bluuebunny/crossref_metadata_2025 huggingface dataset:
An efficient time algorithm for makespan objectives
Authors: Ozturkoglu Yucel
Published: July 2015

This paper focuses on a single machine scheduling subject to machine deterioration with rate-modifying activities (RMA). The motivation for this study stems from the automatic-production line problem with one machine. The main question is to find the sequence in which jobs should be scheduled, how many maintenance activity (RMA) to use, if any, and where to insert them in the schedule during the time interval with optimal makespan objective. This problem is known to be NP-hard and we give concise analyses of the problem and provide polynomial time algorithms to solve the makespan problem. We also propose an algorithm which can be applied to some scheduling problems with the actual processing time of job nonlinearly based on its position.This paper focuses on a single machine scheduling subject to machine deterioration with rate-modifying activities (RMA). The motivation for this study stems from the automatic-production line problem with one machine. The main question is to find the sequence in which jobs should be scheduled, how many maintenance activity (RMA) to use, if any, and where to insert them in the schedule during the time interval with optimal makespan objective. This problem is known to be NP-hard and we give concise analyses of the problem and provide polynomial time algorithms to solve the makespan problem. We also propose an algorithm which can be applied to some scheduling problems with the actual processing time of job nonlinearly based on its position.
Design for Additive Manufacture of Fine Medical Instrumentation—DragonFlex Case Study
Authors: Jelínek Filip , Breedveld Paul
Published: July 2015

The recently popularized domain of additive manufacturing (AM) has much to offer to medical device development, especially to the growing field of minimally invasive surgery (MIS). With the advancements in AM materials, one could soon envision materializing not only the proofs of concept but also the final clinically approved instruments. DragonFlex—the world's first AM steerable MIS instrument prototype—was recently devised with the aim to follow this vision. Apart from the medical device design restrictions, several limitations of AM materials and processes had to be considered. The aim of this paper is to present these insights to those opting for this means of manufacture, serving as a helpful design and material guide. Over the course of its development, DragonFlex has gone through four design generations so far, each differing in the AM material and process used. Due to being a prototype of a MIS instrument of miniature dimensions, the printing processes were limited to stereolithography (SLA), as to achieve the best possible precision and accuracy. Each SLA process and material brought along specific advantages and disadvantages affecting the final printout quality, which needed to be compensated for either at the design stage, during, or after printing itself. The four DragonFlex generations were printed using the following SLA techniques and materials in this order: polymer jetting from Objet VeroBlue™; SLA Digital Light Processing™ (DLP) method from EnvisionTEC® NanoCure RCP30 and R5; conventional SLA from 3D Systems Accura® 60; and DLP based SLA process from a ceramic composite. The material choice and the printing orientation were found to influence the final printout accuracy and integrity of thin features, as well as material's postproduction behavior. The polymeric VeroBlue™ proved structurally sound, although suffering from undermined accuracy and requiring postprocessing, hence recommended for prototyping of upscaled designs of looser manufacturing tolerances or overdimensioned experimental setups. The NanoCure materials are capable of reaching the best accuracy requiring almost no postprocessing, thus ideal for prototyping small intricate features. Yet their mechanical functionality is undermined due to the high brittleness of RCP30 and high flexibility of R5. The transparent Accura® 60 was found to lose its strength and appeal due to high photosensitivity. Finally, the ceramic composite shows the best potential for medical use due to its biocompatibility and superior mechanical properties, yet one has to compensate for the material shrinkage already at the design stage.
A hybrid approach for fusing 4D‐MRI temporal information with 3D‐CT for the study of lung and lung tumor motion
Authors: Yang Y. X. , Teo S.‐K. , Van Reeth E. , Tan C. H. , Tham I. W. K. , Poh C. L.
Published: July 2015

Purpose:

Accurate visualization of lung motion is important in many clinical applications, such as radiotherapy of lung cancer. Advancement in imaging modalities [e.g., computed tomography (CT) and MRI] has allowed dynamic imaging of lung and lung tumor motion. However, each imaging modality has its advantages and disadvantages. The study presented in this paper aims at generating synthetic 4D‐CT dataset for lung cancer patients by combining both continuous three‐dimensional (3D) motion captured by 4D‐MRI and the high spatial resolution captured by CT using the authors’ proposed approach.

Methods:

A novel hybrid approach based on deformable image registration (DIR) and finite element method simulation was developed to fuse a static 3D‐CT volume (acquired under breath‐hold) and the 3D motion information extracted from 4D‐MRI dataset, creating a synthetic 4D‐CT dataset.

Results:

The study focuses on imaging of lung and lung tumor. Comparing the synthetic 4D‐CT dataset with the acquired 4D‐CT dataset of six lung cancer patients based on 420 landmarks, accurate results (average error <2 mm) were achieved using the authors’ proposed approach. Their hybrid approach achieved a 40% error reduction (based on landmarks assessment) over using only DIR techniques.

Conclusions:

The synthetic 4D‐CT dataset generated has high spatial resolution, has excellent lung details, and is able to show movement of lung and lung tumor over multiple breathing cycles.
Regulation of brain glutamate metabolism by nitric oxide and S-nitrosylation
Authors: Raju Karthik , Doulias Paschalis-Thomas , Evans Perry , Krizman Elizabeth N. , Jackson Joshua G. , Horyn Oksana , Daikhin Yevgeny , Nissim Ilana , Yudkoff Marc , Nissim Itzhak , Sharp Kim A. , Robinson Michael B. , Ischiropoulos Harry
Published: July 2015

Protein S-nitrosylation controls the brain’s use of glutamate for energy or neurotransmission.
Function and dysfunction of two-pore channels
Authors: Patel Sandip
Published: July 2015

Channels on endosomal-lysosomal membranes are appealing targets for treatment of Parkinson’s disease and Ebola infection.
Hydrolysis of Clavulanate by Mycobacterium tuberculosis β-Lactamase BlaC Harboring a Canonical SDN Motif
Authors: Soroka Daria , Li de la Sierra-Gallay Inès , Dubée Vincent , Triboulet Sébastien , van Tilbeurgh Herman , Compain Fabrice , Ballell Lluis , Barros David , Mainardi Jean-Luc , Hugonnet Jean-Emmanuel , Arthur Michel
Published: July 2015

Combinations of β-lactams with clavulanate are currently being investigated for tuberculosis treatment. Since
Mycobacterium tuberculosis
produces a broad spectrum β-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of β-lactamase Bla
MAb
of
Mycobacterium abscessus
, which efficiently hydrolyzes clavulanate. Comparison of Bla
MAb
and BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G
132
N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 10
4
-fold increase in
k
cat
(0.41 s
−1
), without affecting the hydrolysis of other β-lactams. Mass spectrometry revealed that acylation of BlaC and of its G
132
N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G
132
N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of β-lactam–clavulanate combinations may be limited by the emergence of resistance. β-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies.
In
Vitro
Efficacy of Antiviral Compounds against Enterovirus D68
Authors: Rhoden Eric , Zhang Mingyu , Nix W. Allan , Oberste M. Steven
Published: July 2015

In 2014, the United States experienced a large outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68). We used a homogeneous, cell-based assay to assess the antiviral activity of compounds developed for EV/rhinovirus infection or other indications. Three of 15 compounds were highly active against all four strains tested (the prototype and three 2014 strains), with 50% effective concentrations of 0.0012 to 0.027 μM. Additional studies are needed to assess their
in vivo
efficacy against EV-D68.
Identification and Characterization of a Novel Family of Selective Antifungal Compounds (CANBEFs) That Interfere with Fungal Protein Synthesis
Authors: Mircus Gabriel , Albert Nathaniel , Ben-Yaakov Dafna , Chikvashvili Dodo , Shadkchan Yona , Kontoyiannis Dimitrios P. , Osherov Nir
Published: July 2015

Invasive mycotic infections have become more common during recent decades, posing an increasing threat to public health. However, despite the growing needs, treatments for invasive fungal infections remain unsatisfactory and are limited to a small number of antifungals. The aim of this study was to identify novel fungal cell wall inhibitors from a library of small chemical compounds using a conditional protein kinase C (PKC)-expressing strain of
Aspergillus nidulans
sensitive to cell wall-active agents. Eight “hit” compounds affecting cell wall integrity were identified from a screen of 35,000 small chemical compounds. Five shared a common basic molecular structure of 4-
c
hloro-6-
a
rylamino-7-
n
itro-
be
nzo
f
urazane (CANBEF). The most potent compound, CANBEF-24, was characterized further and was shown to inhibit the growth of pathogenic
Aspergillus
,
Candida
,
Fusarium
, and
Rhizopus
isolates at micromolar concentrations but not to affect the growth of mammalian cell lines. CANBEF-24 demonstrated strong synergy in combination with caspofungin, an antifungal that inhibits cell wall biosynthesis. Genetic and biochemical analyses with
Aspergillus nidulans
and
Saccharomyces cerevisiae
indicated that CANBEFs selectively inhibit fungal rRNA maturation and protein synthesis, suggesting that their effect on the cell wall is indirect. CANBEFs were nontoxic in insect (
Galleria mellonella
,
Drosophila melanogaster
) and mouse models of fungal infection. Preliminary evidence showing no therapeutic benefit in these models suggests that further cycles of optimization are needed for the development of this novel class of compounds for systemic use.
Population Pharmacokinetic Modeling of Itraconazole and Hydroxyitraconazole for Oral SUBA-Itraconazole and Sporanox Capsule Formulations in Healthy Subjects in Fed and Fasted States
Authors: Abuhelwa Ahmad Y. , Foster David J. R. , Mudge Stuart , Hayes David , Upton Richard N.
Published: July 2015

Itraconazole is an orally active antifungal agent that has complex and highly variable absorption kinetics that is highly affected by food. This study aimed to develop a population pharmacokinetic model for itraconazole and the active metabolite hydroxyitraconazole, in particular, quantifying the effects of food and formulation on oral absorption. Plasma pharmacokinetic data were collected from seven phase I crossover trials comparing the SUBA-itraconazole and Sporanox formulations of itraconazole. First, a model of single-dose itraconazole data was developed, which was then extended to the multidose data. Covariate effects on itraconazole were then examined before extending the model to describe hydroxyitraconazole. The final itraconazole model was a 2-compartment model with oral absorption described by 4-transit compartments. Multidose kinetics was described by total effective daily dose- and time-dependent changes in clearance and bioavailability. Hydroxyitraconazole was best described by a 1-compartment model with mixed first-order and Michaelis-Menten elimination for the single-dose data and a time-dependent clearance for the multidose data. The relative bioavailability of SUBA-itraconazole compared to that of Sporanox was 173% and was 21% less variable between subjects. Food resulted in a 27% reduction in bioavailability and 58% reduction in the transit absorption rate constant compared to that with the fasted state, irrespective of the formulation. This analysis presents the most extensive population pharmacokinetic model of itraconazole and hydroxyitraconazole in the literature performed in healthy subjects. The presented model can be used for simulating food effects on itraconazole exposure and for performing prestudy power analysis and sample size estimation, which are important aspects of clinical trial design of bioequivalence studies.
Contrasting
Ex Vivo
Efficacies of “Reversed Chloroquine” Compounds in Chloroquine-Resistant Plasmodium falciparum and P. vivax Isolates
Authors: Wirjanata Grennady , Sebayang Boni F. , Chalfein Ferryanto , Prayoga  , Handayuni Irene , Noviyanti Rintis , Kenangalem Enny , Poespoprodjo Jeanne Rini , Burgess Steven J. , Peyton David H. , Price Ric N. , Marfurt Jutta
Published: July 2015

Chloroquine (CQ) has been the mainstay of malaria treatment for more than 60 years. However, the emergence and spread of CQ resistance now restrict its use to only a few areas where malaria is endemic. The aim of the present study was to investigate whether a novel combination of a CQ-like moiety and an imipramine-like pharmacophore can reverse CQ resistance
ex vivo
. Between March to October 2011 and January to September 2013, two “reversed chloroquine” (RCQ) compounds (PL69 and PL106) were tested against multidrug-resistant field isolates of
Plasmodium falciparum
(
n
= 41) and
Plasmodium vivax
(
n
= 45) in Papua, Indonesia, using a modified
ex vivo
schizont maturation assay. The RCQ compounds showed high efficacy against both CQ-resistant
P. falciparum
and
P. vivax
field isolates. For
P. falciparum
, the median 50% inhibitory concentrations (IC
50
s) were 23.2 nM for PL69 and 26.6 nM for PL106, compared to 79.4 nM for unmodified CQ (
P
< 0.001 and
P
= 0.036, respectively). The corresponding values for
P. vivax
were 19.0, 60.0, and 60.9 nM (
P
< 0.001 and
P
= 0.018, respectively). There was a significant correlation between IC
50
s of CQ and PL69 (Spearman's rank correlation coefficient [
r
s
] = 0.727,
P
< 0.001) and PL106 (

r
s

= 0.830,
P
< 0.001) in
P. vivax
but not in
P. falciparum
. Both RCQs were equally active against the ring and trophozoite stages of
P. falciparum
, but in
P. vivax
, PL69 and PL106 showed less potent activity against trophozoite stages (median IC
50
s, 130.2 and 172.5 nM) compared to ring stages (median IC
50
s, 17.6 and 91.3 nM). RCQ compounds have enhanced
ex vivo
activity against CQ-resistant clinical isolates of
P. falciparum
and
P. vivax
, suggesting the potential use of reversal agents in antimalarial drug development. Interspecies differences in RCQ compound activity may indicate differences in CQ pharmacokinetics between the two
Plasmodium
species.
In Vitro
Virology Profile of Tenofovir Alafenamide, a Novel Oral Prodrug of Tenofovir with Improved Antiviral Activity Compared to That of Tenofovir Disoproxil Fumarate
Authors: Callebaut Christian , Stepan George , Tian Yang , Miller Michael D.
Published: July 2015

Tenofovir alafenamide (TAF) is an investigational oral prodrug of the HIV-1 nucleotide reverse transcriptase inhibitor tenofovir (TFV). Tenofovir disoproxil fumarate (TDF) is another TFV prodrug, widely used for the treatment of HIV-1 infection. TAF is converted mostly intracellularly to TFV and, in comparison to TDF, achieves higher tenofovir diphosphate (TFV-DP) levels in peripheral blood mononuclear cells. As a result, TAF has demonstrated potent anti-HIV-1 activity at lower doses than TDF in monotherapy studies. Here, the
in vitro
virology profile of TAF was evaluated and compared to that of TDF. TAF displayed potent antiviral activity against all HIV-1 groups/subtypes, as well as HIV-2. TAF exhibited minimal changes in the drug concentration needed to inhibit 50% of viral spread (EC
50
) upon removal of the prodrug, similar to TDF, demonstrating intracellular antiviral persistence. While TAF and TDF exhibited comparable potencies in the absence of serum pretreatment, TAF maintained activity in the presence of human serum, whereas TDF activity was significantly reduced. This result demonstrates TAF's improved plasma stability over TDF, which is driven by the different metabolic pathways of the two prodrugs and is key to TAF's improved
in vivo
antiviral activity. The activity of TAF is specific for HIV, as TAF lacked activity against a large panel of human viruses, with the exception of herpes simplex virus 2, where weak TAF antiviral activity was observed, as previously observed with TFV. Finally,
in vitro
combination studies with antiretroviral drugs from different classes showed additive to synergistic interactions with TAF, consistent with ongoing clinical studies with TAF in fixed-dose combinations with multiple other antiretroviral drugs for the treatment of HIV.
AAC(3)-XI, a New Aminoglycoside 3-
N
-Acetyltransferase from Corynebacterium striatum
Authors: Galimand Marc , Fishovitz Jennifer , Lambert Thierry , Barbe Valérie , Zajicek Jaroslav , Mobashery Shahriar , Courvalin Patrice
Published: July 2015

Corynebacterium striatum
BM4687 was resistant to gentamicin and tobramycin but susceptible to kanamycin A and amikacin, a phenotype distinct among Gram-positive bacteria. Analysis of the entire genome of this strain did not detect any genes for known aminoglycoside resistance enzymes. Yet, annotation of the coding sequences identified 12 putative acetyltransferases or GCN5-related
N
-acetyltransferases. A total of 11 of these coding sequences were also present in the genomes of other
Corynebacterium
spp. The 12th coding sequence had 55 to 60% amino acid identity with acetyltransferases in
Actinomycetales
. The gene was cloned in
Escherichia coli
, where it conferred resistance to aminoglycosides by acetylation. The protein was purified to homogeneity, and its steady-state kinetic parameters were determined for dibekacin and kanamycin B. The product of the turnover of dibekacin was purified, and its structure was elucidated by high-field nuclear magnetic resonance (NMR), indicating transfer of the acetyl group to the amine at the C-3 position. Due to the unique profile of the reaction, it was designated aminoglycoside 3-
N
-acetyltransferase type XI.
Breeding Strategy To Generate Robust Yeast Starter Cultures for Cocoa Pulp Fermentations
Authors: Meersman Esther , Steensels Jan , Paulus Tinneke , Struyf Nore , Saels Veerle , Mathawan Melissa , Koffi Jean , Vrancken Gino , Verstrepen Kevin J.
Published: July 2015

Cocoa pulp fermentation is a spontaneous process during which the natural microbiota present at cocoa farms is allowed to ferment the pulp surrounding cocoa beans. Because such spontaneous fermentations are inconsistent and contribute to product variability, there is growing interest in a microbial starter culture that could be used to inoculate cocoa pulp fermentations. Previous studies have revealed that many different fungi are recovered from different batches of spontaneous cocoa pulp fermentations, whereas the variation in the prokaryotic microbiome is much more limited. In this study, therefore, we aimed to develop a suitable yeast starter culture that is able to outcompete wild contaminants and consistently produce high-quality chocolate. Starting from specifically selected
Saccharomyces cerevisiae
strains, we developed robust hybrids with characteristics that allow them to efficiently ferment cocoa pulp, including improved temperature tolerance and fermentation capacity. We conducted several laboratory and field trials to show that these new hybrids often outperform their parental strains and are able to dominate spontaneous pilot scale fermentations, which results in much more consistent microbial profiles. Moreover, analysis of the resulting chocolate showed that some of the cocoa batches that were fermented with specific starter cultures yielded superior chocolate. Taken together, these results describe the development of robust yeast starter cultures for cocoa pulp fermentations that can contribute to improving the consistency and quality of commercial chocolate production.
Abundant Trimethylornithine Lipids and Specific Gene Sequences Are Indicative of Planctomycete Importance at the Oxic/Anoxic Interface in Sphagnum-Dominated Northern Wetlands
Authors: Moore Eli K. , Villanueva Laura , Hopmans Ellen C. , Rijpstra W. Irene C. , Mets Anchelique , Dedysh Svetlana N. , Sinninghe Damsté Jaap S.
Published: July 2015

Northern wetlands make up a substantial terrestrial carbon sink and are often dominated by decay-resistant
Sphagnum
mosses. Recent studies have shown that planctomycetes appear to be involved in degradation of
Sphagnum
-derived debris. Novel trimethylornithine (TMO) lipids have recently been characterized as abundant lipids in various
Sphagnum
wetland planctomycete isolates, but their occurrence in the environment has not yet been confirmed. We applied a combined intact polar lipid (IPL) and molecular analysis of peat cores collected from two northern wetlands (Saxnäs Mosse [Sweden] and Obukhovskoye [Russia]) in order to investigate the preferred niche and abundance of TMO-producing planctomycetes. TMOs were present throughout the profiles of
Sphagnum
bogs, but their concentration peaked at the oxic/anoxic interface, which coincided with a maximum abundance of planctomycete-specific 16S rRNA gene sequences. The sequences detected at the oxic/anoxic interface were affiliated with the
Isosphaera
group, while sequences present in the anoxic peat layers were related to an uncultured planctomycete group. Pyrosequencing-based analysis identified
Planctomycetes
as the major bacterial group at the oxic/anoxic interface at the Obukhovskoye peat (54% of total 16S rRNA gene sequence reads), followed by
Acidobacteria
(19% reads), while in the Saxnäs Mosse peat,
Acidobacteria
were dominant (46%), and
Planctomycetes
contributed to 6% of the total reads. The detection of abundant TMO lipids in planctomycetes isolated from peat bogs and the lack of TMO production by cultures of acidobacteria suggest that planctomycetes are the producers of TMOs in peat bogs. The higher accumulation of TMOs at the oxic/anoxic interface and the change in the planctomycete community with depth suggest that these IPLs could be synthesized as a response to changing redox conditions at the oxic/anoxic interface.
Seasonal Changes Drive Short-Term Selection for Fitness Traits in the Wheat Pathogen Zymoseptoria tritici
Authors: Suffert Frédéric , Ravigné Virginie , Sache Ivan
Published: July 2015

In a cross-infection experiment, we investigated how seasonal changes can affect adaptation patterns in aZymoseptoria triticipopulation. The fitness of isolates sampled on wheat leaves at the beginning and at the end of a field epidemic was assessed under environmental conditions (temperature and host stage) to which the local pathogen population was successively exposed. Isolates of the final population were more aggressive, and showed greater sporulation intensity under winter conditions and a shorter latency period (earlier sporulation) under spring conditions, than isolates of the initial population. These differences, complemented by lower between-genotype variability in the final population, exhibited an adaptation pattern with three striking features: (i) the pathogen responded synchronously to temperature and host stage conditions; (ii) the adaptation concerned two key fitness traits; (iii) adaptation to one trait (greater sporulation intensity) was expressed under winter conditions while, subsequently, adaptation to the other trait (shorter latency period) was expressed under spring conditions. This can be interpreted as the result of short-term selection, driven by abiotic and biotic factors. This case study cannot yet be generalized but suggests that seasonality may play an important role in shaping the variability of fitness traits. These results further raise the question of possible counterselection during the interepidemic period. While we did not find any trade-off between clonal multiplication on leaves during the epidemic period and clonal spore production on debris, we suggest that final populations could be counterselected by an Allee effect, mitigating the potential impact of seasonal selection on long-term dynamics.
Diversity, Structures, and Collagen-Degrading Mechanisms of Bacterial Collagenolytic Proteases
Authors: Zhang Yu-Zhong , Ran Li-Yuan , Li Chun-Yang , Chen Xiu-Lan
Published: July 2015

Bacterial collagenolytic proteases are important because of their essential role in global collagen degradation and because of their virulence in some human bacterial infections. Bacterial collagenolytic proteases include some metalloproteases of the M9 family from
Clostridium
or
Vibrio
strains, some serine proteases distributed in the S1, S8, and S53 families, and members of the U32 family. In recent years, there has been remarkable progress in discovering new bacterial collagenolytic proteases and in investigating the collagen-degrading mechanisms of bacterial collagenolytic proteases. This review provides comprehensive insight into bacterial collagenolytic proteases, especially focusing on the structures and collagen-degrading mechanisms of representative bacterial collagenolytic proteases in each family. The roles of bacterial collagenolytic proteases in human diseases and global nitrogen cycling, together with the biotechnological and medical applications for these proteases, are also briefly discussed.
Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains
Authors: van den Broek M. , Bolat I. , Nijkamp J. F. , Ramos E. , Luttik M. A. H. , Koopman F. , Geertman J. M. , de Ridder D. , Pronk J. T. , Daran J.-M.
Published: July 2015

Lager brewing strains of
Saccharomyces pastorianus
are natural interspecific hybrids originating from the spontaneous hybridization of
Saccharomyces cerevisiae
and
Saccharomyces eubayanus
. Over the past 500 years,
S. pastorianus
has been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics of
S. pastorianus
genomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed that
S. pastorianus
strain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants in
S. pastorianus
CBS1483. Analysis of eight additional
S. pastorianus
strains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes.
Reconstitution of the
In Vitro
Activity of the Cyclosporine-Specific P450 Hydroxylase from Sebekia benihana and Development of a Heterologous Whole-Cell Biotransformation System
Authors: Ma Li , Du Lei , Chen Hui , Sun Yue , Huang Shan , Zheng Xianliang , Kim Eung-Soo , Li Shengying
Published: July 2015

The cytochrome P450 enzyme CYP-sb21 from
Sebekia benihana
is capable of catalyzing the site-specific hydroxylation of the immunosuppressant cyclosporine (CsA), leading to the single product γ-hydroxy-
N
-methyl-
l
-Leu4-CsA (CsA-4-OH). Unlike authentic CsA, this hydroxylated CsA shows significantly reduced immunosuppressive activity while it retains a side effect of CsA, the hair growth stimulation effect. Although CYP-sb21 was previously identified to be responsible for CsA-specific hydroxylation
in vivo
, the
in vitro
activity of CYP-sb21 has yet to be established for a deeper understanding of this P450 enzyme and further reaction optimization. In this study, we reconstituted the
in vitro
activity of CYP-sb21 by using surrogate redox partner proteins of bacterial and cyanobacterial origins. The highest CsA site-specific hydroxylation activity by CYP-sb21 was observed when it was partnered with the cyanobacterial redox system composed of
se
Fdx and
se
FdR from
Synechococcus elongatus
PCC 7942. The best bioconversion yields were obtained in the presence of 10% methanol as a cosolvent and an NADPH regeneration system. A heterologous whole-cell biocatalyst using
Escherichia coli
was also constructed, and the permeability problem was solved by using
N
-cetyl-
N
,
N
,
N
-trimethylammonium bromide (CTAB). This work provides a useful example for reconstituting a hybrid P450 system and developing it into a promising biocatalyst for industrial application.
Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin
Authors: Lim Chin Giaw , Wong Lynn , Bhan Namita , Dvora Hila , Xu Peng , Venkiteswaran Sankaranarayanan , Koffas Mattheos A. G.
Published: July 2015

Anthocyanins are water-soluble colored pigments found in terrestrial plants and are responsible for the red, blue, and purple coloration of many flowers and fruits. In addition to the plethora of health benefits associated with anthocyanins (cardioprotective, anti-inflammatory, antioxidant, and antiaging properties), these compounds have attracted widespread attention due to their promising potential as natural food colorants. Previously, we reported the biotransformation of anthocyanin, specifically cyanidin 3-
O
-glucoside (C3G), from the substrate (+)-catechin in
Escherichia coli
. In the present work, we set out to systematically improve C3G titers by enhancing substrate and precursor availability, balancing gene expression level, and optimizing cultivation and induction parameters. We first identified
E. coli
transporter proteins that are responsible for the uptake of catechin and secretion of C3G. We then improved the expression of the heterologous pathway enzymes anthocyanidin synthase (ANS) and 3-
O
-glycosyltransferase (3GT) using a bicistronic expression cassette. Next, we augmented the intracellular availability of the critical precursor UDP-glucose, which has been known as the rate-limiting precursor to produce glucoside compounds. Further optimization of culture and induction conditions led to a final titer of 350 mg/liter of C3G. We also developed a convenient colorimetric assay for easy screening of C3G overproducers. The work reported here constitutes a promising foundation to develop a cost-effective process for large-scale production of plant-derived anthocyanin from recombinant microorganisms.
Effects of Trichothecene Production on the Plant Defense Response and Fungal Physiology: Overexpression of the Trichoderma arundinaceum
tri4
Gene in T. harzianum
Authors: Cardoza R. E. , McCormick S. P. , Malmierca M. G. , Olivera E. R. , Alexander N. J. , Monte E. , Gutiérrez S.
Published: July 2015

Trichothecenes are fungal sesquiterpenoid compounds, the majority of which have phytotoxic activity. They contaminate food and feed stocks, resulting in potential harm to animals and human beings.
Trichoderma brevicompactum
and
T. arundinaceum
produce trichodermin and harzianum A (HA), respectively, two trichothecenes that show different bioactive properties. Both compounds have remarkable antibiotic and cytotoxic activities, but in addition, trichodermin is highly phytotoxic, while HA lacks this activity when analyzed
in vivo
. Analysis of
Fusarium
trichothecene intermediates led to the conclusion that most of them, with the exception of the hydrocarbon precursor trichodiene (TD), have a detectable phytotoxic activity which is not directly related to the structural complexity of the intermediate. In the present work, the HA intermediate 12,13-epoxytrichothec-9-ene (EPT) was produced by expression of the
T. arundinaceum
tri4
gene in a transgenic
T. harzianum
strain that already produces TD after transformation with the
T. arundinaceum
tri5
gene. Purified EPT did not show antifungal or phytotoxic activity, while purified HA showed both antifungal and phytotoxic activities. However, the use of the transgenic
T. harzianum
tri4
strain induced a downregulation of defense-related genes in tomato plants and also downregulated plant genes involved in fungal root colonization. The production of EPT by the transgenic
tri4
strain raised levels of
erg1
expression and reduced squalene accumulation while not affecting levels of ergosterol. Together, these results indicate the complex interactions among trichothecene intermediates, fungal antagonists, and host plants.
Public Health Risks of Multiple-Drug-Resistant Enterococcus spp. in Southeast Asia
Authors: Daniel Diane Sunira , Lee Sui Mae , Dykes Gary A. , Rahman Sadequr
Published: July 2015

Enterococci rank as one of the leading causes of nosocomial infections, such as urinary tract infections, surgical wound infections, and endocarditis, in humans. These infections can be hard to treat because of the rising incidence of antibiotic resistance. Enterococci inhabiting nonhuman reservoirs appear to play a critical role in the acquisition and dissemination of antibiotic resistance determinants. The spread of antibiotic resistance has become a major concern in both human and veterinary medicine, especially in Southeast Asia, where many developing countries have poor legislation and regulations to control the supply and excessive use of antimicrobials. This review addresses the occurrence of antibiotic-resistant enterococci in Association of Southeast Asian Nations countries and proposes infection control measures that should be applied to limit the spread of multiple-drug-resistant enterococci.
Dynamics of Photosynthesis in a Glycogen-Deficient
glgC
Mutant of Synechococcus sp. Strain PCC 7002
Authors: Jackson Simon A. , Eaton-Rye Julian J. , Bryant Donald A. , Posewitz Matthew C. , Davies Fiona K.
Published: July 2015

Cyanobacterial glycogen-deficient mutants display impaired degradation of light-harvesting phycobilisomes under nitrogen-limiting growth conditions and secrete a suite of organic acids as a putative reductant-spilling mechanism. This genetic background, therefore, represents an important platform to better understand the complex relationships between light harvesting, photosynthetic electron transport, carbon fixation, and carbon/nitrogen metabolisms. In this study, we conducted a comprehensive analysis of the dynamics of photosynthesis as a function of reductant sink manipulation in a glycogen-deficient
glgC
mutant of
Synechococcus
sp. strain PCC 7002. The
glgC
mutant showed increased susceptibility to photoinhibition during the initial phase of nitrogen deprivation. However, after extended periods of nitrogen deprivation,
glgC
mutant cells maintained higher levels of photosynthetic activity than the wild type, supporting continuous organic acid secretion in the absence of biomass accumulation. In contrast to the wild type, the
glgC
mutant maintained efficient energy transfer from phycobilisomes to photosystem II (PSII) reaction centers, had an elevated PSII/PSI ratio as a result of reduced PSII degradation, and retained a nitrogen-replete-type ultrastructure, including an extensive thylakoid membrane network, after prolonged nitrogen deprivation. Together, these results suggest that multiple global signals for nitrogen deprivation are not activated in the
glgC
mutant, allowing the maintenance of active photosynthetic complexes under conditions where photosynthesis would normally be abolished.
Ecto-5′-Nucleotidase (CD73) Deficiency in Mycobacterium tuberculosis-Infected Mice Enhances Neutrophil Recruitment
Authors: Petit-Jentreau Laetitia , Jouvion Grégory , Charles Patricia , Majlessi Laleh , Gicquel Brigitte , Tailleux Ludovic
Published: July 2015

The immune system needs safeguards that prevent collateral tissue damage mediated by the immune system while enabling an effective response against a pathogen. The purinergic pathway is one such mechanism and finely modulates inflammation by sensing nucleotides in the environment. Extracellular ATP is considered to be a danger signal leading to a proinflammatory response, whereas adenosine is immunosuppressive. CD73, also called ecto-5′-nucleotidase, occupies a strategic position in this pathway, as it is the main enzyme responsible for the generation of adenosine from ATP. Here, we explore the role of CD73 during tuberculosis, a disease characterized by an immune response that is harmful to the host and unable to eradicateMycobacterium tuberculosis. Using CD73 knockout (KO) mice, we found that CD73 regulates the response toM. tuberculosisinfectionin vitroandin vivo. Mycobacterium-infected murine macrophages derived from CD73 KO mice secrete more keratinocyte chemoattractant (KC), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6) and release less vascular endothelial growth factor (VEGF) upon ATP stimulation than do those derived from wild-type (WT) mice.In vivo, CD73 limits the early influx of neutrophils to the lungs without affecting bacterial growth and dissemination. Collectively, our results support the view that CD73 fine-tunes antimycobacterial immune responses.
Role of the Transporter-Like Sensor Kinase CbrA in Histidine Uptake and Signal Transduction
Authors: Zhang Xue-Xian , Gauntlett Jonathan C. , Oldenburg Darby G. , Cook Gregory M. , Rainey Paul B.
Published: July 2015

CbrA is an atypical sensor kinase found in
Pseudomonas
. The autokinase domain is connected to a putative transporter of the sodium/solute symporter family (SSSF). CbrA functions together with its cognate response regulator, CbrB, and plays an important role in nutrient acquisition, including regulation of
hut
genes for the utilization of histidine and its derivative, urocanate. Here we report on the findings of a genetic and biochemical analysis of CbrA with a focus on the function of the putative transporter domain. The work was initiated with mutagenesis of histidine uptake-proficient strains to identify histidine-specific transport genes located outside the
hut
operon. Genes encoding transporters were not identified, but mutations were repeatedly found in
cbrA
. This, coupled with the findings of [
3
H]histidine transport assays and further mutagenesis, implicated CbrA in histidine uptake. In addition, mutations in different regions of the SSSF domain abolished signal transduction. Site-specific mutations were made at four conserved residues: W55 and G172 (SSSF domain), H766 (H box), and N876 (N box). The mutations W55G, G172H, and N876G compromised histidine transport but had minimal effects on signal transduction. The H766G mutation abolished both transport and signal transduction, but the capacity to transport histidine was restored upon complementation with a transport-defective allele of CbrA, most likely due to interdomain interactions. Our combined data implicate the SSSF domain of CbrA in histidine transport and suggest that transport is coupled to signal transduction.

IMPORTANCE
Nutrient acquisition in bacteria typically involves membrane-bound sensors that, via cognate response regulators, determine the activity of specific transporters. However, nutrient perception and uptake are often coupled processes. Thus, from a physiological perspective, it would make sense for systems that couple the process of signaling and transport within a single protein and where transport is itself the stimulus that precipitates signal transduction to have evolved. The CbrA regulator in
Pseudomonas
represents a unique type of sensor kinase whose autokinase domain is connected to a transporter domain. We present genetic and biochemical evidence that suggests that CbrA plays a dual role in histidine uptake and sensing and that transport is dependent on signal transduction.
Mycobacterial spindle cell pseudotumour of the brain in a patient with sarcoidosis
Authors: Ismail Iyad , Carey Martyn , Trotter Simon , Kunst Heinke
Published: July 2015

Mycobacterial spindle cell pseudotumours (MSP) are benign lesions characterised by local proliferation of spindle-shaped histiocytes caused by mycobacterial infections. Cerebral MSP due toMycobacterium avium intracellulare (MAI) infection is rare, and is often misdiagnosed clinically and radiologically as a brain tumour. We present a case with underlying sarcoidosis and known pulmonaryMAIinfection presenting with partial seizures and headaches. Imaging of the brain revealed a solitary extra axial tumour within the right temporal area. Biopsy of the tumour showed evidence of MPS due toMAIinfection. Prolonged treatment with antituberculous therapy showed complete resolution of the cerebral lesion.
Protrusio acetabuli: a rare cause of pulmonary embolus
Authors: Murphy Colm , O'Neill Barry James , Quinlan John Francis , Collins Ronan
Published: July 2015

Protrusio acetabuli is an uncommon complication of total hip arthroplasty, which results in intrapelvic implant migration after erosion of medial acetabular wall. We present a case of severe prosthetic migration with the formation of haematoma and impingement on iliac vessels leading to thrombosis and subsequent pulmonary embolus. This is the first reported case of protrusio acetabuli as a cause of pulmonary embolus.
Liver perforation following foreign body ingestion: an important clinical lesson
Authors: Martel Gareth , Johnston Dorothy , Jones Claire , Scoffield Julie
Published: July 2015

A 25-year-old woman presented to the surgical unit with a 1-week history of ingestion of a pen. Examination revealed a minimally tender epigastrium with no peritonism. Investigations were unremarkable, with normal bloods and no free air on erect chest radiograph. At the time of endoscopy, the pen appeared to have perforated the lesser curve of the stomach. Endoscopic extraction was abandoned and a CT arranged, revealing the tip of the pen lying within the left lobe of the liver. The pen was removed at laparotomy with an uneventful recovery. This case highlights the importance of considering the potential for injury to other structures following gastric perforation, and the importance of having a low threshold for further imaging.
Ipilimumab-associated colitis or refractoryClostridium difficileinfection?
Authors: Gupta Arjun , Khanna Sahil
Published: July 2015

We present a case of a patient with a diagnostic dilemma who was referred for possible faecal microbiota transplantation (FMT) for refractory diarrhoea secondary toClostridium difficileinfection (CDI). On detailed history, the patient was exposed to ipilimumab concomitantly while being treated for CDI, and was instead diagnosed with diarrhoea secondary to superimposed ipilimumab-associated colitis. Ipilimumab is an anti-CTLA4 monoclonal antibody approved for use in metastatic melanoma and under trial for other indications. Ipilimumab is associated with several immune-related adverse effects, of which diarrhoea and colitis are the most common. While FMT has shown tremendous efficacy in managing recurrent and refractory CDI, it was not offered in this case due to negativeC. difficiletesting showing a high degree of suspicion for ipilimumab-associated colitis due to recent drug use. Our patient was successfully managed with fluid resuscitation and steroids, and remains symptom free at last follow-up at 9 months.
A cross-sectional study of the prevalence and associations of iron deficiency in a cohort of patients with chronic obstructive pulmonary disease
Authors: Nickol Annabel H , Frise Matthew C , Cheng Hung-Yuan , McGahey Anne , McFadyen Bethan M , Harris-Wright Tara , Bart Nicole K , Curtis M Kate , Khandwala Shivani , O'Neill David P , Pollard Karen A , Hardinge F Maxine , Rahman Najib M , Armitage Andrew E , Dorrington Keith L , Drakesmith Hal , Ratcliffe Peter J , Robbins Peter A
Published: July 2015

Objectives

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality. Iron deficiency, with or without anaemia, is associated with other chronic conditions, such as congestive heart failure, where it predicts a worse outcome. However, the prevalence of iron deficiency in COPD is unknown. This observational study aimed to determine the prevalence of iron deficiency in COPD and associations with differences in clinical phenotype.

Setting

University hospital outpatient clinic.

Participants

113 adult patients (65% male) with COPD diagnosed according to GOLD criteria (forced expiratory volume in 1 s (FEV1): forced vital capacity (FVC) ratio <0·70 and FEV1<80% predicted); with age-matched and sex-matched control group consisting of 57 healthy individuals.

Main outcome measures

Prevalence of iron deficiency, defined as: any one or more of (1) soluble transferrin receptor >28.1 nmol/L; (2) transferrin saturation <16% and (3) ferritin <12 µg/L. Severity of hypoxaemia, including resting peripheral arterial oxygen saturation (SpO2) and nocturnal oximetry; C reactive protein (CRP); FEV1; self-reported exacerbation rate and Shuttle Walk Test performance.

Results

Iron deficiency was more common in patients with COPD (18%) compared with controls (5%). In the COPD cohort, CRP was higher in patients with iron deficiency (median 10.5 vs 4.0 mg/L, p<0.001), who were also more hypoxaemic than their iron-replete counterparts (median resting SpO292% vs 95%, p<0.001), but haemoglobin concentration did not differ. Patients with iron deficiency had more self-reported exacerbations and a trend towards worse exercise tolerance.

Conclusions

Non-anaemic iron deficiency is common in COPD and appears to be driven by inflammation. Iron deficiency associates with hypoxaemia, an excess of exacerbations and, possibly, worse exercise tolerance, all markers of poor prognosis. Given that it has been shown to be beneficial in other chronic diseases, intravenous iron therapy should be explored as a novel therapeutic option in COPD.