Evaluating the Ribosomal Internal Transcribed Spacer (ITS) as a Candidate Dinoflagellate Barcode Marker

Background

DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates.

Methodology/Principal Findings

The purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name.

Conclusions/Significance

The ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.     

Probing Protein Sequences as Sources for Encrypted Antimicrobial Peptides

Starting from the premise that a wealth of potentially biologically active peptides may lurk within proteins, we describe here a methodology to identify putative antimicrobial peptides encrypted in protein sequences. Candidate peptides were identified using a new screening procedure based on physicochemical criteria to reveal matching peptides within protein databases. Fifteen such peptides, along with a range of natural antimicrobial peptides, were examined using DSC and CD to characterize their interaction with phospholipid membranes. Principal component analysis of DSC data shows that the investigated peptides group according to their effects on the main phase transition of phospholipid vesicles, and that these effects correlate both to antimicrobial activity and to the changes in peptide secondary structure. Consequently, we have been able to identify novel antimicrobial peptides from larger proteins not hitherto associated with such activity, mimicking endogenous and/or exogenous microorganism enzymatic processing of parent proteins to smaller bioactive molecules. A biotechnological application for this methodology is explored. Soybean (Glycine max) plants, transformed to include a putative antimicrobial protein fragment encoded in its own genome were tested for tolerance against Phakopsora pachyrhizi, the causative agent of the Asian soybean rust. This procedure may represent an inventive alternative to the transgenic technology, since the genetic material to be used belongs to the host organism and not to exogenous sources.     

A genetic validation study reveals a role of vitamin D metabolism in the response to interferon-alfa-based therapy of chronic hepatitis C

Background

To perform a comprehensive study on the relationship between vitamin D metabolism and the response to interferon-α-based therapy of chronic hepatitis C.

Methodology/Principal Findings

Associations between a functionally relevant polymorphism in the gene encoding the vitamin D 1α-hydroxylase (CYP27B1-1260 rs10877012) and the response to treatment with pegylated interferon-α (PEG-IFN-α) and ribavirin were determined in 701 patients with chronic hepatitis C. In addition, associations between serum concentrations of 25-hydroxyvitamin D₃ (25[OH]D₃) and treatment outcome were analysed. CYP27B1-1260 rs10877012 was found to be an independent predictor of sustained virologic response (SVR) in patients with poor-response IL28B genotypes (15% difference in SVR for rs10877012 genotype AA vs. CC, p = 0.02, OR = 1.52, 95% CI = 1.061–2.188), but not in patients with favourable IL28B genotype. Patients with chronic hepatitis C showed a high prevalence of vitamin D insufficiency (25[OH]D₃<20 ng/mL) during all seasons, but 25(OH)D₃ serum levels were not associated with treatment outcome.

Conclusions/Significance

Our study suggests a role of bioactive vitamin D (1,25[OH]₂D₃, calcitriol) in the response to treatment of chronic hepatitis C. However, serum concentration of the calcitriol precursor 25(OH)D₃ is not a suitable predictor of treatment outcome.     

Were Multiple Stressors a ‘Perfect Storm’ for Northern Gulf of Mexico Bottlenose Dolphins (Tursiops truncatus) in 2011?

An unusual number of near term and neonatal bottlenose dolphin (Tursiops truncatus) mortalities occurred in the northern Gulf of Mexico (nGOM) in 2011, during the first calving season after two well documented environmental perturbations; sustained cold weather in 2010 and the Deepwater Horizon oil spill (DWHOS). Preceding the stranding event, large volumes of cold freshwater entered the nGOM due to unusually large snowmelt on the adjacent watershed, providing a third potential stressor. We consider the possibility that this extreme cold and freshwater event contributed to the pattern of perinatal dolphin strandings along the nGOM coast. During the 4-month period starting January 2011, 186 bottlenose dolphins, including 46% perinatal calves (nearly double the percentage for the same time period from 2003-2010) washed ashore from Louisiana to western Florida. Comparison of the frequency distribution of strandings to flow rates and water temperature at a monitoring buoy outside Mobile Bay, Alabama (the 4(th) largest freshwater drainage in the U.S.) and along the nGOM coast showed that dolphin strandings peaked in Julian weeks 5, 8, and 12 (February and March), following water temperature minima by 2-3 weeks. If dolphin condition was already poor due to depleted food resources, bacterial infection, or other factors, it is plausible that the spring freshet contributed to the timing and location of the unique stranding event in early 2011. These data provide strong observational evidence to assess links between the timing of the DWHOS, other local environmental stressors, and mortality of a top local predator. Targeted analyses of tissues from stranded dolphins will be essential to define a cause of death, and our findings highlight the importance of considering environmental data along with biological samples to interpret stranding patterns during and after an unusual mortality event.     

WSC-1 and HAM-7 Are MAK-1 MAP Kinase Pathway Sensors Required for Cell Wall Integrity and Hyphal Fusion in Neurospora crassa

A large number of cell wall proteins are encoded in the Neurospora crassa genome. Strains carrying gene deletions of 65 predicted cell wall proteins were characterized. Deletion mutations in two of these genes (wsc-1 and ham-7) have easily identified morphological and inhibitor-based defects. Their phenotypic characterization indicates that HAM-7 and WSC-1 function during cell-to-cell hyphal fusion and in cell wall integrity maintenance, respectively. wsc-1 encodes a transmembrane protein with extensive homology to the yeast Wsc family of sensor proteins. In N. crassa, WSC-1 (and its homolog WSC-2) activates the cell wall integrity MAK-1 MAP kinase pathway. The GPI-anchored cell wall protein HAM-7 is required for cell-to-cell fusion and the sexual stages of the N. crassa life cycle. Like WSC-1, HAM-7 is required for activating MAK-1. A Δwsc-1;Δham-7 double mutant fully phenocopies mutants lacking components of the MAK-1 MAP kinase cascade. The data identify WSC-1 and HAM-7 as the major cell wall sensors that regulate two distinct MAK-1-dependent cellular activities, cell wall integrity and hyphal anastomosis, respectively.     

Incremental effects of endocrine and metabolic biomarkers and abdominal obesity on cardiovascular mortality prediction

Background

Biomarkers may help clinicians predict cardiovascular risk. We aimed to determine if the addition of endocrine, metabolic, and obesity-associated biomarkers to conventional risk factors improves the prediction of cardiovascular and all-cause mortality.

Methodology/Principal Findings

In a population-based cohort study (the Study of Health in Pomerania) of 3,967 subjects (age 20–80 years) free of cardiovascular disease with a median follow-up of 10.0 years (38,638 person-years), we assessed the predictive value of conventional cardiovascular risk factors and the biomarkers thyrotropin; testosterone (in men only); insulin-like growth factor-1 (IGF-1); hemoglobin A1c (HbA1c); creatinine; high-sensitive C-reactive protein (hsCRP); fibrinogen; urinary albumin-to-creatinine ratio; and waist-to-height ratio (WHtR) on cardiovascular and all-cause death.During follow-up, we observed 339 all-cause including 103 cardiovascular deaths. In Cox regression models with conventional risk factors, the following biomarkers were retained as significant predictors of cardiovascular death after backward elimination: HbA1c, IGF-1, and hsCRP. IGF-1 and hsCRP were retained as significant predictors of all-cause death.For cardiovascular death, adding these biomarkers to the conventional risk factors changed the C-statistic from 0.898 to 0.910 (p = 0.02). The net reclassification improvement was 10.6%. For all-cause death, the C-statistic changed from 0.849 to 0.853 (P = 0.09).

Conclusions/Significance

HbA1c, IGF-1, and hsCRP predict cardiovascular death independently of conventional cardiovascular risk factors. These easily assessed endocrine and metabolic biomarkers might improve the ability to predict cardiovascular death.     

Influence of Tryptophan Contained in 1-Methyl-Tryptophan on Antimicrobial and Immunoregulatory Functions of Indoleamine 2,3-Dioxygenase

Indoleamine 2,3-dioxygenase (IDO) has been identified as an important antimicrobial and immunoregulatory effector molecule essential for the establishment of tolerance by regulating local tryptophan (Trp) concentrations. On the other hand, the immunosuppressive capacity of IDO can have detrimental effects for the host as it can lead to deleterious alterations of the immune response by promoting tolerance to some types of tumors. To suppress this disadvantageous IDO effect, the competitive inhibitor 1-Methyl-Tryptophan (1-MT) is being tested in clinical trials. However, it remains inconclusive which stereoisomer of 1-MT is the more effective inhibitor of IDO-mediated immunosuppression. While IDO enzyme activity is more efficiently inhibited by 1-L-MT in cell-free or in vitro settings, 1-D-MT is superior to 1-L-MT in the enhancement of anti-tumor responses in vivo.Here, we present new data showing that commercially available 1-L-MT lots contain tryptophan in amounts sufficient to compensate for the IDO-mediated tryptophan depletion in vitro. The addition of 1-L-MT abrogated IDO-mediated antimicrobial effects and permitted the growth of the tryptophan-auxotroph microorganisms Staphylococcus aureus and Toxoplasma gondii. Consistent with this, the tryptophan within 1-L-MT lots was sufficient to antagonize IDO-mediated inhibition of T cell responses. Mass spectrometry (MS) analysis revealed not only tryptophan within 1-L-MT, but also the incorporation of this tryptophan in bacterial and human proteins that were generated in the presence of 1-L-MT in otherwise tryptophan-free conditions. In summary, these data reveal that tryptophan within 1-L-MT can affect the results of in vitro studies in an L-stereospecific and IDO-independent way.     

The Essential Phosphoinositide Kinase MSS-4 Is Required for Polar Hyphal Morphogenesis,Localizing to Sites of Growth and Cell Fusion in Neurospora crassa

Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P₂. In N. crassa hyphae, PtdIns(4,5)P₂ localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P₂ that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.     

Persurf,a New Method to Improve Surfactant Delivery: A Study in Surfactant Depleted Rats

Purpose

Exogenous surfactant is not very effective in adults with ARDS, since surfactant does not reach atelectatic alveoli. Perfluorocarbons (PFC) can recruit atelectatic areas but do not replace impaired endogenous surfactant. A surfactant-PFC-mixture could combine benefits of both therapies. The aim of the proof-of-principal-study was to produce a PFC-in-surfactant emulsion (Persurf) and to test in surfactant depleted Wistar rats whether Persurf achieves I.) a more homogenous pulmonary distribution and II.) a more homogenous recruitment of alveoli when compared with surfactant or PFC alone.

Methods

Three different PFC were mixed with surfactant and phospholipid concentration in the emulsion was measured. After surfactant depletion, animals either received 30 ml/kg of PF5080, 100 mg/kg of stained (green dye) Curosurf™ or 30 ml/kg of Persurf. Lungs were fixated after 1 hour of ventilation and alveolar aeration and surfactant distribution was estimated by a stereological approach.

Results

Persurf contained 3 mg/ml phospholipids and was stable for more than 48 hours. Persurf-administration improved oxygenation. Histological evaluation revealed a more homogenous surfactant distribution and alveolar inflation when compared with surfactant treated animals.

Conclusions

In surfactant depleted rats administration of PFC-in-surfactant emulsion leads to a more homogenous distribution and aeration of the lung than surfactant alone.     

Canine Recombinant Adenovirus Vector Induces an Immunogenicity-Related Gene Expression Profile in Skin-Migrated CD11b+ -Type DCs

Gene expression profiling of the blood cell response induced early after vaccination has previously been demonstrated to predict the immunogenicity of vaccines. In this study, we evaluated whether the analysis of the gene expression profile of skin-migrated dendritic cells (DCs) could be informative for the in vitro prediction of immunogenicity of vaccine, using canine adenovirus serotype 2 (CAV2) as vaccine vector. CAV2 has been shown to induce immunity to transgenes in several species including sheep and is an interesting alternative to human adenovirus-based vectors, based on the safety records of the parental strain in dogs and the lack of pre-existing immunity in non-host species. Skin-migrated DCs were collected from pseudo-afferent lymph in sheep. Both the CD11b⁺ -type and CD103⁺ -type skin-migrated DCs were transduced by CAV2. An analysis of the global gene response to CAV2 in the two skin DC subsets showed that the gene response in CD11b⁺ -type DCs was far higher and broader than in the CD103⁺ -type DCs. A newly released integrative analytic tool from Ingenuity systems revealed that the CAV2-modulated genes in the CD11b⁺ -type DCs clustered in several activated immunogenicity-related functions, such as immune response, immune cell trafficking and inflammation. Thus gene profiling in skin-migrated DC in vitro indicates that the CD11b⁺ DC type is more responsive to CAV2 than the CD103⁺ DC type, and provides valuable information to help in evaluating and possibly improving viral vector vaccine effectiveness.