bluuebunny/crossref_metadata_2025_split huggingface dataset:
Autotrophic Production of Stable-Isotope-Labeled Arginine in Ralstonia eutropha Strain H16
Authors: Lütte Steffen , Pohlmann Anne , Zaychikov Evgeny , Schwartz Edward , Becher Johannes R. , Heumann Hermann , Friedrich Bärbel 
Published: September 2012

ABSTRACT

With the aim of improving industrial-scale production of stable-isotope (SI)-labeled arginine, we have developed a system for the heterologous production of the arginine-containing polymer cyanophycin in recombinant strains of
Ralstonia eutropha
under lithoautotrophic growth conditions. We constructed an expression plasmid based on the cyanophycin synthetase gene (
cphA
) of
Synechocystis
sp. strain PCC6308 under the control of the strong P

cbbL

promoter of the
R. eutropha
H16

cbb
c

operon (coding for autotrophic CO
2
fixation). In batch cultures growing on H
2
and CO
2
as sole sources of energy and carbon, respectively, the cyanophycin content of cells reached 5.5% of cell dry weight (CDW). However, in the absence of selection (i.e., in antibiotic-free medium), plasmid loss led to a substantial reduction in yield. We therefore designed a novel addiction system suitable for use under lithoautotrophic conditions. Based on the hydrogenase transcription factor HoxA, this system mediated stabilized expression of
cphA
during lithoautotrophic cultivation without the need for antibiotics. The maximum yield of cyanophycin was 7.1% of CDW. To test the labeling efficiency of our expression system under actual production conditions, cells were grown in 10-liter-scale fermentations fed with
13
CO
2
and
15
NH
4
Cl, and the
13
C/
15
N-labeled cyanophycin was subsequently extracted by treatment with 0.1 M HCl; 2.5 to 5 g of [
13
C/
15
N]arginine was obtained per fed-batch fermentation, corresponding to isotope enrichments of 98.8% to 99.4%.
Residual Viral and Bacterial Contamination of Surfaces after Cleaning and Disinfection
Authors: Tuladhar Era , Hazeleger Wilma C. , Koopmans Marion , Zwietering Marcel H. , Beumer Rijkelt R. , Duizer Erwin 
Published: September 2012

ABSTRACT

Environmental surfaces contaminated with pathogens can be sources of indirect transmission, and cleaning and disinfection are common interventions focused on reducing contamination levels. We determined the efficacy of cleaning and disinfection procedures for reducing contamination by noroviruses, rotavirus, poliovirus, parechovirus, adenovirus, influenza virus,
Staphylococcus aureus
, and
Salmonella enterica
from artificially contaminated stainless steel surfaces. After a single wipe with water, liquid soap, or 250-ppm free chlorine solution, the numbers of infective viruses and bacteria were reduced by 1 log
10
for poliovirus and close to 4 log
10
for influenza virus. There was no significant difference in residual contamination levels after wiping with water, liquid soap, or 250-ppm chlorine solution. When a single wipe with liquid soap was followed by a second wipe using 250- or 1,000-ppm chlorine, an extra 1- to 3-log
10
reduction was achieved, and except for rotavirus and norovirus genogroup I, no significant additional effect of 1,000 ppm compared to 250 ppm was found. A reduced correlation between reduction in PCR units (PCRU) and reduction in infectious particles suggests that at least part of the reduction achieved in the second step is due to inactivation instead of removal alone. We used data on infectious doses and transfer efficiencies to estimate a target level to which the residual contamination should be reduced and found that a single wipe with liquid soap followed by a wipe with 250-ppm free chlorine solution was sufficient to reduce the residual contamination to below the target level for most of the pathogens tested.
“Remake” by High-Throughput Sequencing of the Microbiota Involved in the Production of Water Buffalo Mozzarella Cheese
Authors: Ercolini Danilo , De Filippis Francesca , La Storia Antonietta , Iacono Michele 
Published: September 2012

ABSTRACT
Intermediates of production of two batches of traditional mozzarella cheese were analyzed by culture-independent pyrosequencing. The quantitative distribution of taxa within the samples suggested that thermophilic lactic acid bacteria from the natural starter were mainly responsible for the fermentation, while microorganisms found in raw milk did not develop during fermentation.
Epigallocatechin Gallate Induces Upregulation of the Two-Component VraSR System by Evoking a Cell Wall Stress Response in Staphylococcus aureus
Authors: Levinger Oren , Bikels-Goshen Tamar , Landau Elad , Fichman Merav , Shapira Roni 
Published: September 2012

ABSTRACT

We previously found that a short exposure of
Staphylococcus aureus
to subinhibitory (SI) doses of epigallocatechin gallate (EGCG) results in increased cell wall thickness, adaptation, and enhanced tolerance to cell-wall-targeted antibiotics. In this study, the response to EGCG of
sigB
and
vraSR
transcription factor mutants was characterized. We show that in contrast to the results observed for wild-type (WT) strains, an
S. aureus
315
vraSR
null mutant exposed to SI doses of EGCG did not exhibit increased tolerance to EGCG and oxacillin. A diminished increase in tolerance to ampicillin (from 16-fold to 4-fold) and no change in the magnitude of resistance to vancomycin were observed. Preexposure to EGCG enhanced the tolerance of wild-type and
sigB
null mutant cells to lysostaphin, but this enhancement was much weaker in the
vraSR
null mutant. Marked upregulation (about 60-fold) of
vraR
and upregulation of the peptidoglycan biosynthesis-associated genes
murA
,
murF
, and
pbp2
(2-, 5-, and 6-fold, respectively) in response to SI doses of EGCG were determined by quantitative reverse transcription-PCR (qRT-PCR). EGCG also induced the promoter of
sas016
(encoding a cell wall stress protein of unknown function which is not induced in
vraSR
null mutants) in a concentration-dependent manner, showing kinetics comparable to those of cell-wall-targeting antibiotics. Taken together, our results suggest that the two-component VraSR system is involved in modulating the cell response to SI doses of EGCG.
Phe317 Is Essential for Rubber Oxygenase RoxA Activity
Authors: Birke Jakob , Hambsch Nadja , Schmitt Georg , Altenbuchner Josef , Jendrossek Dieter 
Published: September 2012

ABSTRACT

RoxA is an extracellular
c
-type diheme cytochrome secreted by
Xanthomonas sp.
strain 35Y during growth on rubber. RoxA cleaves poly(
cis
-1,4-isoprene) to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). Analysis of the RoxA structure revealed that Phe317 is located in close proximity (≈5 Å) to the N-terminal heme that presumably represents the active site. To find evidence of whether Phe317 is important for catalysis, we changed it to tyrosine, tryptophan, leucine, histidine, or alanine. All five RoxA muteins were expressed after integration of the respective gene into the chromosome of a
Xanthomonas sp.
ΔroxA
strain. Residual clearing zone formation on opaque latex agar was found for
Xanthomonas sp.
strains expressing the Phe317Leu, Phe317Ala, or Phe317His variant (wild type > Leu > Ala > His). Strains in which Phe317 was changed to tyrosine or tryptophan were inactive. Phe317Ala and Phe312Leu RoxA muteins were purified, and polyisoprene cleavage activities were reduced to ≈3% and 10%, respectively. UV-visible spectroscopy of RoxA muteins confirmed that both heme groups were present in an oxidized form, but spectral responses to the addition of low-molecular-weight (inhibitory) ligand molecules such as imidazole and pyridine were different from those of wild-type RoxA. Our results show that residue 317 is involved in interaction with substrates. This is the first report on structure-function analysis of a polyisoprene-cleaving enzyme and on the identification of an amino acid that is essential for polyisoprene cleavage activity.
Colony Organization in the Green Alga Botryococcus braunii (Race B) Is Specified by a Complex Extracellular Matrix
Authors: Weiss Taylor L. , Roth Robyn , Goodson Carrie , Vitha Stanislav , Black Ian , Azadi Parastoo , Rusch Jannette , Holzenburg Andreas , Devarenne Timothy P. , Goodenough Ursula 
Published: September 2012

ABSTRACT

Botryococcus braunii
is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of
B. braunii
cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous β-1, 4- and/or β-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form “drapes” between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.
Structure and Interactions of the Cytoplasmic Domain of the Yersinia Type III Secretion Protein YscD
Authors: Gamez Alicia , Mukerjea Romila , Alayyoubi Maher , Ghassemian Majid , Ghosh Partho 
Published: September 2012

ABSTRACTThe virulence of a large number of Gram-negative bacterial pathogens depends on the type III secretion (T3S) system, which transports select bacterial proteins into host cells. An essential component of theYersiniaT3S system is YscD, a single-pass inner membrane protein. We report here the 2.52-Å resolution structure of the cytoplasmic domain of YscD, called YscDc. The structure confirms that YscDc consists of a forkhead-associated (FHA) fold, which in many but not all cases specifies binding to phosphothreonine. YscDc, however, lacks the structural properties associated with phosphothreonine binding and thus most likely interacts with partners in a phosphorylation-independent manner. Structural comparison highlighted two loop regions, L3 and L4, as potential sites of interactions. Alanine substitutions at L3 and L4 had no deleterious effects on protein structure or stability but abrogated T3S in a dominant negative manner. To gain insight into the function of L3 and L4, we identified proteins associated with YscD by affinity purification coupled to mass spectrometry. The lipoprotein YscJ was found associated with wild-type YscD, as was the effector YopH. Notably, the L3 and L4 substitution mutants interacted with more YopH than did wild-type YscD. These substitution mutants also interacted with SycH (the specific chaperone for YopH), the putative C-ring component YscQ, and the ruler component YscP, whereas wild-type YscD did not. These results suggest that substitutions in the L3 and L4 loops of YscD disrupted the dissociation of SycH from YopH, leading to the accumulation of a large protein complex that stalled the T3S apparatus.
Rapid Depletion of Target Proteins Allows Identification of Coincident Physiological Responses
Authors: Carr Ana C. , Taylor Katherine L. , Osborne Melinda S. , Belous Bradley T. , Myerson Joseph P. , Moore Sean D. 
Published: September 2012

ABSTRACTTargeted protein degradation is a powerful tool that can be used to create unique physiologies depleted of important factors. Current strategies involve modifying a gene of interest such that a degradation peptide is added to an expressed target protein and then conditionally activating proteolysis, either by expressing adapters, unmasking cryptic recognition determinants, or regulating protease affinities using small molecules. For each target, substantial optimization may be required to achieve a practical depletion, in that the target remains present at a normal level prior to induction and is then rapidly depleted to levels low enough to manifest a physiological response. Here, we describe a simplified targeted degradation system that rapidly depletes targets and that can be applied to a wide variety of proteins without optimizing target protease affinities. The depletion of the target is rapid enough that a primary physiological response manifests that is related to the function of the target. Using ribosomal protein S1 as an example, we show that the rapid depletion of this essential translation factor invokes concomitant changes to the levels of several mRNAs, even before appreciable cell division has occurred.
Quantitative cranial ultrasound prediction of severity of disability in premature infants with post-haemorrhagic ventricular dilatation
Authors: Jary Sally , Kmita Grazyna , Wroblewska Jolanta , Whitelaw Andrew 
Published: September 2012

BackgroundInfants with post-haemorrhagic ventricular dilatation (PHVD) have a high risk of severe disability and parenchymal infarction increases this risk. Existing cranial ultrasound (CUS) markers of neurodevelopmental outcome are based on categorical features.ObjectiveTo investigate to what extent quantitative CUS measurements correlated with severity of developmental outcome and the need for ventriculoperitoneal (VP) shunt at 2 years of age.Design69 premature infants with PHVD had lateral ventricle area, intraventricular echodensity and parenchymal lesion dimensions measured at the start of treatment for PHVD. Outcome measures were the Bayley Scales of Infant Development-II and functional ability at 2 years of age. Bayley developmental quotients (DQ) were used in preference to index scores to enable inclusion of severely disabled children.ResultsQuantitative CUS measurements of parenchymal lesion area correlated significantly with later mental and motor DQ. Intraventricular echodensity area correlated with motor DQ in infants with grade 4 intraventricular haemorrhage (IVH). Neither ventricular area nor ventricular width correlated with DQ in grade 3 IVH. Infants who ultimately required a VP shunt had a significantly larger intraventricular echodensity area.ConclusionsCUS measurement of parenchymal lesions in infants with PHVD can increase the precision of predicting severe mental and motor disability, but ventricular size at the start of treatment is not predictive of outcome in infants with PHVD following grade 3 IVH.
An evaluation of a public health advocacy strategy to enhance palliative care provision in Zambia
Authors: Logie Dorothy Elizabeth 
Published: September 2012

BackgroundThe provision of palliative and end-of-life care for patients with progressive disease is inadequate worldwide. The WHO strategy to develop a public health system of palliative care at the national level proposes a quadripartite approach: policy, education, drug availability and implementation. This is particularly necessary in low income countries facing a high disease burden with low resources. To date there have been no published evaluations of advocacy strategies aiming to use the WHO approach to enhance access to palliative care.ObjectiveTo evaluate the Palliative Care Initiative which implements the WHO approach to strategically improve palliative care throughout Zambia, using a multiple methods rapid field evaluation.Methods and resultsEight hospices and palliative care organisations in Zambia were part-funded over 2 years (2009–11) and were thus able to expand their services, offer a reliable supply of drugs, secure fuel for transport, and support home carers. Simultaneously, an extensive programme of rural and urban training resulted in staff being more confident in caring for the dying with improved attitudes to the concerns of very ill patients and their families. A multi-agency morphine pilot project led by the Palliative Care Association of Zambia and the Ministry of Health resolved some of the obstacles and increased morphine availability although its use remains low. Palliative care is now being considered by the technical working group at the Ministry of Health.ConclusionsFor palliative care to thrive in any resource-poor country it must be integrated into the public health system and secure long-term funding.
Lymphatic Reprogramming by Kaposi Sarcoma Herpes Virus Promotes the Oncogenic Activity of the Virus-Encoded G-protein–Coupled Receptor
Authors: Aguilar Berenice , Choi Inho , Choi Dongwon , Chung Hee Kyoung , Lee Sunju , Yoo Jaehyuk , Lee Yong Suk , Maeng Yong Sun , Lee Ha Neul , Park Eunkyung , Kim Kyu Eui , Kim Nam Yoon , Baik Jae Myung , Jung Jae U. , Koh Chester J. , Hong Young-Kwon 
Published: September 2012

Abstract
Kaposi sarcoma, the most common cancer in HIV-positive individuals, is caused by endothelial transformation mediated by the Kaposi sarcoma herpes virus (KSHV)-encoded G-protein–coupled receptor (vGPCR). Infection of blood vascular endothelial cells (BEC) by KSHV reactivates an otherwise silenced embryonic program of lymphatic differentiation. Thus, Kaposi sarcoma tumors express numerous lymphatic endothelial cell (LEC) signature genes. A key unanswered question is how lymphatic reprogramming by the virus promotes tumorigenesis leading to Kaposi sarcoma formation. In this study, we present evidence that this process creates an environment needed to license the oncogenic activity of vGPCR. We found that the G-protein regulator RGS4 is an inhibitor of vGPCR that is expressed in BECs, but not in LECs. RGS4 was downregulated by the master regulator of LEC differentiation PROX1, which is upregulated by KSHV and directs KSHV-induced lymphatic reprogramming. Moreover, we found that KSHV upregulates the nuclear receptor LRH1, which physically interacts with PROX1 and synergizes with it to mediate repression of RGS4 expression. Mechanistic investigations revealed that RGS4 reduced vGPCR-enhanced cell proliferation, migration, VEGF expression, and Akt activation and suppressed tumor formation induced by vGPCR. Our findings resolve long-standing questions about the pathologic impact of KSHV-induced reprogramming of host cell identity, and they offer biologic and mechanistic insights supporting the hypothesis that a lymphatic microenvironment is more favorable for Kaposi sarcoma tumorigenesis. Cancer Res; 72(22); 5833–42. ©2012 AACR.
Prevalence and Types of Pitfall in the Assessment of Mitral Isthmus Linear Conduction Block
Authors: Shah Ashok J. , Pascale Patrizio , Miyazaki Shinsuke , Liu Xingpeng , Roten Laurent , Derval Nicolas , Jadidi Amir S. , Scherr Daniel , Wilton Stephen B. , Pedersen Michala , Knecht Sebastien , Sacher Frédéric , Jaïs Pierre , Haïssaguerre Michel , Hocini Mélèze 
Published: September 2012

Background—
To identify and understand clinically encountered pitfalls in the assessment of transmitral conduction block using differential coronary sinus and left atrial appendage pacing techniques in patients with left mitral isthmus linear ablation.

Methods and Results—
All the available assessments of mitral isthmus block were thoroughly reviewed in 271 mitral isthmus ablation procedures undertaken among 236 patients from October 2008 to April 2011. Bidirectional block was established in 186 of 271 (69%) procedures. Careful evaluation of electrograms recorded on the multipolar coronary sinus and ablation catheters was undertaken to identify and understand the characteristics of pitfall, if any. Pitfall was encountered in 55 of 271 (20%) procedures among 51 patients and categorized into 6 types (types 1, 3, 4, and 5 led to spurious diagnosis of block; types 2 and 6 led to erroneous diagnosis of absence of block). There were 14, 10, 17, 2, 15, and 3 (total=61) cases of pitfall types 1 through 6, respectively. Operator recognized 42 of 61 (69%) pitfalls intraprocedurally. Recognition of types 1 and 5 was difficult because of indiscernible electrograms at usual amplifier settings or presence of slow conduction mimicking block.

Conclusions—
Every fifth assessment of bidirectional block across mitral isthmus linear lesion using differential coronary sinus and left atrial appendage pacing techniques encounters a pitfall, which can lead to erroneous clinical diagnosis of block or absence of block. Recognition of pitfall during the procedure is feasible and necessitates careful distinction of far-field left atrium from the local coronary sinus electrograms besides appropriate adjustments in catheter position and pacing outputs.
Matrix Metalloproteinase-9 Genotype as a Potential Genetic Marker for Abdominal Aortic Aneurysm
Authors: Duellman Tyler , Warren Christopher L. , Peissig Peggy , Wynn Martha , Yang Jay 
Published: September 2012

Background—
Degradation of extracellular matrix support in the large abdominal arteries contribute to abnormal dilation of aorta, leading to abdominal aortic aneurysms, and matrix metalloproteinase-9 (MMP-9) is the predominant enzyme targeting elastin and collagen present in the walls of the abdominal aorta. Previous studies have suggested a potential association between MMP-9 genotype and abdominal aortic aneurysm, but these studies have been limited only to the p-1562 and (CA) dinucleotide repeat microsatellite polymorphisms in the promoter region of the MMP-9 gene. We determined the functional alterations caused by 15 MMP-9 single-nucleotide polymorphisms (SNPs) reported to be relatively abundant in the human genome through Western blots, gelatinase, and promoter–reporter assays and incorporated this information to perform a logistic-regression analysis of MMP-9 SNPs in 336 human abdominal aortic aneurysm cases and controls.

Methods and Results—
Significant functional alterations were observed for 6 exon SNPs and 4 promoter SNPs. Genotype analysis of frequency-matched (age, sex, history of hypertension, hypercholesterolemia, and smoking) cases and controls revealed significant genetic heterogeneity exceeding 20% observed for 6 SNPs in our population of mostly white subjects from Northern Wisconsin. A step-wise logistic-regression analysis with 6 functional SNPs, where weakly contributing confounds were eliminated using Akaike information criteria, gave a final 2 SNP (D165N and p-2502) model with an overall odds ratio of 2.45 (95% confidence interval, 1.06–5.70).

Conclusions—
The combined approach of direct experimental confirmation of the functional alterations of MMP-9 SNPs and logistic-regression analysis revealed significant association between MMP-9 genotype and abdominal aortic aneurysm.