bluuebunny/crossref_metadata_2025_split huggingface dataset:
XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”
Authors: Wu Ming L. , Wessels Hans J. C. T. , Pol Arjan , Op den Camp Huub J. M. , Jetten Mike S. M. , van Niftrik Laura , Keltjens Jan T. 
Published: December 2014

ABSTRACT

“
Candidatus
Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “
Ca
. Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from “
Ca
. Methylomirabilis oxyfera” that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of “
Ca
. Methylomirabilis oxyfera” cells and catalyzed methanol oxidation with appreciable specific activity and affinity (
V
max
of 10 μmol min
−1
mg
−1
protein,

K
m

of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from “
Ca
. Methylomirabilis oxyfera” is the first representative of type 1 XoxF proteins to be described.
Bacterial Communities Associated with Surfaces of Leafy Greens: Shift in Composition and Decrease in Richness over Time
Authors: Dees Merete Wiken , Lysøe Erik , Nordskog Berit , Brurberg May Bente 
Published: December 2014

ABSTRACT

The phyllosphere is colonized by a wide variety of bacteria and fungi; it harbors epiphytes, as well as plant-pathogenic bacteria and even human pathogens. However, little is known about how the bacterial community composition on leafy greens develops over time. The bacterial community of the leafy-green phyllosphere obtained from two plantings of rocket salad (
Diplotaxis tenuifolia
) and three plantings of lettuce (
Lactuca sativa
) at two farms in Norway were profiled by an Illumina MiSeq-based approach. We found that the bacterial richness of the
L. sativa
samples was significantly greater shortly (3 weeks) after planting than at harvest (5 to 7 weeks after planting) for plantings 1 and 3 at both farms. For the second planting, the bacterial diversity remained consistent at the two sites. This suggests that the effect on bacterial colonization of leaves, at least in part must, be seasonally driven rather than driven solely by leaf maturity. The distribution of phyllosphere communities varied between
D. tenuifolia
and
L. sativa
at harvest. The variability between these species at the same location suggests that the leaf-dwelling bacteria are not only passive inhabitants but interact with the host, which shapes niches favoring the growth of particular taxa. This work contributes to our understanding of host plant-specific microbial community structures and shows how these communities change throughout plant development.
Rapid Method Using Two Microbial Enzymes for Detection of
l
-Abrine in Food as a Marker for the Toxic Protein Abrin
Authors: Dodge Anthony G. , Carrasquillo Kelvin , Rivera Luis , Xu Lei , Wackett Lawrence P. , Sadowsky Michael J. 
Published: December 2014

ABSTRACT

Abrin is a toxic protein produced by the ornamental plant
Abrus precatorius
, and it is of concern as a biothreat agent. The small coextracting molecule
N
-methyl-
l
-tryptophan (
l
-abrine) is specific to members of the genus
Abrus
and thus can be used as a marker for the presence or ingestion of abrin. Current methods for the detection of abrin or
l
-abrine in foods and other matrices require complex sample preparation and expensive instrumentation. To develop a fast and portable method for the detection of
l
-abrine in beverages and foods, the
Escherichia coli
proteins
N
-methyltryptophan oxidase (MTOX) and tryptophanase were expressed and purified. The two enzymes jointly degraded
l
-abrine to products that included ammonia and indole, and colorimetric assays for the detection of those analytes in beverage and food samples were evaluated. An indole assay using a modified version of Ehrlich's/Kovac's reagent was more sensitive and less subject to negative interferences from components in the samples than the Berthelot ammonia assay. The two enzymes were added into food and beverage samples spiked with
l
-abrine, and indole was detected as a degradation product, with the visual lower detection limit being 2.5 to 10.0 μM (∼0.6 to 2.2 ppm)
l
-abrine in the samples tested. Results could be obtained in as little as 15 min. Sample preparation was limited to pH adjustment of some samples. Visual detection was found to be about as sensitive as detection with a spectrophotometer, especially in milk-based matrices.
Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle
Authors: Vick Jacob E. , Clomburg James M. , Blankschien Matthew D. , Chou Alexander , Kim Seohyoung , Gonzalez Ramon 
Published: December 2014

ABSTRACT

We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the β-oxidation cycle for fuel and chemical production in
Escherichia coli
(J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541–554, 2012,
http://dx.doi.org/10.1021/sb3000782
). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the
trans
-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the
Euglena gracilis
trans
-enoyl-CoA reductase [
Eg
TER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native
E. coli
enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a β-oxidation reversal. Overexpression of FabI proved as effective as
Eg
TER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of
fabI
, we investigated whether bacterial ENRs from other families were able to complement a
fabI
deletion without promiscuous reduction of crotonyl-CoA. These characteristics from
Bacillus subtilis
FabL enabled Δ
fabI
complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the β-oxidation reversal in
E. coli
.
Mechanisms of Methods for Hepatitis C Virus Inactivation
Authors: Pfaender Stephanie , Brinkmann Janine , Todt Daniel , Riebesehl Nina , Steinmann Joerg , Steinmann Jochen , Pietschmann Thomas , Steinmann Eike 
Published: December 2014

ABSTRACT
Virus inactivation by chemical disinfectants is an important instrument for infection control in medical settings, but the mechanisms involved are poorly understood. In this study, we systematically investigated the effects of several antiviral treatments on hepatitis C virus (HCV) particles as model for enveloped viruses. Studies were performed with authentic cell culture-derived viruses, and the influence of chemical disinfectants, heat, and UV treatment on HCV was analyzed by the determination of infectious particles in a limiting-dilution assay, by quantitative reverse transcription-PCR, by core enzyme-linked immunosorbent assay, and by proteolytic protection assay. All different inactivation methods resulted in a loss of HCV infectivity by targeting different parts of the virus particle. Alcohols such as ethanol and 2-propanol did not affect the viral RNA genome integrity but disrupted the viral envelope membrane in a capsid protection assay. Heat and UV treatment of HCV particles resulted in direct damage of the viral genome since transfection of viral particle-associated RNA into permissive cells did not initiate RNA replication. In addition, heat incubation at 80°C disrupted the HCV envelope, rendering the viral capsid susceptible to proteolytic digest. This study demonstrated the molecular processes of viral inactivation of an enveloped virus and should facilitate the development of effective disinfection strategies in infection control not only against HCV but also against other enveloped viruses.
Diversity of Methane-Cycling Archaea in Hydrothermal Sediment Investigated by General and Group-Specific PCR Primers
Authors: Lever Mark A. , Teske Andreas P. 
Published: December 2014

ABSTRACT

The zonation of anaerobic methane-cycling
Archaea
in hydrothermal sediment of Guaymas Basin was studied by general primer pairs (mcrI, ME1/ME2, mcrIRD) targeting the alpha subunit of methyl coenzyme M reductase gene (
mcrA
) and by new group-specific
mcrA
and 16S rRNA gene primer pairs. The mcrIRD primer pair outperformed the other general
mcrA
primer pairs in detection sensitivity and phylogenetic coverage. Methanotrophic ANME-1
Archaea
were the only group detected with group-specific primers only. The detection of 14
mcrA
lineages surpasses the diversity previously found in this location. Most phylotypes have high sequence similarities to hydrogenotrophs, methylotrophs, and anaerobic methanotrophs previously detected at Guaymas Basin or at hydrothermal vents, cold seeps, and oil reservoirs worldwide. Additionally, five
mcrA
phylotypes belonging to newly defined lineages are detected. Two of these belong to deeply branching new orders, while the others are new species or genera of
Methanopyraceae
and
Methermicoccaceae
. Downcore diversity decreases from all groups detected in the upper 6 cm (∼2 to 40°C, sulfate measurable to 4 cm) to only two groups below 6 cm (>40°C). Despite the presence of hyperthermophilic genera (
Methanopyrus
,
Methanocaldococcus
) in cooler surface strata, no genes were detected below 10 cm (≥60°C). While
mcrA
-based and 16S rRNA gene-based community compositions are generally congruent, the deeply branching
mcrA
cannot be assigned to specific 16S rRNA gene lineages. Our study indicates that even among well-studied metabolic groups and in previously characterized model environments, major evolutionary branches are overlooked. Detecting these groups by improved molecular biological methods is a crucial first step toward understanding their roles in nature.
Angioplasty for renovascular hypertension in 78 children
Authors: Kari Jameela A , Roebuck Derek J , McLaren Clare A , Davis Meryl , Dillon Michael J , Hamilton George , Shroff Rukshana , Marks Stephen D , Tullus Kjell 
Published: December 2014

ObjectivesTo evaluate the outcome of percutaneous transluminal angioplasty (PTA) in children with renovascular hypertension (RVH) treated at a single centre over 29 years.MethodsA retrospective study of the medical charts of all children with RVH who underwent PTA between 1984 and 2012. The primary outcome measurement was blood pressure (BP) achieved after the procedure. The BP before the procedure was compared with that at last available follow-up, 6 (range 0.6–16) years after the initial procedure.ResultsSeventy-eight children with median (range) age of 6.5 (0.5–17) years were studied. Twenty-three (29.5%) had an underlying syndrome, 35 (44.9%) children had bilateral renal artery stenosis (RAS), 18 (23%) intrarenal disease and 11(14%) showed bilateral RAS and intrarenal disease. Twenty (25.6%) children had mid-aortic syndrome and 14 (17.9%) cerebrovascular disease. One hundred and fourteen PTA procedures were carried out including 31 stent insertions. Following PTA, BP was improved in 49 (62.8%) children and of those 18 (23.1%) were cured. Children with involvement of only the main renal arteries showed improved BP control in 79.9% of the children with cure in 39.5%. BP was intentionally maintained above the 95th centile for age and height in four children with coexistent cerebrovascular disease. No change in BP was seen in 18 children despite observed technical success of the PTA, and in seven children due to technical failure of the procedure.ConclusionsPTA provided a clinical benefit in 62.8% of children with RVH.
Improving vitamin A and D intake among Inuit and Inuvialuit in Arctic Canada: evidence from the Healthy Foods North study
Authors: Pakseresht Mohammadreza , Kolahdooz Fariba , Gittelsohn Joel , Roache Cindy , Corriveau André , Sharma Sangita 
Published: December 2014

BackgroundPeople in Arctic Canada are undergoing a nutritional transition and increased prevalence of chronic disease. The Healthy Foods North diet and physical activity intervention was developed in 2007–2008 while working with populations in six communities in Nunavut and the Northwest Territories, Canada.MethodsFour communities received the 1-year intervention (eg, conducting workshops, cooking classes and walking clubs) and two communities served as controls. Among the 263 adult evaluation participants, food frequency questionnaires were used to assess dietary intake at baseline and postintervention. Changes in mean nutrient intakes, nutrient density and dietary adequacy from baseline to postintervention were determined. The intervention impact on nutrient intakes was assessed through multivariate linear regression analysis.ResultsPost-intervention assessment showed a reductions in total fat, saturated, monounsaturated and polyunsaturated fatty acids, and increases in iron intake, only in the intervention group. More than a 4%-increase in the percentage of adherence to vitamins A and D recommendations was observed in the intervention group. After adjusting the regression models, respondents in the intervention communities significantly reduced their energy intake and increased their vitamins A and D intake.ConclusionsThe Healthy Foods North is an effective programme to improve dietary quality among populations of the Canadian Arctic. Long-term interventions are expected to be important factors in the prevention of diet-related chronic diseases in these communities.
IRS2Copy Number Gain,KRASandBRAFMutation Status as Predictive Biomarkers for Response to the IGF-1R/IR Inhibitor BMS-754807 in Colorectal Cancer Cell Lines
Authors: Huang Fei , Chang Han , Greer Ann , Hillerman Stephen , Reeves Karen A. , Hurlburt Warren , Cogswell John , Patel Dharmesh , Qi Zhenhao , Fairchild Craig , Ryseck Rolf-Peter , Wong Tai W. , Finckenstein Friedrich G. , Jackson Jeffrey , Carboni Joan M. 
Published: December 2014

AbstractInsulin-like growth factor receptor 1 (IGF-1R)–targeting therapies are currently at an important crossroad given the low clinical response rates seen in unselected patients. Predictive biomarkers for patient selection are critical for improving clinical benefit. Coupling in vitro sensitivity testing of BMS-754807, a dual IGF-1R/IR inhibitor, with genomic interrogations in 60 human colorectal cancer cell lines, we identified biomarkers correlated with response to BMS-754807. The results showed that cell lines with BRAFV600E or KRASG13D mutation were resistant, whereas cell lines with wild-type of both KRAS and BRAF were particularly sensitive to BMS-754807 if they have either higher RNA expression levels of IR-A or lower levels of IGFBP6. In addition, the cell lines with KRAS mutations, those with either insulin receptor substrate 2 (IRS2) copy number gain (CNG) or higher IGF-1R expression levels, were more sensitive to the drug. Furthermore, cell lines with IRS2 CNG had higher levels of ligand-stimulated activation of IGF-1R and AKT, suggesting that these cell lines with IGF-IR signaling pathways more actively coupled to AKT signaling are more responsive to IGF-1R/IR inhibition. IRS2 siRNA knockdown reduced IRS2 protein expression levels and decreased sensitivity to BMS-754807, providing evidence for the functional involvement of IRS2 in mediating the drug response. The prevalence of IRS2 CNG in colorectal cancer tumors as measured by qPCR-CNV is approximately 35%. In summary, we identified IRS2 CNG, IGF-1R, IR-A, and IGFBP6 RNA expression levels, and KRAS and BRAF mutational status as candidate predictive biomarkers for response to BMS-754807. This work proposed clinical development opportunities for BMS-754807 in colorectal cancer with patient selection to improve clinical benefit. Mol Cancer Ther; 14(2); 620–30. ©2014 AACR.
PD-L1 Expression Correlates with Tumor-Infiltrating Lymphocytes and Response to Neoadjuvant Chemotherapy in Breast Cancer
Authors: Wimberly Hallie , Brown Jason R. , Schalper Kurt , Haack Herbert , Silver Matthew R. , Nixon Christian , Bossuyt Veerle , Pusztai Lajos , Lannin Donald R. , Rimm David L. 
Published: December 2014

AbstractProgrammed death 1 ligand 1 (PD-L1) is an immune regulatory molecule that limits antitumor immune activity. Targeting of PD-L1 and other immune checkpoint proteins has shown therapeutic activity in various tumor types. The expression of PD-L1 and its correlation with response to neoadjuvant chemotherapy in breast cancer has not been studied extensively. Our goal was to assess PD-L1 expression in a cohort of breast cancer patients treated with neoadjuvant chemotherapy. Pretreatment biopsies from 105 patients with breast cancer from Yale New Haven Hospital that subsequently received neoadjuvant chemotherapy were assessed for PD-L1 protein expression by automated quantitative analysis with a rabbit monoclonal antibody (E1L3N) to the cytoplasmic domain of PD-L1. In addition, tumor-infiltrating lymphocytes (TIL) were assessed on hematoxylin and eosin slides. PD-L1 expression was observed in 30% of patients, and it was positively associated with hormone-receptor–negative and triple-negative status and high levels of TILs. Both TILs and PD-L1 measured in the epithelium or stroma predicted pathologic complete response (pCR) to neoadjuvant chemotherapy in univariate and multivariate analyses. However, because they are strongly associated, TILs and PD-L1 cannot both be included in a significant multivariate model. PD-L1 expression is prevalent in breast cancer, particularly hormone-receptor–negative and triple-negative patients, indicating a subset of patients that may benefit from immune therapy. Furthermore, PD-L1 and TILs correlate with pCR, and high PD-L1 predicts pCR in multivariate analysis. Cancer Immunol Res; 3(4); 326–32. ©2014 AACR.
Stereotactic Radiation Therapy Augments Antigen-Specific PD-1–Mediated Antitumor Immune Responses via Cross-Presentation of Tumor Antigen
Authors: Sharabi Andrew B. , Nirschl Christopher J. , Kochel Christina M. , Nirschl Thomas R. , Francica Brian J. , Velarde Esteban , Deweese Theodore L. , Drake Charles G. 
Published: December 2014

AbstractThe immune-modulating effects of radiotherapy (XRT) have gained considerable interest recently, and there have been multiple reports of synergy between XRT and immunotherapy. However, additional preclinical studies are needed to demonstrate the antigen-specific nature of radiation-induced immune responses and elucidate potential mechanisms of synergy with immunotherapy. Here, we demonstrate the ability of stereotactic XRT to induce endogenous antigen-specific immune responses when it is combined with anti–PD-1 checkpoint blockade immunotherapy. Using the small animal radiation research platform (SARRP), image-guided stereotactic XRT delivered to B16-OVA melanoma or 4T1-HA breast carcinoma tumors resulted in the development of antigen-specific T cell– and B cell–mediated immune responses. These immune-stimulating effects of XRT were significantly increased when XRT was combined with either anti–PD-1 therapy or regulatory T cell (Treg) depletion, resulting in improved local tumor control. Phenotypic analyses of antigen-specific CD8 T cells revealed that XRT increased the percentage of antigen-experienced T cells and effector memory T cells. Mechanistically, we found that XRT upregulates tumor-associated antigen–MHC complexes, enhances antigen cross-presentation in the draining lymph node, and increases T-cell infiltration into tumors. These findings demonstrate the ability of XRT to prime an endogenous antigen-specific immune response and provide an additional mechanistic rationale for combining radiation with PD-1 blockade in the clinic. Cancer Immunol Res; 3(4); 345–55. ©2014 AACR.
Demand management: an audit of chemical pathology test rejections by an electronic gate-keeping system at an academic hospital in Cape Town
Authors: Smit Ida , Zemlin Annalise E , Erasmus Rajiv T 
Published: December 2014

Background Demand management is an area of laboratory activity, which is becoming increasingly important. Within the health-care system, demand management can be defined as the use of health resources to maximise its utility. Tygerberg Hospital has introduced an electronic gate-keeping system. Chemistry tests which generate the highest cost are subjected to this system and may be automatically rejected according to a set of rules. This study aimed: (1) to identify the number of chemistry tests rejected by the eGK; (2) to identify which of these rejected tests were subsequently restored and (3) to assess the impact of rejections on clinical outcome and cost-saving. Methods A retrospective audit was conducted to determine the number of chemistry tests rejected and subsequently restored over a 6-month period. The case-notes of patients for whom requested tests previously rejected had been restored were randomly selected and investigated to assess clinical impact. Any cost-saving was calculated. Results A total of 68,480 tests were subjected to gate-keeping, and 4605 tests (6.7%) were rejected while 679 (14.7%) of these were restored by the requestor phoning the laboratory after obtaining authorisation. After examining a subset of clinical notes it was found that in most cases (80%), patient care was unaffected. The total cost saved was £25,387. Conclusions The majority of the rejected tests were unnecessary and following rejection, real savings were made. Electronic gate-keeping is a simple, effective and sustainable method of demand management.