bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid frequency‐dependent changes in free mitochondrial calcium concentration in rat cardiac myocytes
Authors: Wüst Rob C. I. , Helmes Michiel , Martin Jody L. , van der Wardt Thomas J. T. , Musters René J. P. , van der Velden Jolanda , Stienen Ger J. M. 
Published: February 2017

Key points
Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle.
The magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiac myocytes are largely unknown.
Rapid stimulation frequency‐dependent increases but relatively slow decreases in free mitochondrial calcium concentration were observed in rat cardiac myocytes. This asymmetry caused a rise in the mitochondrial calcium concentration with stimulation frequency.
These results provide insight into the mechanisms of mitochondrial calcium uptake and release that are important in healthy and diseased myocardium.
AbstractCalcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. Little is known about the magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiomyocytes. Using adenoviral infection, a ratiometric mitochondrially targeted Förster resonance energy transfer (FRET)‐based calcium indicator (4mtD3cpv, MitoCam) was expressed in cultured adult rat cardiomyocytes and the free mitochondrial calcium concentration ([Ca2+]m) was measured at different stimulation frequencies (0.1–4 Hz) and external calcium concentrations (1.8–3.6 mm) at 37°C. Cytosolic calcium concentrations were assessed under the same experimental conditions in separate experiments using Fura‐4AM. The increases in [Ca2+]m during electrical stimulation at 0.1 Hz were rapid (rise time = 49 ± 2 ms), while the decreases in [Ca2+]m occurred more slowly (decay half time = 1.17 ± 0.07 s). Model calculations confirmed that this asymmetry caused the rise in [Ca2+]m during diastole observed at elevated stimulation frequencies. Inhibition of the mitochondrial sodium–calcium exchanger (mNCE) resulted in a rise in [Ca2+]m at baseline and, paradoxically, in an acceleration of Ca2+ release. In conclusion: rapid increases in [Ca2+]m allow for fast adjustment of mitochondrial ATP production to increases in myocardial demand on a beat‐to‐beat basis and mitochondrial calcium release depends on mNCE activity and mitochondrial calcium buffering.
Analysis of Serial Isolates of
mcr-1
-Positive Escherichia coli Reveals a Highly Active IS
Apl1
Transposon
Authors: Snesrud Erik , Ong Ana C. , Corey Brendan , Kwak Yoon I. , Clifford Robert , Gleeson Todd , Wood Shannon , Whitman Timothy J. , Lesho Emil P. , Hinkle Mary , McGann Patrick 
Published: February 2017

ABSTRACT

The emergence of a transferable colistin resistance gene (
mcr-1
) is of global concern. The insertion sequence IS
Apl1
is a key component in the mobilization of this gene, but its role remains poorly understood. Six
Escherichia coli
isolates were cultured from the same patient over the course of 1 month in Germany and the United States after a brief hospitalization in Bahrain for an unconnected illness. Four carried
mcr-1
as determined by real-time PCR, but two were negative. Two additional
mcr-1
-negative
E. coli
isolates were collected during follow-up surveillance 9 months later. All isolates were analyzed by whole-genome sequencing (WGS). WGS revealed that the six initial isolates were composed of two distinct strains: an initial ST-617
E. coli
strain harboring
mcr-1
and a second, unrelated,
mcr-1
-negative ST-32
E. coli
strain that emerged 2 weeks after hospitalization. Follow-up swabs taken 9 months later were negative for the ST-617 strain, but the
mcr-1
-negative ST-32 strain was still present.
mcr-1
was associated with a single copy of IS
Apl1
, located on a 64.5-kb IncI2 plasmid that shared >95% homology with other
mcr-1
IncI2 plasmids. IS
Apl1
copy numbers ranged from 2 for the first isolate to 6 for the final isolate, but IS
Apl1
movement was independent of
mcr-1
. Some movement was accompanied by gene disruption, including the loss of genes encoding proteins involved in stress responses, arginine catabolism, and
l
-arabinose utilization. These data represent the first comprehensive analysis of IS
Apl1
movement in serial clinical isolates and reveal that, under certain conditions, IS
Apl1
is a highly active IS element whose movement may be detrimental to the host cell.
Novel Bacterial Topoisomerase Inhibitors with Potent Broad-Spectrum Activity against Drug-Resistant Bacteria
Authors: Charrier Cédric , Salisbury Anne-Marie , Savage Victoria J. , Duffy Thomas , Moyo Emmanuel , Chaffer-Malam Nathan , Ooi Nicola , Newman Rebecca , Cheung Jonathan , Metzger Richard , McGarry David , Pichowicz Mark , Sigerson Ralph , Cooper Ian R. , Nelson Gary , Butler Hayley S. , Craighead Mark , Ratcliffe Andrew J. , Best Stuart A. , Stokes Neil R. 
Published: February 2017

ABSTRACT

The novel bacterial topoisomerase inhibitor class is an investigational type of antibacterial inhibitor of DNA gyrase and topoisomerase IV that does not have cross-resistance with the quinolones. Here, we report the evaluation of the
in vitro
properties of a new series of this type of small molecule. Exemplar compounds selectively and potently inhibited the catalytic activities of
Escherichia coli
DNA gyrase and topoisomerase IV but did not block the DNA breakage-reunion step. Compounds showed broad-spectrum inhibitory activity against a wide range of Gram-positive and Gram-negative pathogens, including biodefence microorganisms and
Mycobacterium tuberculosis
. No cross-resistance with fluoroquinolone-resistant
Staphylococcus aureus
and
E. coli
isolates was observed. Measured MIC
90
values were 4 and 8 μg/ml against a panel of contemporary multidrug-resistant isolates of
Acinetobacter baumannii
and
E. coli
, respectively. In addition, representative compounds exhibited greater antibacterial potency than the quinolones against obligate anaerobic species. Spontaneous mutation rates were low, with frequencies of resistance typically <10
−8
against
E. coli
and
A. baumannii
at concentrations equivalent to 4-fold the MIC. Compound-resistant
E. coli
mutants that were isolated following serial passage were characterized by whole-genome sequencing and carried a single Arg38Leu amino acid substitution in the GyrA subunit of DNA gyrase. Preliminary
in vitro
safety data indicate that the series shows a promising therapeutic index and potential for low human ether-a-go-go-related gene (hERG) inhibition (50% inhibitory concentration [IC
50
], >100 μM). In summary, the compounds' distinct mechanism of action relative to the fluoroquinolones, whole-cell potency, low potential for resistance development, and favorable
in vitro
safety profile warrant their continued investigation as potential broad-spectrum antibacterial agents.
Evaluation of
para
-Aminosalicylic Acid as a Substrate of Multiple Solute Carrier Uptake Transporters and Possible Drug Interactions with Nonsteroidal Anti-inflammatory Drugs
In Vitro
Authors: Parvez M. Masud , Shin Ho Jung , Jung Jin Ah , Shin Jae-Gook 
Published: February 2017

ABSTRACT

para
-Aminosalicylic acid (PAS) is a second-line antituberculosis drug that has been used to treat multidrug-resistant and extensively drug-resistant tuberculosis for more than 60 years. Renal secretion and glomerular filtration are the major pathways for the elimination of PAS. We comprehensively studied PAS transport by using cell lines that overexpressed various transporters and found that PAS acts as a novel substrate of an organic anionic polypeptide (OATP1B1), organic cationic transporters (OCT1 and OCT2), and organic anion transporters (OAT1 and OAT3) but is not a substrate of any ATP-binding cassette (ABC) transporters. Net PAS uptake was measured, and the transport affinities (

K
m

values) for OATP1B1, OCT1, OCT2, OAT1, and OAT3 were found to be 50.0, 20.3, 28.7, 78.1, and 100.1 μM, respectively. The net uptake rates suggested that renal OAT1 and OAT3 play relatively major roles in PAS elimination. The representative inhibitors rifampin for OATP1B1, probenecid for OAT1 and OAT3, and verapamil for OCT1 and OCT2 greatly inhibited PAS uptake, suggesting that PAS is dependent on multiple transporters for uptake. We also evaluated nonsteroidal anti-inflammatory drugs (NSAIDs), proton pump inhibitors (PPIs), and metformin for the inhibition of PAS uptake via these transporters. Half-maximal (50%) inhibitory concentrations (IC
50
s) were kinetically determined and used to predict the drug-drug interactions (DDIs) affecting these transporters' activity toward PAS. We found that rifampin, probenecid, ibuprofen, naproxen, cimetidine, and quinidine each exhibited a significant potential for
in vivo
DDIs with PAS. In this study, PAS was found to be a novel substrate of several transporters, and drugs that inhibit these transporters can reduce PAS elimination.
The Emerging Pathogen Candida auris: Growth Phenotype, Virulence Factors, Activity of Antifungals, and Effect of SCY-078, a Novel Glucan Synthesis Inhibitor, on Growth Morphology and Biofilm Formation
Authors: Larkin Emily , Hager Christopher , Chandra Jyotsna , Mukherjee Pranab K. , Retuerto Mauricio , Salem Iman , Long Lisa , Isham Nancy , Kovanda Laura , Borroto-Esoda Katyna , Wring Steve , Angulo David , Ghannoum Mahmoud 
Published: February 2017

ABSTRACT

Candida
auris
, a new multidrug-resistant
Candida
spp. which is associated with invasive infection and high rates of mortality, has recently emerged. Here, we determined the virulence factors (germination, adherence, biofilm formation, phospholipase and proteinase production) of 16
C. auris
isolates and their susceptibilities to 11 drugs belonging to different antifungal classes, including a novel orally bioavailable 1,3-β-
d
-glucan synthesis inhibitor (SCY-078). We also examined the effect of SCY-078 on the growth, ultrastructure, and biofilm-forming abilities of
C. auris
. Our data showed that while the tested strains did not germinate, they did produce phospholipase and proteinase in a strain-dependent manner and had a significantly reduced ability to adhere and form biofilms compared to that of
Candida albicans
(
P
= 0.01).
C. auris
isolates demonstrated reduced susceptibility to fluconazole and amphotericin B, while, in general, they were susceptible to the remaining drugs tested. SCY-078 had an MIC
90
of 1 mg/liter against
C. auris
and caused complete inhibition of the growth of
C. auris
and
C. albicans
. Scanning electron microscopy analysis showed that SCY-078 interrupted
C. auris
cell division, with the organism forming abnormal fused fungal cells. Additionally, SCY-078 possessed potent antibiofilm activity, wherein treated biofilms demonstrated significantly reduced metabolic activity and a significantly reduced thickness compared to the untreated control (
P
< 0.05 for both comparisons). Our study shows that
C. auris
expresses several virulence determinants (albeit to a lesser extent than
C. albicans
) and is resistant to fluconazole and amphotericin B. SCY-078, the new orally bioavailable antifungal, had potent antifungal/antibiofilm activity against
C. auris
, indicating that further evaluation of this antifungal is warranted.
Streptococcus mitis and S. oralis Lack a Requirement for CdsA, the Enzyme Required for Synthesis of Major Membrane Phospholipids in Bacteria
Authors: Adams Hannah M. , Joyce Luke R. , Guan Ziqiang , Akins Ronda L. , Palmer Kelli L. 
Published: February 2017

ABSTRACT

Synthesis and integrity of the cytoplasmic membrane are fundamental to cellular life. Experimental evolution studies have hinted at unique physiology in the Gram-positive bacteria
Streptococcus mitis
and
S. oralis
. These organisms commonly cause bacteremia and infectious endocarditis (IE) but are rarely investigated in mechanistic studies of physiology and evolution. Unlike in other Gram-positive pathogens, high-level (MIC ≥ 256 μg/ml) daptomycin resistance rapidly emerges in
S. mitis
and
S. oralis
after a single drug exposure. In this study, we found that inactivating mutations in
cdsA
are associated with high-level daptomycin resistance in
S. mitis
and
S. oralis
IE isolates. This is surprising given that
cdsA
is an essential gene for life in commonly studied model organisms. CdsA is the enzyme responsible for the synthesis of CDP-diacylglycerol, a key intermediate for the biosynthesis of all major phospholipids in prokaryotes and most anionic phospholipids in eukaryotes. Lipidomic analysis by liquid chromatography-mass spectrometry (LC-MS) showed that daptomycin-resistant strains have an accumulation of phosphatidic acid and completely lack phosphatidylglycerol and cardiolipin, two major anionic phospholipids in wild-type strains, confirming the loss of function of CdsA in the daptomycin-resistant strains. To our knowledge, these daptomycin-resistant streptococci represent the first model organisms whose viability is CdsA independent. The distinct membrane compositions resulting from the inactivation of
cdsA
not only provide novel insights into the mechanisms of daptomycin resistance but also offer unique opportunities to study the physiological functions of major anionic phospholipids in bacteria.
Immunoactive Clostridial Membrane Vesicle Production Is Regulated by a Sporulation Factor
Authors: Obana Nozomu , Nakao Ryoma , Nagayama Kyoko , Nakamura Kouji , Senpuku Hidenobu , Nomura Nobuhiko 
Published: February 2017

ABSTRACT

Recently, many Gram-positive bacteria as well as Gram-negative bacteria have been reported to produce membrane vesicles (MVs), but little is known regarding the regulators involved in MV formation. We found that a Gram-positive anaerobic pathogen,
Clostridium perfringens
, produces MVs predominantly containing membrane proteins and cell wall components. These MVs stimulated proinflammatory cytokine production in mouse macrophage-like cells. We suggested that MVs induced interleukin-6 production through the Toll-like receptor 2 (TLR2) signaling pathway. Thus, the MV could have a role in the bacterium-host interaction and bacterial infection pathogenesis. Moreover, we found that the sporulation master regulator gene
spo0A
was required for vesiculogenesis. A conserved, phosphorylated aspartate residue of Spo0A was indispensable for MV production, suggesting that the phosphorylation of Spo0A triggers MV production. Multiple orphan sensor kinases necessary for sporulation were also required to maximize MV production. These findings imply that
C. perfringens
actively produces immunoactive MVs in response to the environment changing, as recognized by membrane-spanning sensor kinases and by modulating the phosphorylation level of Spo0A.
Raising the Alarmone: Within-Host Evolution of Antibiotic-Tolerant
Enterococcus faecium
Authors: Van Tyne Daria , Gilmore Michael S. 
Published: February 2017

ABSTRACT

Enterococci are ancient commensal bacteria that recently emerged as leading causes of antibiotic-resistant, hospital-acquired infection. Vancomycin-resistant enterococci (VRE) epitomize why drug-resistant enterococcal infections are a problem: VRE readily colonize the antibiotic-perturbed gastrointestinal (GI) tract where they amplify to large numbers, and from there, they infect other body sites, including the bloodstream, urinary tract, and surgical wounds. VRE are resistant to many antimicrobials and host defenses, which facilitates establishment at the site of infection and confounds therapeutic clearance. Having evolved to colonize the GI tract, VRE are comparatively ill adapted to the human bloodstream. A recent study by Honsa and colleagues (E. S. Honsa et al., mBio 8:e02124-16, 2017,
https://doi.org/10.1128/mBio.02124-16
) found that a strain of vancomycin-resistant
Enterococcus faecium
evolved antibiotic tolerance within the bloodstream of an immunocompromised host by activating the stringent response through mutation of
relA
. Precisely how VRE colonize and infect and the selective pressures that led to the outgrowth of
relA
mutants are the subjects of ongoing research.
Mycobacterium tuberculosis
Proteasome Accessory Factor A (PafA) Can Transfer Prokaryotic Ubiquitin-Like Protein (Pup) between Substrates
Authors: Zhang Susan , Burns-Huang Kristin E. , Janssen Guido V. , Li Huilin , Ovaa Huib , Hedstrom Lizbeth , Darwin K. Heran 
Published: February 2017

ABSTRACT

The protein degradation machinery of
Mycobacterium tuberculosis
includes a proteasome and a ubiquitin-like protein (Pup). Proteasome accessory factor A (PafA) attaches Pup to proteins to target them for degradation by the proteasome. Free Pup is unstable and never observed in extracts of
M. tuberculosis
, an observation that led us to hypothesize that PafA may need alternative sources of Pup. Here, we show that PafA can move Pup from one proteasome substrate, inositol 1-phosphate synthetase (Ino1), to two different proteins, malonyl coenzyme A (CoA)-acyl carrier protein transacylase (FabD) and lonely guy (Log). This apparent “transpupylation” reaction required a previously unrecognized depupylase activity in PafA, and, surprisingly, this depupylase activity was much more efficient than the activity of the dedicated depupylase Dop (deamidase of Pup). Thus, PafA can potentially use both newly synthesized Pup and recycled Pup to doom proteins for degradation.

IMPORTANCE
Unlike eukaryotes, which contain hundreds of ubiquitin ligases, Pup-containing bacteria appear to have a single ligase to pupylate dozens if not hundreds of different proteins. The observation that PafA can depupylate and transpupylate
in vitro
offers new insight into how protein stability is regulated in proteasome-bearing bacteria. Importantly, PafA and the dedicated depupylase Dop are each required for the full virulence of
Mycobacterium tuberculosis
. Thus, inhibition of both enzymes may be extremely attractive for the development of therapeutics against tuberculosis.
Fallacy of the Unique Genome: Sequence Diversity within Single
Helicobacter pylori
Strains
Authors: Draper Jenny L. , Hansen Lori M. , Bernick David L. , Abedrabbo Samar , Underwood Jason G. , Kong Nguyet , Huang Bihua C. , Weis Allison M. , Weimer Bart C. , van Vliet Arnoud H. M. , Pourmand Nader , Solnick Jay V. , Karplus Kevin , Ottemann Karen M. 
Published: February 2017

ABSTRACT

Many bacterial genomes are highly variable but nonetheless are typically published as a single assembled genome. Experiments tracking bacterial genome evolution have not looked at the variation present at a given point in time. Here, we analyzed the mouse-passaged
Helicobacter pylori
strain SS1 and its parent PMSS1 to assess intra- and intergenomic variability. Using high sequence coverage depth and experimental validation, we detected extensive genome plasticity within these
H. pylori
isolates, including movement of the transposable element IS
607
, large and small inversions, multiple single nucleotide polymorphisms, and variation in
cagA
copy number. The
cagA
gene was found as 1 to 4 tandem copies located off the
cag
island in both SS1 and PMSS1; this copy number variation correlated with protein expression. To gain insight into the changes that occurred during mouse adaptation, we also compared SS1 and PMSS1 and observed 46 differences that were distinct from the within-genome variation. The most substantial was an insertion in
cagY
, which encodes a protein required for a type IV secretion system function. We detected modifications in genes coding for two proteins known to affect mouse colonization, the HpaA neuraminyllactose-binding protein and the FutB α-1,3 lipopolysaccharide (LPS) fucosyltransferase, as well as genes predicted to modulate diverse properties. In sum, our work suggests that data from consensus genome assemblies from single colonies may be misleading by failing to represent the variability present. Furthermore, we show that high-depth genomic sequencing data of a population can be analyzed to gain insight into the normal variation within bacterial strains.

IMPORTANCE
Although it is well known that many bacterial genomes are highly variable, it is nonetheless traditional to refer to, analyze, and publish “the genome” of a bacterial strain. Variability is usually reduced (“only sequence from a single colony”), ignored (“just publish the consensus”), or placed in the “too-hard” basket (“analysis of raw read data is more robust”). Now that whole-genome sequences are regularly used to assess virulence and track outbreaks, a better understanding of the baseline genomic variation present within single strains is needed. Here, we describe the variability seen in typical working stocks and colonies of pathogen
Helicobacter pylori
model strains SS1 and PMSS1 as revealed by use of high-coverage mate pair next-generation sequencing (NGS) and confirmed by traditional laboratory techniques. This work demonstrates that reliance on a consensus assembly as “the genome” of a bacterial strain may be misleading.
Eighty-three per cent of elite athletes return to preinjury sport after anterior cruciate ligament reconstruction: a systematic review with meta-analysis of return to sport rates, graft rupture rates and performance outcomes
Authors: Lai Courtney C H , Ardern Clare L , Feller Julian A , Webster Kate E 
Published: February 2017

ObjectivesThe primary objective was to calculate the rate of return to sport (RTS) following anterior cruciate ligament (ACL) reconstruction in elite athletes. Secondary objectives were to estimate the time taken to RTS, calculate rates of ACL graft rupture, evaluate postsurgical athletic performance and identify determinants of RTS.DesignPooled RTS and graft rupture rates were calculated using random effects proportion meta-analysis. Time to RTS, performance data and determinants of RTS were synthesised descriptively.Data sourcesMEDLINE, EMBASE, AMED, CINAHL, AMI, PEDro, SPORTDiscus and The Cochrane Library were searched from inception to 19 January 2016. Hand searching of 10 sports medicine journals and reference checking were also performed.Eligibility criteria for selecting studiesStudies were included if they reported the ratio of elite athletes who returned to their preinjury level of sport following ACL reconstruction. Twenty-four studies were included.ResultsThe pooled RTS rate was 83% (95% CI 77% to 88%). The mean time to RTS ranged from 6 to 13 months. The pooled graft rupture rate was 5.2% (95% CI 2.8% to 8.3%). Six out of nine studies that included a noninjured control group found no significant deterioration in athletic performance following ACL reconstruction. Indicators of greater athletic skill or value to the team were associated with RTS.Summary and conclusionsEighty-three per cent of elite athletes returned to sport following ACL reconstruction, while 5.2% sustained a graft rupture. Most athletes who returned to sport performed comparably with matched, uninjured controls. This information may assist in guiding expectations of athletes and clinicians following ACL reconstruction.
Preparation, crystal structure, and dielectric characterization of Li2W2O7ceramic at RF and microwave frequency range
Authors: Chen Jinwu , Li Chunchun , Wang Dan , Xiang Huaicheng , Fang Liang 
Published: February 2017

Single phase Li2W2O7with anorthic structure was prepared by the conventional solid-state reaction method at 550[Formula: see text]C and the anorthic structure was stable up to 660[Formula: see text]C. The dielectric properties at radio frequency (RF) and microwave frequency range were characterized. The sample sintered at 640[Formula: see text]C exhibited the optimum microwave dielectric properties with a relative permittivity of 12.2, a quality factor value of 17,700[Formula: see text]GHz (at 9.8[Formula: see text]GHz), and a temperature coefficient of the resonant frequency of [Formula: see text]232[Formula: see text]ppm/[Formula: see text]C as well as a high relative density [Formula: see text]94.1%. Chemical compatibility measurement indicated Li2W2O7did not react with aluminum electrodes when sintered at 640[Formula: see text]C for 4[Formula: see text]h.