bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transglutaminases locate to calcified areas in advanced human atherosclerotic lesions
Authors: Matlung Hanke , Groen Harald C , de Vos Judith , van Walsum Theo , van der Lugt Aad , Niessen Wiro J , Wentzel Jolanda J , van Bavel Ed , Bakker Erik NTP 
Published: June 2021

We investigated the involvement of the two major transglutaminases (TGs), tissue transglutaminase (TG2) and factor XIIIa (FXIIIA), in human atherosclerotic calcification.MethodsCross‐sections of human carotid atherosclerotic plaques were stained for TG2, FXIIIA, the TG‐induced cross‐link and macrophage marker CD68. The correlation of these staining patterns to calcified areas throughout the specimens was tested by multi‐scale 3D mapping of the microscopic images to μCT images.ResultsThe TG‐induced cross‐link was found in 64 ± 8% (mean ± SEM) of the analyzed areas inside the plaques. Calcified areas correlated with this cross‐link (p<0.001). FXIIIA was found to be the dominant transglutaminase (56 ± 10% of the cross‐link‐positive areas), as compared to 11 ± 3% for TG2. Both transglutaminases correlated with the cross‐link (p=<0.001 and p=0.002 respectively). Staining for FXIIIA correlated with CD68 (p<0.001) on tissue level and also co‐localized with CD68 positive cells as shown by double immunofluorescent staining.ConclusionAreas of calcification locate to the presence and activity of transglutaminases in human atherosclerotic arteries. FXIIIA is dominantly present over TG2 in these plaques and may be derived from local macrophages.Supported by the Netherlands Heart Foundation (NHS 2001T038).
Adeno‐associated Virus 9 Vector in the Gene Therapy of Occlusive Vascular Diseases
Authors: Agrawal Devendra Kumar , Pankajakshan Divya , Del Core Michael , Pipinos Iraklis , Hatzoudis George 
Published: June 2021

We analyzed the efficiency and duration of transfection of AAV9 vector containing SM22α promoter with either eGFP or LacZ as reporter genes. The vectors (1 × 1012 pfu) were incorporated into coronary and carotid arteries of swine using microporous balloon catheter‐based gene delivery approach. Segments from all vessels were excised at various time points and immediately frozen for mRNA/protein expression or were processed for frozen sections for X‐Gal, GFP or H&E staining. GFP was visualized distinctly in the medial layer. Frozen sections were stained and β‐galactosidase gene transfer was assessed by blue‐stained cell nuclei. The endothelial and adventitial layers were carefully scraped off from the harvested vessel and total RNA was isolated from the remaining medial layer for qPCR. In the medial layer of coronary and femoral arteries LacZ mRNA expression was visualized 7 days after vector administration. The GFP mRNA expression was detected at 7 days and lasted for at least 2 months showing the prolonged expression of the AAV9‐vector. The GFP expression was confirmed by Western blot. Control arteries did not show any expression of GFP or LacZ. These findings support the use of AAV9 as a vector to effectively transduce a gene in coronary arteries.(Supported by LB692 Nebraska Tobacco settlement Funds and NHLBI‐sponsored Penn Vector Core, University of Pennsylvania)
Bacterial signaling through Nod2/GSK3b protects the small intestine against ischemia‐reperfusion induced injury in mice
Authors: Jobin Christian , Kim Ji Won 
Published: June 2021

Ischemia/reperfusion (I/R) injury represents an important mucosal injury and is one of the leading causes of death in critically ill patients.AimTo investigate the impact of bacteria/Nod2 signaling in I/R‐mediated injury.MethodsThe human colonic HCT‐116 (expressing endogenous Nod2) cells and rat IEC‐6 cells infected with Ad5Nod2 viral vector were used to study muramyl dipeptide (MDP; 50 ug/mL) signaling. GSK3β (Ser9), histone 3 (Ser10), β‐catenin phosphorylation levels were determined by Western blot or immunohistochemistry analysis. Germ‐free (GF), conventionalized (CNV), Nod2 knock‐out (Nod2−/−) and wild‐type (WT) mice (C57BL/6; 6–8 mice/group) were subjected to intestinal I/R by partial occlusion of the 2 distal branches of the superior mesenteric artery for 60 min, followed by 4 h reperfusion. Proliferation was determined using Ki67 IHC staining.ResultsGSK3β phosphorylation and nuclear accumulation of β‐catenin is induced within 60 min in MDP‐stimulated HCT‐116 cells and Ad5Nod2‐infected IEC‐6 cells. I/R‐induced ileal injury worsen in GF mice compared to CNV mice (1.5±0.22 vs 0.7±0.20, p<0.05) which associated with shorten crypt/villin length (11.9±0.19 μm vs 18.1±0.21 μm, p<0.01). Importantly, I/R‐induced ileal injury is more pronounced in Nod2−/− mice compared to WT mice (1.9±0.23 vs 0.9±0.29, p<0.05), with concomitant shorten crypt/villi length (11.8±0.17 μm vs 18.0±0.16 μm, p<0.05) and decreased IL‐6 mRNA accumulation (18.5±5.7 vs. 54.7±9.4). This increased injury response was accompanied by reduced Ki67 staining, GSK3β and H3 phosphorylation.ConclusionBacteria/Nod2 signaling to GSK3β/β‐catenin pathway represents an important host response to the microbiota and may protect the small intestine against I/R‐induced injury.
Proximal tibia shape and locomotion
Authors: Sylvester Adam D 
Published: June 2021

Since the size and shape of articular surfaces are correlated with habitual postures and movements, joint surfaces can provide important clues about animals' locomotor behaviors. Functional analyses of joint morphology are complicated, however, by the fact that variables other than locomotion influence joint morphology.This presentation considers body size and alignment issues in the context of the primate knee, with particular attention to the morphology of the proximal tibia. Body size has been argued to directly affect joint morphology, with larger animals having flatter surfaces to accommodate their size. The nature of this proposed allometric relationship, however, is not clear. While some researchers have argued for a relationship between body size and articular curvature, I argue that joint morphology is primarily due to locomotor differences which are themselves highly correlated with body size. Thus any apparent allometric relationship is secondary to the effect body size has on locomotion.Another critical issue for functional investigations is the alignment of specimens prior to measurement and analyses. Many analyses align specimens mathematically (e.g. Procrustes) or use functionally meaningless anatomical orientations. Because such alignments do not reflect equivalent in vivo orientations, they can not provide reliable bases for elucidating interspecific functional differences.
Role of the reprogramming factors in the induction of pluripotency
Authors: Sridharan Rupa , Tchieu Jason , Mason Mike , Kuoy Edward , Yachechko Robin , Plath Kathrin 
Published: June 2021

Transcription factor induced reprogramming of somatic cells to induced pluripotent stem (iPS) cells, that are molecularly and functionally similar to ES cells, can be achieved by the expression of Oct4, Sox2, Klf4 and cMyc. We study the mechanism of the remarkable changes that accompany this process, including resetting of the somatic cell transcriptome, epigenome and, in female cells, the reactivation of the silent X‐chromosome. In a recent study, we have found that Oct4, Sox2, Klf4, and cMyc cooperate in binding to activate the embryonic housekeeping program, followed by the specific binding of Oct4, Sox2 and Klf4 to genes encoding pluripotency regulators leading to the self‐sustained, pluripotent state. Thus there are distinct temporal steps to the resetting of the transcription program. Currently, we are investigating the epigenetic roadblocks that impede the conversion of partially reprogrammed cells to complete pluripotency.Grant Funding Source: CIRM
Epigenetic control of neural crest specifier genes by histone demethylases jmjd2A
Authors: Strobl‐Mazzulla Pablo Hernan , Bronner‐Fraser Marianne 
Published: June 2021

The neural crest (NC) is a multipotent, migratory stem cell‐like population that is able to differentiate in many cell derivatives including the peripheral nervous system and facial skeleton of vertebrate embryos. Many studies have been focused on transcriptional regulation underlying NC formation, but less is known about epigenetic influences. Accordingly, we find unexpectedly dynamic epigenetic modifications are critical for proper temporal control of neural crest gene expression in vivo. The histone demethylase, JumonjiD2a (JmjD2A), is expressed in the forming neural folds and its loss of function using morpholinos causes a dramatic down‐regulation of neural crest specifier genes. Importantly, chromatin immunoprecipitation reveals stage‐specific interactions of JmjD2A with regulatory regions of neural crest genes as well as associated stage‐specific modifications in the methylation states of critical lysine residues. The results clearly show that epigenetic events are important for regulating the chromatin structure on neural crest genes, providing the first in vivo evidence for an epigenetic modification directly controlling a critical developmental event in the vertebrate embryo.Grant Funding Source: USPHS grant HD037105
The Role of Mitochondrial Activity in the Differentiation of Human Embryonic Stem Cells (ESCs)
Authors: Marsboom Glenn , Paul Jonathan , Toth Peter T. , Zhang Yanmin , Malik Asrar B , Archer Stephen L. , Rehman Jalees 
Published: June 2021

IntroductionLittle is known about the role of metabolic pathways in regulating stem cell function and fate. We hypothesized that ESCs primarily use non‐mitochondrial glycolytic metabolism to avoid the production of ROS (reactive oxygen species) and promote their self‐renewal potential.MethodsWe studied metabolic gene expression and mitochondrial oxygen consumption in NIH‐approved WA01 human ESCs, adult human aortic smooth muscle cells and spontaneously differentiated ESCs. Mitochondrial membrane potential (ΨΔm) was assessed with the potentiometric dye JC1.ResultsThe expression of the mitochondrial biogenesis transcription factor PGC1a was significantly higher in ESCs when compared to mature vascular cells. ESCs had 16.29±3.94 fold (P<0.05 vs AoSMCs) higher levels of PGC1a. However, oxygen consumption in ESCs was only 0.75nmol oxygen/min per million cells and showed minimal increase with addition of the mitochondrial uncoupler FCCP while AoSMC consumed 4.6nmol/min at baseline and increased to 14 nmol/min with FCCP.ConclusionESCs express high levels of mitochondrial genes and contain substantial mitochondrial mass, but they show minimal evidence of mitochondrial oxygen consumption when compared to mature vascular cells. Understanding the metabolic mechanisms underlying ESC self‐renewal may permit optimization of ESC‐based regenerative therapies by modulating their metabolism.Grant Funding Source: NIH and Heart Research Foundation
Reprogramming of Endothelial Progenitor Cells: Unipotency Towards Multipotency
Authors: Johnson Rajasingh , Yu Nanjia , Gomes Ignatius , Malik Asrar B 
Published: June 2021

The development of autologous stem cells with enhanced lineage plasticity for subsequent testing in myocardial ischemia is an integral part of stem‐cell research. We tested the hypothesis that the treatment of EPC with chromatin modifying agents Trichostatin A (TSA‐ inhibitor for histone deacetylase) and 5Aza‐2‐deoxycytidine (5Aza‐ inhibitor for DNA methylation) influence histone acetylation and DNA methylation respectively thereby modify the chromatin structure and up‐regulates embryonic stem gene expression. In a series of preliminary experiments performed with EPCs that had been treated with 5Aza and TSA, we made the following observations: 1) 5Aza and TSA treatment induced expression of pluripotent genes (Oct4 and Nanog), cardiac stem cell marker Flk1 and down regulated expression of endothelial‐specific genes, 2) treated EPCs differentiated into ectodermal (neuronal) and mesodermal (cardiomyocyte) lineages, 3) aceH3K9 activity was higher and HDAC1 activity was lower in treated EPCs than in control cells, 4) functional and histological evidence from our preliminary studies in mouse AMI model also suggest that intracardiac transplantation of reprogrammed EPC improve left ventricular functions and differentiate into cardiomyocytes and endothelial cell phenotypes. Thus, the epigenetic modifying agents can de‐differentiate EPCs into cells with a more multipotent phenotype, and the de‐differentiated EPCs can subsequently differentiate into multiple cell lineages, including cardiomyocytes that could be used as potential source of cells for treating degenerative diseases.Grant Funding Source: NHLBI
Wnt/beta‐catenin signaling in patterning and differentiation of the vertebrate endoderm
Authors: Murtaugh Lewis Charles 
Published: June 2021

The endoderm gives rise to organs as different in anatomy and function as the lungs, colon and liver, and we want to understand the signaling processes controlling this diversification. Our primary interest is in the pancreas, and our work indicates multiple roles for the Wnt/beta (β)‐catenin signaling pathway in its development. Early, Wnt signaling inhibits pancreas specification within the foregut; later, it promotes expansion of multipotent progenitor cells and their differentiation into exocrine acinar tissue. Until recently, our studies have focused on response to Wnt ligands, mediated by the non‐redundant functions of β‐catenin. To better understand the role of Wnt ligand production, while bypassing redundancy between the ligand‐encoding genes, we have targeted the mouse Porcn gene, homologous to Drosophila porcupine. Porcupine is required for wingless secretion and bioactivity, and we find that mouse cells lacking Porcn cannot produce any of several Wnt ligands tested, but retain normal responsiveness to exogenous Wnts. The Porcn mutant defect thus closely resembles that of fly porcupine, indicating a conserved and non‐redundant role for this gene in Wnt production. Wnt production by Porcn mutant cells is rescued by wildtype human PORCN, but not a PORCN mutant recently identified in a pleiotropic congenital syndrome, Focal Dermal Hypoplasia. Mice carrying a conditional Porcn allele are viable, and will provide a novel genetic tool to understand Wnt function in endoderm development and beyond.Supported by the NIH (R01‐DK075072) and Searle Scholars Program (06‐B‐116).
Forefoot varus malalignment: anatomical origin and association with signs of patellofemoral joint osteoarthritis in cadavers
Authors: Lufler Rebecca , Stefanik Joshua , Niu Jingbo , Hoagland Todd , Foster Timothy , Gross Doug 
Published: June 2021

Forefoot varus (FFV) is a non‐weightbearing foot alignment deformity. FFV can cause foot pronation, which may increase stress in the patellofemoral joint (PFJ). The etiology of FFV may be insufficient valgus torsion of the talar head and is a potential risk factor for PFJ osteoarthritis (OA). The purpose of this study was to determine the relationship of: 1) talar torsion to foot alignment and 2) foot alignment to signs of PFJ OA. Alignment and talar torsion were measured in 50 cadaver feet and cartilage damage was scored 0–4 on patellar and trochlear facets of 45 knees. We dichotomized cartilage damage into presence (≥3) or absence of pathology. Pearson's correlation and logistic regression were used to determine the association of foot alignment to talar torsion and PFJ cartilage damage respectively. Regression analyses were adjusted for gender and age; GEE was used to account for correlation between measurements of the same subject. There was no linear association between foot alignment and talar torsion, r = 0.2, p = 0.2. Those in the highest tertile of foot alignment had 3.9 (95% CI 1.0, 15.3) times the odds of medial PFJ cartilage damage and 2.3 (95% CI 0.7, 7.7) times the odds of lateral PFJ cartilage damage as feet in the lowest tertile. The increased odds of medial PFJ cartilage damage suggest that FFV may present a risk to the PFJ. These findings may have important treatment implications, as FFV is modifiable using orthoses. This research was supported by the American College of Rheumatology Research and Education Foundation.Grant Funding Source: None
Distribution and dynamics of nonmuscle myosins IIs in cardiac and in skeletal muscle cells
Authors: Sanger Joseph W. , Wang Jushuo , White Jennifer , Sanger Jean M. 
Published: June 2021

We have proposed a three‐step model for myofibrillogenesis: premyofibrils to nascent myofibrils to mature myofibrils that involves nonmuscle myosin II presence only in the first two steps. However, it has been reported (Takeda et al., 2000) that antibodies directed against nonmuscle myosin IIs stained the Z‐bands of mature myofibrils and intercalated discs of striated muscle cells. We have used similar nonmuscle myosin II antibodies against intact hearts and cultured cardiac and skeletal muscle cells and did not obtained these results. We have transfected cultures of embryonic avian cardiac and skeletal muscle cells with GFP plasmids encoding the three different isoforms of nonmuscle myosin II (A, B or C). The cells were cotransfected with CeruleanFP plasmids encoding sarcomeric alpha‐actinin, a marker for z‐bodies and Z‐bands. The three different GFP‐nonmuscle myosin II proteins localized to the premyofibrils and nascent myofibrils alternating with z‐bodies (as identified by CeFP‐alpha‐actinin localizations). The nonmuscle myosins never localized to the Z‐bands (identified by CeFP‐alpha‐actinin) or any other region of the mature myofibrils in the muscle cells. Our results support the idea that nonmuscle myosin IIs participate in aligning the z‐bodies and their attached actin filaments in premyofibrils and nascent myofibrils, preceding the formation of the mature myofibril.Research support: NIH.
Impact of aging on dermal wound healing
Authors: Brubaker Aleah L , Mahbub Shegufta , Deburghgrave Cory , Kovacs Elizabeth J 
Published: June 2021

Age‐related changes in wound healing have been characterized by alterations in the early inflammatory, proliferative and late remodeling phases. Aging has also been associated with decline of the innate and adaptive immune response, contributing to morbidity and mortality following infection. This study will characterize the effect of advanced age on the inflammatory response to full‐thickness dermal injury in a murine model. In response to injury, aged mice exhibited decreased neutrophil infiltration into the wound as compared to young mice. Further, macrophage inflammatory protein‐2 (MIP‐2) levels remained elevated in aged mice 72 hours post injury, whereas the levels of this chemokine decrease in young mice after 48 hours. Additionally, the dendritic cell population was decreased by 65% in uninjured aged skin. Interestingly, after injury, the dendritic cell population decreased in young mice and increased in aged mice. These data suggest an age‐related differential response to dermal wound healing that may impact the innate immune response to infectious challenge.Grant Funding Source: National Institute of Health R01 AG506080 (EJK) and the Ralph and Marian C Falk Medical Research Trust
Design of Smart Nanofilms for Regenerative Medicine
Authors: Thangaraj Mridhula , Mills David 
Published: June 2021

Wound healing can be classified into several categories based their severity. There are many modalities to achieve wound healing but, the significant variety in wound types fosters a need to develop more advanced modalities that accelerate the wound healing process. This project developed a smart nanofilm using biocompatible polyelectrolyte nanofilm multilayers (PNMs) with embedded growth factors. PNMs were achieved using nebulization, an expedient tool for nanoassembly. Nebulization significantly reduces deposition time and does note compromise nanofilm characteristics. Oppositely charged PNMs, Chondroitin Sulfate (CS), Protamine Sulfate (PS), Poly‐L‐lysine (PLL) and Poly‐Glutamic acid (PGA), served as effective nanoarchitectures. PNMs (with/without platelet derived growth factor (PDGF) were formed on organtypic keratinocyte cultures (with/without wound creation). PNMs were assembled with PDGF as an active component in deep, double layered, or superficial ten‐layer architectures. AFM analysis demonstrated that heightened surface roughness and thickness is achieved with the nebulization technique when compared to the nanoassembly. Nanofilms further enhanced cell adhesion, proliferation and protein synthesis. The study suggests that bioactive, PNMs, acting as a local drug delivery system and dressing can enhance the initial stages of cutaneous wound healing.Grant Funding Source: National Science Foundation
The extracellular matrix plays an active role in muscle regeneration
Authors: Calve Sarah , Simon Hans‐Georg 
Published: June 2021

In urodele amphibians like the newt, skeletal muscle provides a reservoir for uncommitted stem‐like blastema cells that participate in the regeneration of the lost structure. Using 3D immunohistochemical analyses we found dynamic expression of a transitional extracellular matrix (ECM) rich in hyaluronic acid (HA), tenascin‐C (TN) and fibronectin (FN) around skeletal muscle. To test the hypothesis that the ECM plays key instructive roles early in blastema formation, we investigated the behavior of muscle cells under defined culture conditions using time‐lapse microscopy. Our results revealed that distinct components of the early transitional matrix supported myoblast migration and inhibited fusion (HA and TN) whereas ECM expressed around differentiating muscle induced both proliferation and fusion (FN and laminin). In addition, we developed silicone elastomer substrates with a tunable stiffness that can mimic the transverse stiffness of newt skeletal muscle and blastema (17 kPa and 14 kPa, respectively). By combining defined ECM components with a substrate of appropriate native stiffness, we aim to control the behavior of muscle‐derived cells with high precision. The establishment of a physiologically relevant environment and its controlled manipulation in vitro will provide new insights into the control of the differentiated state of the cell. Funded by NIH, Searle Foundation.Grant Funding Source: NIH, Searle Foundation
A histochemical and immunohistochemical investigation of epimorphic regeneration in the representative lizard, Eublepharis macularius
Authors: McLean Katherine , Vickaryous Matthew 
Published: June 2021

Epimorphic regeneration involves cell proliferation and the formation of a regeneration blastema to replace lost or damaged tissues. Although well described for teleosts and urodeles, this type of regeneration is also characteristic of many lizards. The objective of this study was to investigate the events that characterize epimorphic tail regeneration in the leopard gecko, Eublepharis macularius using both histochemistry and immunohistochemistry. Changes in tissue structure and composition of the regenerating tail were examined at various times post‐tail loss. A phase of wound healing follows immediately after tail loss during which time basal cells of the epidermis proliferate and migrate across the wound site. Once the wound is capped proliferation begins deep to the wound epithelium, giving rise to a localized mass of undifferentiated cells, the regeneration blastema. Blastema formation coincides with the onset of angiogenesis and redevelopment of axons and the ependymal tube of the spinal cord. Skeletal tissues differentiate somewhat later during tail regeneration, matching with the expression of Sox9. We also observe Sox9 expression in both mesenchymal and ependymal cells. Our findings demonstrate the utility of lizard tails as a model system for ongoing studies of epimorphic regeneration.Grant Funding Source: NSERC