bluuebunny/crossref_metadata_2025 huggingface dataset:
A peripheral endocannabinoid mechanism contributes to glucocorticoid-mediated metabolic syndrome
Authors: Bowles Nicole P. , Karatsoreos Ilia N. , Li Xiaosong , Vemuri V. Kiran , Wood Jodi-Anne , Li Zhiying , Tamashiro Kellie L. K. , Schwartz Gary J. , Makriyannis Alexandros M. , Kunos George , Hillard Cecilia J. , McEwen Bruce S. , Hill Matthew N.
Published: December 2014

Significance

Obesity and associated metabolic disorders (e.g., cardiovascular disease and type 2 diabetes) are major public health concerns. These disorders result, in part, from hormonal dysregulation, particularly of glucocorticoids (GCs; central regulators of metabolism and adipogenesis). The specific mechanisms by which GCs modulate these processes remain largely unknown, but GCs increase production of endocannabinoids—potent central and peripheral regulators of appetite, energy balance, and metabolism. Our results show that sustained exposure to GCs produces obesity and metabolic syndrome through a peripheral endocannabinoid mechanism. These data further our understanding of the role of endocannabinoid signaling to promote not only diet-induced, but also, hormonal-mediated obesity and support the argument that peripheral blockade of endocannabinoid signaling could be a potential treatment for obese conditions.
Only three driver gene mutations are required for the development of lung and colorectal cancers
Authors: Tomasetti Cristian , Marchionni Luigi , Nowak Martin A. , Parmigiani Giovanni , Vogelstein Bert
Published: December 2014

Significance

The number of driver events required for human tumorigenesis has remained one of the fundamental issues in cancer research since the seminal studies of Armitage and Doll. This question has become even more important with the recent genome-wide sequencing studies of cancer, whose major goal is the identification of the driver genes responsible for tumor initiation and progression. By using a novel approach that combines conventional epidemiologic studies with genome-wide sequencing data, we show that only three sequential mutations are required to develop lung and colon adenocarcinomas, a number that is lower than what is typically thought to be required for the formation of cancers of these and other organs. This finding has important implications for the design of future cancer genome-sequencing efforts.
mTOR inhibition rescues osteopenia in mice with systemic sclerosis
Authors: Chen Chider , Akiyama Kentaro , Wang Dandan , Xu Xingtian , Li Bei , Moshaverinia Alireza , Brombacher Frank , Sun Lingyun , Shi Songtao
Published: December 2014

Fibrillin-1 (FBN1) deficiency-induced systemic sclerosis is attributed to elevation of interleukin-4 (IL4) and TGF-β, but the mechanism underlying FBN1 deficiency–associated osteopenia is not fully understood. We show that bone marrow mesenchymal stem cells (BMMSCs) from FBN1-deficient (Fbn1+/−) mice exhibit decreased osteogenic differentiation and increased adipogenic differentiation. Mechanistically, this lineage alteration is regulated by IL4/IL4Rα-mediated activation of mTOR signaling to down-regulate RUNX2 and up-regulate PPARγ2, respectively, via P70 ribosomal S6 protein kinase (P70S6K). Additionally, we reveal that activation of TGF-β/SMAD3/SP1 signaling results in enhancement of SP1 binding to the IL4Rα promoter to synergistically activate mTOR pathway in Fbn1+/− BMMSCs. Blockage of mTOR signaling by osteoblastic-specific knockout or rapamycin treatment rescues osteopenia phenotype in Fbn1+/− mice by improving osteogenic differentiation of BMMSCs. Collectively, this study identifies a previously unrecognized role of the FBN1/TGF-β/IL4Rα/mTOR cascade in BMMSC lineage selection and provides experimental evidence that rapamycin treatment may provide an anabolic therapy for osteopenia in Fbn1+/− mice.
PBOOST: a GPU-based tool for parallel permutation tests in genome-wide association studies
Authors: Yang Guangyuan , Jiang Wei , Yang Qiang , Yu Weichuan
Published: December 2014

Motivation: The importance of testing associations allowing for interactions has been demonstrated by Marchini et al. (2005). A fast method detecting associations allowing for interactions has been proposed by Wan et al. (2010a). The method is based on likelihood ratio test with the assumption that the statistic follows the χ2 distribution. Many single nucleotide polymorphism (SNP) pairs with significant associations allowing for interactions have been detected using their method. However, the assumption of χ2 test requires the expected values in each cell of the contingency table to be at least five. This assumption is violated in some identified SNP pairs. In this case, likelihood ratio test may not be applicable any more. Permutation test is an ideal approach to checking the P-values calculated in likelihood ratio test because of its non-parametric nature. The P-values of SNP pairs having significant associations with disease are always extremely small. Thus, we need a huge number of permutations to achieve correspondingly high resolution for the P-values. In order to investigate whether the P-values from likelihood ratio tests are reliable, a fast permutation tool to accomplish large number of permutations is desirable.

Results: We developed a permutation tool named PBOOST. It is based on GPU with highly reliable P-value estimation. By using simulation data, we found that the P-values from likelihood ratio tests will have relative error of >100% when 50% cells in the contingency table have expected count less than five or when there is zero expected count in any of the contingency table cells. In terms of speed, PBOOST completed 107 permutations for a single SNP pair from the Wellcome Trust Case Control Consortium (WTCCC) genome data (Wellcome Trust Case Control Consortium, 2007) within 1 min on a single Nvidia Tesla M2090 device, while it took 60 min in a single CPU Intel Xeon E5-2650 to finish the same task. More importantly, when simultaneously testing 256 SNP pairs for 107 permutations, our tool took only 5 min, while the CPU program took 10 h. By permuting on a GPU cluster consisting of 40 nodes, we completed 1012 permutations for all 280 SNP pairs reported with P-values smaller than 1.6×10−12 in the WTCCC datasets in 1 week.

Availability and implementation: The source code and sample data are available at http://bioinformatics.ust.hk/PBOOST.zip.

Contact:  gyang@ust.hk; eeyu@ust.hk

Supplementary information:  Supplementary data are available at Bioinformatics online.
Clinic staff attitudes towards the use of mHealth technology to conduct perinatal depression screenings: a qualitative study
Authors: Pineros-Leano Maria , Tabb Karen M , Sears Heather , Meline Brandon , Huang Hsiang
Published: December 2014

Background.

The use of mHealth technology is an innovative approach for screening low-income mothers for depression. Past studies show that the use of technology removes barriers such as literacy issues, language challenges, concerns about privacy and lack of transportation and can also increase reliability. However, little is known about staff attitudes and perceptions towards using mHealth technology for screening low-income women for depression in clinics.

Methods.

Four focus groups were conducted with staff members in a supplemental nutrition program for women, infants and children located in a public health clinic. A semi-structured focus group interview guide was used to examine staff perceptions related to depression screening with tablet technology. All interviews were audio recorded and transcribed verbatim. Thematic analysis was used to analyse all focus group data.

Results.

Three major benefits and two major barriers were found. The benefits of using technology for perinatal depression screenings were reduction of literacy and language barriers, reduction of redundancy and errors and increased privacy for clients. The barriers were increased network issues and responsibility for technology, which included fear of the devices being lost, stolen or broken.

Implications.

Before implementing mHealth tablet technology for depression screening in a public health clinic, it is important to address the concerns of staff members to make the transition more effective. This study provides timely information on staff-perceived benefits and barriers when implementing mHealth technology in a public health setting.
Rifampicin and rifapentine significantly reduce concentrations of bedaquiline, a new anti-TB drug
Authors: Svensson Elin M. , Murray Stephen , Karlsson Mats O. , Dooley Kelly E.
Published: December 2014

Objectives

Bedaquiline is the first drug of a new class approved for the treatment of TB in decades. Bedaquiline is metabolized by cytochrome P450 (CYP) 3A4 to a less-active M2 metabolite. Its terminal half-life is extremely long (5–6 months), complicating evaluations of drug–drug interactions. Rifampicin and rifapentine, two anti-TB drugs now being optimized to shorten TB treatment duration, are potent inducers of CYP3A4. This analysis aimed to predict the effect of repeated doses of rifampicin or rifapentine on the steady-state pharmacokinetics of bedaquiline and its M2 metabolite from single-dose data using a model-based approach.

Methods

Pharmacokinetic data for bedaquiline and M2 were obtained from a Phase I study involving 32 individuals each receiving two doses of bedaquiline, alone or together with multiple-dose rifampicin or rifapentine. Sampling was performed over 14 days following each bedaquiline dose. Pharmacokinetic analyses were performed using non-linear mixed-effects modelling. Models were used to simulate potential dose adjustments.

Results

Rifamycin co-administration increased bedaquiline clearance substantially: 4.78-fold [relative standard error (RSE) 9.10%] with rifampicin and 3.96-fold (RSE 5.00%) with rifapentine. Induction of M2 clearance was equally strong. Average steady-state concentrations of bedaquiline and M2 are predicted to decrease by 79% and 75% when given with rifampicin or rifapentine, respectively. Simulations indicated that increasing the bedaquiline dosage to mitigate the interaction would yield elevated M2 concentrations during the first treatment weeks.

Conclusions

Rifamycin antibiotics reduce bedaquiline concentrations substantially. In line with current treatment guidelines for drug-susceptible TB, concomitant use is not recommended, even with dose adjustment.
Yr60, a Gene Conferring Moderate Resistance to Stripe Rust in Wheat
Authors: Herrera-Foessel S. A. , Singh R. P. , Lan C. X. , Huerta-Espino J. , Calvo-Salazar V. , Bansal U. K. , Bariana H. S. , Lagudah E. S.
Published: December 2014

Stripe rust, caused by Puccinia striiformis f. sp. tritici W., is a devastating disease of wheat worldwide. A new stripe rust resistance gene with moderate seedling and adult plant resistance was mapped using an F5 recombinant inbred line (RIL) population developed from the cross of the resistant parent ‘Almop’ with the susceptible parent ‘Avocet’. The parents and RILs were phenotyped for seedling stripe rust response variation in a greenhouse and in field trials at Toluca, Mexico for 2 years. Almop showed moderate levels of resistance at both seedling and adult plant stages compared with the highly susceptible response of Avocet. The distribution of homozygous resistant, homozygous susceptible, and segregating RILs conformed to segregation at a single locus. Seedlings and adult plant responses were correlated, indicating that the same gene conferred resistance at both stages. A bulk segregant analysis approach with widely distributed simple sequence repeat (SSR) markers mapped the resistance gene to the distal region of the long arm of chromosome 4A. The SSR marker wmc776 cosegregated with this gene, whereas markers wmc219 and wmc313 were tightly linked and both located at 0.6 centimorgans. The resistance locus was designated Yr60.
Relationships of Preharvest Weather Conditions and Soil Factors to Susceptibility of Sweetpotato to Postharvest Decay Caused byRhizopus stoloniferandDickeya dadantii
Authors: Edmunds Brooke A. , Clark Christopher A. , Villordon Arthur Q. , Holmes Gerald J.
Published: December 2014

Postharvest soft rots of sweetpotato caused by Rhizopus stolonifer (Rhizopus soft rot) and Dickeya dadantii (bacterial root rot) occur sporadically and can result in significant losses. A 3-year field study related preharvest conditions, including soil texture, chemistry, and fertility; air temperature; soil temperature and moisture; and various cultural practices (153 total variables), to postharvest susceptibility to both diseases in 75 sweetpotato fields in North Carolina and 63 sweetpotato fields in Louisiana. Storage roots were sampled from each field, cured, stored, and inoculated with each pathogen after 100 to 120 days in storage. Disease susceptibility was measured as incidence of diseased storage roots 10 days following inoculation. There was wide variation from field to field in incidence of both diseases (0 to 100% for Rhizopus soft rot and 5 to 95% for bacterial root rot) in both states in each year. Correlations between disease incidence and each of the preharvest variables revealed numerous significant correlations but the variables that correlated with disease incidence were different between North Carolina and Louisiana. Models for both diseases were built by first using forward stepwise regression to identify variables of interest, followed by a mixed-model analysis to produce a final reduced model. For North Carolina fields, postharvest Rhizopus soft rot susceptibility was described by the percentage of the soil cation exchange capacity occupied by calcium, amount of plant-available soil phosphorus, percent soil humic matter, mean air temperature, mean volumetric soil moisture at 40 cm in depth, and mean soil temperature at 2 cm in depth. Postharvest bacterial soft rot susceptibility was described by soil pH and the number of days of high soil temperature late in the season. For Louisiana fields, Rhizopus soft rot susceptibility was described by a complex of variables, including late-season air and soil temperature and late-season days of extreme soil moisture. For bacterial root rot, days of low air temperature and days of high soil temperature late in the season as well as days of low soil moisture best described variation. Although the influence of preharvest variables on postharvest susceptibility was profound for each disease, the complexity of factors involved and differences between the data for the two states makes development of a predictive system extremely difficult.
Nanoparticles modulate surfactant protein A and D mediated protection against influenza A infectionin vitro
Authors: McKenzie Zofi , Kendall Michaela , Mackay Rose-Marie , Tetley Teresa D. , Morgan Cliff , Griffiths Mark , Clark Howard W. , Madsen Jens
Published: December 2014

Numerous epidemiological and toxicological studies have indicated that respiratory infections are exacerbated following enhanced exposure to airborne particulates. Surfactant protein A (SP-A) and SP-D form an important part of the innate immune response in the lung and can interact with nanoparticles to modulate the cellular uptake of these particles. We hypothesize that this interaction will also affect the ability of these proteins to combat infections. TT1, A549 and differentiated THP-1 cells, representing the predominant cell types found in the alveolus namely alveolar type I (ATI) epithelial cells, ATII cells and macrophages, were used to examine the effect of two model nanoparticles, 100 nm amine modified (A-PS) and unmodified polystyrene (U-PS), on the ability of SP-A and SP-D to neutralize influenza A infectionsin vitro. Pre-incubation of low concentrations of U-PS with SP-A resulted in a reduction of SP-A anti-influenza activity in A549 cells, whereas at higher concentrations there was an increase in SP-A antiviral activity. This differential pattern of U-PS concentration on surfactant protein mediated protection against IAV was also shown with SP-D in TT1 cells. On the other hand, low concentrations of A-PS particles resulted in a reduction of SP-A activity in TT1 cells and a reduction in SP-D activity in A549 cells. These results indicate that nanoparticles can modulate the ability of SP-A and SP-D to combat viral challenges. Furthermore, the nanoparticle concentration, surface chemistry and cell type under investigation are important factors in determining the extent of these modulations.
van der Waals forces influencing adhesion of cells
Authors: Kendall K. , Roberts A. D.
Published: December 2014

Adhesion molecules, often thought to be acting by a ‘lock and key’ mechanism, have been thought to control the adhesion of cells. While there is no doubt that a coating of adhesion molecules such as fibronectin on a surface affects cell adhesion, this paper aims to show that such surface contamination is only one factor in the equation. Starting from the baseline idea that van der Waals force is a ubiquitous attraction between all molecules, and thereby must contribute to cell adhesion, it is clear that effects from geometry, elasticity and surface molecules must all add on to the basic cell attractive force. These effects of geometry, elasticity and surface molecules are analysed. The adhesion force measured between macroscopic polymer spheres was found to be strongest when the surfaces were absolutely smooth and clean, with no projecting protruberances. Values of the measured surface energy were then about 35 mJ m
−2
, as expected for van der Waals attractions between the non-polar molecules. Surface projections such as abrasion roughness or dust reduced the molecular adhesion substantially. Water cut the measured surface energy to 3.4 mJ m
−2
. Surface active molecules lowered the adhesion still further to less than 0.3 mJ m
−2
. These observations do not support the lock and key concept.
Nanoparticle growth and surface chemistry changes in cell-conditioned culture medium
Authors: Kendall Michaela , Hodges Nikolas J. , Whitwell Harry , Tyrrell Jess , Cangul Hakan
Published: December 2014

When biomolecules attach to engineered nanoparticle (ENP) surfaces, they confer the particles with a new biological identity. Physical format may also radically alter, changing ENP stability and agglomeration state within seconds. In order to measure which biomolecules are associated with early ENP growth, we studied ENPs in conditioned medium from A549 cell culture, using dynamic light scattering (DLS) and linear trap quadrupole electron transfer dissociation mass spectrometry. Two types of 100 nm polystyrene particles (one uncoated and one with an amine functionalized surface) were used to measure the influence of surface type. In identically prepared conditioned medium, agglomeration was visible in all samples after 1 h, but was variable, indicating inter-sample variability in secretion rates and extracellular medium conditions. In samples conditioned for 1 h or more, ENP agglomeration rates varied significantly. Agglomerate size measured by DLS was well correlated with surface sequestered peptide number for uncoated but not for amine coated polystyrene ENPs. Amine-coated ENPs grew much faster and into larger agglomerates associated with fewer sequestered peptides, but including significant sequestered lactose dehydrogenase. We conclude that interference with extracellular peptide balance and oxidoreductase activity via sequestration is worthy of further study, as increased oxidative stress via this new mechanism may be important for cell toxicity.
‘Bio-nano interactions: new tools, insights and impacts’: summary of the Royal Society discussion meeting
Authors: Lynch Iseult , Feitshans Ilise L. , Kendall Michaela
Published: December 2014

Bio-nano interactions can be defined as the study of interactions between nanoscale entities and biological systems such as, but not limited to, peptides, proteins, lipids, DNA and other biomolecules, cells and cellular receptors and organisms including humans. Studying bio-nano interactions is particularly useful for understandingengineered materialsthat have at least one dimension in the nanoscale. Such materials may consist of discrete particles or nanostructured surfaces. Much of biology functions at the nanoscale; therefore, our ability to manipulate materials such that they are taken up at the nanoscale, and engage biological machinery in a designed and purposeful manner, opens new vistas for more efficient diagnostics, therapeutics (treatments) and tissue regeneration, so-called nanomedicine. Additionally, this ability of nanomaterials to interact with and be taken up by cells allows nanomaterials to be used as probes and tools to advance our understanding of cellular functioning. Yet, as a new technology, assessment of the safety of nanomaterials, and the applicability of existing regulatory frameworks for nanomaterials must be investigated in parallel with development of novel applications. The Royal Society meeting ‘Bio-nano interactions: new tools, insights and impacts' provided an important platform for open dialogue on the current state of knowledge on these issues, bringing together scientists, industry, regulatory and legal experts to concretize existing discourse in science law and policy. This paper summarizes these discussions and the insights that emerged.
Exploiting cell-mediated contraction and adhesion to structure tissues
in vitro
Authors: Wudebwe Uchena N. G. , Bannerman Alistair , Goldberg-Oppenheimer Pola , Paxton Jennifer Z. , Williams Richard L. , Grover Liam M.
Published: December 2014

Progress in tissue engineering is now impacting beyond the field of regenerative medicine. Engineered tissues are now used as tools to evaluate the toxicity of compounds or even to enable the modelling of disease. While many of the materials that are used to facilitate tissue growth are designed to enable cell attachment, many researchers consider that the contraction and modification of these matrices by attached cells is not desirable and take measures to prevent this from occurring. Where substantial alignment of the molecules within tissues, however, is a feature of structure the process of contraction can be exploited to guide new matrix deposition. In this paper, we will demonstrate how we have used the cell contraction process to generate tissues with high levels of organization. The tissues that have been grown in the laboratory have been characterized using a suite of analytical techniques to demonstrate significant levels of matrix organization and mechanical behaviour analogous to natural tissues. This paper provides an overview of research that has been undertaken to determine how tissues have been grown
in vitro
with structuring from the molecular, right through to the macroscopic level.
Rapid evolution of the env gene leader sequence in cats naturally infected with feline immunodeficiency virus
Authors: Bęczkowski Paweł M. , Hughes Joseph , Biek Roman , Litster Annette , Willett Brian J. , Hosie Margaret J.
Published: December 2014

Analysing the evolution of feline immunodeficiency virus (FIV) at the intra-host level is important in order to address whether the diversity and composition of viral quasispecies affect disease progression. We examined the intra-host diversity and the evolutionary rates of the entire env and structural fragments of the env sequences obtained from sequential blood samples in 43 naturally infected domestic cats that displayed different clinical outcomes. We observed in the majority of cats that FIV env showed very low levels of intra-host diversity. We estimated that env evolved at a rate of 1.16×10−3 substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3–V5 fragment of FIV env displayed higher evolutionary rates in healthy cats than in those with terminal illness. Our study provided the first evidence that the leader sequence of env, rather than the V3–V5 sequence, had the highest intra-host diversity and the highest evolutionary rate of all env fragments, consistent with this region being under a strong selective pressure for genetic variation. Overall, FIV env displayed relatively low intra-host diversity and evolved slowly in naturally infected cats. The maximum evolutionary rate was observed in the leader sequence of env. Although genetic stability is not necessarily a prerequisite for clinical stability, the higher genetic stability of FIV compared with human immunodeficiency virus might explain why many naturally infected cats do not progress rapidly to AIDS.
Pollen feeding proteomics: salivary proteins of the passion flower butterfly,Heliconius melpomene
Authors: Harpel Desiree , Cullen Darron A. , Ott Swidbert , Jiggins Chris D. , Walters James R.
Published: December 2014

While most adult Lepidoptera use flower nectar as their primary food source, butterflies in the genusHeliconiushave evolved the novel ability to acquire amino acids from consuming pollen.Heliconiusbutterflies collect pollen on their proboscis, moisten the pollen with saliva, and use a combination of mechanical disruption and chemical degradation to release free amino acids that are subsequently re-ingested in the saliva. Little is known about the molecular mechanisms of this complex pollen feeding adaptation. Here we report an initial shotgun proteomic analysis of saliva fromHeliconius melpomene. Results from liquid-chromatography tandem mass-spectrometry confidently identified 31 salivary proteins, most of which contained predicted signal peptides, consistent with extracellular secretion. Further bioinformatic annotation of these salivary proteins indicated the presence of four distinct functional classes: proteolysis (10 proteins), carbohydrate hydrolysis (5), immunity (6), and “housekeeping”(4). Additionally, six proteins could not be functionally annotated beyond containing a predicted signal sequence. The presence of several salivary proteases is consistent with previous demonstrations thatHeliconiussaliva has proteolytic capacity. It is likely these proteins play a key role in generating free amino acids during pollen digestion. The identification of proteins functioning in carbohydrate hydrolysis is consistent withHeliconiusbutterflies consuming nectar, like other lepidopterans, as well as pollen. Immune-related proteins in saliva are also expected, given that ingestion of pathogens is a very likely route to infection. The few “housekeeping” proteins are likely not true salivary proteins and reflect a modest level of contamination that occurred during saliva collection. Among the unannotated proteins were two sets of paralogs, each seemingly the result of a relatively recent tandem duplication. These results offer a first glimpse into the molecular foundation ofHeliconiuspollen feeding and provide a substantial advance towards comprehensively understanding this striking evolutionary novelty.
Extended X-ray absorption fine structure and multiple-scattering simulation of nickel dithiolene complexes Ni[S2C2(CF3)2]2n(n= −2, −1, 0) and an olefin adduct Ni[S2C2(CF3)2]2(1-hexene)
Authors: Gu Weiwei , Wang Hongxin , Wang Kun
Published: December 2014

A series of Ni dithiolene complexes Ni[S2C2(CF3)]2n(n= −2, −1, 0) (1,2,3) and a 1-hexene adduct Ni[S2C2(CF3)2]2(C6H12) (4) have been examined by NiK-edge X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine-structure (EXAFS) spectroscopies. Ni XANES for1–3reveals clear pre-edge features and approximately +0.7 eV shift in the NiK-edge position for `one-electron' oxidation. EXAFS simulation shows that the Ni—S bond distances for1,2and3(2.11–2.16 Å) are within the typical values for square planar complexes and decrease by ∼0.022 Å for each `one-electron' oxidation. The changes in NiK-edge energy positions and Ni—S distances are consistent with the `non-innocent' character of the dithiolene ligand. The Ni—C interactions at ∼3.0 Å are analyzed and the multiple-scattering parameters are also determined, leading to a better simulation for the overall EXAFS spectra. The 1-hexene adduct4presents no pre-edge feature, and its NiK-edge position shifts by −0.8 eV in comparison with its starting dithiolene complex3. Consistently, EXAFS also showed that the Ni—S distances in4elongate by ∼0.046 Å in comparison with3. The evidence confirms that the neutral complex is `reduced' upon addition of olefin, presumably by olefin donating the π-electron density to the LUMO of3as suggested by UV/visible spectroscopy in the literature.
Expression and crystallization of a bacterial glycoside hydrolase family 116 β-glucosidase fromThermoanaerobacterium xylanolyticum
Authors: Sansenya Sompong , Mutoh Risa , Charoenwattanasatien Ratana , Kurisu Genji , Ketudat Cairns James R.
Published: December 2014

TheThermoanaerobacterium xylanolyticumgene product TxGH116, a glycoside hydrolase family 116 protein of 806 amino-acid residues sharing 37% amino-acid sequence identity over 783 residues with human glucosylceramidase 2 (GBA2), was expressed inEscherichia coli. Purification by heating, immobilized metal-affinity and size-exclusion chromatography produced >90% pure TxGH116 protein with an apparent molecular mass of 90 kDa on SDS–PAGE. The purified TxGH116 enzyme hydrolyzed thep-nitrophenyl (pNP) glycosidespNP-β-D-glucoside,pNP-β-D-galactoside andpNP-N-acetyl-β-D-glucopyranoside, as well as cellobiose and cellotriose. The TxGH116 protein was crystallized using a precipitant consisting of 0.6 Msodium citrate tribasic, 0.1 MTris–HCl pH 7.0 by vapour diffusion with micro-seeding to form crystals with maximum dimensions of 120 × 25 × 5 µm. The TxGH116 crystals diffracted X-rays to 3.15 Å resolution and belonged to the monoclinic space groupP21. Structure solution will allow a structural explanation of the effects of human GBA2 mutations.
Crystallization and preliminary crystallographic studies of a novel noncatalytic carbohydrate-binding module from theRuminococcus flavefacienscellulosome
Authors: Venditto Immacolata , Goyal Arun , Thompson Andrew , Ferreira Luis M. A. , Fontes Carlos M. G. A. , Najmudin Shabir
Published: December 2014

Microbial degradation of the plant cell wall is a fundamental biological process with considerable industrial importance. Hydrolysis of recalcitrant polysaccharides is orchestrated by a large repertoire of carbohydrate-active enzymes that display a modular architecture in which a catalytic domain is connectedvialinker sequences to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus potentiating catalysis. The genome of the most abundant ruminal cellulolytic bacterium,Ruminococcus flavefaciensstrain FD-1, provides an opportunity to discover novel cellulosomal proteins involved in plant cell-wall deconstruction. It encodes a modular protein comprising a glycoside hydrolase family 9 catalytic module (GH9) linked to two unclassified tandemly repeated CBMs (termed CBM-Rf6A and CBM-Rf6B) and a C-terminal dockerin. The novel CBM-Rf6A from this protein has been crystallized and data were processed for the native and a selenomethionine derivative to 1.75 and 1.5 Å resolution, respectively. The crystals belonged to orthorhombic and cubic space groups, respectively. The structure was solved by a single-wavelength anomalous dispersion experiment using theCCP4 program suite andSHELXC/D/E.
Crystallization and preliminary X-ray analysis of the C-terminal fragment of PorM, a subunit of thePorphyromonas gingivalistype IX secretion system
Authors: Stathopulos Julien , Cambillau Christian , Cascales Eric , Roussel Alain , Leone Philippe
Published: December 2014

PorM is a membrane protein involved in the assembly of the type IX secretion system (T9SS) fromPorphyromonas gingivalis, a major bacterial pathogen responsible for periodontal disease in humans. The periplasmic domain of PorM was overexpressed inEscherichia coliand purified. A fragment of the purified protein was obtained by limited proteolysis. Crystals of this fragment belonged to the tetragonal space groupP43212. Native and MAD data sets were recorded to 2.85 and 3.1 Å resolution, respectively, using synchrotron radiation.
Crystallization and preliminary X-ray analysis of the type IV restriction endonuclease ScoMcrA fromStreptomyces coelicolor, which cleaves both Dcm-methylated DNA and phosphorothioated DNA
Authors: Liu Guang , Zhang Zhenyi , Zhao Gong , Deng Zixin , Wu Geng , He Xinyi
Published: December 2014

ScoMcrA is a type IV modification-dependent restriction endonuclease found in the model strainStreptomyces coelicolor. Unlike type I, II and III restriction endonucleases, which cleave unmodified DNA, type IV restriction endonucleases cleave modified DNA, including methylated, hydroxymethylated, glucosyl-hydroxymethylated and phosphorothioated DNA. ScoMcrA targets both Dcm-methylated DNA and phosphorothioated DNA, and makes double-strand breaks 16–28 nt away from the modified nucleotides or the phosphorothioate links. However, the mechanism by which ScoMcrA recognizes these two entirely different types of modification remains unclear. In this study, the ScoMcrA protein was overexpressed, purified and crystallized. The crystals diffracted to 3.35 Å resolution and belonged to space groupP212121. The unit-cell parameters were determined to bea= 130.19,b= 139.36,c= 281.01 Å, α = β = γ = 90°. These results will facilitate the detailed structural analysis of ScoMcrA and further elucidation of its biochemical mechanism.
Expression, purification and crystallization of a membrane-associated, catalytically active type I signal peptidase fromStaphylococcus aureus
Authors: Ting Yi Tian , Batot Gaëlle , Baker Edward N. , Young Paul G.
Published: December 2014

Staphylococcus aureusinfections are becoming increasingly difficult to treat as they rapidly develop resistance to existing antibiotics. Bacterial type I signal peptidases are membrane-associated, cell-surface serine proteases with a unique catalytic mechanism that differs from that of eukaryotic endoplasmic reticulum signal peptidases. They are thus potential antimicrobial targets.S. aureushas a catalytically active type I signal peptidase, SpsB, that is essential for cell viability. To elucidate its structure, thespsBgene fromS. aureusNewman strain was cloned and overexpressed inEscherichia coli. After exploring many different protein-modification constructs, SpsB was expressed as a fusion protein with maltose-binding protein and crystallized by hanging-drop vapour diffusion. The crystals belonged to the monoclinic space groupP21and diffracted to 2.05 Å resolution. The crystal structure of SpsB is anticipated to provide structural insight into Gram-positive signal peptidases and to aid in the development of antibacterial agents that target type I signal peptidases.
Three-dimensional structure of a variant `Termamyl-like'Geobacillus stearothermophilusα-amylase at 1.9 Å resolution
Authors: Offen Wendy A. , Viksoe-Nielsen Anders , Borchert Torben V. , Wilson Keith S. , Davies Gideon J.
Published: December 2014

The enzyme-catalysed degradation of starch is central to many industrial processes, including sugar manufacture and first-generation biofuels. Classical biotechnological platforms involve steam explosion of starch followed by the action of endo-acting glycoside hydrolases termed α-amylases and then exo-acting α-glucosidases (glucoamylases) to yield glucose, which is subsequently processed. A key enzymatic player in this pipeline is the `Termamyl' class of bacterial α-amylases and designed/evolved variants thereof. Here, the three-dimensional structure of one such Termamyl α-amylase variant based upon the parentGeobacillus stearothermophilusα-amylase is presented. The structure has been solved at 1.9 Å resolution, revealing the classical three-domain fold stabilized by Ca2+and a Ca2+–Na+–Ca2+triad. As expected, the structure is similar to theG. stearothermophilusα-amylase but with main-chain deviations of up to 3 Å in some regions, reflecting both the mutations and differing crystal-packing environments.
Structures of theN-acetyltransferase domain ofXylella fastidiosaN-acetyl-L-glutamate synthase/kinase with and without a His tag bound toN-acetyl-L-glutamate
Authors: Zhao Gengxiang , Jin Zhongmin , Allewell Norma M. , Tuchman Mendel , Shi Dashuang
Published: December 2014

Structures of the catalyticN-acetyltransferase (NAT) domain of the bifunctionalN-acetyl-L-glutamate synthase/kinase (NAGS/K) fromXylella fastidiosabound toN-acetyl-L-glutamate (NAG) with and without an N-terminal His tag have been solved and refined at 1.7 and 1.4 Å resolution, respectively. The NAT domain with an N-terminal His tag crystallized in space groupP41212, with unit-cell parametersa=b= 51.72,c= 242.31 Å. Two subunits form a molecular dimer in the asymmetric unit, which contains ∼41% solvent. The NAT domain without an N-terminal His tag crystallized in space groupP21, with unit-cell parametersa= 63.48,b= 122.34,c= 75.88 Å, β = 107.6°. Eight subunits, which form four molecular dimers, were identified in the asymmetric unit, which contains ∼38% solvent. The structures with and without the N-terminal His tag provide an opportunity to evaluate how the His tag affects structure and function. Furthermore, multiple subunits in different packing environments allow an assessment of the plasticity of the NAG binding site, which might be relevant to substrate binding and product release. The dimeric structure of theX. fastidiosaN-acetytransferase (xfNAT) domain is very similar to that of humanN-acetyltransferase (hNAT), reinforcing the notion that mammalian NAGS is evolutionally derived from bifunctional bacterial NAGS/K.
A comprehensive review of the lipid cubic phase orin mesomethod for crystallizing membrane and soluble proteins and complexes
Authors: Caffrey Martin
Published: December 2014

The lipid cubic phase orin mesomethod is a robust approach for crystallizing membrane proteins for structure determination. The uptake of the method is such that it is experiencing what can only be described as explosive growth. This timely, comprehensive and up-to-date review introduces the reader to the practice ofin mesocrystallogenesis, to the associated challenges and to their solutions. A model of how crystallization comes about mechanistically is presented for a more rational approach to crystallization. The possible involvement of the lamellar and inverted hexagonal phases in crystallogenesis and the application of the method to water-soluble, monotopic and lipid-anchored proteins are addressed. How to set up trials manually and automatically with a robot is introduced with reference to open-access online videos that provide a practical guide to all aspects of the method. These range from protein reconstitution to crystal harvesting from the hosting mesophase, which is noted for its viscosity and stickiness. The sponge phase, as an alternative medium in which to perform crystallization, is described. The compatibility of the method with additive lipids, detergents, precipitant-screen components and materials carried along with the protein such as denaturants and reducing agents is considered. The powerful host and additive lipid-screening strategies are described along with how samples that have low protein concentration and cell-free expressed protein can be used. Assaying the protein reconstituted in the bilayer of the cubic phase for function is an important element of quality control and is detailed. Host lipid design for crystallization at low temperatures and for large proteins and complexes is outlined. Experimental phasing by heavy-atom derivatization, soaking or co-crystallization is routine and the approaches that have been implemented to date are described. An overview and a breakdown by family and function of the close to 200 published structures that have been obtained usingin meso-grown crystals are given. Recommendations for conducting the screening process to give a more productive outcome are summarized. The fact that thein mesomethod also works with soluble proteins should not be overlooked. Recent applications of the method forin situserial crystallography at X-ray free-electron lasers and synchrotrons are described. The review ends with a view to the future and to the bright prospects for the method, which continues to contribute to our understanding of the molecular mechanisms of some of nature's most valued proteinaceous robots.
Purification, crystallization and preliminary X-ray crystallographic studies of Rv3899c fromMycobacterium tuberculosis
Authors: Song Yingjia , Liu Jianghui , Li De-Feng , Li Honglin , Wang Shihua , Wang Da-Cheng , Zhou Jie , Bi Lijun
Published: December 2014

Rv3899c, a hypothetical protein fromMycobacterium tuberculosisthat is conserved within the mycobacteria, is predicted to be secreted and has been found in culture filtrates. Here, Rv3899c has been cloned, expressed inEscherichia coliand purified using standard chromatographic techniques. The hanging-drop vapour-diffusion method with PEG 3350 as a precipitant was used to crystallize the protein. N-terminal sequencing results showed that the amino-acid sequence of the crystallized protein began with GATAG, indicating that it is a fragment containing residues 184–410 of Rv3899c. Rv3899c184–410crystals exhibited the symmetry of space groupP212121, with unit-cell parametersa= 49.88,b= 54.72,c= 75.52 Å, α = β = γ = 90°, and diffracted to a resolution of 1.90 Å.
Cloning, expression, refolding, purification and preliminary crystallographic analysis of the sensory domain of theCampylobacterchemoreceptor for aspartate A (CcaA)
Authors: Machuca Mayra A. , Liu Yu C. , Roujeinikova Anna
Published: December 2014

InCampylobacter jejuni, chemotaxis and motility have been identified as important virulence factors that are required for host colonization and invasion. Chemotactic recognition of extracellular signals is mediated by the periplasmic sensory domains of its transducer-like proteins (Tlps). In this study, the sensory domain of theC. jejunichemoreceptor for aspartate A (CcaA) has been expressed inEscherichia coliand purified from inclusion bodies. The urea-denatured protein was refolded and then crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as a precipitating agent. A complete data set has been collected to 1.4 Å resolution using cryocooling conditions and synchrotron radiation. The crystals belonged to space groupP1, with unit-cell parametersa= 39.3,b= 43.3,c= 50.9 Å, α = 92.5, β = 111.4, γ = 114.7°.
SHELXT– Integrated space-group and crystal-structure determination
Authors: Sheldrick George M.
Published: December 2014

The new computer programSHELXTemploys a novel dual-space algorithm to solve the phase problem for single-crystal reflection data expanded to the space groupP1. Missing data are taken into account and the resolution extended if necessary. All space groups in the specified Laue group are tested to find which are consistent with theP1 phases. After applying the resulting origin shifts and space-group symmetry, the solutions are subject to further dual-space recycling followed by a peak search and summation of the electron density around each peak. Elements are assigned to give the best fit to the integrated peak densities and if necessary additional elements are considered. An isotropic refinement is followed for non-centrosymmetric space groups by the calculation of a Flack parameter and, if appropriate, inversion of the structure. The structure is assembled to maximize its connectivity and centred optimally in the unit cell.SHELXThas already solved many thousand structures with a high success rate, and is optimized for multiprocessor computers. It is, however, unsuitable for severely disordered and twinned structures because it is based on the assumption that the structure consists of atoms.
Crystal structure of bis[trans-(ethane-1,2-diamine-κ2N,N′)bis(thiocyanato-κN)chromium(III)] tetrachloridozincate from synchrotron data
Authors: Moon Dohyun , Choi Jong-Ha
Published: December 2014

The structure of the title compound, [Cr(NCS)2(C2H8N2)2]2[ZnCl4], has been determined from synchrotron data. In the asymmetric unit, there are four independent halves of the CrIIIcomplex cations, each of which lies on an inversion centre, and one tetrachloridozincate anion in a general position. The CrIIIatoms are coordinated by the four N atoms of two ethane-1,2-diamine (en) ligands in the equatorial plane and two N-bound NCS−anions in atransarrangement, displaying a slightly distorted octahedral geometry with crystallographic inversion symmetry. The Cr—N(en) and Cr—N(NCS) bond lengths range from 2.0653 (10) to 2.0837 (10) Å and from 1.9811 (10) to 1.9890 (10) Å, respectively. The five-membered metalla-rings are in stablegaucheconformations. The [ZnCl4]2−anion has a distorted tetrahedral geometry. The crystal structure is stabilized by intermolecular hydrogen bonds involving the en NH2or CH2groups as donors and chloride ligands of the anion and S atoms of NCS−ligands as acceptors.
Crystal structure of 4-{[(cyanoimino)(methylsulfanyl)methyl]amino}-1,5-dimethyl-2-phenyl-2,3-dihydro-1H-pyrazol-3-one
Authors: Elgemeie Galal H. , Abouzeid Mamdouh , Jones Peter G.
Published: December 2014

In the title compound, C14H15N5OS, the tautomer present in the solid state is that in which the immediately exocyclic N atom bears the H atom. The central five-membered ring is almost planar (r.m.s. deviation = 0.025 Å), but both its N atoms are significantly pyramidalized. A classical hydrogen bond from the N—H group to the cyanide N atom forms inversion-symmetric dimers, which are further linked by C—H...O interactions.
Pakistan Accumulators (PVT) Limited (PAL) – a third world perspective
Authors: Hussain Umer
Published: December 2014

Subject area

Operational Management, Strategic Management and Marketing Management.

Study level/applicability

The case can be taught in introductory marketing courses and management and organizational policy course and in advanced level in promotion, distribution channels, marketing research, consumer behavior and brand management courses at graduate level. Importance of market and technological research; first mover advantage and disadvantages in new market segment; importance of competition in a market; use of PESTL analysis before pursuing for any segment; types of growth strategies which could be used that is Broad base or Narrow base; how company uses marketing mix strategy; and how managers make decision in dynamic environment (contingency theory approach).

Case overview

This case study relates to a real-life situation the data was collected from primary and secondary sources between 2012 and 2013. The case is of a company Pakistan Accumulators (PAL), having less than 18 years of experience, has been able to grow successfully in the dynamic environment of Pakistan. The decline of the private businesses in Pakistan due to the energy crisis has popped up a new need of power generation alternative equipment in the country. PAL, which is a privately owned company, suppliers of automotive batteries, Uninterruptable Power System (UPS) batteries, lead acid batteries and rechargeable batteries has been able to manage the growth of 20 per cent per year. In this case study, we have highlighted only one market segment of the company that is of UPS batteries, we have focused on what is the future prospect of this particular segment, its attractiveness. Also, the area of focus was the new market segments which can be targeted by the company. Basic issues of the case study: calculation of the market segment value of UPS industry; identification and solution of different challenges faced by PAL in the dynamic Pakistani market (contingency theory); recognition of different future growth prospects for PAL.

Expected learning outcomes

The basic objective of this case is to enhance the analytical and qualitative skills of the students by giving them the real-life perspective of a company working successfully in country like Pakistan which is facing economic and political crises. This case can also be used for understanding the problems of third-world markets and how company can pursue successfully in the long–term.

Supplementary materials

Teaching notes are available for educators only. Please contact your library to gain login details or email support@emeraldinsight.com to request teaching notes.