Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation

Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins

Composition and mixing state of atmospheric aerosols determined by electron microscopy: method development and application to aged Saharan dust deposition in the Caribbean boundary layer

The microphysical properties, composition and mixing state of mineral dust, sea salt and secondary compounds were measured by active and passive aerosol sampling, followed by electron microscopy and X-ray fluorescence in the Caribbean marine boundary layer. Measurements were carried out at Ragged Point, Barbados during June–July 2013 and August 2016. Techniques are presented and evaluated, which allow for statements on atmospheric aerosol concentrations and aerosol mixing state based on collected samples. It became obvious that in the diameter range with the highest dust deposition the deposition velocity models disagree by more than 2 orders of magnitude. Aerosol at Ragged Point was dominated by dust, sea salt and soluble sulfates in varying proportions. The contribution of sea salt was dependent on local wind speed. Sulfate concentrations were linked to long-range transport from Africa and Europe, and South America and the southern Atlantic Ocean. Dust sources were located in western Africa. The dust silicate composition was not significantly varied. Pure feldspar grains were 3&thinsp;% of the silicate particles, of which about a third were K-feldspar. The average dust deposition observed was 10&thinsp;mg&thinsp;m−2&thinsp;d−1 (range of 0.5–47&thinsp;mg&thinsp;m−2&thinsp;d−1), of which 0.67&thinsp;mg&thinsp;m−2&thinsp;d−1 was iron and 0.001&thinsp;mg&thinsp;m−2&thinsp;d−1 phosphorus. Iron deposition was mainly driven by silicate particles from Africa. Dust particles were mixed internally to a minor fraction (10&thinsp;%), mostly with sea salt and less frequently with sulfate. It was estimated that the average dust deposition velocity under ambient conditions is increased by the internal mixture by 30&thinsp;%–140&thinsp;% for particles between 1 and 10&thinsp;µm dust aerodynamic diameter, with approximately 35&thinsp;% at the mass median diameter of deposition (7.0&thinsp;µm). For this size, an effective deposition velocity of 6.4&thinsp;mm&thinsp;s−1 (geometric standard deviation of 3.1 over all individual particles) was observed.</p

Patch-Based Experiments with Object Classification in Video Surveillance

We present a patch-based algorithm for the purpose of object classification in video surveillance. Within detected regions-of-interest (ROIs) of moving objects in the scene, a feature vector is calculated based on template matching of a large set of image patches. Instead of matching direct image pixels, we use Gabor-filtered versions of the input image at several scales. This approach has been adopted from recent experiments in generic object-recognition tasks. We present results for a new typical video surveillance dataset containing over 9,000 object images. Furthermore, we compare our system performance with another existing smaller surveillance dataset. We have found that with 50 training samples or higher, our detection rate is on the average above 95%. Because of the inherent scalability of the algorithm, an embedded system implementation is well within reach

Differential Gene Expression by RamA in Ciprofloxacin-Resistant Salmonella Typhimurium

Overexpression of ramA has been implicated in resistance to multiple drugs in several enterobacterial pathogens. In the present study, Salmonella Typhimurium strain LTL with constitutive expression of ramA was compared to its ramA-deletion mutant by employing both DNA microarrays and phenotype microarrays (PM). The mutant strain with the disruption of ramA showed differential expression of at least 33 genes involved in 11 functional groups. The study confirmed at the transcriptional level that the constitutive expression of ramA was directly associated with increased expression of multidrug efflux pump AcrAB-TolC and decreased expression of porin protein OmpF, thereby conferring multiple drug resistance phenotype. Compared to the parent strain constitutively expressing ramA, the ramA mutant had increased susceptibility to over 70 antimicrobials and toxic compounds. The PM analysis also uncovered that the ramA mutant was better in utilization of 10 carbon sources and 5 phosphorus sources. This study suggested that the constitutive expression of ramA locus regulate not only multidrug efflux pump and accessory genes but also genes involved in carbon metabolic pathways

Probing the Informational and Regulatory Plasticity of a Transcription Factor DNA–Binding Domain

Transcription factors have two functional constraints on their evolution: (1) their binding sites must have enough information to be distinguishable from all other sequences in the genome, and (2) they must bind these sites with an affinity that appropriately modulates the rate of transcription. Since both are determined by the biophysical properties of the DNA–binding domain, selection on one will ultimately affect the other. We were interested in understanding how plastic the informational and regulatory properties of a transcription factor are and how transcription factors evolve to balance these constraints. To study this, we developed an in vivo selection system in Escherichia coli to identify variants of the helix-turn-helix transcription factor MarA that bind different sets of binding sites with varying degrees of degeneracy. Unlike previous in vitro methods used to identify novel DNA binders and to probe the plasticity of the binding domain, our selections were done within the context of the initiation complex, selecting for both specific binding within the genome and for a physiologically significant strength of interaction to maintain function of the factor. Using MITOMI, quantitative PCR, and a binding site fitness assay, we characterized the binding, function, and fitness of some of these variants. We observed that a large range of binding preferences, information contents, and activities could be accessed with a few mutations, suggesting that transcriptional regulatory networks are highly adaptable and expandable

3D Fluid Flow Estimation with Integrated Particle Reconstruction

The standard approach to densely reconstruct the motion in a volume of fluid is to inject high-contrast tracer particles and record their motion with multiple high-speed cameras. Almost all existing work processes the acquired multi-view video in two separate steps, utilizing either a pure Eulerian or pure Lagrangian approach. Eulerian methods perform a voxel-based reconstruction of particles per time step, followed by 3D motion estimation, with some form of dense matching between the precomputed voxel grids from different time steps. In this sequential procedure, the first step cannot use temporal consistency considerations to support the reconstruction, while the second step has no access to the original, high-resolution image data. Alternatively, Lagrangian methods reconstruct an explicit, sparse set of particles and track the individual particles over time. Physical constraints can only be incorporated in a post-processing step when interpolating the particle tracks to a dense motion field. We show, for the first time, how to jointly reconstruct both the individual tracer particles and a dense 3D fluid motion field from the image data, using an integrated energy minimization. Our hybrid Lagrangian/Eulerian model reconstructs individual particles, and at the same time recovers a dense 3D motion field in the entire domain. Making particles explicit greatly reduces the memory consumption and allows one to use the high-res input images for matching. Whereas the dense motion field makes it possible to include physical a-priori constraints and account for the incompressibility and viscosity of the fluid. The method exhibits greatly (~70%) improved results over our recently published baseline with two separate steps for 3D reconstruction and motion estimation. Our results with only two time steps are comparable to those of sota tracking-based methods that require much longer sequences.Comment: To appear in International Journal of Computer Vision (IJCV

Biomimetic self-assembling copolymer-hydroxyapatite nanocomposites with the nanocrystal size controlled by citrate

Citrate binds strongly to the surface of calcium phosphate (apatite) nanocrystals in bone and is thought to prevent crystal thickening. In this work, citrate added as a regulatory element enabled molecular control of the size and stability of hydroxyapatite (HAp) nanocrystals in synthetic nanocomposites, fabricated with self-assembling block copolymer templates. The decrease of the HAp crystal size within the polymer matrix with increasing citrate concentration was documented by solid-state nuclear magnetic resonance (NMR) techniques and wide-angle X-ray diffraction (XRD), while the shapes of HAp nanocrystals were determined by transmission electron microscopy (TEM). Advanced NMR techniques were used to characterize the interfacial species and reveal enhanced interactions between mineral and organic matrix, concomitant with the size effects. The surface-to-volume ratios determined by NMR spectroscopy and long-range 31P{1H} dipolar dephasing show that 2, 10, and 40 mM citrate changes the thicknesses of the HAp crystals from 4 nm without citrate to 2.9, 2.8, and 2.3 nm, respectively. With citrate concentrations comparable to those in body fluids, HAp nanocrystals of sizes and morphologies similar to those in avian and bovine bones have been produced

T2-signal intensity, SSTR expression, and somatostatin analogs efficacy predict response to pasireotide in acromegaly.

peer reviewed[en] OBJECTIVE: T2-signal intensity and somatostatin (SST) receptor expression are recognized predictors of therapy response in acromegaly. We investigated the relationship between these predictors and the hormonal and tumoral responses to long-acting pasireotide (PAS-LAR) therapy, which were also compared with responsiveness to first-generation somatostatin receptor ligands (SRLs). DESIGN: The PAPE study is a cohort study. METHODS: We included 45 acromegaly patients initially receiving SRLs, followed by combination therapy with pegvisomant, and finally PAS-LAR. We assessed tumor volume reduction (≥25% from baseline), IGF-1 levels (expressed as the upper limit of normal), and T2-weighted MRI signal and SST receptor expression of the adenoma. RESULTS: Patients with significant tumor shrinkage during PAS-LAR showed higher IGF-1 levels during PAS-LAR (mean (S.D.): 1.36 (0.53) vs 0.93 (0.43), P = 0.020), less IGF-1 reduction after first-generation SRLs (mean (S.D.): 0.55 (0.71) vs 1.25 (1.07), P = 0.028), and lower SST2 receptor expression (median (IQR): 2.0 (1.0-6.0) vs 12.0 (7.5-12.0), P = 0.040). Overall, T2-signal intensity ratio was increased compared with baseline (mean (S.D.): 1.39 (0.56) vs 1.25 (0.52), P = 0.017) and a higher T2-signal was associated with lower IGF-1 levels during PAS-LAR (β: -0.29, 95% CI: -0.56 to -0.01, P = 0.045). A subset of PAS-LAR treated patients with increased T2-signal intensity achieved greater reduction of IGF-1 (mean (S.D.): 0.80 (0.60) vs 0.45 (0.39), P = 0.016). CONCLUSIONS: Patients unresponsive to SRLs with a lower SST2 receptor expression are more prone to achieve tumor shrinkage during PAS-LAR. Surprisingly, tumor shrinkage is not accompanied by a biochemical response, which is accompanied with a higher T2-signal intensity