Clonal populations of the mouse mammary cell line,COMMA-D,which retain capability of morphogenesis in vivo

Summary Clonal populations were isolated from the mouse mammary cell line, COMMA-D, by transfection with a dominant-selectable gene, pSV2Neo, which confers resistance to the antibiotic, G418. Seven of twenty-four clones isolated retained the ability of the parental line to repopulate cleared mammary fat pads in vivo as ductal-alveolar hyperplasias. Two sublines designated CDNR2 and CDNR4 retained hyperplastic growth potential after multiple passages in vitro with low incidence of tumor formation. A third subpopulation, CDNR1, contained a single integration site for the pSV2Neo plasmid indicating a bonafide clonal origin for this subline. CDNR1 cells displayed heterogeneous growth phenotypes in vivo including hyperplasia, adenocarcinoma, and bone formation. Functional differentiation of CDNR1 cells organized as alveolarlike structures in vivo or on floating collagen gels in vitro was observed as determined by immunoperoxidase staining for the milk-specific protein, casein. Overall, the results indicate that a subset of cells from the COMMA-D cell line may be functionally analogous to stem cells existing in the mammary gland. Supported by NCI research grants CA-38650, CA-33369, CA-39017, and CA-25215.     

Transfer of a marine DNA virus fromEctocarpus toFeldmannia (Ectocarpales,Phaeophyceae): aberrant symptoms and restitution of the host

Summary The marine brown algaEctocarpus siliculosus is invaded by a polyhedric virus, whose genome consists of circular, double-stranded DNA. In laboratory experiments this virus can infect a different host species,Feldmannia simplex. InfectedFeldmannia plants show severe somatic malformations. However, no functional virus particles are formed. SuchFeldmannia plants recover to resume a normal, symptom-free appearance. This result raises the possibility of intergeneric gene transfer in the natural habitat.     

Genome Sequence of Hybrid Vibrio cholerae O1 MJ-1236, B-33, and CIRS101 and Comparative Genomics with V. cholerae

The genomes of Vibrio cholerae O1 Matlab variant MJ-1236, Mozambique O1 El Tor variant B33, and altered O1 El Tor CIRS101 were sequenced. All three strains were found to belong to the phylocore group 1 clade of V. cholerae, which includes the 7th-pandemic O1 El Tor and serogroup O139 isolates, despite displaying certain characteristics of the classical biotype. All three strains were found to harbor a hybrid variant of CTXΦ and an integrative conjugative element (ICE), leading to their establishment as successful clinical clones and the displacement of prototypical O1 El Tor. The absence of strain- and group-specific genomic islands, some of which appear to be prophages and phage-like elements, seems to be the most likely factor in the recent establishment of dominance of V. cholerae CIRS101 over the other two hybrid strains.Vibrio cholerae, a bacterium autochthonous to the aquatic environment, is the causative agent of cholera, a life-threatening disease that causes severe, watery diarrhea. Cholera bacteria are serogrouped based on their somatic O antigens, with more than 200 serogroups identified to date (6). Only toxigenic strains of serogroups O1 and O139 have been identified as agents of cholera epidemics and pandemics; serogroups other than O1 and O139 have the potential to cause mild gastroenteritis or, rarely, local outbreaks. Genes coding for cholera toxin (CTX), ctxAB, and other virulence factors have been shown to reside in bacteriophages and various mobile genetic elements. In addition, V. cholerae serogroup O1 is differentiated into two biotypes, classical and El Tor, by a combination of biochemical traits, by sensitivity to biotype-specific bacteriophages, and more recently by nucleotide sequencing of specific genes and by molecular typing (5, 17, 19).There have been seven pandemics of cholera recorded throughout human history. The seventh and current pandemic began in 1961 in the Indonesian island of Sulawesi and subsequently spread to Asia, Africa, and Latin America; the six previous pandemics are believed to have originated in the Indian subcontinent. Isolates of the sixth pandemic were almost exclusively of the O1 classical biotype, whereas the current (seventh) pandemic is dominated by the V. cholerae O1 El Tor biotype as the causative agent, a transition occurring between 1923 and 1961. Today, the disease continues to remain a scourge in developing countries, confounded by the fact that V. cholerae is native to estuaries and river systems throughout the world (8).Over the past 20 years, several new epidemic lineages of V. cholerae O1 El Tor have emerged (or reemerged). For example, in 1992, a new serogroup, namely, O139 of V. cholerae, was identified as the cause of epidemic cholera in India and Bangladesh (25). The initial concern was that a new pandemic was beginning; however, the geographic range of V. cholerae O139 is currently restricted to Asia. Additionally, V. cholerae O1 hybrids and altered El Tor variants have been isolated repeatedly in Bangladesh (Matlab) (23, 24) and Mozambique (1). Altered V. cholerae O1 El Tor isolates produce cholera toxin of the classical biotype but can be biotyped as El Tor by conventional phenotypic assays, whereas V. cholerae O1 hybrid variants cannot be biotyped based on phenotypic tests and can produce cholera toxin of either biotype. These new variants have subsequently replaced the prototype seventh-pandemic V. cholerae O1 El Tor strains in Asia and Africa, with respect to frequency of isolation from clinical cases of cholera (27).Here, we report the genome sequence of three V. cholerae O1 variants, MJ-1236, a Matlab type I hybrid variant from Bangladesh that cannot be biotyped by conventional methods, CIRS101, an altered O1 El Tor isolate from Bangladesh which harbors ctxB of classical origin, and B33, an altered O1 El Tor isolate from Mozambique which harbors classical CTXΦ, and we compare their genomes with prototype El Tor and classical genomes. From an epidemiological viewpoint, among the three variants characterized in this study, V. cholerae CIRS101 is currently the most “successful” in that strains belonging to this type have virtually replaced the prototype El Tor in Asia and many parts of Africa, notably East Africa. This study, therefore, gives us a unique opportunity to understand why V. cholerae CIRS101 is currently the most successful El Tor variant.     

Prevalence of potentially pathogenic culturable bacteria on eggshells and in cloacae of female Pied Flycatchers in a temperate habitat in central Spain

ABSTRACT Bacteria grow on avian eggshells and thus can potentially cause diseases in developing embryos. Little is known about culturable bacteria colonizing avian eggshells in free‐living birds, with most studies restricted to poultry. Our objective was to examine the culturable bacterial array growing on eggshells during incubation that could negatively affect hatching success of Pied Flycatchers (Ficedula hypoleuca) in a temperate montane habitat in central Spain. Cloacal culturable bacteria of females were also analyzed because bacteria can be vertically transmitted from females to eggs. We used fecal samples as surrogates of cloacal samples due to the small size of sampled birds. We found that eggshells and female cloacae of Pied Flycatchers harbored 24 and 40 bacterial families and species, respectively, but only a few in each clutch and each cloaca. Rod‐shaped gram‐negative bacteria and bacteria in the family Pseudomonadaceae were the most common bacteria on eggshells during early and late incubation and in female cloacae. Although based on small sample sizes, we found that females with rod‐shaped gram‐negative bacteria in their cloacae laid eggs that also had these bacteria, providing possible evidence for vertical transmission. We found no evidence for vertical transmission of Pseudomonadaceae, suggesting a possible environmental source for these bacteria. The prevalence of bacterial morphological types and major taxonomical categories on eggshells did not vary from early to late stages of incubation, providing support for the hypothesis that incubation may have bacteriostatic effects on bacterial proliferation on eggshells. Despite being primary egg invaders in poultry, we detected no effects of culturable Pseudomonadaceae or Pseudomonas luteola on hatching success. Our study represents the first to examine the culturable bacteria growing on the eggshells of a wild bird in a temperate habitat and additional studies based on culture‐independent techniques are required to confirm our results.     

CX3CR1 Is Expressed by Human B Lymphocytes and Meditates CX3CL1 Driven Chemotaxis of Tonsil Centrocytes

Background

Fractalkine/CX₃CL1, a surface chemokine, binds to CX₃CR1 expressed by different lymphocyte subsets. Since CX₃CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX₃CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood.

Methodology/Principal Findings

We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX₃CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX₃CR1 but only germinal centre B cells were attracted by soluble CX₃CL1 in a transwell assay. CX₃CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX₃CR1⁺ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX₃CL1. ELISA assay showed that soluble CX₃CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX₃CL1 did not attract spleen B cells from wild type mice. OVA immunized CX₃CR1⁻/⁻ or CX₃CL1⁻/⁻ mice showed significantly decreased specific IgG production compared to wild type mice.

Conclusion/Significance

We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX₃CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.     

Do Size and Insecticide Treatment Matter? Evaluation of Different Nets against Phlebotomus argentipes,the Vector of Visceral Leishmaniasis in Nepal

In the Indian subcontinent, Leishmania donovani, the parasite causing visceral leishmaniasis (VL) is transmitted by the sand fly vector Phlebotomus argentipes. Long lasting insecticide treated nets (LN) have been postulated as alternative or complement to Indoor Residual Spraying but there are few field studies evaluating the entomological efficacy of different nets against this vector. We conducted two crossover trials in a VL endemic area in Nepal to compare the barrier effect of (1) LN with different mesh sizes (156 holes/inch² vs 625 holes/inch²) and (2) alpha-cypermethrin treated LN and untreated nets having the same mesh size (156 holes/inch²). Each crossover trial had two arms consisting of a sequence of two different nets for 8 nights. We used 10 cattle sheds per trial. A cow placed under the net was used as bait. CDC light traps placed inside the nets were used to evaluate the number of P. argentipes crossing the net barrier. Negative binomial generalized estimating equation (GEE) population-averaged models adjusted by night and sequence were used to estimate the barrier effect of the different nets. The crossover trials conducted in a rural village in Morang district (South-eastern Nepal) demonstrated that reducing the size of the holes in treated nets (625 holes/inch²) increased the barrier effect of LN by 77% (95% confidence interval (CI): 56%–88%) compared with treated nets with larger holes (156 holes/inch²). Treating nets with alpha-cypermethrin reduced the number of P. argentipes captured inside the nets by 77% (95% CI: 27%–93%) compared with untreated nets. The effectiveness and acceptability of finer mesh pyrethroid treated LN should be tested for VL prevention in a randomized controlled trial.     

A C-terminal motif targets Hedgehog to axons, coordinating assembly of the Drosophila eye and brain

The developmental signal Hedgehog is distributed to two receptive fields by the photoreceptor neurons of the developing Drosophila retina. Delivery to the retina propagates ommatidial development across a precursor field. Transport along photoreceptor axons induces the development of postsynaptic neurons in the brain. Hedgehog is composed of N-terminal and C-terminal domains that dissociate in an autoproteolytic reaction that attaches cholesterol to the N-terminal cleavage product. Here, we show that the N-terminal domain is targeted to the retina when synthesized in the absence of the C-terminal domain. In contrast to studies that have focused on cholesterol as a determinant of subcellular localization, we find that the C-terminal domain harbors a conserved motif that overrides retinal localization, sending most of the autocleavage products into vesicles bound for growth cones or synapses. Competition between targeting signals at the opposite ends of Hedgehog apparently controls the match between eye and brain development.