Molecular genetic studies of Hematodinium sp., a parasitic dinoflagellate of the blue crab, Callinectes sapidus

Economically viable fisheries for the American blue crab, Callinectes sapidus, occur in the USA from Delaware Bay to the Gulf of Mexico. Unfortunately, a number of factors, including overexploitation, degraded habitats, and disease, have contributed to the decline of blue crab populations in the USA. In April 2008, the Secretary of Commerce declared the blue crab fishery in the Chesapeake Bay a commercial fishery disaster. One factor potentially contributing to the decline of blue crab populations in high salinity waters is infection with Hematodinium sp., a parasitic dinoflagellate, which can cause significant mortalities in blue crab populations. Since the first report of this parasite infecting blue crabs in 1975, few studies have used molecular techniques to examine life history characteristics, population structure, geographic range, and host specificity of the species of Hematodinium that infects this host. to examine host specificity, a variety of crustaceans were collected from the Delmarva Peninsula, a region that has high prevalence of Hematodinium sp. in blue crabs. Sequence data obtained from the first internal transcribed spacer (ITS1) region of the ribosomal RNA (rRNA) gene complex from Hematodinium sp. in blue crabs and potential alternate hosts was used to determine that this species of Hematodinium is a host generalist, capable of infecting many decapods and possibly amphipods along the Delmarva Peninsula. Additional sequence data obtained from Hematodinium sp. from infected blue crabs collected from Georgia, the Gulf Coast of Florida, and Texas showed that one species of Hematodinium, the same species found in Virginia, infected blue crabs in all of these locations. Due to the lack of variation observed in the ITS1 region sequences, an enriched microsatellite library was developed using a non-clonal primary culture of Hematodinium sp. from an infected blue crab. From this library, eleven microsatellite markers were identified and optimized for Hematodinium sp. to assess intra-specific variation. Eight microsatellite markers were polymorphic and these were used to genotype Hematodinium sp. from infected blue crabs from six locations along the Delmarva Peninsula. Allele frequencies from these microsatellite markers, along with analyses of the multi-locus genotypes recovered from single genotype infections, indicated that the Delmarva Peninsula contains a single population of this parasite. Microsatellite data also indicate that this parasite is haploid in the hemolymph of the blue crab and that multiple infections are common. The observed genotypic variation was extremely high, indicating that sexual reproduction occurs at some point in the life cycle of this parasite, though the exact life history stage that undergoes sexual reproduction is unknown. This is the first report for evidence of ploidy, multiple infections, population structure, and sexual reproduction for this species of Hematodinium

Analysis of Multipath Mitigation Techniques with Land Mobile Satellite Channel Model

Multipath is undesirable for Global Navigation Satellite System (GNSS) receivers, since the reception of multipath can create a significant distortion to the shape of the correlation function leading to an error in the receivers’ position estimate. Many multipath mitigation techniques exist in the literature to deal with the multipath propagation problem in the context of GNSS. The multipath studies in the literature are often based on optimistic assumptions, for example, assuming a static two-path channel or a fading channel with a Rayleigh or a Nakagami distribution. But, in reality, there are a lot of channel modeling issues, for example, satellite-to-user geometry, variable number of paths, variable path delays and gains, Non Line-Of-Sight (NLOS) path condition, receiver movements, etc. that are kept out of consideration when analyzing the performance of these techniques. Therefore, this is of utmost importance to analyze the performance of different multipath mitigation techniques in some realistic measurement-based channel models, for example, the Land Multipath is undesirable for Global Navigation Satellite System (GNSS) receivers, since the reception of multipath can create a significant distortion to the shape of the correlation function leading to an error in the receivers’ position estimate. Many multipath mitigation techniques exist in the literature to deal with the multipath propagation problem in the context of GNSS. The multipath studies in the literature are often based on optimistic assumptions, for example, assuming a static two-path channel or a fading channel with a Rayleigh or a Nakagami distribution. But, in reality, there are a lot of channel modeling issues, for example, satellite-to-user geometry, variable number of paths, variable path delays and gains, Non Line-Of-Sight (NLOS) path condition, receiver movements, etc. that are kept out of consideration when analyzing the performance of these techniques. Therefore, this is of utmost importance to analyze the performance of different multipath mitigation techniques in some realistic measurement-based channel models, for example, the Land Mobile Satellite (LMS) channel model [1]-[4], developed at the German Aerospace Center (DLR). The DLR LMS channel model is widely used for simulating the positioning accuracy of mobile satellite navigation receivers in urban outdoor scenarios. The main objective of this paper is to present a comprehensive analysis of some of the most promising techniques with the DLR LMS channel model in varying multipath scenarios. Four multipath mitigation techniques are chosen herein for performance comparison, namely, the narrow Early-Minus-Late (nEML), the High Resolution Correlator, the C/N0-based two stage delay tracking technique, and the Reduced Search Space Maximum Likelihood (RSSML) delay estimator. The first two techniques are the most popular and traditional ones used in nowadays GNSS receivers, whereas the later two techniques are comparatively new and are advanced techniques, recently proposed by the authors. In addition, the implementation of the RSSML is optimized here for a narrow-bandwidth receiver configuration in the sense that it now requires a significantly less number of correlators and memory than its original implementation. The simulation results show that the reduced-complexity RSSML achieves the best multipath mitigation performance in moderate-to-good carrier-to-noise density ratio with the DLR LMS channel model in varying multipath scenarios

Localization of dexamethasone within dendritic core-multishell (CMS) nanoparticles and skin penetration properties studied by multi-frequency electron paramagnetic resonance (EPR) spectroscopy

The skin and especially the stratum corneum (SC) act as a barrier and protect epidermal cells and thus the whole body against xenobiotica of the external environment. Topical skin treatment requires an efficient drug delivery system (DDS). Polymer-based nanocarriers represent novel transport vehicles for dermal application of drugs. In this study dendritic core-multishell (CMS) nanoparticles were investigated as promising candidates. CMS nanoparticles were loaded with a drug (analogue) and were applied to penetration studies of skin. We determined by dual-frequency electron paramagnetic resonance (EPR) how dexamethasone (Dx) labelled with 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA) is associated with the CMS. The micro-environment of the drug loaded to CMS nanoparticles was investigated by pulsed high-field EPR at cryogenic temperature, making use of the fact that magnetic parameters (g-, A-matrices, and spin-lattice relaxation time) represent specific probes for the micro-environment. Additionally, the rotational correlation time of spin-labelled Dx was probed by continuous wave EPR at ambient temperature, which provides independent information on the drug environment. Furthermore, the penetration depth of Dx into the stratum corneum of porcine skin after different topical applications was investigated. The location of Dx in the CMS nanoparticles is revealed and the function of CMS as penetration enhancers for topical application is shown

Amphiphilic Cyclic Peptide [W\u3csub\u3e4\u3c/sub\u3eKR\u3csub\u3e5\u3c/sub\u3e]-Antibiotics Combinations as Broad-Spectrum Antimicrobial Agents

Linear and cyclic amphiphilic peptides, (W4KR5) and [W4KR5], were evaluated as antibacterial agents against Gram-positive and Gram-negative bacteria, including four multi-drug resistant strains and the corresponding four non-resistant strains. Cyclic peptide [W4KR5] showed higher antibacterial activity than the linear (W4KR5) counterpart. Cyclic [W4KR5] was subjected to combination (physical mixture or covalent conjugation) with meropenem as a model antibiotic to study the impact of the combination on antimicrobial activity. A physical mixture of meropenem and [W4KR5] showed synergistic antibacterial activity against Gram-negative P. aeruginosa (ATCC BAA-1744) and P. aeruginosa (ATCC 27883) strains. [W4KR5] was further subjected to extensive antibacterial studies against additional 10 bacteria strains, showing significant antibacterial efficacy against Gram-positive bacteria strains. Combinations studies of [W4KR5] with an additional 9 commercially available antibiotics showed significant enhancement in antibacterial activity for all tested combinations, especially with tetracycline, tobramycin, levofloxacin, clindamycin, daptomycin, polymyxin, kanamycin, and vancomycin. Time-kill kinetics assay and flow cytometry results exhibited that [W4KR5] had a time-dependent synergistic effect and membrane disruption property. These data indicate that [W4KR5] improves the antibacterial activity, presumably by facilitating the internalization of antibiotics and their interaction with the intracellular targets. This study introduces a potential strategy for treating multidrug-resistant pathogens by combining [W4KR5] and a variety of classical antibiotics to improve the antibacterial effectiveness

Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro

BACKGROUND: Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA. RESULTS: A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology. CONCLUSIONS: This study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA

Functional characterization of the Mycobacterium abscessus genome coupled with condition specific transcriptomics reveals conserved molecular strategies for host adaptation and persistence

Background: Mycobacterium abscessus subsp. abscessus (MAB) is a highly drug resistant mycobacterium and the most common respiratory pathogen among the rapidly growing non-tuberculous mycobacteria. MAB is also one of the most deadly of the emerging cystic fibrosis (CF) pathogens requiring prolonged treatment with multiple antibiotics. In addition to its “mycobacterial” virulence genes, the genome of MAB harbours a large accessory genome, presumably acquired via lateral gene transfer including homologs shared with the CF pathogens Pseudomonas aeruginosa and Burkholderia cepacia. While multiple genome sequences are available there is little functional genomics data available for this important pathogen. Results: We report here the first multi-omics approach to characterize the primary transcriptome, coding potential and potential regulatory regions of the MAB genome utilizing differential RNA sequencing (dRNA-seq), RNA-seq, Ribosome profiling and LC-MS proteomics. In addition we attempt to address the genomes contribution to the molecular systems that underlie MAB’s adaptation and persistence in the human host through an examination of MABs transcriptional response to a number of clinically relevant conditions. These include hypoxia, exposure to sub-inhibitory concentrations of antibiotics and growth in an artificial sputum designed to mimic the conditions within the cystic fibrosis lung. Conclusions: Our integrated map provides the first comprehensive view of the primary transcriptome of MAB and evidence for the translation of over one hundred new short open reading frames (sORFs). Our map will act as a resource for ongoing functional genomics characterization of MAB and our transcriptome data from clinically relevant stresses informs our understanding of MAB’s adaptation to life in the CF lung. MAB’s adaptation to growth in artificial CF sputum highlights shared metabolic strategies with other CF pathogens including P. aeruginosa and mirrors the transcriptional responses that lead to persistence in mycobacteria. These strategies include an increased reliance on amino acid metabolism, and fatty acid catabolism and highlights the relevance of the glyoxylate shunt to growth in the CF lung. Our data suggests that, similar to what is seen in chronically persisting P. aeruginosa, progression towards a biofilm mode of growth would play a more prominent role in a longer-term MAB infection. Finally, MAB’s transcriptional response to antibiotics highlights the role of antibiotic modifications enzymes, active transport and the evolutionarily conserved WhiB7 driven antibiotic resistance regulon

The distribution of lead concentrations and isotope compositions in the eastern Tropical Atlantic Ocean

Anthropogenic emissions have dominated marine Pb sources during the past century. Here we present Pb concentrations and isotope compositions for ocean depth profiles collected in the eastern Tropical Atlantic Ocean (GEOTRACES section GA06), to trace the transfer of anthropogenic Pb into the ocean interior. Variations in Pb concentration and isotope composition were associated with changes in hydrography. Water masses ventilated in the southern hemisphere generally featured lower 206Pb/207Pb and 208Pb/207Pb ratios than those ventilated in the northern hemisphere, in accordance with Pb isotope data of historic anthropogenic Pb emissions. The distributions of Pb concentrations and isotope compositions in northern sourced waters were consistent with differences in their ventilation timescales. For example, a Pb concentration maximum at intermediate depth (600–900 m, 35 pmol kg−1) in waters sourced from the Irminger/Labrador Seas, is associated with Pb isotope compositions (206Pb/207Pb = 1.1818–1.1824, 208Pb/207Pb = 2.4472–2.4483) indicative of northern hemispheric emissions during the 1950s and 1960s close to peak leaded petrol usage, and a transit time of ∼50–60 years. In contrast, North Atlantic Deep Water (2000–4000 m water depth) featured lower Pb concentrations and isotope compositions (206Pb/207Pb = 1.1762–1.184, 208Pb/207Pb = 2.4482–2.4545) indicative of northern hemispheric emissions during the 1910s and 1930s and a transit time of ∼80–100 years. This supports the notion that transient anthropogenic Pb inputs are predominantly transferred into the ocean interior by water mass transport. However, the interpretation of Pb concentration and isotope composition distributions in terms of ventilation timescales and pathways is complicated by (1) the chemical reactivity of Pb in the ocean, and (2) mixing of waters ventilated during different time periods. The complex effects of water mass mixing on Pb distributions is particularly apparent in seawater in the Tropical Atlantic Ocean which is ventilated from the southern hemisphere. In particular, South Atlantic Central Water and Antarctic Intermediate Water were dominated by anthropogenic Pb emitted during the last 50–100 years, despite estimates of much older average ventilation ages in this region

Membrane-Active Cyclic Amphiphilic Peptides: Broad-Spectrum Antibacterial Activity Alone and in Combination with Antibiotics

We designed a library of 24 cyclic peptides containing arginine (R) and tryptophan (W) residues in a sequential manner [RnWn] (n = 2–7) to study the impact of the hydrophilic/hydrophobic ratio, charge, and ring size on the antibacterial activity against Gram-positive and Gram-negative strains. Among peptides, 5a and 6a demonstrated the highest antimicrobial activity. In combination with 11 commercially available antibiotics, 5a and 6a showed remarkable synergism against a large panel of resistant pathogens. Hemolysis (HC50 = 340 μg/mL) and cell viability against mammalian cells demonstrated the selective lethal action of 5a against bacteria over mammalian cells. Calcein dye leakage and scanning electron microscopy studies revealed the membranolytic effect of 5a. Moreover, the stability in human plasma (t1/2 = 3 h) and the negligible ability of pathogens to develop resistance further reflect the potential of 5a for further development as a peptide-based antibiotic

Iron biogeochemistry across marine systems progress from the past decade

Based on an international workshop (Gothenburg, 14–16 May 2008), this review article aims to combine interdisciplinary knowledge from coastal and open ocean research on iron biogeochemistry. The major scientific findings of the past decade are structured into sections on natural and artificial iron fertilization, iron inputs into coastal and estuarine systems, colloidal iron and organic matter, and biological processes. Potential effects of global climate change, particularly ocean acidification, on iron biogeochemistry are discussed. The findings are synthesized into recommendations for future research areas

Towards an ecosystem model of infectious disease

Increasingly intimate associations between human society and the natural environment are driving the emergence of novel pathogens, with devastating consequences for humans and animals alike. Prior to emergence, these pathogens exist within complex ecological systems that are characterized by trophic interactions between parasites, their hosts and the environment. Predicting how disturbance to these ecological systems places people and animals at risk from emerging pathogens-and the best ways to manage this-remains a significant challenge. Predictive systems ecology models are powerful tools for the reconstruction of ecosystem function but have yet to be considered for modelling infectious disease. Part of this stems from a mistaken tendency to forget about the role that pathogens play in structuring the abundance and interactions of the free-living species favoured by systems ecologists. Here, we explore how developing and applying these more complete systems ecology models at a landscape scale would greatly enhance our understanding of the reciprocal interactions between parasites, pathogens and the environment, placing zoonoses in an ecological context, while identifying key variables and simplifying assumptions that underly pathogen host switching and animal-to-human spillover risk. As well as transforming our understanding of disease ecology, this would also allow us to better direct resources in preparation for future pandemics