Miracle-dz/UniFAR-FTUs huggingface dataset:
Alternating Access to the Transmembrane Domain of the ATP-binding Cassette Protein Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7)*

Background: It is expected that the transport pathway of CFTR should alternate between internally and externally accessible conformations. Results: Access to the channel from both sides of the membrane is regulated by opening and closing. Conclusion: The open state is outwardly facing, the closed state inwardly facing. Significance: These findings prompt reevaluation of prevailing models of CFTR conformational changes. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between “outwardly facing” and “inwardly facing” conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)2− ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)2−, access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional (“vestigial”) gates within the CFTR permeation pathway.
Mapping Precursor-binding Site on TatC Subunit of Twin Arginine-specific Protein Translocase by Site-specific Photo Cross-linking*

Background: TatA, TatB, and TatC are subunits of the Tat translocase allowing transport of folded pre-proteins across cellular membranes Results: We identified TatC sites that interact with pre-proteins, TatA, TatB, and TatC Conclusion: The cytosolic N terminus and first cytosolic TatC loop constitute part of a twin arginine recognition site Significance: We developed a working model of how twin arginine pre-protein inserts into Tat translocase. A number of secreted precursor proteins of bacteria, archaea, and plant chloroplasts stand out by a conserved twin arginine-containing sequence motif in their signal peptides. Many of these precursor proteins are secreted in a completely folded conformation by specific twin arginine translocation (Tat) machineries. Tat machineries are high molecular mass complexes consisting of two types of membrane proteins, a hexahelical TatC protein, and usually one or two single-spanning membrane proteins, called TatA and TatB. TatC has previously been shown to be involved in the recognition of twin arginine signal peptides. We have performed an extensive site-specific cross-linking analysis of the Escherichia coli TatC protein under resting and translocating conditions. This strategy allowed us to map the recognition site for twin arginine signal peptides to the cytosolic N-terminal region and first cytosolic loop of TatC. In addition, discrete contact sites between TatC, TatB, and TatA were revealed. We discuss a tentative model of how a twin arginine signal sequence might be accommodated in the Tat translocase.
Impact of the calibration conditions of XR-RV3 films on peak skin dose measurements in interventional radiology

In this study, the impact of radiochromic films' (XR-RV3) calibration on PSD measurements was investigated under various peak kilovoltage (kVp) and additional filtration conditions. Films were calibrated free-in-air for six beam qualities with Allura Xper FD20 system (Philips). Six calibration curves (CCs) were constructed. Each beam quality was characterized in terms of mean energy (ME) in the air, with table, with table and water phantom using Monte Carlo simulations. A cohort of 155 patient films from cardiology (37) and vascular (118) procedures were read with each CC. Routine calibration beam quality was taken as reference (DoseNorm). Overall, it was observed that for a wider ME difference between the exposed film and the CC used, a larger deviation (from -28% to +41%) was observed. The choice of beam quality for the calibration is a key point when additional filtration and kVp are automatically controlled in clinical conditions.
Familial Focal Segmental Glomerulosclerosis (FSGS)-linked α-Actinin 4 (ACTN4) Protein Mutants Lose Ability to Activate Transcription by Nuclear Hormone Receptors*

Background: Mutations in α-ACTN4 are linked with familial FSGS. Results: FSGS linked α-ACTN4 mutants fail to activate transcription mediated by nuclear hormone receptors. Conclusion: The inability of FSGS-linked ACTN4 mutants to potentiate transcriptional activation might be because of their more cytoplasmic localization compared with wild type. Significance: Our results may have implications for understanding the role of α-ACTN4 in the pathophysiology of the kidney disease. Mutations in α-actinin 4 (ACTN4) are linked to familial forms of focal segmental glomerulosclerosis (FSGS), a kidney disease characterized by proteinuria due to podocyte injury. The mechanisms underlying ACTN4 mutant-associated FSGS are not completely understood. Although α-actinins are better known to cross-link actin filaments and modulate cytoskeletal organization, we have previously shown that ACTN4 interacts with transcription factors including estrogen receptor and MEF2s and potentiates their transcriptional activity. Nuclear receptors including retinoic acid receptor (RAR) have been proposed to play a protective role in podocytes. We show here that ACTN4 interacts with and enhances transcriptional activation by RARα. In addition, FSGS-linked ACTN4 mutants not only mislocalized to the cytoplasm, but also lost their ability to associate with nuclear receptors. Consequently, FSGS-linked ACTN4 mutants failed to potentiate transcriptional activation by nuclear hormone receptors in podocytes. In addition, overexpression of these mutants suppressed the transcriptional activity mediated by endogenous wild-type ACTN4 possibly by a cytoplasmic sequestration mechanism. Our data provide the first link between FSGS-linked ACTN4 mutants and transcriptional activation by nuclear receptor such as RARα and peroxisome proliferator-activated receptor γ.
Examination of the effects of AMP-activated protein kinase activation in obese mice

Word Count: 225 words Main Text Word Count: 6357 words Ph.D. Thesis K. Marcinko McMaster University Medical Sciences 89 Abstract Objective: Endurance exercise training reduces insulin resistance, adipose tissue inflammation and non-alcoholic fatty liver disease (NAFLD), an effect often associated with modest weight loss. Recent studies have indicated that highintensity interval training (HIIT) lowers blood glucose in individuals with type 2 diabetes independently of weight loss; however, the organs affected and mechanisms mediating the glucose lowering effects are not known. Intense exercise increases phosphorylation and inhibition of acetyl-CoA carboxylase (ACC) by AMP-activated protein kinase (AMPK) in muscle, adipose tissue and liver. AMPK and ACC are key enzymes regulating fatty acid metabolism, liver fat content, adipose tissue inflammation and insulin sensitivity but the importance of this pathway in regulating insulin sensitivity with HIIT is unknown. Methods: In the current study, the effects of 6 weeks of HIIT were examined using obese mice with serine-alanine knock-in mutations on the AMPK phosphorylation sites of ACC1 and ACC2 (AccDKI) or wild-type (WT) controls. Results: HIIT lowered blood glucose and increased exercise capacity, food intake, basal activity levels, carbohydrate oxidation and liver and adipose tissue insulin sensitivity in HFD-fed WT and AccDKI mice. These changes occurred independently of weight loss or reductions in adiposity, inflammation or liver lipid content. Ph.D. Thesis K. Marcinko McMaster University Medical Sciences 90 Conclusions: These data indicate that HIIT lowers blood glucose levels by improving adipose and liver insulin sensitivity independently of changes in adiposity, inflammation, liver lipid content or AMPK phosphorylation of ACC.
The Contribution of RCK Domains to Human BK Channel Allosteric Activation*

Background: In BK channels, Ca2+ and voltage sensors are allosterically connected to the pore. Results: We optically resolved voltage sensor rearrangements, initiated by Ca2+ binding to the intracellular domains RCK1 and RCK2. Impairing the RCK2 abolished this allosteric effect. Conclusion: The RCK2 Ca2+ sensor is required for the allosteric facilitation of voltage sensor activation. Significance: RCK1 and RCK2 Ca2+ sensors are not functionally homologous. Large conductance voltage- and Ca2+-activated K+ (BK) channels are potent regulators of cellular processes including neuronal firing, synaptic transmission, cochlear hair cell tuning, insulin release, and smooth muscle tone. Their unique activation pathway relies on structurally distinct regulatory domains including one transmembrane voltage-sensing domain (VSD) and two intracellular high affinity Ca2+-sensing sites per subunit (located in the RCK1 and RCK2 domains). Four pairs of RCK1 and RCK2 domains form a Ca2+-sensing apparatus known as the “gating ring. ” The allosteric interplay between voltage- and Ca2+-sensing apparati is a fundamental mechanism of BK channel function. Using voltage-clamp fluorometry and UV photolysis of intracellular caged Ca2+, we optically resolved VSD activation prompted by Ca2+ binding to the gating ring. The sudden increase of intracellular Ca2+ concentration ([Ca2+]i) induced a hyperpolarizing shift in the voltage dependence of both channel opening and VSD activation, reported by a fluorophore labeling position 202, located in the upper side of the S4 transmembrane segment. The neutralization of the Ca2+ sensor located in the RCK2 domain abolished the effect of [Ca2+]i increase on the VSD rearrangements. On the other hand, the mutation of RCK1 residues involved in Ca2+ sensing did not prevent the effect of Ca2+ release on the VSD, revealing a functionally distinct interaction between RCK1 and RCK2 and the VSD. A statistical-mechanical model quantifies the complex thermodynamics interplay between Ca2+ association in two distinct sites, voltage sensor activation, and BK channel opening.
Involvement of Beclin 1 in Engulfment of Apoptotic Cells*

Background: Engulfment of apoptotic cell is facilitated by Rac1-GTPase and phosphatidylinositol 3-kinase (PI3K). Beclin 1 is known to form PI3K complex. Results: Beclin 1 was bound to Rac1, recruited to early phagocytotic cups, and required for apoptotic cell internalization. Conclusion: Beclin 1 regulates apoptotic cell engulfment in cooperation with Rac1. Significance: This report reveals a novel function of Beclin 1 in apoptotic cell clearance. Efficient apoptotic cell engulfment is important for both tissue homeostasis and immune response in mammals. In the present study, we report that Beclin 1 (a regulator of autophagy) is required for apoptotic cell engulfment. The engulfment process was largely abolished in Beclin 1 knock-out cells, and Beclin 1 knockdown significantly decreased apoptotic cell internalization in macrophage and fibroblast cell lines. Beclin 1 was recruited to the early phagocytic cup along with the generation of phosphatidylinositol 3-phosphate and Rac1, which regulates actin dynamics in lamellipodia. No lamellipodia were formed in Beclin 1 knock-out cells, and Beclin 1 knockdown completely inhibited the promotion of engulfment by ectopic expression of Rac1. Beclin 1 was co-immunoprecipitated with Rac1. These data indicate that Beclin 1 coordinates actin dynamics and membrane phospholipid synthesis to promote efficient apoptotic cell engulfment.
G Protein-coupled Receptor Kinase 5 Phosphorylates Nucleophosmin and Regulates Cell Sensitivity to Polo-like Kinase 1 Inhibition*

Background: G protein-coupled receptor kinases (GRKs) are important regulators of receptor signaling although little is known about their functions beyond their receptor modifying activities. Results: GRK5 binds and phosphorylates nucleophosmin. Conclusion: GRK5 and polo-like kinase 1 coordinately regulate nucleophosmin phosphorylation and cell sensitivity to inhibitor-induced apoptosis. Significance: GRKs play an important role in regulating normal cell functions such as cell cycle regulation and apoptosis. G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, leading to their desensitization and endocytosis. GRKs have also been implicated in phosphorylating other classes of proteins and can localize in a variety of cellular compartments, including the nucleus. Here, we attempted to identify potential nuclear substrates for GRK5. Our studies reveal that GRK5 is able to interact with and phosphorylate nucleophosmin (NPM1) both in vitro and in intact cells. NPM1 is a nuclear protein that regulates a variety of cell functions including centrosomal duplication, cell cycle control, and apoptosis. GRK5 interaction with NPM1 is mediated by the N-terminal domain of each protein, and GRK5 primarily phosphorylates NPM1 at Ser-4, a site shared with polo-like kinase 1 (PLK1). NPM1 phosphorylation by GRK5 and PLK1 correlates with the sensitivity of cells to undergo apoptosis with cells having higher GRK5 levels being less sensitive and cells with lower GRK5 being more sensitive to PLK1 inhibitor-induced apoptosis. Taken together, our results demonstrate that GRK5 phosphorylates Ser-4 in nucleophosmin and regulates the sensitivity of cells to PLK1 inhibition.
Paternal duplication of the 11p15 centromeric imprinting control region is associated with increased expression of CDKN1C in a child with Russell–Silver syndrome

Conflict of interest: There are no conflicts of interest to declare. Correspondence to: Yuan Xue, 2165 North Decatur Road, Decatur, GA 30033. Email: yuan.xue@emory.edu Article first published online in Wiley Online Library (wileyonlinelibrary.com): 11 September 2015 DOI 10.1002/ajmg.a. 37371 How to Cite this Article: Xue Y, Shankar S, Cornell K, Dai Z, Wang C-E, Rudd MK, Coffee B. 2015. Paternal duplication of the 11p15 centromeric imprinting control region is associated with increased expression of CDKN1C in a child with Russell–Silver syndrome.
Antimicrobial effect and shelf‐life extension by combined thermal and pulsed electric field treatment of milk

Aims: The impact of a combined hurdle treatment of heat and pulsed electric fields (PEF) was studied on native microbiota used for the inoculation of low‐fat ultra‐high temperature (UHT) milk and whole raw milk. Microbiological shelf‐life of the latter following hurdle treatment or thermal pasteurization was also investigated.
A PIGN mutation responsible for multiple congenital anomalies–hypotonia–seizures syndrome 1 (MCAHS1) in an Israeli–Arab family

Mutations in the PIGN gene involved in the glycosylphoshatidylinositol (GPI) anchor biosynthesis pathway cause Multiple Congenital Anomalies–Hypotonia–Seizures syndrome 1 (MCAHS1). The syndrome manifests developmental delay, hypotonia, and epilepsy, combined with multiple congenital anomalies. We report on the identification of a homozygous novel c.755A>T (p.D252V) deleterious mutation in a patient with Israeli–Arab origin with MCAHS1. The mutated PIGN caused a significant decrease of the overall GPI‐anchored proteins and CD24 expression. Our results, strongly support previously published data, that partial depletion of GPI‐anchored proteins is sufficient to cause severe phenotypic expression. © 2015 Wiley Periodicals, Inc.
4-O-Carboxymethyl Ascochlorin Causes ER Stress and Induced Autophagy in Human Hepatocellular Carcinoma Cells*

Background: 4-O-Carboxymethyl ascochlorin (AS-6), a PPARγ agonist, reduces serum glucose levels in a diabetic mouse model. Results: Proteomics analysis revealed that many proteins up-regulated after AS-6 treatment were associated with ER stress response. Conclusion: AS-6 produces an ER unfolded protein response, inducing autophagy, and activating transcription factor CHOP-dependent apoptosis. Significance: By stimulating autophagy, AS-6 might be effective in blocking proliferation of cancer cells. The synthetic derivative of ascochlorin, 4-O-carboxymethyl ascochlorin (AS-6) is an agonist of the nuclear hormone receptor PPARγ and has been shown to induce differentiation in mouse pre-adipocytes and to ameliorate type II diabetes in a murine model. AS-6 was cytotoxic when added at micromolar concentrations to cultures of three different human cancer cell lines. We used gel electrophoresis and mass spectrometry to identify proteins with altered expression in human hepatocarcinoma cells (HepG2) cells after 12 h in the presence of AS-6 and found 58 proteins that were differentially expressed. Many of the proteins showing increased expression in cells treated with AS-6 are involved in protein quality control, including glucose-regulated protein 78 (GRP78/BiP), a regulator of ER stress responses, and the transcriptional regulator CHOP, which mediates ER stress-induced apoptosis. Cells treated with AS-6 undergo an autophagic response accompanied by increased expression of beclin1, ATG5, and LC3-II and autophagosome formation marked by the appearance of large vesicles containing LC3-II. Grp78 induction was inhibited when the PPARγ antagonist, GW9662, was added together with AS-6, and autophagy and cell death were partially blocked. 3-methyl-adenine (3-MA), an inhibitor of phosphatidyl inositol 3-kinase (PI3-kinase) prevented induction of ATG5 and activation of LC3-II and blocked autophagosome formation. 3-MA also blocked induction of GRP78 and CHOP, suggesting that PI3-kinase, which is known to mediate ER stress-induced autophagy, also plays a role in initiating apoptosis in response to ER stress. Together these data establish that the cytotoxicity of AS-6 operates by a mechanism dependent on ER stress-induced autophagy and apoptosis.
Chemoenzymatic Synthesis of Heparin Oligosaccharides with both Anti-factor Xa and Anti-factor IIa Activities*

Background: Heparin inhibits the activity of factors Xa and IIa in the blood coagulation cascade. Results: A series of size-defined N-sulfated oligosaccharides were synthesized to probe the size requirement for the oligosaccharides displaying anti-IIa activity. Conclusion: Oligosaccharides that display anti-IIa activity are longer than 19 saccharide residues. Significance: The results will direct efforts to prepare synthetic heparin with both anti-Xa and anti-IIa activities. Heparan sulfate (HS) and heparin are highly sulfated polysaccharides. Heparin is a commonly used anticoagulant drug that inhibits the activities of factors Xa and IIa (also known as thrombin) to prevent blood clot formation. Here, we report the synthesis of a series of size-defined oligosaccharides to probe the minimum size requirement for an oligosaccharide with anti-IIa activity. The synthesis was completed by a chemoenzymatic approach involving glycosyltransferases, HS sulfotransferases, and C5-epimerase. We demonstrate the ability to synthesize highly purified N-sulfo-oligosaccharides having up to 21 saccharide residues. The results from anti-Xa and anti-IIa activity measurements revealed that an oligosaccharide longer than 19 saccharide residues is necessary to display anti-IIa activity. The oligosaccharides also exhibit low binding toward platelet factor 4, raising the possibility of preparing a synthetic heparin with a reduced effect of heparin-induced thrombocytopenia. The results from this study demonstrate the ability to synthesize large HS oligosaccharides and provide a unique tool to probe the structure and function relationships of HS that require the use of large HS fragments.
ERK5 Protein Promotes, whereas MEK1 Protein Differentially Regulates, the Toll-like Receptor 2 Protein-dependent Activation of Human Endothelial Cells and Monocytes*

Background: Endothelial cell (EC) Toll-like receptor 2 (TLR2) signaling induces inflammatory events. Results: NF-κB, p38-MAPK, JNK, and ERK5 promote, whereas MEK1 suppresses, EC TLR2 signaling. Conclusion: ERK5 is a newly identified mediator of TLR2 signaling, and TLR2 signaling pathways differ in ECs and monocytes. Significance: TLR2 signaling differences can be exploited therapeutically for endothelial-specific or leukocyte-specific inflammatory responses. Endothelial cell (EC) Toll-like receptor 2 (TLR2) activation up-regulates the expression of inflammatory mediators and of TLR2 itself and modulates important endothelial functions, including coagulation and permeability. We defined TLR2 signaling pathways in EC and tested the hypothesis that TLR2 signaling differs in EC and monocytes. We found that ERK5, heretofore unrecognized as mediating TLR2 activation in any cell type, is a central mediator of TLR2-dependent inflammatory signaling in human umbilical vein endothelial cells, primary human lung microvascular EC, and human monocytes. Additionally, we observed that, although MEK1 negatively regulates TLR2 signaling in EC, MEK1 promotes TLR2 signaling in monocytes. We also noted that activation of TLR2 led to the up-regulation of intracellularly expressed TLR2 and inflammatory mediators via NF-κB, JNK, and p38-MAPK. Finally, we found that p38-MAPK, JNK, ERK5, and NF-κB promote the attachment of human neutrophils to lung microvascular EC that were pretreated with TLR2 agonists. This study newly identifies ERK5 as a key regulator of TLR2 signaling in EC and monocytes and indicates that there are fundamental differences in TLR signaling pathways between EC and monocytes.