bluuebunny/crossref_metadata_2025_split huggingface dataset:
A C-Terminal Proline-Rich Sequence Simultaneously Broadens the Optimal Temperature and pH Ranges and Improves the Catalytic Efficiency of Glycosyl Hydrolase Family 10 Ruminal Xylanases
Authors: Li Zhongyuan , Xue Xianli , Zhao Heng , Yang Peilong , Luo Huiying , Zhao Junqi , Huang Huoqing , Yao Bin 
Published: March 2014

ABSTRACT

Efficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene (
xynA
) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed in
Escherichia coli
. The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more α helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.
Diversity of Two-Domain Laccase-Like Multicopper Oxidase Genes in Streptomyces spp.: Identification of Genes Potentially Involved in Extracellular Activities and Lignocellulose Degradation during Composting of Agricultural Waste
Authors: Lu Lunhui , Zeng Guangming , Fan Changzheng , Zhang Jiachao , Chen Anwei , Chen Ming , Jiang Min , Yuan Yujie , Wu Haipeng , Lai Mingyong , He Yibin 
Published: March 2014

ABSTRACT

Traditional three-domain fungal and bacterial laccases have been extensively studied for their significance in various biotechnological applications. Growing molecular evidence points to a wide occurrence of more recently recognized two-domain laccase-like multicopper oxidase (LMCO) genes in
Streptomyces
spp. However, the current knowledge about their ecological role and distribution in natural or artificial ecosystems is insufficient. The aim of this study was to investigate the diversity and composition of
Streptomyces
two-domain LMCO genes in agricultural waste composting, which will contribute to the understanding of the ecological function of
Streptomyces
two-domain LMCOs with potential extracellular activity and ligninolytic capacity. A new specific PCR primer pair was designed to target the two conserved copper binding regions of
Streptomyces
two-domain LMCO genes. The obtained sequences mainly clustered with
Streptomyces coelicolor
,
Streptomyces violaceusniger
, and
Streptomyces griseus
. Gene libraries retrieved from six composting samples revealed high diversity and a rapid succession of
Streptomyces
two-domain LMCO genes during composting. The obtained sequence types cluster in 8 distinct clades, most of which are homologous with
Streptomyces
two-domain LMCO genes, but the sequences of clades III and VIII do not match with any reference sequence of known streptomycetes. Both lignocellulose degradation rates and phenol oxidase activity at pH 8.0 in the composting process were found to be positively associated with the abundance of
Streptomyces
two-domain LMCO genes. These observations provide important clues that
Streptomyces
two-domain LMCOs are potentially involved in bacterial extracellular phenol oxidase activities and lignocellulose breakdown during agricultural waste composting.
Aerobic Granules: Microbial Landscape and Architecture, Stages, and Practical Implications
Authors: Gonzalez-Gil Graciela , Holliger Christof 
Published: March 2014

ABSTRACT
For the successful application of aerobic granules in wastewater treatment, granules containing an appropriate microbial assembly able to remove contaminants should be retained and propagated within the reactor. To manipulate and/or optimize this process, a good understanding of the formation and dynamic architecture of the granules is desirable. Models of granules often assume a spherical shape with an outer layer and an inner core, but limited information is available regarding the extent of deviations from such assumptions. We report on new imaging approaches to gain detailed insights into the structural characteristics of aerobic granules. Our approach stained all components of the granule to obtain a high quality contrast in the images; hence limitations due to thresholding in the image analysis were overcome. A three-dimensional reconstruction of the granular structure was obtained that revealed the mesoscopic impression of the cavernlike interior of the structure, showing channels and dead-end paths in detail. In “old” granules, large cavities allowed for the irrigation and growth of dense microbial colonies along the path of the channels. Hence, in some areas, paradoxically higher biomass content was observed in the inner part of the granule compared to the outer part. Microbial clusters “rooting” from the interior of the mature granule structure indicate that granules mainly grow via biomass outgrowth and not by aggregation of small particles. We identify and discuss phenomena contributing to the life cycle of aerobic granules. With our approach, volumetric tetrahedral grids are generated that may be used to validate complex models of granule formation.
Deletion Mutant Library for Investigation of Functional Outputs of Cyclic Diguanylate Metabolism in Pseudomonas aeruginosa PA14
Authors: Ha Dae-Gon , Richman Megan E. , O'Toole George A. 
Published: March 2014

ABSTRACT

We constructed a library of in-frame deletion mutants targeting each gene in
Pseudomonas aeruginosa
PA14 predicted to participate in cyclic di-GMP (c-di-GMP) metabolism (biosynthesis or degradation) to provide a toolkit to assist investigators studying c-di-GMP-mediated regulation by this microbe. We present phenotypic assessments of each mutant, including biofilm formation, exopolysaccharide (EPS) production, swimming motility, swarming motility, and twitch motility, as a means to initially characterize these mutants and to demonstrate the potential utility of this library.
Identification of
opsA
, a Gene Involved in Solute Stress Mitigation and Survival in Soil, in the Polycyclic Aromatic Hydrocarbon-Degrading Bacterium Novosphingobium sp. Strain LH128
Authors: Fida Tekle Tafese , Breugelmans Philip , Lavigne Rob , van der Meer Jan Roelof , De Mot René , Vaysse Pierre-Joseph , Springael Dirk 
Published: March 2014

ABSTRACT

The aim of this study was to identify genes involved in solute and matric stress mitigation in the polycyclic aromatic hydrocarbon (PAH)-degrading
Novosphingobium
sp. strain LH128. The genes were identified using plasposon mutagenesis and by selection of mutants that showed impaired growth in a medium containing 450 mM NaCl as a solute stress or 10% (wt/vol) polyethylene glycol (PEG) 6000 as a matric stress. Eleven and 14 mutants showed growth impairment when exposed to solute and matric stresses, respectively. The disrupted sequences were mapped on a draft genome sequence of strain LH128, and the corresponding gene functions were predicted. None of them were shared between solute and matric stress-impacted mutants. One NaCl-affected mutant (i.e., NA7E1) with a disruption in a gene encoding a putative outer membrane protein (OpsA) was susceptible to lower NaCl concentrations than the other mutants. The growth of NA7E1 was impacted by other ions and nonionic solutes and by sodium dodecyl sulfate (SDS), suggesting that
opsA
is involved in osmotic stress mitigation and/or outer membrane stability in strain LH128. NA7E1 was also the only mutant that showed reduced growth and less-efficient phenanthrene degradation in soil compared to the wild type. Moreover, the survival of NA7E1 in soil decreased significantly when the moisture content was decreased but was unaffected when soluble solutes from sandy soil were removed by washing.
opsA
appears to be important for the survival of strain LH128 in soil, especially in the case of reduced moisture content, probably by mitigating the effects of solute stress and retaining membrane stability.
Biochemical and Kinetic Characterization of Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase 2 (Xfp2) from Cryptococcus neoformans
Authors: Glenn Katie , Ingram-Smith Cheryl , Smith Kerry S. 
Published: March 2014

ABSTRACT

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogen
Cryptococcus neoformans
, which has two
XFP
genes (designated
XFP1
and
XFP2
). Our kinetic characterization of
C. neoformans
Xfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics.
C. neoformans
Xfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inhibitory concentration (IC
50
) of 0.6 mM. PEP and OAA were found to share the same or have overlapping allosteric binding sites, while ATP binds at a separate site. AMP acts as a very potent activator; as little as 20 μM AMP is capable of increasing Xfp2 activity by 24.8% ± 1.0% (mean ± standard error of the mean), while 50 μM prevented inhibition caused by 0.6 mM ATP. AMP and PEP/OAA operated independently, with AMP activating Xfp2 and PEP/OAA inhibiting the activated enzyme. This study provides valuable insight into the metabolic role of Xfp within fungi, specifically the fungal pathogen
Cryptococcus neoformans
, and suggests that at least some Xfps display substrate cooperative binding and allosteric regulation.
UPC2
Is Universally Essential for Azole Antifungal Resistance in Candida albicans
Authors: Vasicek Erin M. , Berkow Elizabeth L. , Flowers Stephanie A. , Barker Katherine S. , Rogers P. David 
Published: March 2014

ABSTRACT

In
Candida albicans
, the transcription factor Upc2 is central to the regulation of ergosterol biosynthesis.
UPC2
-activating mutations contribute to azole resistance, whereas disruption increases azole susceptibility. In the present study, we investigated the relationship of
UPC2
to fluconazole susceptibility, particularly in azole-resistant strains. In addition to the reduced fluconazole MIC previously observed with
UPC2
disruption, we observed a lower minimum fungicidal concentration (MFC) for a
upc2
Δ/Δ mutant than for its azole-susceptible parent, SC5314. Moreover, the
upc2
Δ/Δ mutant was unable to grow on a solid medium containing 10 μg/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed higher fungistatic activity against the
upc2
Δ/Δ mutant than against SC5314.
UPC2
disruption in strains carrying specific resistance mutations also resulted in reduced MICs and MFCs.
UPC2
disruption in a highly azole resistant clinical isolate containing multiple resistance mechanisms likewise resulted in a reduced MIC and MFC. This mutant was unable to grow on a solid medium containing 10 μg/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed increased fungistatic activity against the
upc2
Δ/Δ mutant in the resistant background. Microarray analysis showed attenuated induction by fluconazole of genes involved in sterol biosynthesis, iron transport, or iron homeostasis in the absence of
UPC2
. Taken together, these data demonstrate that the
UPC2
transcriptional network is universally essential for azole resistance in
C. albicans
and represents an attractive target for enhancing azole antifungal activity.
Recognition and Killing of Autologous, Primary Glioblastoma Tumor Cells by Human Cytomegalovirus pp65-Specific Cytotoxic T Cells
Authors: Nair Smita K. , De Leon Gabriel , Boczkowski David , Schmittling Robert , Xie Weihua , Staats Janet , Liu Rebecca , Johnson Laura A. , Weinhold Kent , Archer Gary E. , Sampson John H. , Mitchell Duane A. 
Published: March 2014

Abstract
Purpose: Despite aggressive conventional therapy, glioblastoma (GBM) remains uniformly lethal. Immunotherapy, in which the immune system is harnessed to specifically attack malignant cells, offers a treatment option with less toxicity. The expression of cytomegalovirus (CMV) antigens in GBM presents a unique opportunity to target these viral proteins for tumor immunotherapy. Although the presence of CMV within malignant gliomas has been confirmed by several laboratories, its relevance as an immunologic target in GBM has yet to be established. The objective of this study was to explore whether T cells stimulated by CMV pp65 RNA-transfected dendritic cells (DC) target and eliminate autologous GBM tumor cells in an antigen-specific manner.
Experimental Design: T cells from patients with GBM were stimulated with autologous DCs pulsed with CMV pp65 RNA, and the function of the effector CMV pp65-specific T cells was measured.
Results: In this study, we demonstrate the ability to elicit CMV pp65-specific immune responses in vitro using RNA-pulsed autologous DCs generated from patients with newly diagnosed GBM. Importantly, CMV pp65-specific T cells lyse autologous, primary GBM tumor cells in an antigen-specific manner. Moreover, T cells expanded in vitro using DCs pulsed with total tumor RNA demonstrated a 10- to 20-fold expansion of CMV pp65-specific T cells as assessed by tetramer analysis and recognition and killing of CMV pp65-expressing target cells.
Conclusion: These data collectively demonstrate that CMV-specific T cells can effectively target glioblastoma tumor cells for immunologic killing and support the rationale for the development of CMV-directed immunotherapy in patients with GBM. Clin Cancer Res; 20(10); 2684–94. ©2014 AACR.
CD4
+
T Cells Promote the Transition From Hypertrophy to Heart Failure During Chronic Pressure Overload
Authors: Laroumanie Fanny , Douin-Echinard Victorine , Pozzo Joffrey , Lairez Olivier , Tortosa Florence , Vinel Claire , Delage Christine , Calise Denis , Dutaur Marianne , Parini Angelo , Pizzinat Nathalie 
Published: March 2014

Background—
The mechanisms by which the heart adapts to chronic pressure overload, producing compensated hypertrophy and eventually heart failure (HF), are still not well defined. We aimed to investigate the involvement of T cells in the progression to HF using a transverse aortic constriction (TAC) model.

Methods and Results—

Chronic HF was associated with accumulation of T lymphocytes and activated/effector CD4
+
T cells within cardiac tissue. After TAC, enlarged heart mediastinal draining lymph nodes showed a high density of both CD4
+
and CD8
+
T-cell subsets. To investigate the role of T cells in HF, TAC was performed on mice deficient for recombination activating gene 2 expression (RAG2KO) lacking B and T lymphocytes. Compared with wild-type TAC mice, RAG2KO mice did not develop cardiac dilation and showed improved contractile function and blunted adverse remodeling. Reconstitution of the T-cell compartment into RAG2KO mice before TAC enhanced contractile dysfunction, fibrosis, collagen accumulation, and cross-linking. To determine the involvement of a specific T-cell subset, we performed TAC on mice lacking CD4
+
(MHCIIKO) and CD8
+
T-cell subsets (CD8KO). In contrast to CD8KO mice, MHCIIKO mice did not develop ventricular dilation and dysfunction. MHCIIKO mice also displayed very low fibrosis, collagen accumulation, and cross-linking within cardiac tissue. Interestingly, mice with transgenic CD4
+
T-cell receptor specific for ovalbumin failed to develop HF and adverse remodeling.

Conclusions—

These results demonstrate for the first time a crucial role of CD4
+
T cells and specific antigen recognition in the progression from compensated cardiac hypertrophy to HF.
Greenhouse Gas Emissions from International Shipping: The Response from China’s Shipping Industry to the Regulatory Initiatives of the International Maritime Organization
Authors: Shi Yubing 
Published: March 2014

Abstract
The International Maritime Organization (imo) received its mandate to regulate shipping greenhouse gas (ghg) emissions from the Kyoto Protocol. However, the imo Convention and the un Convention on the Law of the Sea also provide it with competence in this area. In exercising its mandate, the imo has developed regulatory initiatives. China’s shipping industry is playing a growing role in the international shipping market, and its response to these initiatives will have a substantial effect on the future application of these regulations. This article analyses the ghg mandate of the imo, examines the main outcomes achieved within the organization on this issue, and assesses the response from China’s shipping industry to this issue. It concludes that the interests of the shipping industries from developing countries will need to be taken into account in the development of regulatory efforts if a consensus is to be achieved in the global reduction of ghg emissions from ships.
Water Governance in the Aftermath of Rio+20
Authors: Sindico Francesco 
Published: March 2014

Abstract
This article focuses on water governance in the aftermath of the 2012 United Nations Conference on Sustainable Development (uncsd or Rio+20). Water governance is defined here as a process in which law has a transformational role by allowing policy goals to become tangible and enforceable rights and obligations. Against this conceptual background global water governance appears to be still fragmented and incoherent. More coordination efforts and further harmonisation is needed, but more importantly global institutions are required to allow international law to operate effectively. It is within this context that the uncsd can be seen as an international agenda setting process and three key water related topics appear to be on such agenda: water and sanitation; water and ecosystem services and water and climate change. The article concludes with a call in favour of considering water not just as a public good, but mainly as a driver for sustainable development.
Associations between beliefs about the causes of mental disorders and stigmatising attitudes: Results of a national survey of the Australian public
Authors: Reavley Nicola J , Jorm Anthony F 
Published: March 2014

Objective: To examine the associations between beliefs about the causes of depression, schizophrenia, social phobia and post-traumatic stress disorder (PTSD) and stigmatising attitudes towards people with these disorders. Methods: In 2011, telephone interviews were carried out with 6019 Australians aged 15 or over. Participants were presented with a case vignette describing either depression, depression with suicidal thoughts, early schizophrenia, chronic schizophrenia, social phobia or PTSD. Participants were asked about their beliefs about the causes of these disorders and about their personally held stigmatising attitudes, stigmatising attitudes perceived in others and the desire for social distance from the person described in the vignette. Results: Belief in a weak or nervous personality as the cause of mental disorders was most consistently associated with personal stigma, perceived stigma and desire for social distance across vignettes. Belief in biogenetic causes was associated with a decreased belief in mental disorders as due to weakness rather than sickness, but was not linked to either a decreased or increased belief in dangerousness and unpredictability or desire for social distance. Belief in physical causes was associated with an increased belief in mental disorders as due to weakness rather than sickness. Belief in psychosocial causes had no consistent associations with stigma. Conclusions: Explaining mental disorders as due to personality characteristics is a more important factor in stigma than either biogenetic or psychosocial explanations.
The accuracy of colonoscopic localisation of colorectal tumours: a prospective, multi-centred observational study
Authors: Johnstone MS , Moug SJ ,   on behalf of The West of Scotland Surgical Research Network
Published: March 2014

Background and aims Colonoscopy is essential for accurate pre-operative colorectal tumour localisation, but its accuracy for localisation remains undetermined due to limitations of previous work. This study aimed to establish the accuracy of colonoscopic localisation and to determine how frequently inaccuracy results in altered surgical management. Method A prospective, multi-centred, powered observational study recruited 79 patients with colorectal tumours that underwent curative surgical resection. Patient and colonoscopic factors were recorded. Pre-operative colonoscopic and radiological lesion localisations were compared to intra-operative localisation using pre-defined anatomical bowel segments to determine accuracy, with changes in planned surgical management documented. Results Colonoscopy accurately located the colorectal tumour in 64/79 patients (81%). Five out of 15 inaccurately located patients required on-table alteration in planned surgical management. Pre-operative imaging was unable to visualise the primary tumour in 23.1% of cases, a finding that was more prevalent amongst bowel screener patients compared to symptomatic patients (45.8% vs. 13%; p = 0.003). Conclusion Colonoscopic lesion localisation is inaccurate in 19.0% of cases and occurred throughout the colon with a change in on-table surgical management in 6.3%. With CT unable to visualise lesions in just under a quarter of cases, particularly in the screening population, preoperative localisation is heavily reliant on colonoscopy.
Cluster of influenza A cases in vaccinated population of adults in Virology Laboratory in Glasgow in December 2012
Authors: Bradley-Stewart A , Miller RS , MacLean A , Aitken C , Whittaker L , Gregory V , Daniels R , Gunson R 
Published: March 2014

Background and aims The majority of influenza infections during the 2012/2013 influenza season in Scotland have been due to influenza A H3N2. We report an outbreak of influenza A H3N2 in a vaccinated population of adults in the Regional Virology Laboratory in Glasgow. This investigation was carried out to confirm the epidemiological link between cases. Methods and results Staff with clinical symptoms of influenza-like illness were included. Samples were tested by real-time polymerase chain reaction and sequencing. Staff were interviewed to obtain information regarding symptom onset and vaccination status. Eight confirmed cases and six clinically diagnosed cases were reported, which all occurred within 4 days of a lunchtime Christmas quiz. The eight samples subtyped as H3 virus. The haemagglutinin gene in the confirmed cases was sequenced and shown to be identical. Most of the attendees had been immunised against influenza with the same vaccine batch at least 6 weeks earlier. Conclusion This outbreak appears to have been an isolated incident, which arose due to a social event that provided the ideal conditions for transmission of a respiratory disease. It may have been compounded by low-vaccine effectiveness this season. Sequence data supported the epidemiological link.