bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Good Clinical Practice guideline and its interpretation – perceptions of clinical trial teams in sub‐Saharan Africa
Authors: Vischer N. , Pfeiffer C. , Joller A. , Klingmann I. , Ka A. , Kpormegbe S. K. , Burri C. 
Published: June 2016

AbstractObjectivesTo explore the advantages and challenges of working with the Good Clinical Practice (GCP)‐International Conference of Harmonization (ICH) E6 guideline and its interpretation from the perspective of clinical trial teams based in sub‐Saharan Africa.MethodsWe conducted 60 key informant interviews with clinical trial staff at different levels in clinical research centres in Kenya, Ghana, Burkina Faso and Senegal and thematically analysed the responses.ResultsClinical trial teams perceived working with ICH‐GCP as highly advantageous and regarded ICH‐GCP as applicable to their setting and efficiently applied. Only for informed consent did some clinical trial staff (one‐third) perceive the guideline as insufficiently applicable. Specific challenges included meeting the requirements for written and individual consent, conditions for impartial witnesses for illiterates or legally acceptable representatives for children, guaranteeing voluntary participation and ensuring full understanding of the consent given. It was deemed important to have ICH‐GCP compliance monitored by relevant ethics committees and regulatory authorities, without having guidelines applied overcautiously.ConclusionClinical trial teams in sub‐Saharan Africa perceived GCP as a helpful guideline, despite having been developed by northern organisations and despite the high administrative burden of implementing it. To mitigate consent challenges, we suggest adapting GCP and making use of the flexibility it offers.
Quantifying Variability in Growth and Thermal Inactivation Kinetics of Lactobacillus plantarum
Authors: Aryani D. C. , den Besten H. M. W. , Zwietering M. H. 
Published: June 2016

ABSTRACT

The presence and growth of spoilage organisms in food might affect the shelf life. In this study, the effects of experimental, reproduction, and strain variabilities were quantified with respect to growth and thermal inactivation using 20
Lactobacillus plantarum
strains. Also, the effect of growth history on thermal resistance was quantified. The strain variability in μ
max
was similar (
P
> 0.05) to reproduction variability as a function of pH, a
w
, and temperature, while being around half of the reproduction variability (
P
< 0.05) as a function of undissociated lactic acid concentration [HLa]. The cardinal growth parameters were estimated for the
L. plantarum
strains, and the pH
min
was between 3.2 and 3.5, the
a
w,min
was between 0.936 and 0.953, the [HLa
max
], at pH 4.5, was between 29 and 38 mM, and the
T
min
was between 3.4 and 8.3°C. The average
D
values ranged from 0.80 min to 19 min at 55°C, 0.22 to 3.9 min at 58°C, 3.1 to 45 s at 60°C, and 1.8 to 19 s at 63°C. In contrast to growth, the strain variability in thermal resistance was on average six times higher than the reproduction variability and more than ten times higher than the experimental variability. The strain variability was also 1.8 times higher (
P
< 0.05) than the effect of growth history. The combined effects of strain variability and growth history on
D
value explained all of the variability as found in the literature, although with bias. Based on an illustrative milk-processing chain, strain variability caused ∼2-log
10
differences in growth between the most and least robust strains and >10-log
10
differences after thermal treatment.

IMPORTANCE
Accurate control and realistic prediction of shelf life is complicated by the natural diversity among microbial strains, and limited information on microbiological variability is available for spoilage microorganisms. Therefore, the objectives of the present study were to quantify strain variability, reproduction (biological) variability, and experimental variability with respect to the growth and thermal inactivation kinetics of
Lactobacillus plantarum
and to quantify the variability in thermal resistance attributed to growth history. The quantitative knowledge obtained on experimental, reproduction, and strain variabilities can be used to improve experimental designs and to adequately select strains for challenge growth and inactivation tests. Moreover, the integration of strain variability in prediction of microbial growth and inactivation kinetics will result in more realistic predictions of
L. plantarum
dynamics along the food production chain.
Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus
Authors: Daou Marianne , Piumi François , Cullen Daniel , Record Eric , Faulds Craig B. 
Published: June 2016

ABSTRACT

The genome of the white rot fungus
Pycnoporus cinnabarinus
includes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated from
Phanerochaete chrysosporium
. The glyoxal oxidase of
P. chrysosporium
is physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H
2
O
2
and utilizes an array of aldehydes and α-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases of
P. cinnabarinus
, GLOX1 (
Pci
GLOX1) and GLOX2 (
Pci
GLOX2), were heterologously produced in
Aspergillus niger
strain D15#26 (
pyrG
negative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein for
Pci
GLOX1 and
Pci
GLOX2, respectively. Both proteins were approximately 60 kDa in size and N-glycosylated. The optimum temperature for the activity of these enzymes was 50°C, and the optimum pH was 6. The enzymes retained most of their activity after incubation at 50°C for 4 h. The highest relative activity and the highest catalytic efficiency of both enzymes occurred with glyoxylic acid as the substrate. The two
P. cinnabarinus
enzymes generally exhibited similar substrate preferences, but
Pci
GLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyde.

IMPORTANCE
This study addresses the poorly understood role of how fungal peroxidases obtain an
in situ
supply of hydrogen peroxide to enable them to oxidize a variety of organic and inorganic compounds. This cooperative activity is intrinsic in the living organism to control the amount of toxic H
2
O
2
in its environment, thus providing a feed-on-demand scenario, and can be used biotechnologically to supply a cheap source of peroxide for the peroxidase reaction. The secretion of multiple glyoxal oxidases by filamentous fungi as part of a lignocellulolytic mechanism suggests a controlled system, especially as these enzymes utilize fungal metabolites as the substrates. Two glyoxal oxidases have been isolated and characterized to date, and the differentiation of the substrate specificity of the two enzymes produced by
Pycnoporus cinnabarinus
illustrates the alternative mechanisms existing in a single fungus, together with the utilization of these enzymes to prepare platform chemicals for industry.
Biochemical Characteristics and Substrate Degradation Pattern of a Novel Exo-Type β-Agarase from the Polysaccharide-Degrading Marine Bacterium Flammeovirga sp. Strain MY04
Authors: Han Wenjun , Cheng Yuanyuan , Wang Dandan , Wang Shumin , Liu Huihui , Gu Jingyan , Wu Zhihong , Li Fuchuan 
Published: June 2016

ABSTRACT

Exo-type agarases release disaccharide units (3,6-anhydro-
l
-galactopyranose-α-1,3-
d
-galactose) from the agarose chain and, in combination with endo-type agarases, play important roles in the processive degradation of agarose. Several exo-agarases have been identified. However, their substrate-degrading patterns and corresponding mechanisms are still unclear because of a lack of proper technologies for sugar chain analysis. Herein, we report the novel properties of AgaO, a disaccharide-producing agarase identified from the genus
Flammeovirga
. AgaO is a 705-amino-acid protein that is unique to strain MY04. It shares sequence identities of less than 40% with reported GH50 β-agarases. Recombinant AgaO (rAgaO) yields disaccharides as the sole final product when degrading agarose and associated oligosaccharides. Its smallest substrate is a neoagarotetraose, and its disaccharide/agarose conversion ratio is 0.5. Using fluorescence labeling and two-stage mass spectrometry analysis, we demonstrate that the disaccharide products are neoagarobiose products instead of agarobiose products, as verified by
13
C nuclear magnetic resonance spectrum analysis. Therefore, we provide a useful oligosaccharide sequencing method to determine the patterns of enzyme cleavage of glycosidic bonds. Moreover, AgaO produces neoagarobiose products by gradually cleaving the units from the nonreducing end of fluorescently labeled sugar chains, and so our method represents a novel biochemical visualization of the exolytic pattern of an agarase. Various truncated AgaO proteins lost their disaccharide-producing capabilities, indicating a strict structure-function relationship for the whole enzyme. This study provides insights into the novel catalytic mechanism and enzymatic properties of an exo-type β-agarase for the benefit of potential future applications.

IMPORTANCE
Exo-type agarases can degrade agarose to yield disaccharides almost exclusively, and therefore, they are important tools for disaccharide preparation. However, their enzymatic mechanisms and agarose degradation patterns are still unclear due to the lack of proper technologies for sugar chain analysis. In this study, AgaO was identified as an exo-type agarase of agarose-degrading
Flammeovirga
bacteria, representing a novel branch of glycoside hydrolase family 50. Using fluorescence labeling, high-performance liquid chromatography, and mass spectrum analysis technologies, we provide a useful oligosaccharide sequencing method to determine the patterns of enzyme cleavage of glycosidic bonds. We also demonstrate that AgaO produces neoagarobiose by gradually cleaving disaccharides from the nonreducing end of fluorescently labeled sugars. This study will benefit future enzyme applications and oligosaccharide studies.
Phylogenetically Structured Differences in rRNA Gene Sequence Variation among Species of Arbuscular Mycorrhizal Fungi and Their Implications for Sequence Clustering
Authors: House Geoffrey L. , Ekanayake Saliya , Ruan Yang , Schütte Ursel M. E. , Kaonongbua Wittaya , Fox Geoffrey , Ye Yuzhen , Bever James D. 
Published: June 2016

ABSTRACT

Arbuscular mycorrhizal (AM) fungi form mutualisms with plant roots that increase plant growth and shape plant communities. Each AM fungal cell contains a large amount of genetic diversity, but it is unclear if this diversity varies across evolutionary lineages. We found that sequence variation in the nuclear large-subunit (LSU) rRNA gene from 29 isolates representing 21 AM fungal species generally assorted into genus- and species-level clades, with the exception of species of the genera
Claroideoglomus
and
Entrophospora
. However, there were significant differences in the levels of sequence variation across the phylogeny and between genera, indicating that it is an evolutionarily constrained trait in AM fungi. These consistent patterns of sequence variation across both phylogenetic and taxonomic groups pose challenges to interpreting operational taxonomic units (OTUs) as approximations of species-level groups of AM fungi. We demonstrate that the OTUs produced by five sequence clustering methods using 97% or equivalent sequence similarity thresholds failed to match the expected species of AM fungi, although OTUs from AbundantOTU, CD-HIT-OTU, and CROP corresponded better to species than did OTUs from mothur or UPARSE. This lack of OTU-to-species correspondence resulted both from sequences of one species being split into multiple OTUs and from sequences of multiple species being lumped into the same OTU. The OTU richness therefore will not reliably correspond to the AM fungal species richness in environmental samples. Conservatively, this error can overestimate species richness by 4-fold or underestimate richness by one-half, and the direction of this error will depend on the genera represented in the sample.

IMPORTANCE
Arbuscular mycorrhizal (AM) fungi form important mutualisms with the roots of most plant species. Individual AM fungi are genetically diverse, but it is unclear whether the level of this diversity differs among evolutionary lineages. We found that the amount of sequence variation in an rRNA gene that is commonly used to identify AM fungal species varied significantly between evolutionary groups that correspond to different genera, with the exception of two genera that are genetically indistinguishable from each other. When we clustered groups of similar sequences into operational taxonomic units (OTUs) using five different clustering methods, these patterns of sequence variation caused the number of OTUs to either over- or underestimate the actual number of AM fungal species, depending on the genus. Our results indicate that OTU-based inferences about AM fungal species composition from environmental sequences can be improved if they take these taxonomically structured patterns of sequence variation into account.
Microbial Transformations of Selenium Species of Relevance to Bioremediation
Authors: Eswayah Abdurrahman S. , Smith Thomas J. , Gardiner Philip H. E. 
Published: June 2016

ABSTRACTSelenium species, particularly the oxyanions selenite (SeO32−) and selenate (SeO42−), are significant pollutants in the environment that leach from rocks and are released by anthropogenic activities. Selenium is also an essential micronutrient for organisms across the tree of life, including microorganisms and human beings, particularly because of its presence in the 21st genetically encoded amino acid, selenocysteine. Environmental microorganisms are known to be capable of a range of transformations of selenium species, including reduction, methylation, oxidation, and demethylation. Assimilatory reduction of selenium species is necessary for the synthesis of selenoproteins. Dissimilatory reduction of selenate is known to support the anaerobic respiration of a number of microorganisms, and the dissimilatory reduction of soluble selenate and selenite to nanoparticulate elemental selenium greatly reduces the toxicity and bioavailability of selenium and has a major role in bioremediation and potentially in the production of selenium nanospheres for technological applications. Also, microbial methylation after reduction of Se oxyanions is another potentially effective detoxification process if limitations with low reaction rates and capture of the volatile methylated selenium species can be overcome. This review discusses microbial transformations of different forms of Se in an environmental context, with special emphasis on bioremediation of Se pollution.
Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis
Authors: Westbrook Adam W. , Moo-Young Murray , Chou C. Perry 
Published: June 2016

ABSTRACT

The establishment of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system for strain construction in
Bacillus subtilis
is essential for its progression toward industrial utility. Here we outline the development of a CRISPR-Cas9 tool kit for comprehensive genetic engineering in
B. subtilis
. In addition to site-specific mutation and gene insertion, our approach enables continuous genome editing and multiplexing and is extended to CRISPR interference (CRISPRi) for transcriptional modulation. Our tool kit employs chromosomal expression of Cas9 and chromosomal transcription of guide RNAs (gRNAs) using a gRNA transcription cassette and counterselectable gRNA delivery vectors. Our design obviates the need for multicopy plasmids, which can be unstable and impede cell viability. Efficiencies of up to 100% and 85% were obtained for single and double gene mutations, respectively. Also, a 2.9-kb hyaluronic acid (HA) biosynthetic operon was chromosomally inserted with an efficiency of 69%. Furthermore, repression of a heterologous reporter gene was achieved, demonstrating the versatility of the tool kit. The performance of our tool kit is comparable with those of systems developed for
Escherichia coli
and
Saccharomyces cerevisiae
, which rely on replicating vectors to implement CRISPR-Cas9 machinery.

IMPORTANCE
In this paper, as the first approach, we report implementation of the CRISPR-Cas9 system in
Bacillus subtilis
, which is recognized as a valuable host system for biomanufacturing. The study enables comprehensive engineering of
B. subtilis
strains with virtually any desired genotypes/phenotypes and biochemical properties for extensive industrial application.
Effects of Carbohydrate Source on Genetic Competence in Streptococcus mutans
Authors: Moye Zachary D. , Son Minjun , Rosa-Alberty Ariana E. , Zeng Lin , Ahn Sang-Joon , Hagen Stephen J. , Burne Robert A. 
Published: June 2016

ABSTRACT

The capacity to internalize and catabolize carbohydrates is essential for dental caries pathogens to persist and cause disease. The expression of many virulence-related attributes by
Streptococcus mutans
, an organism strongly associated with human dental caries, is influenced by the peptide signaling pathways that control genetic competence. Here, we demonstrate a relationship between the efficiency of competence signaling and carbohydrate source. A significant increase in the activity of the promoters for
comX
,
comS
, and
comYA
after exposure to competence-stimulating peptide (CSP) was observed in cells growing on fructose, maltose, sucrose, or trehalose as the primary carbohydrate source, compared to cells growing on glucose. However, only cells grown in the presence of trehalose or sucrose displayed a significant increase in transformation frequency. Notably, even low concentrations of these carbohydrates in the presence of excess glucose could enhance the expression of
comX
, encoding a sigma factor needed for competence, and the effects on competence were dependent on the cognate sugar:phosphotransferase permease for each carbohydrate. Using green fluorescent protein (GFP) reporter fusions, we observed that growth in fructose or trehalose resulted in a greater proportion of the population activating expression of
comX
and
comS
, encoding the precursor of
comX
-inducing peptide (XIP), after addition of CSP, than growth in glucose. Thus, the source of carbohydrate significantly impacts the stochastic behaviors that regulate subpopulation responses to CSP, which can induce competence in
S. mutans
.

IMPORTANCE
The signaling pathways that regulate development of genetic competence in
Streptococcus mutans
are intimately intertwined with the pathogenic potential of the organism, impacting biofilm formation, stress tolerance, and expression of known virulence determinants. Induction of the gene for the master regulator of competence, ComX, by competence-stimulating peptide (CSP) occurs in a subpopulation of cells. Here, we show that certain carbohydrates that are common in the human diet enhance the ability of CSP to activate transcription of
comX
and that a subset of these carbohydrates stimulates progression to the competent state. The cognate sugar:phosphotransferase permeases for each sugar are needed for these effects. Interestingly, single-cell analysis shows that the carbohydrates that increase
com
gene expression do so by enhancing the proportion of cells that respond to CSP. A mathematical model is developed to explain how carbohydrates modulate bistable behavior in the system via the ComRS pathway and ComX stability.
Periplasmic Cytophaga hutchinsonii Endoglucanases Are Required for Use of Crystalline Cellulose as the Sole Source of Carbon and Energy
Authors: Zhu Yongtao , Han Lanlan , Hefferon Kathleen L. , Silvaggi Nicholas R. , Wilson David B. , McBride Mark J. 
Published: June 2016

ABSTRACTThe soil bacteriumCytophaga hutchinsoniiactively digests crystalline cellulose by a poorly understood mechanism. Genome analyses identified nine genes predicted to encode endoglucanases with roles in this process. No predicted cellobiohydrolases, which are usually involved in the utilization of crystalline cellulose, were identified. Chromosomal deletions were performed in eight of the endoglucanase-encoding genes:cel5A,cel5B,cel5C,cel9A,cel9B,cel9C,cel9E, andcel9F. Each mutant retained the ability to digest crystalline cellulose, although the deletion ofcel9Ccaused a modest decrease in cellulose utilization. Strains with multiple deletions were constructed to identify the critical cellulases. Cells of a mutant lacking bothcel5Bandcel9Cwere completely deficient in growth on cellulose. Cell fractionation and biochemical analyses indicate that Cel5B and Cel9C are periplasmic nonprocessive endoglucanases. The requirement of periplasmic endoglucanases for cellulose utilization suggests that cellodextrins are transported across the outer membrane during this process. Bioinformatic analyses predict that Cel5A, Cel9A, Cel9B, Cel9D, and Cel9E are secreted across the outer membrane by the type IX secretion system, which has been linked to cellulose utilization. These secreted endoglucanases may perform the initial digestion within amorphous regions on the cellulose fibers, releasing oligomers that are transported into the periplasm for further digestion by Cel5B and Cel9C. The results suggest that both cell surface and periplasmic endoglucanases are required for the growth ofC. hutchinsoniion cellulose and that novel cell surface proteins may solubilize and transport cellodextrins across the outer membrane.IMPORTANCEThe bacteriumCytophaga hutchinsoniidigests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by genetic analyses. Intracellular (periplasmic) nonprocessive endoglucanases performed an important role in cellulose utilization. The results suggest a model involving partial digestion at the cell surface, solubilization and uptake of cellodextrins across the outer membrane by an unknown mechanism, and further digestion within the periplasm. The ability to sequester cellodextrins and digest them intracellularly may limit losses of soluble cellobiose to other organisms.C. hutchinsoniiuses an unusual approach to digest cellulose and is a potential source of novel proteins to increase the efficiency of conversion of cellulose into soluble sugars and biofuels.