183 research outputs found
The MerR-like regulator BrlR confers biofilm tolerance by activating multidrug-efflux pumps in Pseudomonas aeruginosa biofilms
A defining characteristic of biofilms is antibiotic tolerance that can be up to 1,000-fold greater than that of planktonic cells. In Pseudomonas aeruginosa, biofilm tolerance to antimicrobial agents requires the biofilm-specific MerR-type transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm tolerance has not been elucidated. Genome-wide transcriptional profiling indicated that brlR was required for maximal expression of genes associated with antibiotic resistance, in particular those encoding the multidrug efflux pumps MexAB-OprM and MexEF-OprN. Chromatin immunoprecipitation (ChIP) analysis revealed a direct regulation of these genes by BrlR, with DNA binding assays confirming BrlR binding to the promoter regions of the mexAB-oprM and mexEF-oprN operons. Quantitative reverse transcriptase PCR (qRT-PCR) analysis further indicated BrlR to be an activator of mexAB-oprM and mexEF-oprN gene expression. Moreover, immunoblot analysis confirmed increased MexA abundance in cells overexpressing brlR. Inactivation of both efflux pumps rendered biofilms significantly more susceptible to five different classes of antibiotics by affecting MIC but not the recalcitrance of biofilms to killing by bactericidal agents. Overexpression of either efflux pump in a âŹbrlR strain partly restored tolerance of âŹbrlR biofilms to antibiotics. Expression of brlR in mutant biofilms lacking both efflux pumps partly restored antimicrobial tolerance of biofilms to wild-type levels. Our results indicate that BrlR acts as an activator of multidrug efflux pumps to confer tolerance to P. aeruginosa biofilms and to resist the action of antimicrobial agents
The Anti-Sigma Factor MucA of Pseudomonas aeruginosa: Dramatic Differences of a mucA22 vs. a ÎmucA Mutant in Anaerobic Acidified Nitrite Sensitivity of Planktonic and Biofilm Bacteria in vitro and During Chronic Murine Lung Infection
Mucoid mucA22 Pseudomonas aeruginosa (PA) is an opportunistic lung pathogen of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients that is highly sensitive to acidified nitrite (A-NO2-). In this study, we first screened PA mutant strains for sensitivity or resistance to 20 mM A-NO2- under anaerobic conditions that represent the chronic stages of the aforementioned diseases. Mutants found to be sensitive to A-NO2- included PA0964 (pmpR, PQS biosynthesis), PA4455 (probable ABC transporter permease), katA (major catalase, KatA) and rhlR (quorum sensing regulator). In contrast, mutants lacking PA0450 (a putative phosphate transporter) and PA1505 (moaA2) were A-NO2- resistant. However, we were puzzled when we discovered that mucA22 mutant bacteria, a frequently isolated mucA allele in CF and to a lesser extent COPD, were more sensitive to A-NO2- than a truncated ÎmucA deletion (Î157â194) mutant in planktonic and biofilm culture, as well as during a chronic murine lung infection. Subsequent transcriptional profiling of anaerobic, A-NO2--treated bacteria revealed restoration of near wild-type transcript levels of protective NO2- and nitric oxide (NO) reductase (nirS and norCB, respectively) in the ÎmucA mutant in contrast to extremely low levels in the A-NO2--sensitive mucA22 mutant. Proteins that were S-nitrosylated by NO derived from A-NO2- reduction in the sensitive mucA22 strain were those involved in anaerobic respiration (NirQ, NirS), pyruvate fermentation (UspK), global gene regulation (Vfr), the TCA cycle (succinate dehydrogenase, SdhB) and several double mutants were even more sensitive to A-NO2-. Bioinformatic-based data point to future studies designed to elucidate potential cellular binding partners for MucA and MucA22. Given that A-NO2- is a potentially viable treatment strategy to combat PA and other infections, this study offers novel developments as to how clinicians might better treat problematic PA infections in COPD and CF airway diseases
Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa
Background
Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (Ď22) to initiate transcription of the alginate biosynthetic operon.
Results
In the current study, we mapped the mucE transcriptional start site, and determined that PmucE activity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in PmucE activity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ÎalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE.
Conclusions
Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa
Methods and Compositions Based on Culturing Microorganisms in Low Sedimental Fluid Shear Conditions
The benefits of applying a low sedimental fluid shear environment to manipulate microorganisms were examined. Microorganisms obtained from a low sedimental fluid shear culture, which exhibit modified phenotypic and molecular genetic characteristics, are useful for the development of novel and improved diagnostics, therapeutics, vaccines, and bio-industrial products. Furthermore, application of low sedimental fluid conditions to microorganisms permits identification of molecules uniquely expressed under these conditions, providing a basis for the design of new therapeutic targets
The AllWISE Motion Survey, Part 2
We use the AllWISE Data Release to continue our search for WISE-detected
motions. In this paper, we publish another 27,846 motion objects, bringing the
total number to 48,000 when objects found during our original AllWISE motion
survey are included. We use this list, along with the lists of confirmed
WISE-based motion objects from the recent papers by Luhman and by Schneider et
al. and candidate motion objects from the recent paper by Gagne et al. to
search for widely separated, common-proper-motion systems. We identify 1,039
such candidate systems. All 48,000 objects are further analyzed using
color-color and color-mag plots to provide possible characterizations prior to
spectroscopic follow-up. We present spectra of 172 of these, supplemented with
new spectra of 23 comparison objects from the literature, and provide
classifications and physical interpretations of interesting sources. Highlights
include: (1) the identification of three G/K dwarfs that can be used as
standard candles to study clumpiness and grain size in nearby molecular clouds
because these objects are currently moving behind the clouds, (2) the
confirmation/discovery of several M, L, and T dwarfs and one white dwarf whose
spectrophotometric distance estimates place them 5-20 pc from the Sun, (3) the
suggestion that the Na 'D' line be used as a diagnostic tool for interpreting
and classifying metal-poor late-M and L dwarfs, (4) the recognition of a triple
system including a carbon dwarf and late-M subdwarf, for which model fits of
the late-M subdwarf (giving [Fe/H] ~ -1.0) provide a measured metallicity for
the carbon star, and (5) a possible 24-pc-distant K5 dwarf + peculiar red L5
system with an apparent physical separation of 0.1 pc.Comment: 62 pages with 80 figures, accepted for publication in The
Astrophysical Journal Supplement Series, 23 Mar 2016; second version fixes a
few small typos and corrects the footnotes for Table
Discovery of Two Nearby, Peculiar L Dwarfs from the 2MASS Proper Motion Survey: Young or Metal-Rich?
We present the discovery of two nearby L dwarfs from our 2MASS proper motion
search, which uses multi-epoch 2MASS observations covering ~4700 square degrees
of sky. 2MASS J18212815+1414010 and 2MASS J21481628+4003593 were overlooked by
earlier surveys due to their faint optical magnitudes and their proximity to
the Galactic Plane (10 degrees < |b| < 15 degrees). Assuming that both dwarfs
are single, we derive spectrophotometric distances of ~10 pc, thus increasing
the number of known L dwarfs within 10 pc to 10. In the near-infrared, 2MASS
J21481628+4003593 shows a triangular-shaped H-band spectrum, strong CO
absorption, and a markedly red J-Ks color (2.38+/-0.06) for its L6 optical
spectral type. 2MASS J18212815+1414010 also shows a triangular-shaped H-band
spectrum and a slightly red J-Ks color (1.78+/-0.05) for its L4.5 optical
spectral type. Both objects show strong silicate absorption at 9-11 microns.
Cumulatively, these features imply an unusually dusty photosphere for both of
these objects. We examine several scenarios to explain the underlying cause for
their enhanced dust content and find that a metal-rich atmosphere or a
low-surface gravity are consistent with these results. 2MASS J18212815+1414010
may be young (and therefore have a low-surface gravity) based on its low
tangential velocity of 10 km/s. On the other hand, 2MASS J21481628+4003593 has
a high tangential velocity of 62 km/s and is therefore likely old. Hence, high
metallicity and low-surface gravity may lead to similar effects.Comment: 9 pages, 4 tables, 13 figures. Accepted to Ap
Mitochondrial genome sequence analysis: A custom bioinformatics pipeline substantially improves Affymetrix MitoChip v2.0 call rate and accuracy
BACKGROUND: Mitochondrial genome sequence analysis is critical to the diagnostic evaluation of mitochondrial disease. Existing methodologies differ widely in throughput, complexity, cost efficiency, and sensitivity of heteroplasmy detection. Affymetrix MitoChip v2.0, which uses a sequencing-by-genotyping technology, allows potentially accurate and high-throughput sequencing of the entire human mitochondrial genome to be completed in a cost-effective fashion. However, the relatively low call rate achieved using existing software tools has limited the wide adoption of this platform for either clinical or research applications. Here, we report the design and development of a custom bioinformatics software pipeline that achieves a much improved call rate and accuracy for the Affymetrix MitoChip v2.0 platform. We used this custom pipeline to analyze MitoChip v2.0 data from 24 DNA samples representing a broad range of tissue types (18 whole blood, 3 skeletal muscle, 3 cell lines), mutations (a 5.8 kilobase pair deletion and 6 known heteroplasmic mutations), and haplogroup origins. All results were compared to those obtained by at least one other mitochondrial DNA sequence analysis method, including Sanger sequencing, denaturing HPLC-based heteroduplex analysis, and/or the Illumina Genome Analyzer II next generation sequencing platform.
RESULTS: An average call rate of 99.75% was achieved across all samples with our custom pipeline. Comparison of calls for 15 samples characterized previously by Sanger sequencing revealed a total of 29 discordant calls, which translates to an estimated 0.012% for the base call error rate. We successfully identified 4 known heteroplasmic mutations and 24 other potential heteroplasmic mutations across 20 samples that passed quality control.
CONCLUSIONS: Affymetrix MitoChip v2.0 analysis using our optimized MitoChip Filtering Protocol (MFP) bioinformatics pipeline now offers the high sensitivity and accuracy needed for reliable, high-throughput and cost-efficient whole mitochondrial genome sequencing. This approach provides a viable alternative of potential utility for both clinical diagnostic and research applications to traditional Sanger and other emerging sequencing technologies for whole mitochondrial genome analysis
Discoveries from a Near-infrared Proper Motion Survey using Multi-epoch 2MASS Data
We have conducted a 4030-square-deg near-infrared proper motion survey using
multi-epoch data from the Two Micron All-Sky Survey (2MASS). We find 2778
proper motion candidates, 647 of which are not listed in SIMBAD. After
comparison to DSS images, we find that 107 of our proper motion candidates lack
counterparts at B-, R-, and I-bands and are thus 2MASS-only detections. We
present results of spectroscopic follow-up of 188 targets that include the
infrared-only sources along with selected optical-counterpart sources with
faint reduced proper motions or interesting colors. We also establish a set of
near-infrared spectroscopic standards with which to anchor near-infrared
classifications for our objects. Among the discoveries are six young field
brown dwarfs, five "red L" dwarfs, three L-type subdwarfs, twelve M-type
subdwarfs, eight "blue L" dwarfs, and several T dwarfs. We further refine the
definitions of these exotic classes to aid future identification of similar
objects. We examine their kinematics and find that both the "blue L" and "red
L" dwarfs appear to be drawn from a relatively old population. This survey
provides a glimpse of the kinds of research that will be possible through
time-domain infrared projects such as the UKIDSS Large Area Survey, various
VISTA surveys, and WISE, and also through z- or y-band enabled, multi-epoch
surveys such as Pan-STARRS and LSST.Comment: To appear in the September 2010 issue of The Astrophysical Journal,
Supplement Serie
The Phylogeny of the Four Pan-American MtDNA Haplogroups: Implications for Evolutionary and Disease Studies
Only a limited number of complete mitochondrial genome sequences belonging to Native American haplogroups were available until recently, which left America as the continent with the least amount of information about sequence variation of entire mitochondrial DNAs. In this study, a comprehensive overview of all available complete mitochondrial DNA (mtDNA) genomes of the four pan-American haplogroups A2, B2, C1, and D1 is provided by revising the information scattered throughout GenBank and the literature, and adding 14 novel mtDNA sequences. The phylogenies of haplogroups A2, B2, C1, and D1 reveal a large number of sub-haplogroups but suggest that the ancestral Beringian population(s) contributed only six (successful) founder haplotypes to these haplogroups. The derived clades are overall starlike with coalescence times ranging from 18,000 to 21,000 years (with one exception) using the conventional calibration. The average of about 19,000 years somewhat contrasts with the corresponding lower age of about 13,500 years that was recently proposed by employing a different calibration and estimation approach. Our estimate indicates a human entry and spread of the pan-American haplogroups into the Americas right after the peak of the Last Glacial Maximum and comfortably agrees with the undisputed ages of the earliest Paleoindians in South America. In addition, the phylogenetic approach also indicates that the pathogenic status proposed for various mtDNA mutations, which actually define branches of Native American haplogroups, was based on insufficient grounds
Good outcomes after repeated pediatric liver retransplantations: A justified procedure even in times of organ shortage
Background
Pediatric liver transplantations generally represent advanced surgery for selected patients. In case of acute or chronic graft failure, biliary or vessel complications, a retransplantation (reLT) can be necessary. In these situations massive adhesions, critical patient condition or lack of good vessels for anastomosis often are problematic.
Methods
Between 2008 and 2021, 208 pediatric patients received a liver transplantation at our center. Retrospectively, all cases with at least one retransplantation were identified and stored in a database. Indication, intra- and postoperative course and overall survival (OS) were analyzed.
Results
Altogether 31 patients (14.9%) received a reLT. In 22 cases only one reLT was done, 8 patients received 2 reLTs and 1 patient needed a fourth graft. Median age for primary transplantation, first, second and third reLT was 14 (range: 1â192âmonths), 60.5 (range: 1â215âmonths), 58.5 (range: 14â131âmonths) and 67âmonths, respectively. Although biliary atresia (42%) and acute liver failure (23%) represented the main indications for the primary liver transplantation, acute and chronic graft failure (1st reLT: 36%, 2nd reLT: 38%), hepatic artery thrombosis (1st reLT: 29%, 2nd reLT: 25%, 3rd reLT: 100%) and biliary complications (1st reLT: 26%, 2nd reLT: 37%) were the most frequent indications for reLT. OS was 81.8% for patients with 1 reLT, 87.5% with 2 reLTs and 100% with 3 reLTs.
Conclusion
Pediatric liver retransplantation is possible with a good outcome even after multiple retransplantations in specialized centers. Nevertheless, careful patient and graft selection, as well as good preoperative conditioning, are essential
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