TY - CONF TI - The Chicken Gut Microbiome and Salmonella AU - Hassan, H.M. T2 - United State Animal Health Association – Annual Meeting C2 - 2014/10/21/ CY - Arkansas, MO DA - 2014/10/21/ PY - 2014/10/21/ ER - TY - CONF TI - Development of the chick microbiome: How early exposure influences future microbial diversity AU - Ballou, A.L. AU - Ali, R. AU - Mendoza, M. AU - Hassan, H. AU - Koci, M.D. T2 - Symposium on Gut Health C2 - 2014/// CY - St Louis, MO DA - 2014/// PY - 2014/11// ER - TY - CONF TI - Comparative analysis of 16S amplicon sequencing data from Roche 454 and Ion Torrent PGM platforms AU - Allali, Imane AU - Cadenas, Maria Belen AU - Ballou, Anne AU - Mendoza, Mary AU - Ali, Rizwana AU - Hassan, Hosni AU - Koci, Matthew AU - Azcarate-Peril, M. Andrea T2 - UNC Research Computing Symposium C2 - 2014/5/20/ DA - 2014/5/20/ PY - 2014/5/20/ ER - TY - RPRT TI - A vector for vaccination against Lyme Disease and other insect transmitted diseases using a live Salmonella vaccine AU - Hassan, H. DA - 2014/5/5/ PY - 2014/5/5/ M3 - Invention Disclosure ER - TY - JOUR TI - ORNL hosts the participants of the 4thNeutrons in Structural Biology Workshop and the IMAGINE single crystal neutron diffractometer first external users AU - Meilleur, Flora T2 - Neutron News AB - "ORNL hosts the participants of the 4th Neutrons in Structural Biology Workshop and the IMAGINE single crystal neutron diffractometer first external users." Neutron News, 25(1), p. 12 DA - 2014/1/2/ PY - 2014/1/2/ DO - 10.1080/10448632.2014.870435 VL - 25 IS - 1 SP - 12-12 ER - TY - JOUR TI - The Neutrons in Structural Biology Workshop celebrates its 5th edition AU - Meilleur, Flora T2 - Neutron News AB - "The Neutrons in Structural Biology Workshop celebrates its 5th edition." Neutron News, 25(4), p. 11 DA - 2014/10/2/ PY - 2014/10/2/ DO - 10.1080/10448632.2014.955418 VL - 25 IS - 4 SP - 11-11 ER - TY - JOUR TI - A genome-scale resource for the functional characterization of Arabidopsis transcription factors. T2 - Cell reports AB - Extensive transcriptional networks play major roles in cellular and organismal functions. Transcript levels are in part determined by the combinatorial and overlapping functions of multiple transcription factors (TFs) bound to gene promoters. Thus, TF-promoter interactions provide the basic molecular wiring of transcriptional regulatory networks. In plants, discovery of the functional roles of TFs is limited by an increased complexity of network circuitry due to a significant expansion of TF families. Here, we present the construction of a comprehensive collection of Arabidopsis TFs clones created to provide a versatile resource for uncovering TF biological functions. We leveraged this collection by implementing a high-throughput DNA binding assay and identified direct regulators of a key clock gene (CCA1) that provide molecular links between different signaling modules and the circadian clock. The resources introduced in this work will significantly contribute to a better understanding of the transcriptional regulatory landscape of plant genomes. DA - 2014/7/17/ PY - 2014/7/17/ DO - 10.1016/j.celrep.2014.06.033 UR - http://europepmc.org/articles/PMC4125603 ER - TY - JOUR TI - Relative Roles of CD90 and c-Kit to the Regenerative Efficacy of Cardiosphere-Derived Cells in Humans and in a Mouse Model of Myocardial Infarction AU - Cheng, Ke AU - Ibrahim, Ahmed AU - Hensley, M. Taylor AU - Shen, Deliang AU - Sun, Baiming AU - Middleton, Ryan AU - Liu, Weixin AU - Smith, Rachel R. AU - Marban, Eduardo T2 - JOURNAL OF THE AMERICAN HEART ASSOCIATION AB - Background The regenerative potential of cardiosphere‐derived cells ( CDC s) for ischemic heart disease has been demonstrated in mice, rats, pigs, and a recently completed clinical trial ( CADUCEUS ). CDC s are CD 105 + stromal cells of intrinsic cardiac origin, but the antigenic characteristics of the active fraction remain to be defined. CDC s contain a small minority of c‐kit + cells, which have been argued to be cardiac progenitors, and a variable fraction of CD 90 + cells whose bioactivity is unclear. Methods We performed a retrospective analysis of data from the CADUCEUS trial and a prospective mouse study to elucidate the roles of c‐kit + and CD 90 + cells in human CDC s. Here, we show, surprisingly, that c‐kit expression has no relationship to CDC s' therapeutic efficacy in humans, and depletion of c‐kit + cells does not undermine the structural and functional benefits of CDC s in a mouse model of myocardial infarction (MI). In contrast, CD 90 expression negatively correlates with the therapeutic benefit of CDC s in humans (ie, higher CD 90 expression associated with lower efficacy). Depletion of CD 90 + cells augments the functional potency of CDC s in murine MI. CD 90 − CDC s secrete lower levels of inflammatory cytokines and can differentiate into cardiomyocytes in vitro and in vivo. Conclusion The majority population of CDC s ( CD 105 + / CD 90 − /c‐kit − ) constitutes the active fraction, both in terms of therapeutic efficacy and in the ability to undergo cardiomyogenic differentiation. The c‐kit + fraction is neither necessary for, nor contributory to, the regenerative efficacy of CDC s. DA - 2014/10// PY - 2014/10// DO - 10.1161/jaha.114.001260 VL - 3 IS - 5 SP - SN - 2047-9980 KW - cardiosphere-derived cells KW - CD90 KW - ckit KW - myocardial infarction ER - TY - JOUR TI - Modifying Caspase-3 Activity by Altering Allosteric Networks AU - Cade, Christine AU - Swartz, Paul AU - MacKenzie, Sarah H. AU - Clark, A. Clay T2 - BIOCHEMISTRY AB - Caspases have several allosteric sites that bind small molecules or peptides. Allosteric regulators are known to affect caspase enzyme activity, in general, by facilitating large conformational changes that convert the active enzyme to a zymogen-like form in which the substrate-binding pocket is disordered. Mutations in presumed allosteric networks also decrease activity, although large structural changes are not observed. Mutation of the central V266 to histidine in the dimer interface of caspase-3 inactivates the enzyme by introducing steric clashes that may ultimately affect positioning of a helix on the protein surface. The helix is thought to connect several residues in the active site to the allosteric dimer interface. In contrast to the effects of small molecule allosteric regulators, the substrate-binding pocket is intact in the mutant, yet the enzyme is inactive. We have examined the putative allosteric network, in particular the role of helix 3, by mutating several residues in the network. We relieved steric clashes in the context of caspase-3(V266H), and we show that activity is restored, particularly when the restorative mutation is close to H266. We also mimicked the V266H mutant by introducing steric clashes elsewhere in the allosteric network, generating several mutants with reduced activity. Overall, the data show that the caspase-3 native ensemble includes the canonical active state as well as an inactive conformation characterized by an intact substrate-binding pocket, but with an altered helix 3. The enzyme activity reflects the relative population of each species in the native ensemble. DA - 2014/12/9/ PY - 2014/12/9/ DO - 10.1021/bi500874k VL - 53 IS - 48 SP - 7582-7595 SN - 0006-2960 ER - TY - JOUR TI - Loop Electrostatics Modulates the Intersubunit Interactions in Ferritin AU - Bernacchioni, Caterina AU - Ghini, Veronica AU - Pozzi, Cecilia AU - Di Pisa, Flavio AU - Theil, Elizabeth C. AU - Turano, Paola T2 - ACS CHEMICAL BIOLOGY AB - Functional ferritins are 24-mer nanocages that self-assemble with extended contacts between pairs of 4-helix bundle subunits coupled in an antiparallel fashion along the C2 axes. The largest intersubunit interaction surface in the ferritin nanocage involves helices, but contacts also occur between groups of three residues midway in the long, solvent-exposed L-loops of facing subunits. The anchor points between intersubunit L-loop pairs are the salt bridges between the symmetry-related, conserved residues Asp80 and Lys82. The resulting quaternary structure of the cage is highly soluble and thermostable. Substitution of negatively charged Asp80 with a positively charged Lys in homopolymeric M ferritin introduces electrostatic repulsions that inhibit the oligomerization of the ferritin subunits. D80K ferritin was present in inclusion bodies under standard overexpressing conditions in E. coli, contrasting with the wild type protein. Small amounts of fully functional D80K nanocages formed when expression was slowed. The more positively charged surface results in a different solubility profile and D80K crystallized in a crystal form with a low density packing. The 3D structure of D80K variant is the same as wild type except for the side chain orientations of Lys80 and facing Lys82. When three contiguous Lys groups are introduced in D80KI81K ferritin variant the nanocage assembly is further inhibited leading to lower solubility and reduced thermal stability. Here, we demonstrate that the electrostatic pairing at the center of the L-loops has a specific kinetic role in the self-assembly of ferritin nanocages. DA - 2014/11// PY - 2014/11// DO - 10.1021/cb500431r VL - 9 IS - 11 SP - 2517-2525 SN - 1554-8937 ER - TY - JOUR TI - Definition of the Cattle Killer Cell Ig-like Receptor Gene Family: Comparison with Aurochs and Human Counterparts AU - Sanderson, Nicholas D. AU - Norman, Paul J. AU - Guethlein, Lisbeth A. AU - Ellis, Shirley A. AU - Williams, Christina AU - Breen, Matthew AU - Park, Steven D. E. AU - Magee, David A. AU - Babrzadeh, Farbod AU - Warry, Andrew AU - Watson, Mick AU - Bradley, Daniel G. AU - MacHugh, David E. AU - Parham, Peter AU - Hammond, John A. T2 - JOURNAL OF IMMUNOLOGY AB - Abstract Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MHC class I evolved convergently in different species of placental mammal. Unexpectedly, diversified killer cell Ig–like receptors (KIRs) are shared by simian primates, including humans, and cattle, but not by other species. Whereas much is known of human KIR genetics and genomics, knowledge of cattle KIR is limited to nine cDNA sequences. To facilitate comparison of the cattle and human KIR gene families, we determined the genomic location, structure, and sequence of two cattle KIR haplotypes and defined KIR sequences of aurochs, the extinct wild ancestor of domestic cattle. Larger than its human counterpart, the cattle KIR locus evolved through successive duplications of a block containing ancestral KIR3DL and KIR3DX genes that existed before placental mammals. Comparison of two cattle KIR haplotypes and aurochs KIR show the KIR are polymorphic and the gene organization and content appear conserved. Of 18 genes, 8 are functional and 10 were inactivated by point mutation. Selective inactivation of KIR3DL and activating receptor genes leaves a functional cohort of one inhibitory KIR3DL, one activating KIR3DX, and six inhibitory KIR3DX. Functional KIR diversity evolved from KIR3DX in cattle and from KIR3DL in simian primates. Although independently evolved, cattle and human KIR gene families share important function-related properties, indicating that cattle KIR are NK cell receptors for cattle MHC class I. Combinations of KIR and MHC class I are the major genetic factors associated with human disease and merit investigation in cattle. DA - 2014/12/15/ PY - 2014/12/15/ DO - 10.4049/jimmunol.1401980 VL - 193 IS - 12 SP - 6016-6030 SN - 1550-6606 ER - TY - JOUR TI - Physiologic and biochemical assessments of koi (Cyprinus carpio) following immersion in propofol AU - Oda, Ayako AU - Bailey, Kate M. AU - Lewbart, Gregory A. AU - Griffith, Emily H. AU - Posner, Lysa P. T2 - JAVMA-JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION AB - Abstract Objective —To determine efficacy of propofol as an immersion agent to induce general anesthesia in koi ( Cyprinus carpio ). Design —Prospective, crossover study. Animals —10 adult koi (mean ± SD weight, 325 ± 81 g). Procedures —Koi were exposed to each of 4 concentrations of propofol (1, 2.5, 5, and 10 mg/L) with a 1-week washout period between trials. In a subsequent trial, koi were anesthetized with propofol (5 mg/L) and anesthesia was maintained with propofol (3 mg/L) for 20 minutes. Response to a noxious stimulus was assessed by means of needle insertion into an epaxial muscle. Results —At a propofol concentration of 1 mg/L, koi were sedated but never anesthetized. At propofol concentrations of 2.5, 5, and 10 mg/L, mean ± SD anesthetic induction times were 13.4 ± 3.3, 3.8 ± 1.1, and 2.3 ± 0.9 minutes, respectively; mean recovery times were 12.9 ± 8.3, 11.0 ± 6.3, and 18.1 ± 13.0 minutes; mean heart rates were 57 ± 25, 30 ± 14, and 22 ± 14 beats/min; mean opercular rates were 58 ± 18, 68 ± 15, and 48 ± 22 beats/min; and 1 of 10, 2 of 10, and 0 of 10 fish responded to needle insertion. All fish recovered satisfactorily. Following 20 minutes of anesthesia, 2 fish had recovery times > 4 hours and 1 fish died. Conclusions and Clinical Relevance —Immersion in propofol at concentrations ≥ 2.5 mg/L induced general anesthesia in koi. Maintenance of anesthesia with propofol for 20 minutes was associated with prolonged recovery times in 2 of 9 and death in 1 of 9 koi. DA - 2014/12/1/ PY - 2014/12/1/ DO - 10.2460/javma.245.11.1286 VL - 245 IS - 11 SP - 1286-1291 SN - 1943-569X ER - TY - JOUR TI - Surgical treatment of synovial osteochondromatosis in the middle carpal joint of a pony AU - Newman, J. C. AU - Lustgarten, M. AU - Berman, K. G. AU - Vivrette, S. AU - Redding, W. R. T2 - EQUINE VETERINARY EDUCATION AB - Summary A 9‐year‐old P aint pony gelding presented for signs of left carpal swelling of 1–2 weeks' duration. Radiographic, ultrasonographic and arthroscopic evaluation of the left carpus was consistent with synovial osteochondromatosis. This presumptive clinical diagnosis was confirmed histopathologically. Arthroscopic removal of the osteochondral bodies resulted in resolution of the carpal effusion and return to previous athletic activity by 4.5 months post operatively. Arthroscopic removal of osteochondral bodies is the treatment of choice in cases of suspected synovial osteochondromatosis. DA - 2014/8// PY - 2014/8// DO - 10.1111/eve.12093 VL - 26 IS - 8 SP - 395-399 SN - 2042-3292 KW - horse KW - synovial osteochondromatosis KW - arthroscopic KW - carpus ER - TY - JOUR TI - Structure and DNA-Binding Traits of the Transition State Regulator AbrB AU - Olson, Andrew L. AU - Tucker, Ashley T. AU - Bobay, Benjamin G. AU - Soderblom, Erik J. AU - Moseley, M. Arthur AU - Thompson, Richele J. AU - Cavanagh, John T2 - STRUCTURE AB - The AbrB protein from Bacillus subtilis is a DNA-binding global regulator controlling the onset of a vast array of protective functions under stressful conditions. Such functions include biofilm formation, antibiotic production, competence development, extracellular enzyme production, motility, and sporulation. AbrB orthologs are known in a variety of prokaryotic organisms, most notably in all infectious strains of Clostridia, Listeria, and Bacilli. Despite its central role in bacterial response and defense, its structure has been elusive because of its highly dynamic character. Orienting its N- and C-terminal domains with respect to one another has been especially problematic. Here, we have generated a structure of full-length, tetrameric AbrB using nuclear magnetic resonance, chemical crosslinking, and mass spectrometry. We note that AbrB possesses a strip of positive electrostatic potential encompassing its DNA-binding region and that its C-terminal domain aids in DNA binding. DA - 2014/11/4/ PY - 2014/11/4/ DO - 10.1016/j.str.2014.08.018 VL - 22 IS - 11 SP - 1650-1656 SN - 1878-4186 ER - TY - JOUR TI - Small-Molecule Suppression of beta-Lactam Resistance in Multidrug-Resistant Gram-Negative Pathogens AU - Brackett, Christopher M. AU - Melander, Roberta J. AU - An, Il Hwan AU - Krishnamurthy, Aparna AU - Thompson, Richele J. AU - Cavanagh, John AU - Melander, Christian T2 - JOURNAL OF MEDICINAL CHEMISTRY AB - Recent efforts toward combating antibiotic resistance in bacteria have focused on Gram-positive bacteria; however, multidrug-resistant Gram-negative bacteria pose a significant risk to public health. An orthogonal approach to the development of new antibiotics is to develop adjuvant compounds that enhance the susceptibility of drug-resistant strains of bacteria to currently approved antibiotics. This paper describes the synthesis and biological activity of a library of aryl amide 2-aminoimidazoles based on a lead structure from an initial screen. A small molecule was identified from this library that is capable of lowering the minimum inhibitory concentration of β-lactam antibiotics by up to 64-fold. DA - 2014/9/11/ PY - 2014/9/11/ DO - 10.1021/jm501050e VL - 57 IS - 17 SP - 7450-7458 SN - 1520-4804 ER - TY - JOUR TI - Loading and Release Mechanism of Red Clover Necrotic Mosaic Virus Derived Plant Viral Nanoparticles for Drug Delivery of Doxorubicin AU - Cao, Jing AU - Guenther, Richard H. AU - Sit, Tim L. AU - Opperman, Charles H. AU - Lommel, Steven A. AU - Willoughby, Julie A. T2 - SMALL AB - Loading and release mechanisms of Red clover necrotic mosaicvirus (RCNMV) derived plant viral nanoparticle (PVN) are shown for controlled delivery of the anticancer drug, doxorubicin (Dox). Previous studies demonstrate that RCNMV's structure and unique response to divalent cation depletion and re‐addition enables Dox infusion to the viral capsid through a pore formation mechanism. However, by controlling the net charge of RCNMV outer surface and accessibility of RCNMV interior cavity, tunable release of PVN is possible via manipulation of the Dox loading capacity and binding locations (external surface‐binding or internal capsid‐encapsulation) with the RCNMV capsid. Bimodal release kinetics is achieved via a rapid release of surface‐Dox followed by a slow release of encapsulated Dox. Moreover, the rate of Dox release and the amount of released Dox increases with an increase in environmental pH or a decrease in concentration of divalent cations. This pH‐responsive Dox release from PVN is controlled by Fickian diffusion kinetics where the release rate is dependent on the location of the bound or loaded active molecule. In summary, controllable release of Dox‐loaded PVNs is imparted by 1) formulation conditions and 2) driven by the capsid's pH‐ and ion‐ responsive functions in a given environment. DA - 2014/12/29/ PY - 2014/12/29/ DO - 10.1002/smll.201400558 VL - 10 IS - 24 SP - 5126-5136 SN - 1613-6829 ER - TY - JOUR TI - Establishing ion ratio thresholds based on absolute peak area for absolute protein quantification using protein cleavage isotope dilution mass spectrometry AU - Loziuk, Philip L. AU - Sederoff, Ronald R. AU - Chiang, Vincent L. AU - Muddiman, David C. T2 - ANALYST AB - Relative abundance values and their associated variability are dynamic and dependent on absolute abundance. DA - 2014/// PY - 2014/// DO - 10.1039/c4an00567h VL - 139 IS - 21 SP - 5439-5450 SN - 1364-5528 ER - TY - JOUR TI - Magnetic targeting of cardiosphere-derived stem cells with ferumoxytol nanoparticles for treating rats with myocardial infarction AU - Vandergriff, Adam C. AU - Hensley, Taylor M. AU - Henry, Eric T. AU - Shen, Deliang AU - Anthony, Shirena AU - Zhang, Jinying AU - Cheng, Ke T2 - BIOMATERIALS AB - Stem cell transplantation is a promising therapeutic strategy for acute or chronic ischemic cardiomyopathy. A major limitation to efficacy in cell transplantation is the low efficiency of retention and engraftment, due at least in part to significant early "wash-out" of cells from coronary blood flow and heart contraction. We sought to enhance cell retention and engraftment by magnetic targeting. Human cardiosphere-derived stem cells (hCDCs) were labeled with FDA-approved ferumoxytol nanoparticles Feraheme(®) (F) in the presence of heparin (H) and protamine (P). FHP labeling is nontoxic to hCDCs. FHP-labeled rat CDCs (FHP-rCDCs) were intracoronarily infused into syngeneic rats, with and without magnetic targeting. Magnetic resonance imaging, fluorescence imaging, and quantitative PCR revealed magnetic targeting increased cardiac retention of transplanted FHP-rCDCs. Neither infusion of FHP-rCDCs nor magnetic targeting exacerbated cardiac inflammation or caused iron overload. The augmentation of acute cell retention translated into more attenuated left ventricular remodeling and greater therapeutic benefit (ejection fraction) 3 weeks after treatment. Histology revealed enhanced cell engraftment and angiogenesis in hearts from the magnetic targeting group. FHP labeling is safe to cardiac stem cells and facilitates magnetically-targeted stem cell delivery into the heart which leads to augmented cell engraftment and therapeutic benefit. DA - 2014/10// PY - 2014/10// DO - 10.1016/j.biomaterials.2014.06.031 VL - 35 IS - 30 SP - 8528-8539 SN - 1878-5905 KW - Cardiac stem cells KW - Magnetic targeting KW - Myocardial infarction KW - Ferumoxytol KW - MRI KW - Superparamagnetic iron oxide nanoparticles ER - TY - JOUR TI - SnRK1 Phosphorylation of AL2 Delays Cabbage Leaf Curl Virus Infection in Arabidopsis AU - Shen, Wei AU - Dallas, Mary Beth AU - Goshe, Michael B. AU - Hanley-Bowdoin, Linda T2 - JOURNAL OF VIROLOGY AB - Geminivirus AL2/C2 proteins play key roles in establishing infection and causing disease in their plant hosts. They are involved in viral gene expression, counter host defenses by suppressing transcriptional gene silencing, and interfere with the host signaling involved in pathogen resistance. We report here that begomovirus and curtovirus AL2/C2 proteins interact strongly with host geminivirus Rep-interacting kinases (GRIKs), which are upstream activating kinases of the protein kinase SnRK1, a global regulator of energy and nutrient levels in plants. We used an in vitro kinase system to show that GRIK-activated SnRK1 phosphorylates recombinant AL2/C2 proteins from several begomoviruses and to map the SnRK1 phosphorylation site to serine-109 in the AL2 proteins of two New World begomoviruses: Cabbage Leaf Curl Virus (CaLCuV) and Tomato mottle virus. A CaLCuV AL2 S109D phosphomimic mutation did not alter viral DNA levels in protoplast replication assays. In contrast, the phosphomimic mutant was delayed for symptom development and viral DNA accumulation during infection of Arabidopsis thaliana, demonstrating that SnRK1 contributes to host defenses against CaLCuV. Our observation that serine-109 is not conserved in all AL2/C2 proteins that are SnRK1 substrates in vitro suggested that phosphorylation of viral proteins by plant kinases contributes to the evolution of geminivirus-host interactions.Geminiviruses are single-stranded DNA viruses that cause serious diseases in many crops. Dicot-infecting geminiviruses carry genes that encode multifunctional AL2/C2 proteins that are essential for infection. However, it is not clear how AL2/C2 proteins are regulated. Here, we show that the host protein kinase SnRK1, a central regulator of energy balance and nutrient metabolism in plants, phosphorylates serine-109 in AL2 proteins of three subgroups of New World begomoviruses, resulting in a delay in viral DNA accumulation and symptom appearance. Our results support SnRK1's antiviral role and reveal a novel mechanism underlying this function. Phylogenetic analysis suggested that AL2 S109 evolved as begomoviruses migrated from the Old World to the New World and may have provided a selective advantage as begomoviruses adapted to a different environment and different plant hosts. This study provides new insights into the interaction of viral pathogens with their plant hosts at the level of viral protein modification by the host. DA - 2014/9// PY - 2014/9// DO - 10.1128/jvi.00761-14 VL - 88 IS - 18 SP - 10598-10612 SN - 1098-5514 ER - TY - JOUR TI - Canine Prostate Cancer Cell Line (Probasco) Produces Osteoblastic Metastases In Vivo AU - Simmons, Jessica K. AU - Dirksen, Wessel P. AU - Hildreth, Blake E., III AU - Dorr, Carlee AU - Williams, Christina AU - Thomas, Rachael AU - Breen, Matthew AU - Toribio, Ramiro E. AU - Rosol, Thomas J. T2 - PROSTATE AB - Abstract BACKGROUND In 2012, over 240,000 men were diagnosed with prostate cancer and over 28,000 died from the disease. Animal models of prostate cancer are vital to understanding its pathogenesis and developing therapeutics. Canine models in particular are useful due to their similarities to late‐stage, castration‐resistant human disease with osteoblastic bone metastases. This study established and characterized a novel canine prostate cancer cell line that will contribute to the understanding of prostate cancer pathogenesis. METHODS A novel cell line (Probasco) was derived from a mixed breed dog that had spontaneous prostate cancer. Cell proliferation and motility were analyzed in vitro. Tumor growth in vivo was studied by subcutaneous, intratibial, and intracardiac injection of Probasco cells into nude mice. Tumors were evaluated by bioluminescent imaging, Faxitron radiography, µCT, and histology. RT‐PCR and genome‐wide DNA copy number profiling were used to characterize the cell line. RESULTS The Probasco cells grew in vitro (over 75 passages) and were tumorigenic in nude mice. Probasco cells expressed high levels of BMP2, CDH1, MYOF, FOLH1, RUNX2, and SMAD5 modest CXCL12, SLUG, and BMP, and no PTHrP mRNA. Following intracardiac injection, Probasco cells metastasized primarily to the appendicular skeleton, and both intratibial and intracardiac injections produced osteoblastic tumors in bone. Comparative genomic hybridization demonstrated numerous DNA copy number aberrations throughout the genome, including large losses and gains in multiple chromosomes. CONCLUSIONS The Probasco prostate cancer cell line will be a valuable model to investigate the mechanisms of prostate cancer pathogenesis and osteoblastic bone metastases. Prostate 74: 1251–1265, 2014 . © 2014 Wiley Periodicals, Inc. DA - 2014/9// PY - 2014/9// DO - 10.1002/pros.22838 VL - 74 IS - 13 SP - 1251-1265 SN - 1097-0045 KW - prostate cancer KW - osteoblastic metastases KW - canine KW - dog ER - TY - JOUR TI - Magnetic antibody-linked nanomatchmakers for therapeutic cell targeting AU - Cheng, Ke AU - Shen, Deliang AU - Hensley, M. Taylor AU - Middleton, Ryan AU - Sun, Baiming AU - Liu, Weixin AU - De Couto, Geoffrey AU - Marban, Eduardo T2 - NATURE COMMUNICATIONS AB - Stem cell transplantation is a promising strategy for therapeutic cardiac regeneration, but current therapies are limited by inefficient interaction between potentially beneficial cells (either exogenously transplanted or endogenously recruited) and the injured tissue. Here we apply targeted nanomedicine to achieve in vivo cell-mediated tissue repair, imaging and localized enrichment without cellular transplantation. Iron nanoparticles are conjugated with two types of antibodies (one against antigens on therapeutic cells and the other directed at injured cells) to produce magnetic bifunctional cell engager (MagBICE). The antibodies link the therapeutic cells to the injured cells, whereas the iron core of MagBICE enables physical enrichment and imaging. We treat acute myocardial infarction by targeting exogenous bone marrow-derived stem cells (expressing CD45) or endogenous CD34-positive cells to injured cardiomyocytes (expressing myosin light chain. Targeting can be further enhanced by magnetic attraction, leading to augmented functional benefits. MagBICE represents a generalizable platform technology for regenerative medicine. Cell therapy requires sufficient amounts of therapeutic cells to be delivered to the injured tissue. Here the authors use magnetic iron nanoparticles conjugated with antibodies that bind therapeutic cells and cardiomyocytes to treat myocardial ischemia/reperfusion injury in rats and show that targeting to the heart is enhanced upon local application of a magnetic field. DA - 2014/9// PY - 2014/9// DO - 10.1038/ncomms5880 VL - 5 SP - SN - 2041-1723 ER - TY - JOUR TI - Distinct regulatory mechanisms of the human ferritin gene by hypoxia and hypoxia mimetic cobalt chloride at the transcriptional and post-transcriptional levels AU - Huang, Bo-Wen AU - Miyazawa, Masaki AU - Tsuji, Yoshiaki T2 - CELLULAR SIGNALLING AB - Cobalt chloride has been used as a hypoxia mimetic because it stabilizes hypoxia inducible factor-1α (HIF1-α) and activates gene transcription through a hypoxia responsive element (HRE). However, differences between hypoxia and hypoxia mimetic cobalt chloride in gene regulation remain elusive. Expression of ferritin, the major iron storage protein, is regulated at the transcriptional and posttranscriptional levels through DNA and RNA regulatory elements. Here we demonstrate that hypoxia and cobalt chloride regulate ferritin heavy chain (ferritin H) expression by two distinct mechanisms. Both hypoxia and cobalt chloride increased HIF1-α but a putative HRE in the human ferritin H gene was not activated. Instead, cobalt chloride but not hypoxia activated ferritin H transcription through an antioxidant responsive element (ARE), to which Nrf2 was recruited. Intriguingly, cobalt chloride downregulated ferritin H protein expression while it upregulated other ARE-regulated antioxidant genes in K562 cells. Further characterization demonstrated that cobalt chloride increased interaction between iron regulatory proteins (IRP1 and IRP2) and iron responsive element (IRE) in the 5′UTR of ferritin H mRNA, resulting in translational block of the accumulated ferritin H mRNA. In contrast, hypoxia had marginal effect on ferritin H transcription but increased its translation through decreased IRP1–IRE interaction. These results suggest that hypoxia and hypoxia mimetic cobalt chloride employ distinct regulatory mechanisms through the interplay between DNA and mRNA elements at the transcriptional and post-transcriptional levels. DA - 2014/12// PY - 2014/12// DO - 10.1016/j.cellsig.2014.08.018 VL - 26 IS - 12 SP - 2702-2709 SN - 1873-3913 KW - Hypoxia KW - Cobalt chloride KW - Hypoxia inducible factor (HIF) KW - Iron regulatory protein (IRP) KW - Nrf2 KW - Ferritin ER - TY - JOUR TI - Crystallization and preliminary X-ray diffraction analysis of Hypocrea jecorina Cel7A in two new crystal forms AU - Bodenheimer, A.M. AU - Cuneo, M.J. AU - Swartz, P.D. AU - He, J. AU - O'Neill, H.M. AU - Myles, D.A. AU - Evans, B.R. AU - Meilleur, Flora AU - Section, F. T2 - ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS AB - Cel7A (previously known as cellobiohydrolase I) from Hypocrea jecorina was crystallized in two crystalline forms, neither of which have been previously reported. Both forms co-crystallize under the same crystallization conditions. The first crystal form belonged to space group C2, with unit-cell parameters a=152.5, b=44.9, c=57.6 Å, β=101.2°, and diffracted X-rays to 1.5 Å resolution. The second crystal form belonged to space group P6₃22, with unit-cell parameters a=b≃155, c≃138 Å, and diffracted X-rays to 2.5 Å resolution. The crystals were obtained using full-length Cel7A, which consists of a large 434-residue N-terminal catalytic domain capable of cleaving cellulose, a 27-residue flexible linker and a small 36-residue C-terminal carbohydrate-binding module (CBM). However, a preliminary analysis of the electron-density maps suggests that the linker and CBM are disordered in both crystal forms. Complete refinement and structure analysis are currently in progress. DA - 2014/6// PY - 2014/6// DO - 10.1107/s2053230x14008851 VL - 70 IS - Pt 6 SP - 773-776 SN - 2053-230X UR - http://europepmc.org/abstract/med/24915091 ER - TY - JOUR TI - A simple improved-throughput xylem protoplast system for studying wood formation AU - Lin, Ying-Chung AU - Li, Wei AU - Chen, Hao AU - Li, Quanzi AU - Sun, Ying-Hsuan AU - Shi, Rui AU - Lin, Chien-Yuan AU - Wang, Jack P. AU - Chen, Hsi-Chuan AU - Chuang, Ling AU - Qu, Guan-Zheng AU - Sederoff, Ronald R. AU - Chiang, Vincent L. T2 - NATURE PROTOCOLS DA - 2014/9// PY - 2014/9// DO - 10.1038/nprot.2014.147 VL - 9 IS - 9 SP - 2194-2205 SN - 1750-2799 ER - TY - JOUR TI - A robust chromatin immunoprecipitation protocol for studying transcription factor-DNA interactions and histone modifications in wood-forming tissue AU - Li, Wei AU - Lin, Ying-Chung AU - Li, Quanzi AU - Shi, Rui AU - Lin, Chien-Yuan AU - Chen, Hao AU - Chuang, Ling AU - Qu, Guan-Zheng AU - Sederoff, Ronald R. AU - Chiang, Vincent L. T2 - NATURE PROTOCOLS DA - 2014/9// PY - 2014/9// DO - 10.1038/nprot.2014.146 VL - 9 IS - 9 SP - 2180-2193 SN - 1750-2799 ER - TY - JOUR TI - Redesigning the procaspase-8 dimer interface for improved dimerization AU - Ma, C. X. AU - MacKenzie, S. H. AU - Clark, A. C. T2 - Protein Science DA - 2014/// PY - 2014/// VL - 23 IS - 4 SP - 442-453 ER - TY - JOUR TI - Live Attenuated Tetravalent Dengue Virus Host Range Vaccine Is Immunogenic in African Green Monkeys following a Single Vaccination AU - Briggs, Caitlin M. AU - Smith, Katherine M. AU - Piper, Amanda AU - Huitt, Emerson AU - Spears, Carla J. AU - Quiles, Michelle AU - Ribeiro, Mariana AU - Thomas, Malcolm E. AU - Brown, Dennis T. AU - Hernandez, Raquel T2 - JOURNAL OF VIROLOGY AB - ABSTRACT The causative agent of dengue fever, dengue virus (DENV), is transmitted by mosquitoes, and as distribution of these insects has expanded, so has dengue-related disease. DENV is a member of the Flaviviridae family and has 4 distinct serotypes (DENV-1, -2, -3, and -4). No lasting cross protection is afforded to heterologous serotypes following infection by any one of the individual serotypes. The presence of nonneutralizing antibodies to one serotype can facilitate the occurrence of more-severe dengue hemorrhagic fever through immune enhancement upon infection with a second serotype. For this reason, the development of a safe, tetravalent vaccine to produce a balanced immune response to all four serotypes is critical. We have developed a novel approach to produce safe and effective live-attenuated vaccines for DENV and other insect-borne viruses. Host range (HR) mutants of each DENV serotype were created by truncating transmembrane domain 1 of the E protein and selecting for strains of DENV that replicated well in insect cells but not mammalian cells. These vaccine strains were tested for immunogenicity in African green monkeys (AGMs). No vaccine-related adverse events occurred. The vaccine strains were confirmed to be attenuated in vivo by infectious center assay (ICA). Analysis by 50% plaque reduction neutralization test (PRNT 50 ) established that by day 62 postvaccination, 100% of animals seroconverted to DENV-1, -2, -3, and -4. Additionally, the DENV HR tetravalent vaccine (HR-Tet) showed a tetravalent anamnestic immune response in 100% (16/16) of AGMs after challenge with wild-type (WT) DENV strains. IMPORTANCE We have generated a live attenuated viral (LAV) vaccine capable of eliciting a strong immune response in African green monkeys (AGMs) in a single dose. This vaccine is delivered by injecting one of four attenuated serotypes into each limb of the animal. 100% of animals given the vaccine generated antibodies against all 4 serotypes, and this response was found to be balanced in nature. This is also one of the first studies of dengue in AGMs, and our study suggests that viremia and antibody response in AGMs may be similar to those seen in DENV infection in humans. DA - 2014/6// PY - 2014/6// DO - 10.1128/jvi.00541-14 VL - 88 IS - 12 SP - 6729-6742 SN - 1098-5514 ER - TY - JOUR TI - Data-driven modeling reconciles kinetics of ERK phosphorylation, localization, and activity states AU - Ahmed, Shoeb AU - Grant, Kyle G. AU - Edwards, Laura E. AU - Rahman, Anisur AU - Cirit, Murat AU - Goshe, Michael B. AU - Haugh, Jason M. T2 - MOLECULAR SYSTEMS BIOLOGY AB - Abstract The extracellular signal‐regulated kinase ( ERK ) signaling pathway controls cell proliferation and differentiation in metazoans. Two hallmarks of its dynamics are adaptation of ERK phosphorylation, which has been linked to negative feedback, and nucleocytoplasmic shuttling, which allows active ERK to phosphorylate protein substrates in the nucleus and cytosol. To integrate these complex features, we acquired quantitative biochemical and live‐cell microscopy data to reconcile phosphorylation, localization, and activity states of ERK . While maximal growth factor stimulation elicits transient ERK phosphorylation and nuclear translocation responses, ERK activities available to phosphorylate substrates in the cytosol and nuclei show relatively little or no adaptation. Free ERK activity in the nucleus temporally lags the peak in nuclear translocation, indicating a slow process. Additional experiments, guided by kinetic modeling, show that this process is consistent with ERK 's modification of and release from nuclear substrate anchors. Thus, adaptation of whole‐cell ERK phosphorylation is a by‐product of transient protection from phosphatases. Consistent with this interpretation, predictions concerning the dose‐dependence of the pathway response and its interruption by inhibition of MEK were experimentally confirmed. DA - 2014/1// PY - 2014/1// DO - 10.1002/msb.134708 VL - 10 IS - 1 SP - SN - 1744-4292 KW - growth factor receptors KW - mitogen-activated protein kinases KW - negative feedback KW - nucleocytoplasmic shuttling KW - mathematical model ER - TY - JOUR TI - Coordinating subdomains of ferritin protein cages with catalysis and biomineralization viewed from the C (4) cage axes AU - Theil, Elizabeth C. AU - Turano, Paola AU - Ghini, Veronica AU - Allegrozzi, Marco AU - Bernacchioni, Caterina T2 - JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY AB - Integrated ferritin protein cage function is the reversible synthesis of protein-caged, solid Fe2O3·H2O minerals from Fe2+ for metabolic iron concentrates and oxidant protection; biomineral order differs in different ferritin proteins. The conserved 432 geometric symmetry of ferritin protein cages parallels the subunit dimer, trimer, and tetramer interfaces, and coincides with function at several cage axes. Multiple subdomains distributed in the self-assembling ferritin nanocages have functional relationships to cage symmetry such as Fe2+ transport though ion channels (threefold symmetry), biomineral nucleation/order (fourfold symmetry), and mineral dissolution (threefold symmetry) studied in ferritin variants. On the basis of the effects of natural or synthetic subunit dimer cross-links, cage subunit dimers (twofold symmetry) influence iron oxidation and mineral dissolution. 2Fe2+/O2 catalysis in ferritin occurs in single subunits, but with cooperativity (n = 3) that is possibly related to the structure/function of the ion channels, which are constructed from segments of three subunits. Here, we study 2Fe2+ + O2 protein catalysis (diferric peroxo formation) and dissolution of ferritin Fe2O3·H2O biominerals in variants with altered subunit interfaces for trimers (ion channels), E130I, and external dimer surfaces (E88A) as controls, and altered tetramer subunit interfaces (L165I and H169F). The results extend observations on the functional importance of structure at ferritin protein twofold and threefold cage axes to show function at ferritin fourfold cage axes. Here, conserved amino acids facilitate dissolution of ferritin-protein-caged iron biominerals. Biological and nanotechnological uses of ferritin protein cage fourfold symmetry and solid-state mineral properties remain largely unexplored. DA - 2014/6// PY - 2014/6// DO - 10.1007/s00775-014-1103-z VL - 19 IS - 4-5 SP - 615-622 SN - 1432-1327 KW - Ferritin KW - Protein nanocage KW - Ferric oxo KW - Di-iron protein KW - Nanobiomineral ER - TY - JOUR TI - A maize root tip system to study DNA replication programmes in somatic and endocycling nuclei during plant development AU - Bass, Hank W. AU - Wear, Emily E. AU - Lee, Tae-Jin AU - Hoffman, Gregg G. AU - Gumber, Hardeep K. AU - Allen, George C. AU - Thompson, William F. AU - Hanley-Bowdoin, Linda T2 - JOURNAL OF EXPERIMENTAL BOTANY AB - The progress of nuclear DNA replication is complex in both time and space, and may reflect several levels of chromatin structure and 3-dimensional organization within the nucleus. To understand the relationship between DNA replication and developmental programmes, it is important to examine replication and nuclear substructure in different developmental contexts including natural cell-cycle progressions in situ. Plant meristems offer an ideal opportunity to analyse such processes in the context of normal growth of an organism. Our current understanding of large-scale chromosomal DNA replication has been limited by the lack of appropriate tools to visualize DNA replication with high resolution at defined points within S phase. In this perspective, we discuss a promising new system that can be used to visualize DNA replication in isolated maize (Zea mays L.) root tip nuclei after in planta pulse labelling with the thymidine analogue, 5-ethynyl-2'-deoxyuridine (EdU). Mixed populations of EdU-labelled nuclei are then separated by flow cytometry into sequential stages of S phase and examined directly using 3-dimensional deconvolution microscopy to characterize spatial patterns of plant DNA replication. Combining spatiotemporal analyses with studies of replication and epigenetic inheritance at the molecular level enables an integrated experimental approach to problems of mitotic inheritance and cellular differentiation. DA - 2014/6// PY - 2014/6// DO - 10.1093/jxb/ert470 VL - 65 IS - 10 SP - 2747-2756 SN - 1460-2431 KW - DNA replication KW - EdU KW - endocycle KW - flow cytometry KW - high-resolution microscopy KW - maize KW - mitotic cell cycle KW - root development KW - S phase ER - TY - JOUR TI - A DNA Mimic: The Structure and Mechanism of Action for the Anti-Repressor Protein AbbA AU - Tucker, Ashley T. AU - Bobay, Benjamin G. AU - Banse, Allison V. AU - Olson, Andrew L. AU - Soderblom, Erik J. AU - Moseley, M. Arthur AU - Thompson, Richele J. AU - Varney, Kristen M. AU - Losick, Richard AU - Cavanagh, John T2 - JOURNAL OF MOLECULAR BIOLOGY AB - Bacteria respond to adverse environmental conditions by switching on the expression of large numbers of genes that enable them to adapt to unfavorable circumstances. In Bacillus subtilis, many adaptive genes are under the negative control of the global transition state regulator, the repressor protein AbrB. Stressful conditions lead to the de-repression of genes under AbrB control. Contributing to this de-repression is AbbA, an anti-repressor that binds to and blocks AbrB from binding to DNA. Here, we have determined the NMR structure of the functional AbbA dimer, confirmed that it binds to the N-terminal DNA-binding domain of AbrB, and have provided an initial description for the interaction using computational docking procedures. Interestingly, we show that AbbA has structural and surface characteristics that closely mimic the DNA phosphate backbone, enabling it to readily carry out its physiological function. DA - 2014/5/1/ PY - 2014/5/1/ DO - 10.1016/j.jmb.2014.02.010 VL - 426 IS - 9 SP - 1911-1924 SN - 1089-8638 KW - transition state regulator KW - AbbA KW - DNA mimic KW - molecular docking KW - NMR ER - TY - JOUR TI - Lignin extraction from biomass with protic ionic liquids AU - Achinivu, Ezinne C. AU - Howard, Reagan M. AU - Li, Guoqing AU - Gracz, Hanna AU - Henderson, Wesley A. T2 - GREEN CHEMISTRY AB - A simple, highly effective method for lignin extraction from biomass is reported using PILs which can easily be distilled/recovered. DA - 2014/// PY - 2014/// DO - 10.1039/c3gc42306a VL - 16 IS - 3 SP - 1114-1119 SN - 1463-9270 ER - TY - JOUR TI - Decoding the massive genome of loblolly pine using haploid DNA and novel assembly strategies AU - Neale, David B. AU - Wegrzyn, Jill L. AU - Stevens, Kristian A. AU - Zimin, Aleksey V. AU - Puiu, Daniela AU - Crepeau, Marc W. AU - Cardeno, Charis AU - Koriabine, Maxim AU - Holtz-Morris, Ann E. AU - Liechty, John D. AU - Martinez-Garcia, Pedro J. AU - Vasquez-Gross, Hans A. AU - Lin, Brian Y. AU - Zieve, Jacob J. AU - Dougherty, William M. AU - Fuentes-Soriano, Sara AU - Wu, Le-Shin AU - Gilbert, Don AU - Marcais, Guillaume AU - Roberts, Michael AU - Holt, Carson AU - Yandell, Mark AU - Davis, John M. AU - Smith, Katherine E. AU - Dean, Jeffrey F. D. AU - Lorenz, W. Walter AU - Whetten, Ross W. AU - Sederoff, Ronald AU - Wheeler, Nicholas AU - McGuire, Patrick E. AU - Main, Doreen AU - Loopstra, Carol A. AU - Mockaitis, Keithanne AU - deJong, Pieter J. AU - Yorke, James A. AU - Salzberg, Steven L. AU - Langley, Charles H. T2 - GENOME BIOLOGY AB - The size and complexity of conifer genomes has, until now, prevented full genome sequencing and assembly. The large research community and economic importance of loblolly pine, Pinus taeda L., made it an early candidate for reference sequence determination.We develop a novel strategy to sequence the genome of loblolly pine that combines unique aspects of pine reproductive biology and genome assembly methodology. We use a whole genome shotgun approach relying primarily on next generation sequence generated from a single haploid seed megagametophyte from a loblolly pine tree, 20-1010, that has been used in industrial forest tree breeding. The resulting sequence and assembly was used to generate a draft genome spanning 23.2 Gbp and containing 20.1 Gbp with an N50 scaffold size of 66.9 kbp, making it a significant improvement over available conifer genomes. The long scaffold lengths allow the annotation of 50,172 gene models with intron lengths averaging over 2.7 kbp and sometimes exceeding 100 kbp in length. Analysis of orthologous gene sets identifies gene families that may be unique to conifers. We further characterize and expand the existing repeat library based on the de novo analysis of the repetitive content, estimated to encompass 82% of the genome.In addition to its value as a resource for researchers and breeders, the loblolly pine genome sequence and assembly reported here demonstrates a novel approach to sequencing the large and complex genomes of this important group of plants that can now be widely applied. DA - 2014/// PY - 2014/// DO - 10.1186/gb-2014-15-3-r59 VL - 15 IS - 3 SP - SN - 1474-760X ER - TY - JOUR TI - A role for iron and oxygen chemistry in preserving soft tissues, cells and molecules from deep time AU - Schweitzer, M. H. AU - Zheng, W. X. AU - Cleland, T. P. AU - Goodwin, M. B. AU - Boatman, E. AU - Theil, E. AU - Marcus, M. A. AU - Fakra, S. C. T2 - Proceedings of the Royal Society of London. Series B DA - 2014/// PY - 2014/// VL - 281 IS - 1775 ER - TY - JOUR TI - Systems Biology of Lignin Biosynthesis in Populus trichocarpa: Heteromeric 4-Coumaric Acid: Coenzyme A Ligase Protein Complex Formation, Regulation, and Numerical Modeling AU - Chen, Hsi-Chuan AU - Song, Jina AU - Wang, Jack P. AU - Lin, Ying-Chung AU - Ducoste, Joel AU - Shuford, Christopher M. AU - Liu, Jie AU - Li, Quanzi AU - Shi, Rui AU - Nepomuceno, Angelito AU - Isik, Fikret AU - Muddiman, David C. AU - Williams, Cranos AU - Sederoff, Ronald R. AU - Chiang, Vincent L. T2 - PLANT CELL AB - As a step toward predictive modeling of flux through the pathway of monolignol biosynthesis in stem differentiating xylem of Populus trichocarpa, we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro to form a heterotetrameric protein complex. This conclusion is based on laser microdissection, coimmunoprecipitation, chemical cross-linking, bimolecular fluorescence complementation, and mass spectrometry. The tetramer is composed of three subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. This protein–protein interaction affects the direction and rate of metabolic flux for monolignol biosynthesis in P. trichocarpa. A mathematical model was developed for the behavior of 4CL3 and 4CL5 individually and in mixtures that form the enzyme complex. The model incorporates effects of mixtures of multiple hydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition, and self-inhibition, along with characteristic of the substrates, the enzyme isoforms, and the tetrameric complex. Kinetic analysis of different ratios of the enzyme isoforms shows both inhibition and activation components, which are explained by the mathematical model and provide insight into the regulation of metabolic flux for monolignol biosynthesis by protein complex formation. DA - 2014/3// PY - 2014/3// DO - 10.1105/tpc.113.119685 VL - 26 IS - 3 SP - 876-893 SN - 1532-298X ER - TY - JOUR TI - Redirecting intracellular trafficking and the secretion pattern of FSH dramatically enhances ovarian function in mice AU - Wang, Huizhen AU - Larson, Melissa AU - Jablonka-Shariff, Albina AU - Pearl, Christopher A. AU - Miller, William L. AU - Conn, P. Michael AU - Boime, Irving AU - Kumar, T. Rajendra T2 - PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA AB - FSH and luteinizing hormone (LH) are secreted constitutively or in pulses, respectively, from pituitary gonadotropes in many vertebrates, and regulate ovarian function. The molecular basis for this evolutionarily conserved gonadotropin-specific secretion pattern is not understood. Here, we show that the carboxyterminal heptapeptide in LH is a gonadotropin-sorting determinant in vivo that directs pulsatile secretion. FSH containing this heptapeptide enters the regulated pathway in gonadotropes of transgenic mice, and is released in response to gonadotropin-releasing hormone, similar to LH. FSH released from the LH secretory pathway rescued ovarian defects in Fshb-null mice as efficiently as constitutively secreted FSH. Interestingly, the rerouted FSH enhanced ovarian follicle survival, caused a dramatic increase in number of ovulations, and prolonged female reproductive lifespan. Furthermore, the rerouted FSH vastly improved the in vivo fertilization competency of eggs, their subsequent development in vitro and when transplanted, the ability to produce offspring. Our study demonstrates the feasibility to fine-tune the target tissue responses by modifying the intracellular trafficking and secretory fate of a pituitary trophic hormone. The approach to interconvert the secretory fate of proteins in vivo has pathophysiological significance, and could explain the etiology of several hormone hyperstimulation and resistance syndromes. DA - 2014/4/15/ PY - 2014/4/15/ DO - 10.1073/pnas.1321404111 VL - 111 IS - 15 SP - 5735-5740 SN - 0027-8424 KW - protein sorting KW - regulated secretion KW - dense core granules KW - folliculogenesis ER - TY - JOUR TI - Moving Fe2+ from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates AU - Behera, Rabindra K. AU - Theil, Elizabeth C. T2 - PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA AB - Ferritin biominerals are protein-caged metabolic iron concentrates used for iron-protein cofactors and oxidant protection (Fe(2+) and O2 sequestration). Fe(2+) passage through ion channels in the protein cages, like membrane ion channels, required for ferritin biomineral synthesis, is followed by Fe(2+) substrate movement to ferritin enzyme (Fox) sites. Fe(2+) and O2 substrates are coupled via a diferric peroxo (DFP) intermediate, λmax 650 nm, which decays to [Fe(3+)-O-Fe(3+)] precursors of caged ferritin biominerals. Structural studies show multiple conformations for conserved, carboxylate residues E136 and E57, which are between ferritin ion channel exits and enzymatic sites, suggesting functional connections. Here we show that E136 and E57 are required for ferritin enzyme activity and thus are functional links between ferritin ion channels and enzymatic sites. DFP formation (Kcat and kcat/Km), DFP decay, and protein-caged hydrated ferric oxide accumulation decreased in ferritin E57A and E136A; saturation required higher Fe(2+) concentrations. Divalent cations (both ion channel and intracage binding) selectively inhibit ferritin enzyme activity (block Fe(2+) access), Mn(2+) << Co(2+) < Cu(2+) < Zn(2+), reflecting metal ion-protein binding stabilities. Fe(2+)-Cys126 binding in ferritin ion channels, observed as Cu(2+)-S-Cys126 charge-transfer bands in ferritin E130D UV-vis spectra and resistance to Cu(2+) inhibition in ferritin C126S, was unpredicted. Identifying E57 and E136 links in Fe(2+) movement from ferritin ion channels to ferritin enzyme sites completes a bucket brigade that moves external Fe(2+) into ferritin enzymatic sites. The results clarify Fe(2+) transport within ferritin and model molecular links between membrane ion channels and cytoplasmic destinations. DA - 2014/6/3/ PY - 2014/6/3/ DO - 10.1073/pnas.1318417111 VL - 111 IS - 22 SP - 7925-7930 SN - 0027-8424 KW - iron traffic KW - oxidoreductase enzyme activity KW - antioxidant KW - ferrihydrite KW - BioIron ER - TY - JOUR TI - In Vivo Mapping of Arabidopsis Scaffold/Matrix Attachment Regions Reveals Link to Nucleosome-Disfavoring Poly(dA:dT) Tracts AU - Pascuzzi, Pete E. AU - Flores-Vergara, Miguel A. AU - Lee, Tae-Jin AU - Sosinski, Bryon AU - Vaughn, Matthew W. AU - Hanley-Bowdoin, Linda AU - Thompson, William F. AU - Allen, George C. T2 - PLANT CELL AB - Scaffold or matrix attachment regions (S/MARs) are found in all eukaryotes. The pattern of distribution and genomic context of S/MARs is thought to be important for processes such as chromatin organization and modulation of gene expression. Despite the importance of such processes, much is unknown about the large-scale distribution and sequence content of S/MARs in vivo. Here, we report the use of tiling microarrays to map 1358 S/MARs on Arabidopsis thaliana chromosome 4 (chr4). S/MARs occur throughout chr4, spaced much more closely than in the large plant and animal genomes that have been studied to date. Arabidopsis S/MARs can be divided into five clusters based on their association with other genomic features, suggesting a diversity of functions. While some Arabidopsis S/MARs may define structural domains, most occur near the transcription start sites of genes. Genes associated with these S/MARs have an increased probability of expression, which is particularly pronounced in the case of transcription factor genes. Analysis of sequence motifs and 6-mer enrichment patterns show that S/MARs are preferentially enriched in poly(dA:dT) tracts, sequences that resist nucleosome formation, and the majority of S/MARs contain at least one nucleosome-depleted region. This global view of S/MARs provides a framework to begin evaluating genome-scale models for S/MAR function. DA - 2014/1// PY - 2014/1// DO - 10.1105/tpc.113.121194 VL - 26 IS - 1 SP - 102-120 SN - 1532-298X ER - TY - JOUR TI - Chemical shift assignments and secondary structure prediction of the phosphorelay protein VanU from Vibrio anguillarum AU - Bobay, Benjamin G. AU - Thompson, Richele J. AU - Milton, Debra L. AU - Cavanagh, John T2 - BIOMOLECULAR NMR ASSIGNMENTS AB - Vibrio anguillarum is a biofilm forming Gram-negative bacterium that survives prolonged periods in seawater and causes vibriosis in marine life. A quorum-sensing signal transduction pathway initiates biofilm formation in response to environmental stresses. The phosphotransferase protein VanU is the focal point of the quorum-sensing pathway and facilitates the regulation between independent phosphorelay systems that activate or repress biofilm formation. Here we report the 1H, 13C, and 15N backbone and side chain resonance assignments and secondary structure prediction for VanU from V. anguillarum. DA - 2014/4// PY - 2014/4// DO - 10.1007/s12104-013-9478-2 VL - 8 IS - 1 SP - 177-179 SN - 1874-270X KW - VanU KW - NMR KW - Vibrio anguillarum KW - Phosphorelay ER - TY - JOUR TI - Chemical shift assignments and secondary structure prediction of the master biofilm regulator, SinR, from Bacillus subtilis AU - Stowe, Sean D. AU - Olson, Andrew L. AU - Losick, Richard AU - Cavanagh, John T2 - BIOMOLECULAR NMR ASSIGNMENTS AB - Bacillus subtilis is a soil-dwelling Gram-positive bacterial species that has been extensively studied as a model of biofilm formation and stress-induced cellular differentiation. The tetrameric protein, SinR, has been identified as a master regulator for biofilm formation and linked to the regulation of the early transition states during cellular stress response, such as motility and biofilm-linked biosynthetic genes. SinR is a 111-residue protein that is active as a dimer of dimers, composed of two distinct domains, a DNA-binding helix-turn-helix N-terminus domain and a C-terminal multimerization domain. In order for biofilm formation to proceed, the antagonist, SinI, must inactivate SinR. This interaction results in a dramatic structural rearrangement of both proteins. Here we report the full-length backbone and side chain chemical shift values in addition to the experimentally derived secondary structure predictions as the first step towards directly studying the complex interaction dynamics between SinR and SinI. DA - 2014/4// PY - 2014/4// DO - 10.1007/s12104-013-9473-7 VL - 8 IS - 1 SP - 155-158 SN - 1874-270X KW - NMR KW - SinR KW - Bacillus subtilis KW - Biofilm regulation ER - TY - JOUR TI - Chemical shift assignments and secondary structure prediction of the C-terminal domain of the response regulator BfmR from Acinetobacter baumannii AU - Olson, Andrew L. AU - Thompson, Richele J. AU - Melander, Christian AU - Cavanagh, John T2 - BIOMOLECULAR NMR ASSIGNMENTS AB - Acinetobacter baumannii is a Gram-negative pathogen responsible for severe nocosomial infections by forming biofilms in healthcare environments. The two-domain response regulator BfmR has been shown to be the master controller for biofilm formation. Inactivation of BfmR resulted in an abolition of pili production and consequently biofilm creation. Here we report backbone and sidechain resonance assignments and secondary structure prediction for the C-terminal domain of BfmR (residues 130–238) from A. baumannii. DA - 2014/4// PY - 2014/4// DO - 10.1007/s12104-012-9454-2 VL - 8 IS - 1 SP - 67-70 SN - 1874-270X KW - NMR KW - BfmR KW - Acinetobacter baumannii KW - Assignment ER - TY - JOUR TI - Carboxylation of cytosine (5caC) in the CG dinucleotide in the E-box motif (CGCAG vertical bar GTG) increases binding of the Tcf3 vertical bar Ascl1 helix-loop-helix heterodimer 10-fold AU - Golla, Jaya Prakash AU - Zhao, Jianfei AU - Mann, Ishminder K. AU - Sayeed, Syed K. AU - Mandal, Ajeet AU - Rose, Robert B. AU - Vinson, Charles T2 - BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS AB - Three oxidative products of 5-methylcytosine (5mC) occur in mammalian genomes. We evaluated if these cytosine modifications in a CG dinucleotide altered DNA binding of four B-HLH homodimers and three heterodimers to the E-Box motif CGCAG|GTG. We examined 25 DNA probes containing all combinations of cytosine in a CG dinucleotide and none changed binding except for carboxylation of cytosine (5caC) in the strand CGCAG|GTG. 5caC enhanced binding of all examined B-HLH homodimers and heterodimers, particularly the Tcf3|Ascl1 heterodimer which increased binding ~10-fold. These results highlight a potential function of the oxidative products of 5mC, changing the DNA binding of sequence-specific transcription factors. DA - 2014/6/27/ PY - 2014/6/27/ DO - 10.1016/j.bbrc.2014.05.018 VL - 449 IS - 2 SP - 248-255 SN - 1090-2104 KW - Carboxylation KW - E-Box motif KW - CG dinucleotide KW - Basic-helix-loop-helix KW - DNA binding KW - Tcf3 vertical bar Ascl1 heterodimer ER - TY - JOUR TI - A Model for the Flexibility of the Distal Histidine in Dehaloperoxidase-Hemoglobin A Based on X-ray Crystal Structures of the Carbon Monoxide Adduct AU - Zhao, Junjie AU - Serrano, Vesna AU - Franzen, Stefan T2 - BIOCHEMISTRY AB - Dehaloperoxidase hemoglobin A (DHP A) is a multifunctional hemoglobin that appears to have evolved oxidative pathways for the degradation of xenobiotics as a protective function that complements the oxygen transport function. DHP A possesses at least two internal binding sites, one for substrates and one for inhibitors, which include various halogenated phenols and indoles. Herein, we report the X-ray crystallographic structure of the carbonmonoxy complex (DHPCO). Unlike other DHP structures with 6-coordinated heme, the conformation of the distal histidine (H55) in DHPCO is primarily external or solvent exposed, despite the fact that the heme Fe is 6-coordinated. As observed generally in globins, DHP exhibits two distal histidine conformations (one internal and one external). In previous structural studies, we have shown that the distribution of H55 conformations is weighted strongly toward the external position when the DHP heme Fe is 5-coordinated. The large population of the external conformation of the distal histidine observed in DHPCO crystals at pH 6.0 indicates that some structural factor in DHP must account for the difference from other globins, which exhibit a significant external conformation only when pH < 4.5. While the original hypothesis suggested that interaction with a heme-Fe-bound ligand was the determinant of H55 conformation, the current study forces a refinement of that hypothesis. The external or open conformation of H55 is observed to have interactions with two propionate groups in heme, at distances of 3.82 and 2.73 Å, respectively. A relatively weak hydrogen bonding interaction between H55 and CO, combined with strong interactions with heme propionate (position 6), is hypothesized to strengthen the external conformation of H55. Density function theory (DFT) calculations were conducted to test whether there is a weaker hydrogen bond interaction between H55 and heme bonded CO or O2. Molecular dynamics simulations were conducted to examine how the tautomeric forms of H55 affect the dynamic motions of the distal histidine that govern the switching between open and closed conformations. The calculations support the modified hypothesis suggesting a competition between the strength of interactions with heme ligand and the heme propionates as the factors that determine the conformation of the distal histidine. DA - 2014/4/22/ PY - 2014/4/22/ DO - 10.1021/bi5001905 VL - 53 IS - 15 SP - 2474-2482 SN - 0006-2960 ER - TY - JOUR TI - Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions AU - Khan, Mateen A. AU - Ma, Jia AU - Walden, William E. AU - Merrick, William C. AU - Theil, Elizabeth C. AU - Goss, Dixie J. T2 - NUCLEIC ACIDS RESEARCH AB - Metal ion binding was previously shown to destabilize IRE-RNA/IRP1 equilibria and enhanced IRE-RNA/eIF4F equilibria. In order to understand the relative importance of kinetics and stability, we now report rapid rates of protein/RNA complex assembly and dissociation for two IRE-RNAs with IRP1, and quantitatively different metal ion response kinetics that coincide with the different iron responses in vivo. kon, for FRT IRE-RNA binding to IRP1 was eight times faster than ACO2 IRE-RNA. Mn2+ decreased kon and increased koff for IRP1 binding to both FRT and ACO2 IRE-RNA, with a larger effect for FRT IRE-RNA. In order to further understand IRE-mRNA regulation in terms of kinetics and stability, eIF4F kinetics with FRT IRE-RNA were determined. kon for eIF4F binding to FRT IRE-RNA in the absence of metal ions was 5-times slower than the IRP1 binding to FRT IRE-RNA. Mn2+ increased the association rate for eIF4F binding to FRT IRE-RNA, so that at 50 µM Mn2+ eIF4F bound more than 3-times faster than IRP1. IRP1/IRE-RNA complex has a much shorter life-time than the eIF4F/IRE-RNA complex, which suggests that both rate of assembly and stability of the complexes are important, and that allows this regulatory system to respond rapidly to change in cellular iron. DA - 2014/// PY - 2014/// DO - 10.1093/nar/gku248 VL - 42 IS - 10 SP - 6567-6577 SN - 1362-4962 ER - TY - JOUR TI - Protein Molecular Data from Ancient (> 1 million years old) Fossil Material: Pitfalls, Possibilities and Grand Challenges AU - Schweitzer, Mary Higby AU - Schroeter, Elena R. AU - Goshe, Michael B. T2 - ANALYTICAL CHEMISTRY AB - Advances in resolution and sensitivity of analytical techniques have provided novel applications, including the analyses of fossil material. However, the recovery of original proteinaceous components from very old fossil samples (defined as >1 million years (1 Ma) from previously named limits in the literature) is far from trivial. Here, we discuss the challenges to recovery of proteinaceous components from fossils, and the need for new sample preparation techniques, analytical methods, and bioinformatics to optimize and fully utilize the great potential of information locked in the fossil record. We present evidence for survival of original components across geological time, and discuss the potential benefits of recovery, analyses, and interpretation of fossil materials older than 1 Ma, both within and outside of the fields of evolutionary biology. DA - 2014/7/15/ PY - 2014/7/15/ DO - 10.1021/ac500803w VL - 86 IS - 14 SP - 6731-6740 SN - 1520-6882 ER - TY - JOUR TI - Chemical Changes during Anaerobic Decomposition of Hardwood, Softwood, and Old Newsprint under Mesophilic and Thermophilic Conditions AU - Cruz, Florentino B. AU - Yelle, Daniel J. AU - Gracz, Hanna S. AU - Barlaz, Morton A. T2 - JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY AB - The anaerobic decomposition of plant biomass is an important aspect of global organic carbon cycling. While the anaerobic metabolism of cellulose and hemicelluloses to methane and carbon dioxide are well-understood, evidence for the initial stages of lignin decomposition is fragmentary. The objective of this study was to look for evidence of chemical transformations of lignin in woody tissues [hardwood (HW), softwood (SW), and old newsprint (ONP)] after anaerobic decomposition using Klason and acid-soluble lignin, CuO oxidation, and 2D NMR. Tests were conducted under mesophilic and thermophilic conditions, and lignin associations with structural carbohydrates are retained. For HW and ONP, the carbon losses could be attributed to cellulose and hemicelluloses, while carbon loss in SW was attributable to an uncharacterized fraction (e.g., extractives etc.). The 2D NMR and chemical degradation methods revealed slight reductions in β-O-4 linkages for HW and ONP, with no depolymerization of lignin in any substrate. DA - 2014/7/9/ PY - 2014/7/9/ DO - 10.1021/jf501653h VL - 62 IS - 27 SP - 6362-6374 SN - 1520-5118 KW - anaerobic decomposition KW - CuO oxidation KW - lignin KW - HSQC KW - NMR ER - TY - JOUR TI - Leishmanicidal activity of a daucane sesquiterpene isolated from Eryngium foetidum AU - Rojas-Silva, Patricio AU - Graziose, Rocky AU - Vesely, Brian AU - Poulev, Alexander AU - Mbeunkui, Flaubert AU - Grace, Mary H. AU - Kyle, Dennis E. AU - Lila, Mary Ann AU - Raskin, Ilya T2 - PHARMACEUTICAL BIOLOGY AB - Eryngium foetidum L. (Apiaceae) is a traditional herb that has been used for numerous medicinal applications, including as a treatment for parasitic infections, especially in the Neotropics from where it originates.This study evaluates the in vitro leishmanicidal and cytotoxicity activities of isolated compounds based on a bioassay-guided fractionation approach.Defatted aerial parts of E. foetidum were subjected to extraction with methanol followed by partitioning with n-hexane, ethyl acetate and 50% methanol. Then, the first two fractions were subsequently fractionated by column chromatography and HPLC. Compound identity was confirmed by mass spectrometry and NMR spectroscopy. Leishmania tarentolae (promastigotes) and L. donovani (amastigotes) were used as testing parasites. L6 rat myoblasts were used for cytotoxicity. All extracts and fractions were tested at 20 μg/mL.The initial methanol extract showed 20% growth inhibition of L. tarentolae. Then, the n-hexane and ethyl acetate fractions were also active showing approximately 40% growth inhibition. From these two fractions, the following compounds were isolated: lasidiol p-methoxybenzoate (1), a daucane sesquiterpene; and 4-hydroxy-1,1,5-trimethyl-2-formyl-cyclohexadien-(2,5)-[α-acetoxymethyl-cis-crotonate] (2), a terpene aldehyde ester derivative. Compound 1 inhibited the growth of both L. tarentolae and L. donovani with IC₅₀ values of 14.33 and 7.84 μM, respectively; and showed no cytotoxicity (IC₅₀ > 50 μM). Compound 2 was inactive in the L. tarentolae assay (IC₅₀ > 50 μM).This study presented the bioassay-guided fractionation with the leishmanicidal and cytotoxicity activities of two compounds isolated for the first time from an Eryngium species. DA - 2014/3// PY - 2014/3// DO - 10.3109/13880209.2013.837077 VL - 52 IS - 3 SP - 398-401 SN - 1744-5116 KW - Apiaceae KW - bioassay guided fractionation KW - cytotoxicity KW - leishmaniasis KW - LC-MS KW - NMR KW - sesquiterpenoids ER - TY - JOUR TI - ELASTOGRAPHIC CHARACTERISTICS OF THE METACARPAL TENDONS IN HORSES WITHOUT CLINICAL EVIDENCE OF TENDON INJURY AU - Lustgarten, Meghann AU - Redding, W. Rich AU - Labens, Raphael AU - Morgan, Michel AU - Davis, Weston AU - Seiler, Gabriela S. T2 - VETERINARY RADIOLOGY & ULTRASOUND AB - Tendon and ligament injuries are common causes of impaired performance in equine athletes. Gray‐scale ultrasonography is the current standard method for diagnosing and monitoring these injuries, however this modality only provides morphologic information. Elastography is an ultrasound technique that allows detection and measurement of tissue strain, and may provide valuable mechanical information about equine tendon and ligament injuries. The purpose of this study was to determine the feasibility, reproducibility, and repeatability of elastography; and to describe elastographic characteristics of metacarpal tendons in sound horses. Nineteen legs for 17 clinically sound horses without evidence of musculoskeletal pathology were included. Elastographic images of the superficial and deep digital flexor tendons and the branches of the suspensory ligament (tendon of the interosseous muscle) were described quantitatively and qualitatively. There was no statistically significant difference between operators ( P = 0.86) nor within operators ( P = 0.93). For qualitative assessments, reproducibility (0.46) was moderate and repeatability (0.78) was good. Similar to human Achilles tendons, equine tendons were classified as predominantly hard using elastography. There was no statistically significant difference in stiffness of the flexor tendons ( P = 0.96). No significant difference in stiffness was found with altered leg position during standing ( P = 0.84) and while nonweight bearing ( P = 0.61). The flexor tendons were softer when imaged in longitudinal versus transverse planes ( P < 0.01) however, the suspensory branches were not ( P = 0.67). Findings supported future clinical application of elastography as a noninvasive “stall‐side” imaging modality for evaluation of the tendons and ligaments of the distal forelimb in horses. DA - 2014/1// PY - 2014/1// DO - 10.1111/vru.12104 VL - 55 IS - 1 SP - 92-101 SN - 1740-8261 KW - elastography KW - equine KW - musculoskeletal KW - ultrasound ER - TY - JOUR TI - Spectroscopic Studies of Single and Double Variants of M Ferritin: Lack of Conversion of a Biferrous Substrate Site into a Cofactor Site for O-2 Activation AU - Kwak, Yeonju AU - Schwartz, Jennifer K. AU - Haldar, Suranjana AU - Behera, Rabindra K. AU - Tosha, Takehiko AU - Theil, Elizabeth C. AU - Solomon, Edward I. T2 - BIOCHEMISTRY AB - Ferritin has a binuclear non-heme iron active site that functions to oxidize iron as a substrate for formation of an iron mineral core. Other enzymes of this class have tightly bound diiron cofactor sites that activate O2 to react with substrate. Ferritin has an active site ligand set with 1-His/4-carboxylate/1-Gln rather than the 2-His/4-carboxylate set of the cofactor site. This ligand variation has been thought to make a major contribution to this biferrous substrate rather than cofactor site reactivity. However, the Q137E/D140H double variant of M ferritin, has a ligand set that is equivalent to most of the diiron cofactor sites, yet did not rapidly react with O2 or generate the peroxy intermediate observed in the cofactor sites. Therefore, in this study, a combined spectroscopic methodology of circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD has been applied to evaluate the factors required for the rapid O2 activation observed in cofactor sites. This methodology defines the coordination environment of each iron and the bridging ligation of the biferrous active sites in the double and corresponding single variants of frog M ferritin. Based on spectral changes, the D140H single variant has the new His ligand binding, and the Q137E variant has the new carboxylate forming a μ-1,3 bridge. The spectra for the Q137E/D140H double variant, which has the cofactor ligand set, however, reflects a site that is more coordinately saturated than the cofactor sites in other enzymes including ribonucleotide reductase, indicating the presence of additional water ligation. Correlation of this double variant and the cofactor sites to their O2 reactivities indicates that electrostatic and steric changes in the active site and, in particular, the hydrophobic nature of a cofactor site associated with its second sphere protein environment, make important contributions to the activation of O2 by the binuclear non-heme iron enzymes. DA - 2014/1/28/ PY - 2014/1/28/ DO - 10.1021/bi4013726 VL - 53 IS - 3 SP - 473-482 SN - 0006-2960 ER - TY - JOUR TI - Refinement of macromolecular structures against neutron data with SHELXL2013 AU - Gruene, Tim AU - Hahn, Hinrich W. AU - Luebben, Anna V. AU - Meilleur, Flora AU - Sheldrick, George M. T2 - JOURNAL OF APPLIED CRYSTALLOGRAPHY AB - Some of the improvements in SHELX2013 make SHELXL convenient to use for refinement of macromolecular structures against neutron data without the support of X-ray data. The new NEUT instruction adjusts the behaviour of the SFAC instruction as well as the default bond lengths of the AFIX instructions. This work presents a protocol on how to use SHELXL for refinement of protein structures against neutron data. It includes restraints extending the Engh & Huber [Acta Cryst. (1991), A47, 392-400] restraints to H atoms and discusses several of the features of SHELXL that make the program particularly useful for the investigation of H atoms with neutron diffraction. SHELXL2013 is already adequate for the refinement of small molecules against neutron data, but there is still room for improvement, like the introduction of chain IDs for the refinement of macromolecular structures. DA - 2014/1// PY - 2014/1// DO - 10.1107/s1600576713027659 VL - 47 SP - 462-466 SN - 1600-5767 ER - TY - JOUR TI - Neutron structure of the cyclic glucose-bound xylose isomerase E186Q mutant AU - Munshi, P. AU - Snell, E.H. AU - Woerd AU - Mj, Judge AU - Ra, Myles AU - Da, Ren AU - Z, Meilleur AU - F, Acta Crystallographica Section AU - D. T2 - ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY AB - Ketol-isomerases catalyze the reversible isomerization between aldoses and ketoses. D-Xylose isomerase carries out the first reaction in the catabolism of D-xylose, but is also able to convert D-glucose to D-fructose. The first step of the reaction is an enzyme-catalyzed ring opening of the cyclic substrate. The active-site amino-acid acid/base pair involved in ring opening has long been investigated and several models have been proposed. Here, the structure of the xylose isomerase E186Q mutant with cyclic glucose bound at the active site, refined against joint X-ray and neutron diffraction data, is reported. Detailed analysis of the hydrogen-bond networks at the active site of the enzyme suggests that His54, which is doubly protonated, is poised to protonate the glucose O5 position, while Lys289, which is neutral, promotes deprotonation of the glucose O1H hydroxyl group via an activated water molecule. The structure also reveals an extended hydrogen-bonding network that connects the conserved residues Lys289 and Lys183 through three structurally conserved water molecules and residue 186, which is a glutamic acid to glutamine mutation. DA - 2014/2// PY - 2014/2// DO - 10.1107/s1399004713029684 VL - 70 IS - Pt 2 SP - 414-420 SN - 2059-7983 UR - http://europepmc.org/abstract/med/24531475 ER - TY - JOUR TI - Pyruvate Protects Pathogenic Spirochetes from H2O2 Killing AU - Troxell, Bryan AU - Zhang, Jun-Jie AU - Bourret, Travis J. AU - Zeng, Melody Yue AU - Blum, Janice AU - Gherardini, Frank AU - Hassan, Hosni M. AU - Yang, X. Frank T2 - PLoS ONE AB - Pathogenic spirochetes cause clinically relevant diseases in humans and animals, such as Lyme disease and leptospirosis. The causative agent of Lyme disease, Borrelia burgdorferi, and the causative agent of leptospirosis, Leptospria interrogans, encounter reactive oxygen species (ROS) during their enzootic cycles. This report demonstrated that physiologically relevant concentrations of pyruvate, a potent H2O2 scavenger, and provided passive protection to B. burgdorferi and L. interrogans against H2O2. When extracellular pyruvate was absent, both spirochetes were sensitive to a low dose of H2O2 (≈0.6 µM per h) generated by glucose oxidase (GOX). Despite encoding a functional catalase, L. interrogans was more sensitive than B. burgdorferi to H2O2 generated by GOX, which may be due to the inherent resistance of B. burgdorferi because of the virtual absence of intracellular iron. In B. burgdorferi, the nucleotide excision repair (NER) and the DNA mismatch repair (MMR) pathways were important for survival during H2O2 challenge since deletion of the uvrB or the mutS genes enhanced its sensitivity to H2O2 killing; however, the presence of pyruvate fully protected ΔuvrB and ΔmutS from H2O2 killing further demonstrating the importance of pyruvate in protection. These findings demonstrated that pyruvate, in addition to its classical role in central carbon metabolism, serves as an important H2O2 scavenger for pathogenic spirochetes. Furthermore, pyruvate reduced ROS generated by human neutrophils in response to the Toll-like receptor 2 (TLR2) agonist zymosan. In addition, pyruvate reduced neutrophil-derived ROS in response to B. burgdorferi, which also activates host expression through TLR2 signaling. Thus, pathogenic spirochetes may exploit the metabolite pyruvate, present in blood and tissues, to survive H2O2 generated by the host antibacterial response generated during infection. DA - 2014/1/2/ PY - 2014/1/2/ DO - 10.1371/journal.pone.0084625 VL - 9 IS - 1 SP - e84625 J2 - PLoS ONE LA - en OP - SN - 1932-6203 UR - http://dx.doi.org/10.1371/journal.pone.0084625 DB - Crossref ER - TY - JOUR TI - Ferric Uptake Regulator-Dependent Antinitrosative Defenses in Salmonella enterica Serovar Typhimurium Pathogenesis AU - Husain, Maroof AU - Jones-Carson, Jessica AU - Liu, Lin AU - Song, Miryoung AU - Saah, J. Royden AU - Troxell, Bryan AU - Mendoza, Mary AU - Hassan, Hosni AU - Vazquez-Torresa, Andres T2 - INFECTION AND IMMUNITY AB - Herein we report an important role for the ferric uptake regulator (Fur) in the resistance of Salmonella enterica serovar Typhimurium to the reactive nitrogen species produced by inducible nitric oxide (NO) synthase in an NRAMP1(r) murine model of acute systemic infection. The expression of fur protected Salmonella grown under normoxic and hypoxic conditions against the bacteriostatic activity of NO. The hypersusceptibility of fur-deficient Salmonella to the cytotoxic actions of NO coincides with a marked repression of respiratory activity and the reduced ability of the bacteria to detoxify NO. A fur mutant Salmonella strain contained reduced levels of the terminal quinol oxidases of the electron transport chain. Addition of the heme precursor δ-aminolevulinic acid restored the cytochrome content, respiratory activity, NO consumption, and wild-type growth in bacteria undergoing nitrosative stress. The innate antinitrosative defenses regulated by Fur added to the adaptive response associated with the NO-detoxifying activity of the flavohemoprotein Hmp. Our investigations indicate that, in addition to playing a critical role in iron homeostasis, Fur is an important antinitrosative determinant of Salmonella pathogenesis. DA - 2014/1// PY - 2014/1// DO - 10.1128/iai.01201-13 VL - 82 IS - 1 SP - 333-340 SN - 1098-5522 ER - TY - JOUR TI - DRoP: A Water Analysis Program Identifies Ras-GTP-Specific Pathway of Communication between Membrane-Interacting Regions and the Active Site AU - Kearney, Bradley Ni. AU - Johnson, Christian W. AU - Roberts, Daniel M. AU - Swartz, Paul AU - Mattos, Carla T2 - JOURNAL OF MOLECULAR BIOLOGY AB - Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras. DA - 2014/2/6/ PY - 2014/2/6/ DO - 10.1016/j.jmb.2013.10.036 VL - 426 IS - 3 SP - 611-629 SN - 1089-8638 KW - crystallographic water analysis KW - Ras GTPase KW - water-mediated networks in protein structure KW - protein hydration KW - DRoP software ER -