Identification of an estrogen-regulated circadian mechanism necessary for breast acinar morphogenesis

Altered estrogen receptor α (ERA) signaling and altered circadian rhythms are both features of breast cancer. By using a method to entrain circadian oscillations in human cultured cells, we recently reported that the expression of key clock genes oscillates in a circadian fashion in ERA-positive breast epithelial cells but not in breast cancer cells, regardless of their ERA status. Moreover, we reported that ERA mRNA oscillates in a circadian fashion in ERA-positive breast epithelial cells, but not in ERA-positive breast cancer cells. By using ERA-positive HME1 breast epithelial cells, which can be both entrained in vitro and can form mammary gland-like acinar structures in three-dimensional (3D) culture, first we identified a circuit encompassing ERA and an estrogen-regulated loop consisting of two circadian clock genes, PER2 and BMAL1. Further, we demonstrated that this estrogen-regulated circuit is necessary for breast epithelial acinar morphogenesis. Disruption of this circuit due to ERA-knockdown, negatively affects the estrogen-sustained circadian PER2-BMAL1 mechanism as well as the formation of 3D HME1 acini. Conversely, knockdown of either PER2 or BMAL1, by hampering the PER2-BMAL1 loop of the circadian clock, negatively affects ERA circadian oscillations and 3D breast acinar morphogenesis. To our knowledge, this study provides the first evidence of the implication of an ERA-circadian clock mechanism in the breast acinar morphogenetic process.     

The multi-locus H-2D w16 region has an organization distinct from the D d region

Genomic DNA blot analyses using probes derived from the BALB/c 3 flanking region of the L ^d gene (L ^d 3 fl-C) and from near the BALB/c D3 ^d gene (50.2A) indicate that the B10.GAA37 mouse strain has a multi-locus D (D ^w16) region distinct from the five-gene organization observed in the D ^d and D ^q regions. To isolate the D ^w16 region class I genes, a genomic B10. GAA37-EMBL3 library was generated and screened with probes that preferentially hybridize to K and D region class I genes. Hybridization analyses of the isolated clones with L ^d derived oligonucleotide probes suggested that one of the clones contained the L ^w16 gene, whereas several other clones contained the L ^w16 gene. The sequence of the D ^w16 gene is most similar to that of the D ^p gene, particularly in the 3 half. Furthermore, the L ^w16 gene is quite similar in the 5 half and virtually identical in the 3 half to the L ^d gene, indicating that L ^w16, but not D ^w16, is a member of the L ^d gene family. Collectively, these data suggest that, through a D region recombination event, the novel D ^w16 region may have been assembled from primordial counterparts of the D ^p and L ^d genes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M60774-M60776, and M62759.     

Evaluation of biorational insecticides and DNA barcoding as tools to improve insect pest management in lablab bean (Lablab purpureus) in Bangladesh

Lablab bean (Lablab purpureus) is a popular vegetable crop in Bangladesh, but farmers growing this crop experience significant losses to insect pests despite heavy reliance on conventional insecticides. We conducted field studies to improve pest management in lablab bean by testing biorational insecticides as alternatives to conventional insecticides for the control of pod borers (Maruca vitrata) and aphids (Aphis craccivora), and characterizing flower-inhabiting thrips as an emerging pest in this crop. In field experiments, spinosad was the most promising biorational we tested, suppressing pod-boring caterpillars more effectively than thiamethoxam or quinalphos. In contrast, azadirachtin (neem) did not significantly suppress any of the insect pests we measured, although target aphid populations were generally low at our research site. Using DNA barcoding at the coxI locus combined with morphological identification, we found eight thrips taxa inhabiting lablab bean flowers, dominated by Megalurothrips usitatus and M. distalis/peculiaris. A preliminary regression analysis indicated that these flower-inhabiting thrips reduced lablab bean yield. Our results suggest that spinosad may be useful within lablab bean IPM programs, and that these programs will likely need to incorporate tactics against thrips to effectively protect yield. Finally, we found that DNA barcoding was a valuable tool for pest identification in an understudied crop and region, but that to reach its full potential will require an investment in more comprehensive reference libraries.     

Morphological and mitochondrial DNA variation revealed an undescribed lineage under the genus Sperata (Bagridae) in Bangladesh

Catfishes Sperata are popular, known for its taste and nutritional value, and are found naturally in wide variety of freshwaters in South Asia. The taxonomy of Sperata spp., sampled from Hakaluki Haor in Bangladesh, was re‐evaluated based on morphological variation and DNA barcoding. The collective variation in morphometric characters and mitochondrial DNA revealed an undescribed old and well‐separated lineage under the genus Sperata along with two previously known Sperata aor and Sperata seenghala in Bangladesh. Analyses of morphological traits suggested significant differentiation among Sperata species. The variation in mitochondrial DNA supported the distinct lineage and taxonomical discrimination. Sperata aor diverged earlier from the new lineage and Sperata seenghala with a divergence of 5.39 (CI: 3.91–7.19) Mya (PP > 90). Sperata seenghala and the new lineage shared a most recent common ancestry, which diverged from each other around 3.41 (CI: 2.24–4.62) Mya (PP > 90). Thus, the newly identified lineage could be a subspecies of S. seenghala or even a species under the genus Sperata. The information of the study will be useful for conservation, sustainable management and selective breeding of the putative species, including previously reported S. aor and S. seenghala in Bangladesh.     

Efficient enhancement of gallic acid accumulation in cell suspension cultures of <Emphasis Type="Italic">Barringtonia racemosa</Emphasis> L. by elicitation

The bioactive compound, bacoside A, has immense importance for the treatment of memory disorders and Alzheimer’s disease. Due to the growing commercial interest in the herb, Bacopa monnieri, it has been listed as highly endangered species. The present study was aimed at enhancing the production of bacoside A using an alternative technology of plant cell suspension culture. Initial experiments of docking simulations using bacoside A showed good inhibition of acetyl cholinesterase (binding energy value of ??20 kcal/mol), when comparison was made with other phytocompounds and the synthetic drug for Alzheimer’s disease. In vitro experiments established that B. monnieri cell suspension culture can be developed in Murashige and Skoog medium containing containing 0.1 mg/L benzylaminopurine and 0.5 mg/L naphthalene acetic acid. Plackett–Burman studies predicted that the most effective factors for maximum biomass production were inoculum size (t-value of 4.87), sucrose concentration (t-value of 0.25) and KH₂PO₄ concentration (t-value of 0.007). The nitrate to ammonium ratio (t-value of ? 0.42) did not have significant effect on the cell suspension biomass. The optimum concentration of the crucial variables obtained from a central composite design were—inoculum size of 2 g/L, sucrose concentration of 30 g/L and KH₂PO₄ concentration of 1.24 mM in one-sixth strength MS medium. The best model for optimum production of biomass and bacoside A was experimentally verified and the correlation between the predicted and actual values was found to be 99% for biomass and 94% for bacoside A production. The experimental results have been discussed in the present work.     

Rubrioxytricha guamensis nov. spec. (Ciliophora,Spirotricha), a Novel Hypotrich Ciliate from Guam (United States), Micronesia

The oxytrichid ciliate Rubrioxytricha guamensis nov. spec. isolated from water samples collected from a small freshwater pond near the Hagåtña River in Hagåtña, Guam (United States territory), Micronesia, was investigated, using live observation and protargol impregnation. The morphology, ontogenesis, and molecular phylogeny inferred from the small‐subunit rRNA gene sequences were studied. The new species is mainly characterized by a cell size of about 100 × 35 μm in vivo, two elongate ellipsoidal macronuclear nodules and two micronuclei, a single contractile vacuole, a colorless cytoplasm, yellowish cortical granules, arranged in short rows and in small groups, an adoral zone occupying about 34% of body length and comprising 27 membranelles on average, about 27 cirri each in the right and left marginal rows, 18 frontoventral transverse cirri, four dorsal kineties including one dorsomarginal row, and one or two caudal cirri at the posterior end of dorsal kinety 3. The ontogenesis of the new species is similar to that of Rubrioxytricha indica Naqvi et al., 2006. Phylogenetic analyses based on SSU rRNA gene sequences consistently place the new species within the family Oxytrichidae Ehrenberg, 1838, where it clustered with other Rubrioxytricha species, viz., R. tsinlingensis, R. ferruginea, and R. haematoplasma.     

Distribution of 5S and 18S–28S rDNA loci in a tetraploid cotton (Gossypium hirsutum L.) and its putative diploid ancestors

The most widely cultivated species of cotton,Gossypium hirsutum, is a disomic tetraploid (2n=4x=52). It has been proposed previously that extant A- and D-genome species are most closely related to the diploid progenitors of the tetraploid. We used fluorescent in situ hybridization (FISH) to determine the distribution of 5S and 18S-28S rDNA loci in the A-genome speciesG. herbaceum andG. arboreum, the D-genome speciesG. raimondii andG. thurberi, and the AD tetraploidG. hirsutum. High signal-to-noise, single-label FISH was used to enumerate rDNA loci, and simultaneous, dual-label FISH was used to determine the syntenic relationships of 5S rDNA loci relative to 18S–28S rDNA loci. These techniques provided greater sensitivity than our previous methods and permitted detection of six newG. hirsutum 18S–28S rDNA loci, bringing the total number of observed loci to 11. Differences in the intensity of the hybrizization signal at these loci allowed us to designate them as major, intermediate, or minor 18–28S loci. Using genomic painting with labeled A-genome DNA, five 18S–28S loci were localized to theG. hirsutum A-subgenome and six to the D-subgenome. Four of the 11 18S–28S rDNA loci inG. hirsutum could not be accounted for in its presumed diploid progenitors, as both A-genome species has three loci and both D-genome species had four.G. hirsutum has two 5S rDNA loci, both of which are syntenic to major 18S–28S rDNA loci. All four of the diploid genomes wer examined contained a single 5S locus. InG. herbaceum (A₁) andG. thurberi (D₁), the 5S locus is syntenic to a major 18S–28S locus, but inG. arboreum (A₂) andG. raimondii (D₅), the proposed D-genome progenitor ofG. hirsutum, the 5S loci are syntenic tominor and intermediate 18S–28S loci, respecitively. The multiplicity, variation in size and site number, and lack of additivity between the tetraploid species and its putative diploid ancestors indicate that the behavior of rDNA loci in cotton is nondogmatic, and considerably more complex and dynamic than previously envisioned. The relative variability of 18S–28S rDNA loci versus 5S rDNA loci suggests that the behavior of tandem repearts can differ widely. Edited by: R. Appels     

The proliferation of fungal hyphae in soils supporting mycorrhizal and non-mycorrhizal plants

This study investigated the impact of mycorrhizal plants, non-mycorrhizal plants and soil organic matter on the relative abundance of soil hyphae perceived to belong to indigenous arbuscular mycorrhizal (AM) plants. The mycorrhizal plants corn (Zea mays L.) and barley (Hordeum vulgare L.) and a non-mycorrhizal plant, canola (Brassica napus L.), were grown in unsterilized soil in pots inoculated with mycorrhizal corn root fragments. The abundance of hyphae was measured after 5 weeks and the response of fungal growth to the addition of corn residues in the absence of plants was assessed. The abundance of hyphae was higher in the presence of the mycorrhizal plants than in the other treatments. AM hyphae present under mycorrhizal plants accounted for more than 83% of the measured hyphae. The levels of root colonization of 32% in corn and 27% in barley confirmed the mycorrhizal status of the experimental plants. Only a few points of entry were observed in canola, the non-host plant. The percentage of mycorrhizal colonization was positively related (R ²?=?0.85) to the abundance of soil hyphae, indicating that AM hyphae were the major component of the soil hyphae in the presence of mycorrhizal plants in this study.