Scale‐dependent post‐establishment spread and genetic diversity in an invading mollusc in South America

Aims Our study aimed to characterize the dispersal dynamics and population genetic structure of the introduced golden mussel Limnoperna fortunei throughout its invaded range in South America and to determine how different dispersal methods, that is, human‐mediated dispersal and downstream natural dispersal, contribute to genetic variation among populations. Location Paraná–Uruguay–Río de la Plata watershed in Argentina, Brazil, Paraguay and Uruguay. Methods We performed genetic analyses based on a comprehensive sampling strategy encompassing 22 populations (N = 712) throughout the invaded range in South America, using the mitochondrial cytochrome c oxidase subunit I (COI) gene and eight polymorphic nuclear microsatellites. We employed both population genetics and phylogenetic analyses to clarify the dispersal dynamics and population genetic structure. Results We detected relatively high genetic differentiation between populations (F_ST = ?0.041 to 0.111 for COI, ?0.060 to 0.108 for microsatellites) at both fine and large geographical scales. Bayesian clustering and three‐dimensional factorial correspondence analyses consistently revealed two genetically distinct clusters, highlighting genetic discontinuities in the invaded range. Results of all genetic analyses suggest ship‐mediated ‘jump’ dispersal as the dominant mode of spread of golden mussels in South America, while downstream natural dispersal has had limited effects on contemporary genetic patterns. Main conclusions Our study provides new evidence that post‐establishment dispersal dynamics and genetic patterns vary across geographical scales. While ship‐mediated ‘jump’ dispersal dominates post‐establishment spread of golden mussels in South America, once colonies become established in upstream locations, larvae produced may be advected downstream to infill patchy distributions. Moreover, genetic structuring at fine geographical scales, especially within the same drainages, suggests a further detailed understanding of dynamics of larval dispersal and settlement in different water systems. Knowledge of the mechanisms by which post‐establishment spread occurs can, in some cases, be used to limit dispersal of golden mussels and other introduced species.     

Cryo X-ray nano-tomography of vaccinia virus infected cells

We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels.     

Effects of dietary Bacillus subtilis, Tetraselmis chuii, and Phaeodactylum tricornutum, singularly or in combination, on the immune response and disease resistance of sea bream (Sparus aurata L.)

Combined or individual effects of two microalgae (Phaeodactylum tricornutum and Tetraselmis chuii) and Bacillus subtilis on immune response, gene expression, and survival to challenge with Photobacterium damselae subsp. piscicida of gilthead sea bream were investigated. To test the capacity of B. subtilis to grow employing the microalgae polysaccharides as energy and carbon source, an in vitro assay was defined, and demonstrated that the digestion product of microalgae, mainly P. tricornutum, supported the growth of B. subtilis much better than glucose. For the in vivo study, fish were distributed in six equal groups (each of two replicates) and received one of the following experimental diets: C) control, non-supplemented diet; T) T. chuii 100 g kg(-1); P) P. tricornutum 100 g kg(-1); B) B. subtilis (10(7) cfu g(-1)); BT) B. subtilis (10(7) cfu g(-1))+T. chuii (100 g kg(-1)); and BP) B. subtilis (10(7) cfu g(-1))+P. tricornutum (100 g kg(-1)). The complement activity, serum IgM level, respiratory burst, phagocytic activity, and expression of seven selected immune-related genes in head-kidney were evaluated following two and four weeks of treatment. At the end of the feeding trial, fish were challenged by intraperitoneal injection of LD(50) concentration of P. damselae subsp. piscicida and mortality was recorded. This is the first study testing the immunomodulatory capacity of the microalgae used in the present work. The dietary applications of B. subtilis, T. chuii, and P. tricornutum, singly or in combination, may exhibit up-regulating effects on gilthead sea bream immune parameters. P. tricornutum demonstrated the highest immunostimulant activity. There were no significant differences between combination feeding and feeding ingredients separately. Our results demonstrated the potential of microalgae as immunostimulants for fish, although further studies regarding the implications and effects of a stimulated immune system against pathogens, especially the protective capacity against specific diseases, are necessary.     

Engineering and adaptive evolution of Escherichia coli for d-lactate fermentation reveals GatC as a xylose transporter

Despite the abundance of xylose in nature, the production of chemicals from C5 sugars remains challenging in metabolic engineering. By deleting xylFGH genes and using adaptive evolution, an efficient E. coli strain capable of producing d-lactate from xylose was engineered. Quantitative proteomics and genome sequencing were used to understand the new phenotype and the metabolic limitations of xylose conversion to d-lactate. Proteomics identified major changes in enzyme concentration in the glycolytic and tricarboxylic acid pathways. Whole genome sequencing of the evolved strain identified a point mutation in the gatC gene, which resulted in a change from serine to leucine at position 184 of the GatC protein. The knockout of gatC in a number of strains and the insertion of the mutation in the non-evolved strain confirmed its activity as a xylose transporter and demonstrated that the mutation is responsible for the high xylose consumption phenotype in the evolved strain. The newly found xylose transporter is a candidate for future strain engineering for converting C5-C6 syrups into valuable chemicals.     

Identification of a unique gene cluster of Brucella spp. that mediates adhesion to host cells

Brucella, the causative agent of brucellosis, a major zoonotic disease affecting a broad range of mammals, is a gram-negative bacterium whose virulence is dependent on the capacity to attach and invade different cells of the host. The bacterium is able to infect through a diverse repertoire of epitheliums: skin, airways or gastric. Although much has been studied on the mechanisms Brucella uses to establish an intracellular replication niche, almost none is known on how the bacterium adheres and invades host cells. We report here the identification of a pathogenicity island that harbors a gene homologous to proteins with bacterial immunoglobulin-like domains present in other pathogens that play a role in attachment and invasion. Deletion of the entire island results in a mutant with a reduced attachment capacity measured by intracellular replication and adhesion assays. Intraperitoneal and oral experimental infection of mice strongly suggests that this island plays a role during the oral infection probably mediating attachment and trespassing of the gastric epithelium to establish a systemic infection.     

High quality long-term CD4+ and CD8+ effector memory populations stimulated by DNA-LACK/MVA-LACK regimen in Leishmania major BALB/c model of infection

Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4(+) and CD8(+) T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4(+) and CD8(+) effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4(+) and CD8(+) T cells. Anti-vector responses were largely CD8(+)-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4(+) and CD8(+) T cells with an effector phenotype, could be relevant in protection against leishmaniasis.     

Silencing of miR-370 in Human Cholangiocarcinoma by Allelic Loss and Interleukin-6 Induced Maternal to Paternal Epigenotype Switch

Cholangiocarcinoma (CCA) is a highly lethal malignant tumor arising from the biliary tract epithelium. Interleukin-6 (IL-6) is a major mediator of inflammation and contributor to carcinogenesis within the biliary tree. Previous studies suggested that enforced IL-6 contributes to cholangiocarcinogenesis through hypermethylation of several genes implicated in CCA. However, the precise mechanisms of IL-6 effects in CCA remain unclear. We now demonstrate that microRNA (miR)-370 is underexpressed in a large cohort of human CCA vs. normal liver tissues. In addition, we show that IL-6 induces a time-dependent silencing of miR-370. In addition, demethylation of CCA cells results in upregulation of miR-370. Furthermore, we demonstrate that miR-370 is imprinted, and that the Intergenic Differentially Methylated Region (IG-DMR) responsible for imprinting regulation of this genomic locus is hypermethylated in response to IL-6 treatment. In addition, the IG-DMR is hypermethylated in human CCA specimens compared to normal matched controls, in the same location as the IL-6 induced hypermethylation. Finally, miR-370 was found to regulate WNT10B in luciferase as well as western blotting experiments. Our data indicate that the paternal allele of miR-370 is normally silenced through genomic imprinting and that the overexpression of IL-6 in CCA effectively suppresses the expression of miR-370 from the maternal allele, lending support to the theory that miR-370 silencing in human CCA follows a classic two-hit mechanism.     

Breast feeding, parity and breast cancer subtypes in a Spanish cohort

Background

Differences in the incidence and outcome of breast cancer among Hispanic women compared with white women are well documented and are likely explained by ethnic differences in genetic composition, lifestyle, or environmental exposures.

Methodolgy/Principal Findings

A population-based study was conducted in Galicia, Spain. A total of 510 women diagnosed with operable invasive breast cancer between 1997 and 2010 participated in the study. Data on demographics, breast cancer risk factors, and clinico-pathological characteristics were collected. The different breast cancer tumor subtypes were compared on their clinico-pathological characteristics and risk factor profiles, particularly reproductive variables and breastfeeding. Among the 501 breast cancer patients (with known ER and PR receptors), 85% were ER+/PR+ and 15% were ER-&PR-. Among the 405 breast cancer with known ER, PR and HER2 status, 71% were ER+/PR+/HER2- (luminal A), 14% were ER+/PR+/HER2+ (luminal B), 10% were ER−/PR−/HER2- (triple negative breast cancer, TNBC), and 5% were ER−/PR−/HER2+ (non-luminal). A lifetime breastfeeding period equal to or longer than 7 months was less frequent in case patients with TNBC (OR = 0.25, 95% CI = 0.08–0.68) compared to luminal A breast cancers. Both a low (2 or fewer pregnancies) and a high (3–4 pregnancies) number of pregnancies combined with a long breastfeeding period were associated with reduced odds of TNBC compared with luminal A breast cancer, although the association seemed to be slightly more pronounced among women with a low number of pregnancies (OR = 0.09, 95% CI = 0.005–0.54).

Conclusions/Significance

In case-case analyses with the luminal A cases as the reference group, we observed a lower proportion of TNBC among women who breastfed 7 or more months. The combination of longer breastfeeding duration and lower parity seemed to further reduce the odds of having a TNBC compared to a luminal A breast cancer.