Indications of diverse behavioural ecologies in the morphologically conservative Australian land snails Pommerhelix and Meridolum (Stylommatophora: Camaenidae)

The behavioural ecology of terrestrial molluscs is poorly known. We consider the behavioural ecology of three morphologically similar, parapatric camaenids of southeast Australia: Meridolum corneovirens, Meridolum middenense, and Pommerhelix duralensis. We critically review available literature on their behaviour and address knowledge gaps through field and laboratory studies, including spool-line tracking data for P. duralensis and M. corneovirens. These data are supplemented by in situ observation for each species and ex situ dietary preference studies for P. duralensis and M. corneovirens. Our results suggest a complex behavioural ecology for each species, with some inter-specific behavioural differences within the morphologically conservative group. Implications for invertebrate conservation planning are briefly discussed.     

Role of Bacterial Communities in the Natural Suppression of Rhizoctonia solani Bare Patch Disease of Wheat (Triticum aestivum L.)

Rhizoctonia bare patch and root rot disease of wheat, caused by Rhizoctonia solani AG-8, develops as distinct patches of stunted plants and limits the yield of direct-seeded (no-till) wheat in the Pacific Northwest of the United States. At the site of a long-term cropping systems study near Ritzville, WA, a decline in Rhizoctonia patch disease was observed over an 11-year period. Bacterial communities from bulk and rhizosphere soil of plants from inside the patches, outside the patches, and recovered patches were analyzed by using pyrosequencing with primers designed for 16S rRNA. Taxa in the class Acidobacteria and the genus Gemmatimonas were found at higher frequencies in the rhizosphere of healthy plants outside the patches than in that of diseased plants from inside the patches. Dyella and Acidobacteria subgroup Gp7 were found at higher frequencies in recovered patches. Chitinophaga, Pedobacter, Oxalobacteriaceae (Duganella and Massilia), and Chyseobacterium were found at higher frequencies in the rhizosphere of diseased plants from inside the patches. For selected taxa, trends were validated by quantitative PCR (qPCR), and observed shifts of frequencies in the rhizosphere over time were duplicated in cycling experiments in the greenhouse that involved successive plantings of wheat in Rhizoctonia-inoculated soil. Chryseobacterium soldanellicola was isolated from the rhizosphere inside the patches and exhibited significant antagonism against R. solani AG-8 in vitro and in greenhouse tests. In conclusion, we identified novel bacterial taxa that respond to conditions affecting bare patch disease symptoms and that may be involved in suppression of Rhizoctonia root rot and bare batch disease.     

Mitochondrial genome sequences effectively reveal the phylogeny of Hylobates gibbons

Background

Uniquely among hominoids, gibbons exist as multiple geographically contiguous taxa exhibiting distinctive behavioral, morphological, and karyotypic characteristics. However, our understanding of the evolutionary relationships of the various gibbons, especially among Hylobates species, is still limited because previous studies used limited taxon sampling or short mitochondrial DNA (mtDNA) sequences. Here we use mtDNA genome sequences to reconstruct gibbon phylogenetic relationships and reveal the pattern and timing of divergence events in gibbon evolutionary history.

Methodology/Principal Findings

We sequenced the mitochondrial genomes of 51 individuals representing 11 species belonging to three genera (Hylobates, Nomascus and Symphalangus) using the high-throughput 454 sequencing system with the parallel tagged sequencing approach. Three phylogenetic analyses (maximum likelihood, Bayesian analysis and neighbor-joining) depicted the gibbon phylogenetic relationships congruently and with strong support values. Most notably, we recover a well-supported phylogeny of the Hylobates gibbons. The estimation of divergence times using Bayesian analysis with relaxed clock model suggests a much more rapid speciation process in Hylobates than in Nomascus.

Conclusions/Significance

Use of more than 15 kb sequences of the mitochondrial genome provided more informative and robust data than previous studies of short mitochondrial segments (e.g., control region or cytochrome b) as shown by the reliable reconstruction of divergence patterns among Hylobates gibbons. Moreover, molecular dating of the mitogenomic divergence times implied that biogeographic change during the last five million years may be a factor promoting the speciation of Sundaland animals, including Hylobates species.     

Identification of a Human Cyclin D1-Derived Peptide that Induces Human Cytotoxic CD4 T Cells

Cyclin D1 is over-expressed in various human tumors and therefore can be a potential oncogenic target antigen. However, only a limited number of T cell epitopes has been characterized. We aimed at identifying human cyclin D1-derived peptides that include both CD4 and CD8 T cell epitopes and to test if such multi-epitope peptides could yield improved cytotoxic CD8 T cell responses as well as cytotoxic CD4 T cells. Five HLA-DR.B1-binding peptides containing multiple overlapping CD4 epitopes and HLA-A0201-restricted CD8 T cell epitopes were predicted by computer algorithms. Immunogenicity of the synthetic peptides was assessed by stimulating T cells from healthy donors in vitro and the epitope recognition was measured by IFN-γ ELISPOT and ⁵¹Chromium release assays. A HLA-DR.B1 peptide, designed “DR-1”, in which a HLA-A0201-binding epitopes (D1-1) was imbedded, induced CD3 T cell responses against both DR-1 and D1-1 peptides in IFN-γ ELISPOT assay. This suggested processing of the shorter D1-1 epitope from the DR-1 sequence. However, only DR-1-stimulated CD4 or CD3 T cells possessed cytotoxicity against peptide-pulsed autologous DCs and a cancer cell line, that expresses a high level of cyclin D1. Monoclonal antibody to HLA-DR abrogated the epitope-specific responses of both CD3 and CD4 T cells, demonstrating class II-mediated killing. Our studies suggest a possible role of CD4 T cells in anti-tumor immunity as cytotoxic effectors against HLA-DR expressing cancers and provide a rationale for designing peptide vaccines that include CD4 epitopes.     

Effects of concrete properties and nutrients on fungal colonization and fouling

This study describes the fouling of concrete surfaces by diverse fungal genera under controlled laboratory conditions. A circulating flow-through chamber was designed for testing the effects of different concrete compositions and exogenously added nutrients on fungal colonization and fouling. Fungal strains belonging to the genera Alternaria, Cladosporium, Epicoccum, Fusarium, Mucor, Penicillium, Pestalotiopsis, and Trichoderma were cultured directly from visibly fouled concrete structures and used individually and in a mix to inoculate mortar tiles varying in cement composition, supplementary cementitious material additions, water-to-cement ratio, and surface roughness. A strong positive relationship was observed between tile water-to-cement ratio and the amount of biofouling. In addition, cement containing photocatalytic titanium dioxide and exposed to artificial sunlight strongly inhibited fungal colonization and fouling. Mortar tiles coated with form-release oil and incubated with sterile rainwater were also capable of supporting fungal colonization. Our results indicate that the fouling of concrete surfaces by fungi can be influenced by variations in concrete composition variations and available nutrients.     

Expression, purification, and human antibody response to a 67 kDa vaccine candidate for schistosomiasis japonica

Schistosomiasis remains a leading cause of morbidity and mortality in the developing tropical world, and vaccines to prevent these infections remain a scientific and public health priority. Sj67 is a 67 kDa Schistosoma japonicum surface membrane protein homologous to a family of actin-binding proteins. Sj67 is recognized by a mouse monoclonal antibody (mAb 6) that confers resistance to challenge infection in passive transfer experiments. These data support Sj67 as a potential vaccine candidate for schistosomiasis japonica. In the present study, we report the ligation-independent cloning of a cDNA encoding thioredoxin/elastin-like polypeptide (ELP)/rSj67 into a pET-32 Xa/LIC vector. Soluble recombinant fusion protein (Thio-ELP-rSj67) was expressed and purified using anion-exchange and size exclusion chromatography. rSj67 was cleaved from the Thio-ELP fusion partner by digestion with Factor Xa protease and purified using hydroxyapatite column chromatography. Endotoxin was reduced by absorption to a polymyxin support. Purified rSj67 had a molecular weight of 67 kDa and N-terminal sequencing confirmed that the first five amino acids of the recombinant protein matched the predicted sequence for the Sj67 gene. In Western blot analysis, rSj67 was recognized by the Sj67 specific mAb 6 antibody. IgG antibodies in sera from schistosomiasis infected volunteers living in an endemic area of the Philippines (n = 13) recognized rSj67 with 4.7-fold greater median fluorescence compared to uninfected North American controls (n = 5) (p < 0.009). Together, these data confirm the expression and purification of recombinant Sj67 and its immuno-reactivity with sera from S. japonicum infected humans.     

Role of the interactions between the active site base and the substrate Schiff base in amine oxidase catalysis. Evidence from structural and spectroscopic studies of the 2-hydrazinopyridine adduct of Escherichia coli amine oxidase

2-Hydrazinopyridine (2HP) is an irreversible inhibitor of copper amine oxidases (CAOs). 2HP reacts directly at the C5 position of the TPQ cofactor, yielding an intense chromophore with lambda(max) approximately 430 nm (adduct I) in Escherichia coli amine oxidase (ECAO). The adduct I form of wild type (WT-ECAO) was assigned as a hydrazone on the basis of the X-ray crystal structure. The hydrazone adduct appears to be stabilized by two key hydrogen-bonding interactions between the TPQ-2HP moiety and two active site residues: the catalytic base (D383) and the conserved tyrosine residue (Y369). In this work, we have synthesized a model compound (2) for adduct I from the reaction of a TPQ model compound (1) and 2HP. NMR spectroscopy and X-ray crystallography show that 2 exists predominantly as the azo form (lambda(max) at 414 nm). Comparison of the UV-vis and resonance Raman spectra of 2 with adduct I in WT, D383E, D383N, and Y369F forms of ECAO revealed that adduct I in WT and D383N is a tautomeric mixture where the hydrazone form is favored. In D383E adduct I, the equilibrium is further shifted in favor of the hydrazone form. UV-vis spectroscopic pH titrations of adduct I in WT, D383N, D383E, and 2 confirmed that D383 in WT adduct I is protonated at pH 7 and stabilizes the hydrazone tautomer by a short hydrogen-bonding interaction. The deprotonation of D383 (pKa approximately 9.7) in adduct I resulted in conversion of adduct I to the azo tautomer with a blue shift of the lambda(max) to 420 nm, close to that of 2. In contrast, adduct I in D383N and D383E is stable and did not show any pH-dependent spectral changes. In Y369F, adduct I was not stable and gradually converted into a new species with lambda(max) at approximately 530 nm (adduct II). A detailed mechanism for the adduct I formation in WT has been proposed that is consistent with the mechanism proposed for the oxidation of substrate by CAOs but addresses some key differences in the active site chemistry of hydrazine inhibitors and substrate amines.     

High prevalence of Schistosoma japonicum by perfusion in naturally exposed water buffalo in a region of the Philippines endemic for human schistosomiasis

In the past decade, ecological surveys emphasized rats and dogs as the most significant animal reservoirs for Schistosoma japonicum (S.j) in the Philippines. However, recent studies demonstrated 51–91% prevalence of schistosomiasis among water buffalo using qPCR in the Sj endemic regions in the Philippines. In order to resolve the inconsistency of reported surveys regarding Sj endemicity among carabao, a domestic water buffalo that is the most important draught animal, we introduced 42 schistosome negative water buffalo to Macanip, Jaro municipality, Leyte, the Philippines, a subsistence rice-farming village that has been the focus of schistosomiasis japonica studies of our group for the past 20 years. We conducted perfusion to the remaining 34 buffalo that survived 10 months of nature exposure and Typhoon Haiyan. Thirty-three water buffalo were found to be positive with at least 1 pair of worms from the mesenteric vein. The infection rate is 97%, with the worm burden of 94 (95% confidence interval, 49–138 worms) worms. To our knowledge, this is the first report about S. japonicum worm burden in naturally infected water buffalo in the Philippines. The fact that with less than one-year of exposure, in this human schistosomiasis endemic area, only 1 out of 34 water buffalo was uninfected is striking. Urgent attention is needed for a cost-effective technique for monitoring Sj infection in animals and humans. Meanwhile, intervention implementation, including water buffalo treatment and vaccination, should be taken into consideration.     

Resistance to ROS1 Inhibition Mediated by EGFR Pathway Activation in Non-Small Cell Lung Cancer

The targeting of oncogenic ‘driver’ kinases with small molecule inhibitors has proven to be a highly effective therapeutic strategy in selected non-small cell lung cancer (NSCLC) patients. However, acquired resistance to targeted therapies invariably arises and is a major limitation to patient care. ROS1 fusion proteins are a recently described class of oncogenic driver, and NSCLC patients that express these fusions generally respond well to ROS1-targeted therapy. In this study, we sought to determine mechanisms of acquired resistance to ROS1 inhibition. To accomplish this, we analyzed tumor samples from a patient who initially responded to the ROS1 inhibitor crizotinib but eventually developed acquired resistance. In addition, we generated a ROS1 inhibition-resistant derivative of the initially sensitive NSCLC cell line HCC78. Previously described mechanisms of acquired resistance to tyrosine kinase inhibitors including target kinase-domain mutation, target copy number gain, epithelial-mesenchymal transition, and conversion to small cell lung cancer histology were found to not underlie resistance in the patient sample or resistant cell line. However, we did observe a switch in the control of growth and survival signaling pathways from ROS1 to EGFR in the resistant cell line. As a result of this switch, ROS1 inhibition-resistant HCC78 cells became sensitive to EGFR inhibition, an effect that was enhanced by co-treatment with a ROS1 inhibitor. Our results suggest that co-inhibition of ROS1 and EGFR may be an effective strategy to combat resistance to targeted therapy in some ROS1 fusion-positive NSCLC patients.