Miracle-dz/UniFAR-FTUs huggingface dataset:
A Pore-blocking Hydrophobic Motif at the Cytoplasmic Aperture of the Closed-state Nav1.7 Channel Is Disrupted by the Erythromelalgia-associated F1449V Mutation*

Sodium channel Nav1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Nav1.7 produce painful disorders, whereas loss-of-function Nav1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Nav1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Nav1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pore's cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Nav1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Nav1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Nav1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Nav1.7.
Proteomic signatures of extracellular vesicles secreted by nonmineralizing and mineralizing human osteoblasts and stimulation of tumor cell growth

Beyond forming bone, osteoblasts play pivotal roles in various biologic processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have been implicated in intercellular communication via transfer of proteins and nucleic acids between cells. We focused on the proteomic characterization of nonmineralizing (NMOBs) and mineralizing (MOBs) human osteoblast (SV‐HFOs) EVs and investigated their effect on human prostate cancer (PC3) cells by microscopic, proteomic, and gene expression analyses. Proteomic analysis showed that 97% of the proteins were shared among NMOB and MOB EVs, and 30% were novel osteoblast‐specific EV proteins. Label‐free quantification demonstrated mineralization stage‐dependent 5‐fold enrichment of 59 and 451 EV proteins in NMOBs and MOBs, respectively. Interestingly, bioinformatic analyses of the osteoblast EV proteomes and EV‐regulated prostate cancer gene expression profiles showed that they converged on pathways involved in cell survival and growth. This was verified by in vitro proliferation assays where osteoblast EV uptake led to 2‐fold increase in PC3 cell growth compared to cell‐free culture medium‐derived vesicle controls. Our findings elucidate the mineralization stage‐specific protein content of osteoblast‐secreted EVs, show a novel way by which osteoblasts communicate with prostate cancer, and open up innovative avenues for therapeutic intervention. —Morhayim, J., van de Peppel, J., Demmers, J. A. A. , Kocer, G. , Nigg, A. L. , van Driel, M. , Chiba, H. , van Leeuwen, J. P., Proteomic signatures of extracellular vesicles secreted by nonmineralizing and mineralizing human osteoblasts and stimulation of tumor cell growth. FASEB J. 29, 274–285 (2015). www.fasebj. org
Collagen matrix‐induced expression of integrin αVβ3 in circulating angiogenic cells can be targeted by matricellular protein CCN1 to enhance their function

Circulating angiogenic cells (CACs) play an important role in vascular homeostasis and hold therapeutic promise for treating a variety of cardiovascular diseases. However, further improvements are needed because the effects of CAC therapy remain minimal or transient. The regenerative potential of these cells can be improved by culture on a collagen‐based matrix through the up‐regulation of key integrin proteins. We found that human CAC function was enhanced by using the matricellular protein CCN1 (CYR61/CTGF/NOV family member 1) to target integrin αV and β3, which are up‐regulated on matrix. Compared to matrix‐cultured CACs, CCN1‐matrix CACs exhibited a 2.2‐fold increase in cell proliferation, 1.8‐fold greater migration toward VEGF, and 1.7‐fold more incorporation into capillary‐like structures in an angiogenesis assay. In vivo, intramuscular injection of CCN1‐matrix‐cultured CACs into ischemic hind limbs of CD‐1 nude mice resulted in blood flow recovery to 80% of baseline, which was greater than matrix‐cultured CACs (66%) and PBS (35%) treatment groups. Furthermore, transplanted CCN1‐matrix CACs exhibited greater engraftment (11‐fold) and stimulated the up‐regulation of survival and angiogenic genes (>3‐fold). These findings reveal the importance of cell‐matrix interactions in regulating CAC function and also reveal a mechanism by which these may be exploited to enhance cell therapies for ischemic disease. —McNeill, B. , Vulesevic, B. , Ostojic, A. , Ruel, M. , Suuronen, E. J. Collagen matrix‐induced expression of integrin αVβ3 in circulating angiogenic cells can be targeted by matricellular protein CCN1 to enhance their function. FASEB J. 29, 1198‐1207 (2015). www.fasebj. org
Absence of nicotinic acetylcholine receptor α7 subunit amplifies inflammation and accelerates onset of fibrosis: an inflammatory kidney model

Inflammation is regulated by endogenous mechanisms, including anti‐inflammatory cytokines, adenosine, and the nicotinic acetylcholine receptor α7 subunit (α7nAChR). We investigated the role of α7nAChR in protection against the progression of tissue injury in a model of severe, macrophage‐mediated, cytokine‐dependent anti‐glomerular basement membrane (GBM) glomerulonephritis (GN), in α7nAChR‐deficient (α7‐/‐) mice. At d7 after the injection of anti‐GBM antibody, kidneys from α7‐/‐ mice displayed severe glomeruli (P < 0.0001) and tubulointerstitial lesions (P < 0.001) compared to kidneys from WT mice. An important finding was the presence of severe glomerulosclerosis in α7‐/‐ mice in this early phase of the disease. Kidneys of α7‐/‐ mice showed greater accumulation of inflammatory cells and higher expression of chemokines and cytokines than did those of WT mice. In addition, in α7‐/‐ fibrotic kidneys, the expression of fibrin, collagen, TGF‐β, and tissue inhibitor of metalloproteinase (TIMP)‐2 increased, and the expression of TIMP3 declined. The increase in counterregulatory responses to inflammation in α7‐/‐ nephritic kidneys did not compensate for the lack of α7nAChR. These findings indicate that α7nAChR plays a key role in regulating the inflammatory response in anti‐GBM GN and that disruption of the endogenous protective α7nAChR amplifies inflammation to accelerate kidney damage and fibrosis. —Truong, L. D. , Trostel. J., Garcia, G. E. Absence of nicotinic acetylcholine receptor α7 subunit amplifies inflammation and accelerates onset of fibrosis: an inflammatory kidney model. FASEB J. 29, 3558‐3570 (2015). www.fasebj. org
Spatial Structure and pH-dependent Conformational Diversity of Dimeric Transmembrane Domain of the Receptor Tyrosine Kinase EphA1*

Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel α-helical bundle, region (544-569)2, through the N-terminal glycine zipper motif A550X3G554X3G558. Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu547, whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A560X3G564. This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pKa value of Glu547 and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an “extracellular pH sensor,” and can have relevant physiological implications.
Dynamic changes in the cardiac methylome during postnatal development

Relatively little is known about the epigenetic control mechanisms that guide postnatal organ maturation. The goal of this study was to determine whether DNA methylation plays an important role in guiding transcriptional changes during the first 2 wk of mouse heart development, which is an important period for cardiomyocyte maturation, loss of proliferative capacity and loss of regenerative potential. Gene expression profiling (RNA‐seq) and genome‐wide sequencing of methylated DNA (MBD‐seq) identified dynamic changes in the cardiac methylome during postnatal development [2545 differentially methylated regions (DMRs) from P1 to P14 in the mouse]. The vast majority (~80%) of DMRs were hypermethylated between P1 and P14, and these hyper‐methylated regions were associated with transcriptional shut down of important developmental signaling pathways, including Hedgehog, bone morphogenetic protein, TGF‐β, fibroblast growth factor, and Wnt/β‐catenin signaling. Postnatal inhibition of DNA methylation with 5‐aza‐2'‐deoxycytidine induced a marked increase (~3‐fold) in cardiomyocyte proliferation and ~50% reduction in the percentage of binucleated cardiomyocytes compared with saline‐treated controls. This study provides novel evidence for widespread alterations in DNA methylation during postnatal heart maturation and suggests that cardiomyocyte cell cycle arrest during the neonatal period is subject to regulation by DNA methylation. —Sim, C. B. , Ziemann, M. , Kaspi, A. , Harikrishnan, K. N. , Ooi, J., Khurana, I. , Chang, L. , Hudson, J. E. , El‐Osta, A. , Porrello, E. R. Dynamic changes in the cardiac methylome during postnatal development. FASEB J. 29, 1329‐1343 (2015). www.fasebj. org
The relationship between inflammation‐induced neuronal excitability and disrupted motor activity in the guinea pig distal colon

Background Colitis is associated with increased excitability of afterhyperpolarization neurons (AH neurons) and facilitated synaptic transmission in the myenteric plexus. These changes are accompanied by disrupted propulsive motility, particularly in ulcerated regions. This study examined the relationship between myenteric AH neuronal hyperexcitability and disrupted propulsive motility.
Deposition of bioactive human epidermal growth factor in the egg white of transgenic hens using an oviduct‐specific minisynthetic promoter

Currently, transgenic animals have found a wide range of industrial applications and are invaluable in various fields of basic research. Notably, deposition of transgene‐encoded proteins in the egg white (EW) of hens affords optimal production of genetically engineered biomaterials. In the present study, we developed a minisynthetic promoter modulating transgene transcription specifically in the hen's oviduct, and assayed the bioactivity of human epidermal growth factor (hEGF) driven by that promoter, after partial purification of epidermal growth factor (EGF) from transgenic hen eggs. Our minisynthetic promoter driving expression of chicken codonoptimized human epidermal growth factor (cEGF) features 2 consecutive estrogen response elements of the ovalbumin (OV) promoter, ligated with a 3.0 kb OV promoter region carrying OV regulatory elements, and a 5′‐UTR. Subsequently, a 3′‐UTR carrying the poly‐A tail sequence of the OV gene was added after incorporation of the cEGF transgene. Finally, we partially purified cEGF from transgenic hen eggs and evaluated the biofunctional activities thereof in vitro and in vivo. In the in vitro assay, EW‐derived hEGF exhibited a proliferative effect on HeLa cells similar to that of commercial hEGF. In the in vivo assay, compared to the nontreated control, transgenic hen egg‐derived EGF afforded slightly higher levels of re‐epithelialization (via fibroplasia) and neovascularization of wounded skin of miniature pigs than did the commercial material. In conclusion, transgenic hens may be used to produce genetically engineered bioactive biomaterials driven by an oviduct‐specific minisynthetic promoter. —Park, T. S. , Lee, H. G. , Moon, J. K. , Lee, H. J., Yoon, J. W. , Yun, B. N. R. , Kang, S. ‐C. , Kim, J., Kim, H. , Han, J. Y. , Han, B. K. Deposition of bioactive human epidermal growth factor in the egg white of transgenic hens using an oviduct‐specific minisynthetic promoter. FASEB J. 29, 2386‐2396 (2015). www.fasebj. org
Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis*

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only ∼100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.
Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein‐2

Promoting bone regeneration and repair of bone defects is a need that has not been well met to date. We have previously found that adenosine, acting via receptors (A2AR) promotes wound healing and inhibits inflammatory osteolysis and hypothesized that A2AR might be a novel target to promote bone regeneration. Therefore, we determined whether direct A2AR stimulation or increasing endogenous adenosine concentrations via purine transport blockade with dipyridamole regulates bone formation. We determined whether coverage of a 3 mm trephine defect in a mouse skull with a collagen scaffold soaked in saline, bone morphogenetic protein‐2 (BMP‐2; 200 ng), 1 μM CGS21680 (A2AR agonist, EC50 = 160 nM), or 1 μM dipyridamole (EC50 = 32 nM) promoted bone regeneration. Microcomputed tomography examination demonstrated that CGS21680 and dipyridamole markedly enhanced bone regeneration as well as BMP‐2 8 wk after surgery (60 ± 2%, 79 ± 2%, and 75 ± 1% bone regeneration, respectively, vs. 32 ± 2% in control, P < 0.001). Blockade by a selective A2AR antagonist (ZM241385, 1 μM) or deletion of A2AR abrogated the effect of CGS21680 and dipyridamole on bone regeneration. Both CGS21680 and dipyridamole treatment increased alkaline phosphatase‐positive osteoblasts and diminished tartrate resistance acid phosphatase‐positive osteoclasts in the defects. In vivo imaging with a fluorescent dye for new bone formation revealed a strong fluorescent signal in treated animals that was equivalent to BMP‐2. In conclusion, stimulation of A2AR by specific agonists or by increasing endogenous adenosine levels stimulates new bone formation as well as BMP‐2 and represents a novel approach to stimulating bone regeneration. —Mediero, A. , Wilder, T. , Perez‐Aso, M. , Cronstein, B. N. Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein‐2. FASEB J. 29, 1577‐1590 (2015). www.fasebj. org
Glucocorticoids entrain molecular clock components in human peripheral cells

In humans, shift work induces a desynchronization between the circadian system and the outside world, which contributes to shift work‐associated medical disorders. Using a simulated night shift experiment, we previously showed that 3 d of bright light at night fully synchronize the central clock to the inverted sleep schedule, whereas the peripheral clocks located in peripheral blood mononuclear cells (PBMCs) took longer to reset. This underlines the need for testing the effects of synchronizers on both the central and peripheral clocks. Glucocorticoids display circadian rhythms controlled by the central clock and are thought to act as synchronizers of rodent peripheral clocks. In the present study, we tested whether the human central and peripheral clocks were sensitive to exogenous glucocorticoids (Cortef) administered in the late afternoon. We showed that 20 mg Cortef taken orally acutely increased PER1 expression in PBMC peripheral clocks. After 6 d of Cortef administration, the phases of central markers were not affected, whereas those of PER2‐3 and BMAL1 expression in PBMCs were shifted by ~9.5‐11.5 h. These results demonstrate, for the first time, that human peripheral clocks are entrained by glucocorticoids. Importantly, they suggest innovative interventions for shift workers and jet‐lag travelers, combining synchronizing agents for the central and peripheral clocks. —Cuesta, M. , Cermakian, N. , Boivin, D. B. Glucocorticoids entrain molecular clock components in human peripheral cells. FASEB J. 29, 1360‐1370 (2015). www.fasebj. org
Therapeutic Pancreatic Endoscopy

Therapeutic endoscopic retrograde cholangiopancreatography (ERCP) has established itself as a valuable tool for the treatment of a variety of biliary tract disorders. Fueled by an ever-increasing demand for minimally invasive therapy, pancreatic endoscopy was the logical frontier for expansion of therapeutic ERCP. Endoscopic treatment of benign pancreatic disorders can be a cost-effective alternative to more invasive radiological or surgical methods. Conditions such as acute and chronic pancreatitis, pancreatic calculi, and pseudocysts can in many cases be managed endoscopically. This review summarizes the current state of the art in therapeutic pancreatic endoscopy.
Follistatin-like 1, a Secreted Muscle Protein, Promotes Endothelial Cell Function and Revascularization in Ischemic Tissue through a Nitric-oxide Synthase-dependent Mechanism*

Myogenic Akt signaling coordinates blood vessel recruitment with normal tissue growth. Here, we investigated the role of Follistatin-like 1 (Fstl1) in the regulation of endothelial cell function and blood vessel growth in muscle. Transgenic Akt1 overexpression in skeletal muscle led to myofiber growth that was coupled to an increase in muscle capillary density. Myogenic Akt signaling or ischemic hind limb surgery led to the induction of Fstl1 in muscle and increased circulating levels of Fstl1. Intramuscular administration of an adenoviral vector expressing Fstl1 (Ad-Fstl1) accelerated flow recovery and increased capillary density in the ischemic hind limbs of wild-type mice, and this was associated with an increase in endothelial nitric oxide synthase (eNOS) phosphorylation at residue Ser-1179. In cultured endothelial cells, Ad-Fstl1 stimulated migration and differentiation into network structures and inhibited apoptosis under conditions of serum deprivation. These cell responses were associated with the activating phosphorylation of Akt and eNOS. Conversely, transduction with dominant-negative Akt or LY294002 blocked Fstl1-stimulated eNOS phosphorylation and inhibited Fstl1-stimulated cellular responses. Treatment with the eNOS inhibitor NG-nitro-l-arginine methyl ester also reduced endothelial cell migration and differentiation induced by Ad-Fstl1. The stimulatory effect of Ad-Fstl1 on ischemic limb reperfusion was abolished in mice lacking eNOS. These data indicate that Fstl1 is a secreted muscle protein or myokine that can function to promote endothelial cell function and stimulates revascularization in response to ischemic insult through its ability to activate Akt-eNOS signaling.
AMPKα is critical for enhancing skeletal muscle fatty acid utilization during in vivo exercise in mice

The importance of AMPK in regulation of fatty acid (FA) oxidation in skeletal muscle with contraction/exercise is unresolved. Using a mouse model lacking both AMPKα1 and ‐α2 in skeletal muscle specifically (mdKO), we hypothesized that FA utilization would be impaired in skeletal muscle. AMPKα mdKO mice displayed normal respiratory exchange ratio (RER) when fed chow or a high‐fat diet, or with prolonged fasting. However, in vivo treadmill exercise at the same relative intensity induced a higher RER in AMPKα mdKO mice compared to wild‐type (WT = 0.81 ± 0.01 (sem); mdKO = 0.87 ± 0.02 (sem); P < 0.01), indicating a decreased utilization of FA. Further, ex vivo contraction‐induced FA oxidation was impaired in AMPKα mdKO muscle, suggesting that the increased RER during exercise originated from decreased skeletal muscle FA oxidation. A decreased muscle protein expression of CD36 (cluster of differentiation 36) and FABPpm (plasma membrane fatty acid binding protein) (by ~17‐40%), together with fully abolished TBC1D1 (tre‐2/USP6, BUB2, cdc16 domain family member 1) Ser237 phosphorylation during contraction/exercise in AMPKα mdKO mice, may impair FA transport capacity and FA transport protein translocation to sarcolemma, respectively. AMPKα is thus required for normal FA metabolism during exercise and muscle contraction. —Fentz, J., Kjøbsted, R. , Birk, J. B. , Jordy, A. B. , Jeppesen, J., Thorsen, K. , Schjerling, P., Kiens, B. , Jessen, N. , Viollet, B. , Wojtaszewski, J. F. P. AMPKα is critical for enhancing skeletal muscle fatty acid utilization during in vivo exercise in mice. FASEB J. 29, 1725‐1738 (2015). www.fasebj. org