Paracentrin 1, a synthetic antimicrobial peptide from the sea-urchin Paracentrotus lividus, interferes with staphylococcal and Pseudomonas aeruginosa biofilm formation

The rise of antibiotic-resistance as well as the reduction of investments by pharmaceutical companies in the development of new antibiotics have stimulated the investigation for alternative strategies to conventional antibiotics. Many antimicrobial peptides show a high specificity for prokaryotes and a low toxicity for eukaryotic cells and, due to their mode of action the development of resistance is considered unlikely. We recently characterised an antimicrobial peptide that was called Paracentrin 1 from the 5-kDa peptide fraction from the coelomocyte cytosol of the Paracentrotus lividus. In this study, the chemically synthesised Paracentrin 1, was tested for its antimicrobial and antibiofilm properties against reference strains of Gram positive and Gram negative. The Paracentrin 1 was active against planktonic form of staphylococcal strains (reference and isolates) and Pseudomonas aeruginosa ATCC 15442 at concentrations ranging from 12.5 to 6.2 mg/ml. The Paracentrin 1 was able to inhibit biofilm formation of staphylococcal and Pseudomonas aeruginosa strains at concentrations ranging from 3.1 to 0.75 mg/ml. We consider the tested peptide as a good starting molecule for novel synthetic derivatives with improved pharmaceutical potentia

Title: Water Structure as a Function of Temperature from X-ray Scattering Experiments and Ab Initio Molecular Dynamics

We present high-quality x-ray scattering experiments on pure water taken over a temperature range of 2°C to 77°C using a synchrotron beam line at the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory. The ALS x-ray scattering intensities are qualitatively different in trend of maximum intensity over this temperature range compared to older x-ray experiments. While the common procedure is to report both the intensity curve and radial distribution function(s), the proper extraction of the real-space pair correlation functions from the experimental scattering is very difficult due to uncertainty introduced in the experimental corrections, the proper weighting of OO, OH, and HH contributions, and numerical problems of Fourier transforming truncated data in Q-space. Instead we consider the direct calculation of xray scattering spectra using electron densities derived from density functional theory based on real-space configurations generated with classical water models. The simulation of the experimental intensity is therefore definitive for determining radial distribution functions over a smaller Q-range. We find that the TIP4P, TIP5P and polarizable TIP4P-Pol2 water models, with DFT-LDA densities, show very good agreement with the experimental intensities, and TIP4P-Pol2 in particular shows quantitative agreement over the full temperature range. The resulting radial distribution functions from TIP4P-Pol2 provide the current best benchmarks for real-space water structure over the biologically relevant temperature range studied here. 1 Introduction Liquid water structure is characterized by x-ray (or neutron) diffraction that measures experimental intensities as a function of momentum transfer, Q=4πsin(θ/2)/λ, where λ is the wavelength and θ is the scattering angle with respect to the incident beam. The most recent x-ray data taken at ambient conditions at the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory exhibited significant differences when compared to the scattering curves of past x-ray experiments The intensity is the true experimental observable in which error-bars are well-defined. However, it is typical practice for water scattering experiments to also report radial distributions in addition to the intensity profile, primarily because it is more convenient and practical to consider water structure in terms of real-space distribution functions (1) ( In this study we consider the direct calculation of x-ray scattering spectra using ab-initio density functional theory with the LDA functional over the temperature range studied by experiment. The generation of the real-space "snapshots" could come from either a first 3 principles molecular dynamics calculation Experimental Methods Experimental setup. The data collection was performed at the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory on beam line 7.3.3. Doubly distilled and degassed water was used in each experiment, and several data sets with independent fillings of the sample holder were collected at each temperature (2°C, 11°C, 22°C, 33°C, 44°C, 55°C, 66°C, and 77°C). A silicon monochrometer was used to produce an x-ray beam with spot size on the order of 100µm, with 99% of the energy at 12.800 ± 0.001 keV or a wavelength of 0.9686Å. This wavelength provided the best compromise between sufficient flux and maximizing our 4 accessible Q-range. Data sets were collected with a flat slab water sample using a transmisson geometry, with the sample tilted with respect to the incoming x-ray beam by an angle of 30 o . A Bruker Charge Coupled Device (CCD) area detector, mounted on a Huber diffractometer and with dimensions of 9.6cm x 9.6cm collected the diffracted x-rays. In order to realize the full range of 0.1Å -1 < Q <11.1Å -1 provided in this study, the data were collected with the detector in three different positions which were pieced together for the final result. The sample to detector positions were determined to 100µm precision while the angle between the xray beam and the normal to detector face were characterized on the order of mrads. The geometries at each detector position were determined by collecting PbS powder patterns with the powder placed within the sample holder and fitting the resulting sharp Bragg rings to determine all the geometric parameters. A more detailed description of this procedure can be found in previous work Experimental corrections. The collected raw intensity data was transformed to a circularly integrated scattering cross-section on a per electron scale versus Q. The following divides the corrections into two parts. The first deals with corrections that must be applied generally to all detector positions such as absorption, geometric corrections, and polarization of the radiation. The second set of corrections deals with overlapping data from different panels and thus the contribution of background and sample holder scattering. Corrections to the intensities due to absorption by air, water, and window material are given by the form where I o is the intensity if there were no absorption in the sample, I is the measured intensity, t is the thickness, τ is the angle between the plane of the sample and the incident x-ray beam, and ν has the form ν=cosθsinτ+sinθcosφcosτ. The absorption coefficients, µ ρ , can be obtained from http://www-cxro.lbl.gov/optical_constants using a tabulated format based on 6 When the incident radiation is plane-polarized as in our experiment, the in-plane and outof-plane polarization is treated separately, and the measured intensities must be rescaled by the factor ( The data were also corrected for the 1/r 2 fall off of intensity, and correction for pixel orientation with respect to the incident radiation. We discovered that detector manufacturers attempt to provide a correction of this sort in their bundled software for data read-out. Corrections to the image collected by the CCD includes distortions introduced by the fiber optic tapers connecting the plate to the chip, a dark current correction that takes into account photoelectrons ejected by thermal motions in the CCDs and any low-level background radiation in the experimental hutch, and finally a flat field correction that provides a correction for variations in pixel sensitivity. This correction involves a calibration measurement that collects a reference image intensity using either a radioactive source or scattering from a fluorescent material at a set distance from the detector. The image is used to generate a scale factor for each pixel such that, when applied to the flat field image, each pixel measures the same intensity. The set distance with which this correction is calibrated varies among manufacturers. If the calibration experiment involves a relatively small source to detector distance, then the geometric corrections (1/r 2 fall off, pixel orientation) are implicitly taken into account by the manufacturer in the flat field correction. This is the case for the Bruker detector, so we need to "uncorrect" the collected image for the manufacturers geometry, and reapply the correct geometric corrections for our experiment. This is an insidious problem since these detector corrections are applied 7 before the "raw image" is available to the experimentalist, and the correction is not documented in the manufacturer literature. We next turn to the procedure for the subtraction of background that is required in order to match segments of the intensity over the full range of Q in this experiment

Hydrogeological Features and Sustainable Use of Geothermal Resources: Selected Case Studies in Italy

Within the framework of the activities aimed at studying current and future exploitation and the related sustainability of geothermal resources, and considering the key role hydrogeology plays in the study of geothermal systems and design of installations, in October 2012 the Working Group IDROGEOTER was set-up within the IAH (International Association of Hydrogeologists) Italian Chapter. The first activity of IDROGEOTER‘s workplan is the analysis of state of the art in current use of low-to high enthalpy geothermal resources in Italy and of the hydrogeological settings resulting from features (e.g.: hydrostratigraphy, hydraulic and hydrodynamic conditions, hydrogeochemistry, …) influencing the availability of the resource and the potential of the systems. Detailed studies supporting the possible optimization of the use of geothermal resources, carried out in different areas (see figure) and under different hydrogeological conditions in Italy, are described in the paper. In the Piedmont Region (NW Italy) several experimental sites have been investigated in order to assess the potential subsurface effects of open-loop Groundwater Heat Pumps (GW-HPs) plants for the cooling and heating of buildings. A comparison between field measures and numerical modelling results reveals that the most important aquifer parameters affecting the developing of the Thermal Affected Zone (TAZ) around the injection wells are those related to advective heat transfer. The Lombardy Region (N Italy) is currently the most populated and industrialized region in Italy and therefore the area where the highest number of GWHP plants (open and closed loop) are installed, from which a representative sample will be selected with the objective of identifying the critical hydrogeological factors contributing both to the geothermal potential and to a sustainable use of the resource. In the Veneto Region (NE Italy), the Euganean Geothermal Field is the most important thermal field in northern Italy (about 250 active wells) and the thermal waters (65-86 °C) are mainly used for spas; recently, a new conceptual model of the Euganean Geothermal System has been proposed and tested in a numerical model; with regard to low enthalpy, studies on sites potentially suitable for closed-loop and open-loop systems, together with data from automated monitoring of several wells, could be used for advanced analysis of different hydrogeothermal systems. Research activities in the Lazio Region (Central Italy) focus also on low enthalpy and are specifically aimed at the mapping of the geothermal potential of aquifers, and at pilot studies of sites characterized by gravel aquifers, alluvial Holocene deposits of the Tevere River and alluvial pre-volcanic Pleistocene deposits. In the Campania Region (S Italy - Mondragone plain), in the framework of the geothermal exploration programme “VIGOR” (Evaluation of Geothermal Potential in Convergence Region), a groundwater balance, verifying the recharge area of thermal springs (temperature 33-54 °C) connected to a large carbonate aquifer, has permitted the identification of the most suitable area in which to drill a geothermal well. In the Apulia Region (SE Italy) the thermal field trends consequent to groundwater advection and the influence of seawater intrusion have been reconstructed for two karstic coastal aquifers (Murgia and Salento) at various elevation between -5 and -100 m amsl. These reconstructions are intended to provide required base knowledge for correctly implementing low enthalpy HP plants in the saturated zones. Further studies and inventory of data and applications will be part of the activities of IDROGEOTER, which will also include the preparation of a proposal of guidelines for hydro-geothermal studies

Controlled synthesis of the DSF cell–cell signal is required for biofilm formation and virulence in Xanthomonas campestris

Virulence of the black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is regulated by cell–cell signalling involving the diffusible signal factor DSF. Synthesis and perception of DSF require products of genes within the rpf cluster (for regulation of pathogenicity factors). RpfF directs DSF synthesis whereas RpfC and RpfG are involved in DSF perception. Here we have examined the role of the rpf/DSF system in biofilm formation in minimal medium using confocal laser-scanning microscopy of GFP-labelled bacteria. Wild-type Xcc formed microcolonies that developed into a structured biofilm. In contrast, an rpfF mutant (DSF-minus) and an rpfC mutant (DSF overproducer) formed only unstructured arrangements of bacteria. A gumB mutant, defective in xanthan biosynthesis, was also unable to develop the typical wild-type biofilm. Mixed cultures of gumB and rpfF mutants formed a typical biofilm in vitro. In contrast, in mixed cultures the rpfC mutant prevented the formation of the structured biofilm by the wild-type and did not restore wild-type biofilm phenotypes to gumB or rpfF mutants. These effects on structured biofilm formation were correlated with growth and disease development by Xcc strains in Nicotiana benthamiana leaves. These findings suggest that DSF signalling is finely balanced during both biofilm formation and virulence

SARS-CoV-2 Infection Incidence and Outcome Before and After Full Vaccination in Patients With Monoclonal Gammopathy of Undetermined Significance

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A high-density consensus map of A and B wheat genomes

A durum wheat consensus linkage map was developed by combining segregation data from six mapping populations. All of the crosses were derived from durum wheat cultivars, except for one accession of T. ssp. dicoccoides. The consensus map was composed of 1,898 loci arranged into 27 linkage groups covering all 14 chromosomes. The length of the integrated map and the average marker distance were 3,058.6 and 1.6 cM, respectively. The order of the loci was generally in agreement with respect to the individual maps and with previously published maps. When the consensus map was aligned to the deletion bin map, 493 markers were assigned to specific bins. Segregation distortion was found across many durum wheat chromosomes, with a higher frequency for the B genome. This high-density consensus map allowed the scanning of the genome for chromosomal rearrangements occurring during the wheat evolution. Translocations and inversions that were already known in literature were confirmed, and new putative rearrangements are proposed. The consensus map herein described provides a more complete coverage of the durum wheat genome compared with previously developed maps. It also represents a step forward in durum wheat genomics and an essential tool for further research and studies on evolution of the wheat genome. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00122-012-1939-y) contains supplementary material, which is available to authorized users

Predictive Criteria to Study the Pathogenesis of Malaria-Associated ALI/ARDS in Mice

Malaria-associated acute lung injury/acute respiratory distress syndrome (ALI/ARDS) often results in morbidity and mortality. Murine models to study malaria-associated ALI/ARDS have been described; we still lack a method of distinguishing which mice will develop ALI/ARDS before death. This work aimed to characterize malaria-associated ALI/ARDS in a murine model and to demonstrate the first method to predict whether mice are suffering from ALI/ARDS before death. DBA/2 mice infected with Plasmodium berghei ANKA developing ALI/ARDS or hyperparasitemia (HP) were compared using histopathology, PaO2 measurement, pulmonary X-ray, breathing capacity, lung permeability, and serum vascular endothelial growth factor (VEGF) levels according to either the day of death or the suggested predictive criteria. We proposed a model to predict malaria-associated ALI/ARDS using breathing patterns (enhanced pause and frequency respiration) and parasitemia as predictive criteria from mice whose cause of death was known to retrospectively diagnose the sacrificed mice as likely to die of ALI/ARDS as early as 7 days after infection. Using this method, we showed increased VEGF levels and increased lung permeability in mice predicted to die of ALI/ARDS. This proposed method for accurately identifying mice suffering from ALI/ARDS before death will enable the use of this model to study the pathogenesis of this disease.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ São Paulo, Inst Ciencias Biomed, Dept Imunol, BR-05508900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, BR-09972270 Diadema, SP, BrazilUniv São Paulo, Inst Med Trop São Paulo, BR-05403000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, BR-09972270 Diadema, SP, BrazilUniv São Paulo, Inst Ciencias Biomed, Dept Parasitol, BR-05508000 São Paulo, BrazilUniv São Paulo, Fac Med Vet & Zootecn, Dept Cirurgia, BR-05508270 São Paulo, BrazilUniv São Paulo, Fac Med Vet & Zootecn, Dept Med Vet Prevent & Saude Anim, BR-05508270 São Paulo, BrazilUniv São Paulo, Fac Ciencias Farmaceut, Dept Anal Clin & Toxicol, BR-05508000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, BR-09972270 Diadema, SP, BrazilUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, BR-09972270 Diadema, SP, BrazilFAPESP: 2009/53256-7FAPESP: 2009/53889-0CNPq: 306668/2012-2CNPq: 470590/2009-2Web of Scienc

A Rhizobium leguminosarum CHDL- (Cadherin-Like-) lectin participates in assembly and remodeling of the biofilm matrix

In natural environments most bacteria live in multicellular structures called biofilms. These cell aggregates are enclosed in a self-produced polymeric extracellular matrix, which protects the cells, provides mechanical stability and mediates cellular cohesion and adhesion to surfaces. Although important advances were made in the identification of the genetic and extracellular factors required for biofilm formation, the mechanisms leading to biofilm matrix assembly, and the roles of extracellular proteins in these processes are still poorly understood. The symbiont Rhizobium leguminosarum requires the synthesis of the acidic exopolysaccharide and the PrsDE secretion system to develop a mature biofilm. PrsDE is responsible for the secretion of the Rap family of proteins that share one or two Ra/CHDL (cadherin-like-) domains. RapA2 is a calcium-dependent lectin with a cadherin-like β sheet structure that specifically recognizes the exopolysaccharide, either as a capsular polysaccharide (CPS) or in its released form [extracellular polysaccharide (EPS)]. In this study, using gain and loss of function approaches combined with phenotypic and microscopic studies we demonstrated that RapA lectins are involved in biofilm matrix development and cellular cohesion. While the absence of any RapA protein increased the compactness of bacterial aggregates, high levels of RapA1 expanded distances between cells and favored the production of a dense matrix network. Whereas endogenous RapA(s) are predominantly located at one bacterial pole, we found that under overproduction conditions, RapA1 surrounded the cell in a way that was reminiscent of the capsule. Accordingly, polysaccharide analyses showed that the RapA lectins promote CPS formation at the expense of lower EPS production. Besides, polysaccharide analysis suggests that RapA modulates the EPS size profile. Collectively, these results show that the interaction of RapA lectins with the polysaccharide is involved in rhizobial biofilm matrix assembly and remodeling.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula