2. Next, hPSCs induced greater implant vascularization than the unsorted stromal vascular fraction from patient-matched samples. Finally, we observed an additive effect on implant vascularization with hPSC+NELL-1 by histomorphometry and immunohistochemistry, accompanied by in vitro elaboration of vasculogenic growth factors. These findings hold significant implications for the cell/protein combination therapy hPSC+NELL-1 in the development of strategies for vascularized bone regeneration.

Perivascular stem cells: a prospectively purified mesenchymal stem cell population for bone tissue engineering.

Adipose tissue is an ideal source of mesenchymal stem cells for bone tissue engineering: it is largely dispensable and readily accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which leads to unreliable bone formation. In the present study, we prospectively purified human perivascular stem cells (PSCs) from adipose tissue and compared their bone-forming capacity with that of traditionally derived SVF. PSCs are a population (sorted by fluorescence-activated cell sorting) of pericytes (CD146+CD34-CD45-) and adventitial cells (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. Here, we found that PSCs underwent osteogenic differentiation in vitro and formed bone after intramuscular implantation without the need for predifferentiation. We next sought to optimize PSCs for in vivo bone formation, adopting a demineralized bone matrix for osteoinduction and tricalcium phosphate particle formulation for protein release. Patient-matched, purified PSCs formed significantly more bone in comparison with traditionally derived SVF by all parameters. Recombinant bone morphogenetic protein 2 increased in vivo bone formation but with a massive adipogenic response. In contrast, recombinant Nel-like molecule 1 (NELL-1; a novel osteoinductive growth factor) selectively enhanced bone formation. These studies suggest that adipose-derived human PSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, PSCs are a stem cell-based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy. Finally, NELL-1 is a candidate growth factor able to induce human PSC osteogenesis.

Use of human perivascular stem cells for bone regeneration.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized). FSDs are bicortical and are stabilized with a polyethylene bar and K-wires. The FSD described is also a critical size defect, which does not significantly heal on its own. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Additive effects of sonic hedgehog and Nell-1 signaling in osteogenic versus adipogenic differentiation of human adipose-derived stromal cells.

A theoretical inverse relationship exists between osteogenic (bone forming) and adipogenic (fat forming) mesenchymal stem cell (MSC) differentiation. This inverse relationship in theory partially underlies the clinical entity of osteoporosis, in which marrow MSCs have a preference for adipose differentiation that increases with age. Two pro-osteogenic cytokines have been recently studied that each also possesses antiadipogenic properties: Sonic Hedgehog (SHH) and NELL-1 proteins. In the present study, we assayed the potential additive effects of the biologically active N-terminus of SHH (SHH-N) and NELL-1 protein on osteogenic and adipogenic differentiation of human primary adipose-derived stromal cell (hASCs). We observed that both recombinant SHH-N and NELL-1 protein significantly enhanced osteogenic differentiation and reduced adipose differentiation across all markers examined (alkaline phosphatase, Alizarin red and Oil red O staining, and osteogenic gene expression). Moreover, SHH-N and NELL-1 directed signaling produced additive effects on the pro-osteogenic and antiadipogenic differentiation of hASCs. NELL-1 treatment increased Hedgehog signaling pathway expression; coapplication of the Smoothened antagonist Cyclopamine reversed the pro-osteogenic effect of NELL-1. In summary, Hedgehog and Nell-1 signaling exert additive effects on the pro-osteogenic and antiadipogenic differentiation of ASCs. These studies suggest that the combination cytokines SHH-N+NELL-1 may represent a viable future technique for inducing the osteogenic differentiation of MSCs.