Miracle-dz/UniFAR-FTUs huggingface dataset:
EZH2 deletion in early mesenchyme compromises postnatal bone microarchitecture and structural integrity and accelerates remodeling

In this study, we examined the functional importance of EZH2 during skeletal development and homeostasis using the conditional deletion of Ezh2 (Ezh2fl/fl) in early mesenchyme with the use of a Prrx‐1‐cre driver mouse (Ezh2+/+). Heterozygous (Ezh2+/−) newborn and 4‐wk‐old mice exhibited increased skeletal size, growth plate size, and weight when compared to the wild‐type control (Ezh2+/+), whereas homozygous deletion of Ezh2 (Ezh2−/−)resultedin skeletal deformities and reduced skeletal size, growth plate size, and weight in newborn and 4‐wk‐old mice. Ezh2−/− mice exhibited enhanced trabecular patterning. Osteogenic cortical and trabecular bone formation was enhanced in Ezh2+/− and Ezh2−/− animals. Ezh2+/− and Ezh2−/− mice displayed thinner cortical bone and decreased mechanical strength compared to the wild‐type control. Differences in cortical bone thickness were attributed to an increased number of osteoclasts, corresponding with elevated levels of the bone turnover markers cross‐linked C‐telopeptide‐1 and tartrate‐resistant acid phosphatase, detected within serum. Moreover, Ezh2+/− mice displayed increased osteoclastogenic potential coinciding with an upregulation of Rankl and M‐csf expression by mesenchymal stem cells (MSCs). MSCs isolated from Ezh2+/− mice also exhibited increased trilineage potential compared with wild‐type bone marrow stromal/stem cells (BMSCs). Gene expression studies confirmed the upregulation of known Ezh2 target genes in Ezh2−/− bone tissue, many of which are involved in Wnt/BMP signaling as promoters of osteogenesis and inhibitors of adipogenesis. In summary, EZH2 appears to be an important orchestrator of skeletal development, postnatal bone remodelling and BMSC fate determination in vitro and in vivo. —Hemming, S. , Cakouros, D. , Codrington, J., Vandyke, K. , Arthur, A. , Zannettino, A. , Gronthos, S. EZH2 deletion in early mesenchyme compromises postnatal bone microarchitecture and structural integrity and accelerates remodeling. FASEB J. 31, 1011–1027 (2017). www.fasebj. org
Effects of Combined Phosphorylation at Ser-617 and Ser-1179 in Endothelial Nitric-oxide Synthase on EC50(Ca2+) Values for Calmodulin Binding and Enzyme Activation*

We have investigated the possible biochemical basis for enhancements in NO production in endothelial cells that have been correlated with agonist- or shear stress-evoked phosphorylation at Ser-1179. We have found that a phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC50(Ca2+) values for calmodulin binding and enzyme activation from the control values of 182 ± 2 and 422 ± 22 nm to 116 ± 2 and 300 ± 10 nm. These are similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q. K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry 47, 7557–7566). Although combining substitutions at Ser-617 and Ser-1179 has no additional effect on maximal synthase activity, cooperativity between the two substitutions completely disinhibits reductase activity and further reduces the EC50(Ca2+) values for calmodulin binding and enzyme activation to 77 ± 2 and 130 ± 5 nm. We have confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 and phosphomimetic substitutions at these positions have similar functional effects. Changes in the biochemical properties of eNOS produced by combined phosphorylation at Ser-617 and Ser-1179 are predicted to substantially increase synthase activity in cells at a typical basal free Ca2+ concentration of 50–100 nm.
Hey1 Basic Helix-Loop-Helix Protein Plays an Important Role in Mediating BMP9-induced Osteogenic Differentiation of Mesenchymal Progenitor Cells*

Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We previously demonstrated that bone morphogenetic protein (BMP) 9 is one of the most potent and yet least characterized BMPs that are able to induce osteogenic differentiation of MSCs both in vitro and in vivo. Here, we conducted gene expression-profiling analysis and identified that Hey1 of the hairy/Enhancer of split-related repressor protein basic helix-loop-helix family was among the most significantly up-regulated early targets in BMP9-stimulated MSCs. We demonstrated that Hey1 expression was up-regulated at the immediate early stage of BMP9-induced osteogenic differentiation. Chromatin immunoprecipitation analysis indicated that Hey1 may be a direct target of the BMP9-induced Smad signaling pathway. Silencing Hey1 expression diminished BMP9-induced osteogenic differentiation both in vitro and in vivo and led to chondrogenic differentiation. Likewise, constitutive Hey1 expression augmented BMP9-mediated bone matrix mineralization. Hey1 and Runx2 were shown to act synergistically in BMP9-induced osteogenic differentiation, and Runx2 expression significantly decreased in the absence of Hey1, suggesting that Runx2 may function downstream of Hey1. Accordingly, the defective osteogenic differentiation caused by Hey1 knockdown was rescued by exogenous Runx2 expression. Thus, our findings suggest that Hey1, through its interplay with Runx2, may play an important role in regulating BMP9-induced osteoblast lineage differentiation of MSCs.
Epigenetic memory in response to environmental stressors

Exposure to environmental stressors, toxicants, and nutrient deficiencies can affect DNA in several ways. Some exposures cause damage and alter the structure of DNA, but there is increasing evidence that the same or other environmental exposures, including those that occur during fetal development in utero, can cause epigenetic effects that modulate DNA function and gene expression. Some epigenetic changes to DNA that affect gene transcription are at least partially reversible (i.e., they can be enzymatically reversed after cessation of exposure to environmental agents), but some epigenetic modifications seem to persist, even for decades. To explain the effects of early life experiences (such as famine and exposures to other stressors) on the long‐term persistence of specific patterns of epigenetic modifications, such as DNA methylation, we propose an analogy with immune memory. We propose that an epigenetic memory can be established and maintained in self‐renewing stem cell compartments. We suggest that the observations on early life effects on adult diseases and the persistence of methylation changes in smokers support our hypothesis, for which a mechanistic basis, however, needs to be further clarified. We outline a new model based on methylation changes. Although these changes seem to be mainly adaptive, they are also implicated in the pathogenesis and onset of diseases, depending on individual genotypic background and types of subsequent exposures. Elucidating the relationships between the adaptive and maladaptive consequences of the epigenetic modifications that result from complex environmental exposures is a major challenge for current and future research in epigenetics. —Vineis, P., Chatziioannou, A. , Cunliffe, V. T. , Flanagan, J. M. , Hanson, M. , Kirsch‐Volders, M. , Kyrtopoulos, S. Epigenetic memory in response to environmental stressors. FASEB J. 31, 2241–2251 (2017). www.fasebj. org
Connexin‐purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation

Morphine is one of the most widely used drugs for the treatment of pain. However, side effects, including persistent constipation and antinociceptive tolerance, limit its clinical efficacy. Prolonged morphine treatment results in a “leaky” gut, predisposing to colonic inflammation that is facilitated by microbial dysbiosis and associated bacterial translocation. In this study, we examined the role of enteric glia in mediating this secondary inflammatory response to prolonged treatment with morphine. We found that purinergic P2X receptor activity was significantly enhanced in enteric glia that were isolated from mice with long‐term morphine treatment (in vivo) but not upon direct exposure of glia to morphine (in vitro). LPS, a major bacterial product, also increased ATP‐induced currents, as well as expression of P2X4, P2X7, IL6, IL‐1β mRNA in enteric glia. LPS increased connexin43 (Cx43) expression and enhanced ATP release from enteric glia cells. LPS‐induced P2X currents and proinflammatory cytokine mRNA expression were blocked by the Cx43 blockers Gap26 and carbenoxolone. Likewise, colonic inflammation related to prolonged exposure to morphine was significantly attenuated by carbenoxolone (25 mg/kg). Carbenoxolone also prevented gut wall disruption and significantly reduced morphine‐induced constipation. These findings imply that enteric glia activation is a significant modulator of morphine‐related inflammation and constipation. —Bhave, S. , Gade, A. , Kang, M. , Hauser, K. F. , Dewey, W. L. , Akbarali, H. I. Connexin‐purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation. FASEB J. 31, 2649–2660 (2017). www.fasebj. org
A Novel Small Molecule Regulator of Guanine Nucleotide Exchange Activity of the ADP-ribosylation Factor and Golgi Membrane Trafficking*

An image-based phenotypic screen was developed to identify small molecule regulators of intracellular traffic. Using this screen we found that AG1478, a previously known inhibitor of epidermal growth factor receptor, had epidermal growth factor receptor-independent activity in inducing the disassembly of the Golgi in human cells. Similar to brefeldin A (BFA), a known disrupter of the Golgi, AG1478 inhibits the activity of small GTPase ADP-ribosylation factor. Unlike BFA, AG1478 exhibits low cytotoxicity and selectively targets the cis-Golgi without affecting endosomal compartment. We show that AG1478 inhibits GBF1, a large nucleotide exchange factor for the ADP-ribosylation factor, in a Sec7 domain-dependent manner and mimics the phenotype of a GBF1 mutant that has an inactive mutation. The treatment with AG1478 leads to the recruitment of GBF1 to the vesicular-tubular clusters adjacent to the endoplasmic reticulum exit sites, a step only transiently observed previously in the presence of BFA. We propose that the treatment with AG1478 delineates a membrane trafficking intermediate step that depends upon the Sec7 domain.
Generation of Insulin-secreting Islet-like Clusters from Human Skin Fibroblasts*♦

Increasing evidence suggests that islet cell transplantation for patients with type I diabetes holds great promise for achieving insulin independence. However, the extreme shortage of matched organ donors and the necessity for chronic immunosuppression has made it impossible for this treatment to be used for the general diabetic population. Recent success in generating insulin-secreting islet-like cells from human embryonic stem (ES) cells, in combination with the success in deriving human ES cell-like induced pluripotent stem (iPS) cells from human fibroblasts by defined factors, have raised the possibility that patient-specific insulin-secreting islet-like cells might be derived from somatic cells through cell fate reprogramming using defined factors. Here we confirm that human ES-like iPS cells can be derived from human skin cells by retroviral expression of OCT4, SOX2, c-MYC, and KLF4. Importantly, using a serum-free protocol, we successfully generated insulin-producing islet-like clusters (ILCs) from the iPS cells under feeder-free conditions. We demonstrate that, like human ES cells, skin fibroblast-derived iPS cells have the potential to be differentiated into islet-like clusters through definitive and pancreatic endoderm. The iPS-derived ILCs not only contain C-peptide-positive and glucagon-positive cells but also release C-peptide upon glucose stimulation. Thus, our study provides evidence that insulin-secreting ILCs can be generated from skin fibroblasts, raising the possibility that patient-specific iPS cells could potentially provide a treatment for diabetes in the future.
Foxo4‐ and Stat3‐dependent IL‐10 production by progranulin in regulatory T cells restrains inflammatory arthritis

Progranulin (PGRN) restrains inflammation and is therapeutic against inflammatory arthritis; however, the underlying immunological mechanism remains unknown. In this study, we demonstrated that anti‐inflammatory cytokine IL‐10 was a critical mediator for PGRN‐mediated anti‐inflammation in collagen‐induced arthritis by using PGRN and IL‐10 genetically modified mouse models. IL‐10 green fluorescent protein reporter mice revealed that regulatory T (Treg) cells were the predominant source of IL‐10 in response to PGRN. In addition, PGRN‐mediated expansion and activation of Treg cells, as well as IL‐10 production, depends on JNK signaling, but not on known PGRN‐activated ERK and PI3K pathways. Furthermore, microarray and chromatin immunoprecipitation sequencing screens led to the discovery of forkhead box protein O4 and signal transducer and activator of transcription 3 as the transcription factors required for PGRN induction of IL‐10 in Treg cells. These findings define a previously unrecognized signaling pathway that underlies IL‐10 production by PGRN in Treg cells and present new insights into the mechanisms by which PGRN resolves inflammation in inflammatory conditions and autoimmune diseases, particularly inflammatory arthritis. —Fu, W. , Hu, W. , Shi, L. , Mundra, J. J. Xiao, G. , Dustin, M. L. , Liu, C. Foxo4‐ and Stat3‐dependent IL‐10 production by progranulin in regulatory T cells restrains inflammatory arthritis. FASEB J. 31, 1354–1367 (2017) www.fasebj. org
THE INCIDENCE OF COINCIDENT PROSTATIC CALCULI, PROSTATIC HYPERPLASIA AND CARCINOMA OF THE PROSTATE GLAND

Previous to 1927 it was the opinion of most men interested in urology that prostatic calculi were rarely if ever associated with prostatic carcinoma. It was not until Kretschmer 1 reported 76 cases of prostatic calculi (in 5 of which prostatic carcinoma also was present) that it became generally appreciated that prostatic carcinoma and calculi could occur together. The practical significance of estimating the incidence of prostatic calculi associated with prostatic hyperplasia and carcinoma of the prostate gland lies largely in the differential diagnosis of prostatic calculi and prostatic carcinoma. For years it has been emphasized that it may be impossible to distinguish a prostatic calculus from prostatic carcinoma by palpation. This problem of differential diagnosis was believed to have been solved by the use of roentgenographic studies of the prostatic region. This was particularly true in the minds of those who believed that prostatic calculi and carcinoma rarely if
Anterior cruciate ligament injuries in female athletes: Anatomy, physiology, and motor control

Anterior cruciate ligament (ACL) injuries are common in sports. Most ACL injuries are noncontact in nature and frequently occur in certain athletic tasks. In sports such as soccer, basketball and volleyball, female athletes have higher risk for ACL injuries than their male counterparts. Previous studies on the proposed risk factors for noncontact ACL injuries focused mainly on nonmodifiable static factors that are remote from the injury events, without consideration of movements. Little convincing evidence, theoretical or experimental, has demonstrated the association between these static factors and the risk of noncontact ACL injuries. Female athletes have altered knee motion patterns in athletic tasks that place the ACL at risk. These altered motion patterns by female athletes tend to increase the load on the ACL, and may be a major contributor to the elevated frequency of ACL injuries in female athletes.
A Binding Domain on Mesothelin for CA125/MUC16*

Ovarian cancer and malignant mesothelioma frequently express both mesothelin and CA125 (also known as MUC16) at high levels on the cell surface. The interaction between mesothelin and CA125 may facilitate the implantation and peritoneal spread of tumors by cell adhesion, whereas the detailed nature of this interaction is still unknown. Here, we used truncated mutagenesis and alanine replacement techniques to identify a binding site on mesothelin for CA125. We examined the molecular interaction by Western blot overlay assays and further quantitatively analyzed by enzyme-linked immunosorbent assay. We also evaluated the binding on cancer cells by flow cytometry. We identified the region (296–359) consisting of 64 amino acids at the N-terminal of cell surface mesothelin as the minimum fragment for complete binding activity to CA125. We found that substitution of tyrosine 318 with an alanine abolished CA125 binding. Replacement of tryptophan 321 and glutamic acid 324 with alanine could partially decrease binding to CA125, whereas mutation of histidine 354 had no effect. These results indicate that a conformation-sensitive structure of the region (296–359) is required and sufficient for the binding of mesothelin to CA125. In addition, we have shown that a single chain monoclonal antibody (SS1) recognizes this CA125-binding domain and blocks the mesothelin-CA125 interaction on cancer cells. The identified CA125-binding domain significantly inhibits cancer cell adhesion and merits evaluation as a new therapeutic agent for preventing or treating peritoneal malignant tumors.
Maternal injuries during the periconceptional period and the risk of birth defects, National Birth Defects Prevention Study, 1997-2005.

Maternal injuries during pregnancy are common (∼7% prevalence). However, few studies have examined the association between maternal injuries and birth defects. The National Birth Defects Prevention Study is a population-based case-control study of birth defects in 10 U.S. states. Cases were ascertained through surveillance; controls were randomly selected from infants delivered without major birth defects in the study regions. Mothers completed a telephone interview on exposures before and during pregnancy, including injuries. We assessed associations between periconceptional (month before until the end of the third month of pregnancy) maternal injuries and birth defects. We used logistic regression to estimate adjusted odds ratios (AORs) and 95% confidence intervals (CI). Periconceptional injuries were associated with interrupted aortic arch type B [AOR = 5.2, 95% CI 1.2, 23.2]; atrioventricular septal defect [AOR = 2.2, 95% CI 1.1, 4.4]; pulmonary atresia [AOR = 3.2, 95% CI 1.6, 6.4]; tricuspid atresia [AOR = 2.8, 95% CI 1.2, 6.7]; hypoplastic left heart syndrome [AOR = 2.0, 95% CI 1.1, 3.4]; anorectal atresia/stenosis [AOR = 1.7, 95% CI 1.0, 2.7]; longitudinal limb deficiency [AOR = 2.1, 95% CI 1.1, 3.9]; and gastroschisis [AOR = 1.8, 95% CI 1.2, 2.8]. Associations with longitudinal limb deficiency, gastroschisis and hypoplastic left heart syndrome were stronger for intentional injuries. Our results suggest maternal injury during the periconceptional period, particularly those inflicted intentionally, may be associated with select birth defects. This analysis was hypothesis-generating, with many associations tested. Further research is warranted.
Receptor for Advanced Glycation End Products Is Subjected to Protein Ectodomain Shedding by Metalloproteinases*

The receptor for advanced glycation end products (RAGE) is a 55-kDa type I membrane glycoprotein of the immunoglobulin superfamily. Ligand-induced up-regulation of RAGE is involved in various pathophysiological processes, including late diabetic complications and Alzheimer disease. Application of recombinant soluble RAGE has been shown to block RAGE-mediated pathophysiological conditions. After expression of full-length RAGE in HEK cells we identified a 48-kDa soluble RAGE form (sRAGE) in the culture medium. This variant of RAGE is smaller than a 51-kDa soluble version derived from alternative splicing. The release of sRAGE can be induced by the phorbol ester PMA and the calcium ionophore calcimycin via calcium-dependent protein kinase C subtypes. Hydroxamic acid-based metalloproteinase inhibitors block the release of sRAGE, and by RNA interference experiments we identified ADAM10 and MMP9 to be involved in RAGE shedding. In protein biotinylation experiments we show that membrane-anchored full-length RAGE is the precursor of sRAGE and that sRAGE is efficiently released from the cell surface. We identified cleavage of RAGE to occur close to the cell membrane. Ectodomain shedding of RAGE simultaneously generates sRAGE and a membrane-anchored C-terminal RAGE fragment (RAGE-CTF). The amount of RAGE-CTF increases when RAGE-expressing cells are treated with a γ-secretase inhibitor, suggesting that RAGE-CTF is normally further processed by γ-secretase. Identification of these novel mechanisms involved in regulating the availability of cell surface-located RAGE and its soluble ectodomain may influence further research in RAGE-mediated processes in cell biology and pathophysiology.
Neuron‐specific caveolin‐1 overexpression improves motor function and preserves memory in mice subjected to brain trauma

Studies in vitro and in vivo demonstrate that membrane/lipid rafts and caveolin (Cav) organize progrowth receptors, and, when overexpressed specifically in neurons, Cav‐1 augments neuronal signaling and growth and improves cognitive function in adult and aged mice; however, whether neuronal Cav‐1 overexpression can preserve motor and cognitive function in the brain trauma setting is unknown. Here, we generated a neuron‐targeted Cav‐1–overexpressing transgenic (Tg) mouse [synapsin‐driven Cav‐1 (SynCav1 Tg)] and subjected it to a controlled cortical impact model of brain trauma and measured biochemical, anatomic, and behavioral changes. SynCav1 Tg mice exhibited increased hippocampal expression of Cav‐1 and membrane/lipid raft localization of postsynaptic density protein 95, NMDA receptor, and tropomyosin receptor kinase B. When subjected to a controlled cortical impact, SynCav1 Tg mice demonstrated preserved hippocampus‐dependent fear learning and memory, improved motor function recovery, and decreased brain lesion volume compared with wild‐type controls. Neuron‐targeted overexpression of Cav‐1 in the adult brain prevents hippocampus‐dependent learning and memory deficits, restores motor function after brain trauma, and decreases brain lesion size induced by trauma. Our findings demonstrate that neuron‐targeted Cav‐1 can be used as a novel therapeutic strategy to restore brain function and prevent trauma‐associated maladaptive plasticity. —Egawa, J., Schilling, J. M. , Cui, W. , Posadas, E. , Sawada, A. , Alas, B. , Zemljic‐Harpf, A. E. , Fannon‐Pavlich, M. J., Mandyam, C. D. , Roth, D. M. , Patel, H. H. , Patel, P. M. , Head, B. P. Neuron‐specific caveolin‐1 overexpression improves motor function and preserves memory in mice subjected to brain trauma. FASEB J. 31, 3403–3411 (2017). www.fasebj. org