Molecular basis for pacemaker cells in epithelia

Intercellular signaling is highly coordinated in excitable tissues such as heart, but the organization of intercellular signaling in epithelia is less clear. We examined Ca(2+) signaling in hepatoma cells expressing the hepatocyte gap junction protein connexin32 (cx32) or the cardiac gap junction protein cx43, plus a fluorescently tagged V(1a) vasopressin receptor (V(1a)R). Release of inositol 1,4,5-trisphosphate (InsP(3)) in wild type cells increased Ca(2+) in the injected cell but not in neighboring cells, while the Ca(2+) signal spread to neighbors when gap junctions were expressed. Photorelease of caged Ca(2+) rather than InsP(3) resulted in a small increase in Ca(2+) that did not spread to neighbors with or without gap junctions. However, photorelease of Ca(2+) in cells stimulated with low concentrations of vasopressin resulted in a much larger increase in Ca(2+), which spread to neighbors via gap junctions. Cells expressing tagged V(1a)R similarly had increased sensitivity to vasopressin, and could signal to neighbors via gap junctions. Higher concentrations of vasopressin elicited Ca(2+) signals in all cells. In cx32 or cx43 but not in wild type cells, this signaling was synchronized and began in cells expressing the tagged V(1a)R. Thus, intercellular Ca(2+) signals in epithelia are organized by three factors: 1) InsP(3) must be generated in each cell to support a Ca(2+) signal in that cell; 2) gap junctions are necessary to synchronize Ca(2+) signals among cells; and 3) cells with relatively increased expression of hormone receptor will initiate Ca(2+) signals and thus serve as pacemakers for their neighbors. Together, these factors may allow epithelia to act in an integrated, organ-level fashion rather than as a collection of isolated cells.     

Do saline taxa evolve faster? Comparing relative rates of molecular evolution between freshwater and marine eukaryotes

The major branches of life diversified in the marine realm, and numerous taxa have since transitioned between marine and freshwaters. Previous studies have demonstrated higher rates of molecular evolution in crustaceans inhabiting continental saline habitats as compared with freshwaters, but it is unclear whether this trend is pervasive or whether it applies to the marine environment. We employ the phylogenetic comparative method to investigate relative molecular evolutionary rates between 148 pairs of marine or continental saline versus freshwater lineages representing disparate eukaryote groups, including bony fish, elasmobranchs, cetaceans, crustaceans, mollusks, annelids, algae, and other eukaryotes, using available protein‐coding and noncoding genes. Overall, we observed no consistent pattern in nucleotide substitution rates linked to habitat across all genes and taxa. However, we observed some trends of higher evolutionary rates within protein‐coding genes in freshwater taxa—the comparisons mainly involving bony fish—compared with their marine relatives. The results suggest no systematic differences in substitution rate between marine and freshwater organisms.     

Contrasting effects of necrotrophic and biotrophic plant pathogens on the aphid Aphis fabae

Phytophagous insects have to contend with a wide variation in food quality brought about by a variety of factors intrinsic and extrinsic to the plant. One of the most important factors is infection by plant pathogenic fungi. Necrotrophic and biotrophic plant pathogenic fungi may have contrasting effects on insect herbivores due to their different infection mechanisms and induction of different resistance pathways, although this has been little studied and there has been no study of their combined effect. We studied the effect of the biotrophic rust fungus Uromyces viciae‐fabae (Pers.) Schroet (Basidiomycota: Uredinales: Pucciniaceae) and the necrotrophic fungus Botrytis cinerea Pers. (Ascomycota: Helotiales: Sclerotiniaceae) singly and together on the performance of the aphid Aphis fabae Scopoli (Hemiptera: Aphididae) on Vicia faba (L.) (Fabaceae). Alone, botrytis had an inhibitory effect on individual A. fabae development, survival, and fecundity, whereas rust infection consistently enhanced individual aphids' performance. These effects varied in linear relation to lesion or pustule density. However, whole‐plant infection by either pathogen resulted in a smaller aphid population of smaller aphids than on uninfected plants, indicating a lowering of aphid carrying capacity with infection. When both fungi were applied simultaneously to a leaf they generally cancelled the effect of each other out, resulting in most performance parameters being similar to the controls, although fecundity was reduced. However, sequential plant infection (pathogens applied 5 days apart) led to a 70% decrease in fecundity and 50% reduction in intrinsic rate of increase. The application of rust before botrytis had a greater inhibitory effect on aphids than applying botrytis before rust. Rust infection increased leaf total nitrogen concentration by 30%, whereas infection by botrytis with or without rust led to a 38% decrease. The aphids' responses to the two plant pathogens individually is consistent with the alteration in plant nutrient content by infection and also the induction of different plant defence pathways and the possible cross‐talk between them. This is the first demonstration of the complex effects of the dual infection of a plant by contrasting pathogens on insect herbivores.     

Burkholderia cepacia Complex Phage-Antibiotic Synergy (PAS): Antibiotics Stimulate Lytic Phage Activity

The Burkholderia cepacia complex (Bcc) is a group of at least 18 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. Bcc organisms possess high levels of innate antimicrobial resistance, and alternative therapeutic strategies are urgently needed. One proposed alternative treatment is phage therapy, the therapeutic application of bacterial viruses (or bacteriophages). Recently, some phages have been observed to form larger plaques in the presence of sublethal concentrations of certain antibiotics; this effect has been termed phage-antibiotic synergy (PAS). Those reports suggest that some antibiotics stimulate increased production of phages under certain conditions. The aim of this study is to examine PAS in phages that infect Burkholderia cenocepacia strains C6433 and K56-2. Bcc phages KS12 and KS14 were tested for PAS, using 6 antibiotics representing 4 different drug classes. Of the antibiotics tested, the most pronounced effects were observed for meropenem, ciprofloxacin, and tetracycline. When grown with subinhibitory concentrations of these three antibiotics, cells developed a chain-like arrangement, an elongated morphology, and a clustered arrangement, respectively. When treated with progressively higher antibiotic concentrations, both the sizes of plaques and phage titers increased, up to a maximum. B. cenocepacia K56-2-infected Galleria mellonella larvae treated with phage KS12 and low-dose meropenem demonstrated increased survival over controls treated with KS12 or antibiotic alone. These results suggest that antibiotics can be combined with phages to stimulate increased phage production and/or activity and thus improve the efficacy of bacterial killing.     

A hypoallergenic vaccine obtained by tail-to-head restructuring of timothy grass pollen profilin, Phl p 12, for the treatment of cross-sensitization to profilin

Profilins are highly cross-reactive allergens in pollens and plant food. In a paradigmatic approach, the cDNA coding for timothy grass pollen profilin, Phl p 12, was used as a template to develop a new strategy for engineering an allergy vaccine with low IgE reactivity. Non-IgE-reactive fragments of Phl p 12 were identified by synthetic peptide chemistry and restructured (rs) as a new molecule, Phl p 12-rs. It comprised the C terminus of Phl p 12 at its N terminus and the Phl p 12 N terminus at its C terminus. Phl p 12-rs was expressed in Escherichia coli and purified to homogeneity. Determination of secondary structure by circular dichroism indicated that the restructuring process had reduced the IgE-reactive alpha-helical contents of the protein but retained its beta-sheet conformation. Phl p 12-rs exhibited reduced IgE binding capacity and allergenic activity but preserved T cell reactivity in allergic patients. IgG Abs induced by immunization of mice and rabbits with Phl p 12-rs cross-reacted with pollen and food-derived profilins. Recombinant Phl p 12-rs, rPhl p 12-rs, induced less reaginic IgE to the wild-type allergen than rPhl p 12. However, the rPhl p 12-rs-induced IgGs inhibited allergic patients' IgE Ab binding to profilins to a similar degree as those induced by immunization with the wild type. Phl p 12-rs specific IgG inhibited profilin-induced basophil degranulation. In conclusion, a restructured recombinant vaccine was developed for the treatment of profilin-allergic patients. The strategy of tail-to-head reassembly of hypoallergenic allergen fragments within one molecule represents a generally applicable strategy for the generation of allergy vaccines.     

COB231 targets amyloid plaques in post‐mortem human brain tissue and in an Alzheimer mouse model

Previous works have shown the interest of naturally fluorescent proflavine derivatives to label Abeta deposits in vitro. This study aimed to further characterize the properties of the proflavine 3‐acetylamino‐6‐[3‐(propargylamino)propanoyl]aminoacridine (COB231) derivative as a probe. This compound was therefore evaluated on human post‐mortem and mice brain slices and in vivo in 18‐month‐old triple transgenic mice APPswe, PS1M146V and tauP301L (3xTgAD) mice presenting the main characteristics of Alzheimer's disease (AD). COB231 labelled amyloid plaques on brain slices of AD patients, and 3xTgAD mice at 10 and 0.1 μM respectively. However, no labelling of the neurofibrillary tangle‐rich areas was observed either at high concentration or in the brain of fronto‐temporal dementia patients. The specificity of this mapping was attested in mice using Thioflavin S and IMPY as positive controls of amyloid deposits. After intravenous injection of COB231 in old 3xTgAD mice, fluorescent amyloid plaques were detected in the cortex and hippocampus, demonstrating COB231 blood–brain barrier permeability. We also controlled the cellular localization of COB231 on primary neuronal cultures and showed that COB231 accumulates into the cytoplasm and not into the nucleus. Finally, using a viability assay, we only detected a slight cytotoxic effect of COB231 (< 10%) for the highest concentration (100 μM).

     

Emergence and Diversification of Dengue 2 Cosmopolitan Genotype in Pakistan, 2011

Major dengue epidemics have been observed in the Indian subcontinent since the 1980s and have occurred with increased hospitalizations and mortality. In 2011, the first major epidemic of dengue occurred in Lahore, the second largest city in Pakistan, and resulted in 21,685 confirmed cases and 350 deaths. To investigate the possible viral causes for the increased epidemic activity, we determined the predominant serotype and characterized the viruses genetically. Of 50 patients carefully selected as probable dengue fever or dengue hemorrhagic fever, 34 were positive by virologic testing (i.e. PCR and/or virus isolation). DENV-2 was detected in 32 patients and DENV-1 in two. A total of 24 partial and three full DENV genomes were sequenced. Phylogenetic analyses of the capsid (C), pre-membrane (prM), and envelope genes comprising 2500 nucleotides in length indicated that all DENV-2 isolates in Pakistan since 2007 form a monophyletic lineage that is endemic in the country. These viruses were all of the cosmopolitan genotype (IV) and most closely related to viruses isolated in India and Sri Lanka in the past two decades. Phylogenetic analyses of data currently available in GenBank suggest that the Cosmopolitan genotype has diverged into two geographically distinct sub-lineages: sub-lineage IV-a has only been observed in Southeast Asia, China and Oceania, while IV-b is prevalent in the Indian subcontinent. These results highlight the increased diversity of dengue viruses as they spread geographically within the region.     

Gene editing the phytoene desaturase alleles of Cavendish banana using CRISPR/Cas9

Bananas are a staple food source and a major export commodity worldwide. The Cavendish dessert banana is a triploid AAA genome type and accounts for around 47% of global production. Being essentially sterile, genetic modification is perhaps the only pathway available to improve this cultivar. In this study, we used the CRISPR/Cas9 gene editing system to deliver a self-cleaving polycistronic guide RNA (gRNA) designed to target exon 1 of the Phytoene desaturase (PDS) gene in the Cavendish cultivar “Williams”. Genotyping of 19 independent events showed a 100% PDS modification rate primarily in the form of insertions (1–105 nt) or deletions (1–55 nt) (indels) at the predicted cleavage site. Tri-allelic disruptive modifications were observed in 63% of plants and resulted in both albinism and dwarfing. Pale green (16%) and wildtype green (21%) phenotypes generally correlated with in-frame indels in at least one of the three PDS alleles. Editing efficiency was dependent on both target site selection and Cas9 abundance. This is the first report of a highly effective CRISPR/Cas9 modification system using a polycistronic gRNA in Cavendish banana. Such an editing platform will be of considerable utility for the development of disease resistance and novel agro-traits in this commercially important cultivar into the future.     

Gender-specific effects of exercise on cardiac pathology in Na+/H+ exchanger overexpressing mice

The Na(+)/H(+) exchanger isoform 1 (NHE1) has been implicated in various cardiac pathologies including ischemia/reperfusion damage to the myocardium and cardiac hypertrophy. It is known that NHE1 levels increase in cardiac disease and we have recently demonstrated that expression of an activated NHE1 protein promotes cardiac hypertrophy in the mouse myocardium. We examined the gender-specific effects of exercise in combination with elevated cardiac expression of NHE1 on the myocardium in mice. Control mice and transgenic mice expressing elevated levels of wild type NHE1 and activated NHE1 were examined. There were gender-specific differences in the effects of NHE1 with exercise. Exercised wild type male mice showed a tendency toward increased heart weight. This was not apparent in female mice expressing elevated NHE1 levels. In some transgenic female mice, there was a significant decrease in the size of the exercised hearts, which was different from what occurred with male mice. Body weight was maintained in exercised control and transgenic male mice; however, it decreased in female mice with exercise more so in transgenic female mice expressing elevated levels of NHE1. Female mice expressing activated NHE1 had elevated HW/BW ratios compared to males, and this was exaggerated by exercise. These results suggest that gender-specific activation of NHE1 may be critical and that NHE1 plays a more critical role in promoting some types of hypertrophy in females in comparison with males.