Tri-locus sequence data reject a “Gondwanan origin hypothesis” for the African/South Pacific crab genus Hymenosoma

Crabs of the family Hymenosomatidae are common in coastal and shelf regions throughout much of the southern hemisphere. One of the genera in the family, Hymenosoma, is represented in Africa and the South Pacific (Australia and New Zealand). This distribution can be explained either by vicariance (presence of the genus on the Gondwanan supercontinent and divergence following its break-up) or more recent transoceanic dispersal from one region to the other. We tested these hypotheses by reconstructing phylogenetic relationships among the seven presently-accepted species in the genus, as well as examining their placement among other hymenosomatid crabs, using sequence data from two nuclear markers (Adenine Nucleotide Transporter [ANT] exon 2 and 18S rDNA) and three mitochondrial markers (COI, 12S and 16S rDNA). The five southern African representatives of the genus were recovered as a monophyletic lineage, and another southern African species, Neorhynchoplax bovis, was identified as their sister taxon. The two species of Hymenosoma from the South Pacific neither clustered with their African congeners, nor with each other, and should therefore both be placed into different genera. Molecular dating supports a post-Gondwanan origin of the Hymenosomatidae. While long-distance dispersal cannot be ruled out to explain the presence of the family Hymenosomatidae on the former Gondwanan land-masses and beyond, the evolutionary history of the African species of Hymenosoma indicates that a third means of speciation may be important in this group: gradual along-coast dispersal from tropical towards temperate regions, with range expansions into formerly inhospitable habitat during warm climatic phases, followed by adaptation and speciation during subsequent cooler phases.     

A new xeromorphic conifer from the Cretaceous of East China

Newly discovered coniferous shoots from the Cretaceous of Nanjing are attributed to the xeromorphic coniferous genus Glenrosa Watson and Fisher, which is found in China as well as in Eurasia for the first time. G. nanjingensis sp. nov. is known from shoots bearing scale-like leaves with thick epidermal cuticle and hypodermis. Stomata are confined to the abaxial surface deeply sunken in stomatal chambers which are filled with papillae. The cuticle, which has been sectioned for TEM, consists of an outer pale amorphous cuticle proper, usually with a peripheral polylamellate zone, and an inner dark cuticular layer containing reticulate fibrillae. G. nanjingensis sp. nov. bears a close resemblance to the type species G. texensis (Fontaine) Watson and Fisher from North America in gross morphology and especially in the characteristic stomatal chambers, but differs in phyllotaxis and in having the hypostomatic leaf with an obtuse apex. G. nanjingensis sp. nov. is believed to have grown near inland marshes or water bodies under hot and arid climates.     

Protein kinase C decreases the apparent affinity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel which plays a major role in Ca2+ signalling. Three isoforms of IP3R have been identified (IP3R-1, IP3R-2 and IP3R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP3R are poorly known. RINm5F cells who express almost exclusively (approximately 90%) the IP3R-3, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) may influence IP3R-3-mediated Ca2+ release. With an immunoprecipitation approach we confirmed that RINm5F cells express almost exclusively the IP3R-3 isoform. With an in vitro phosphorylation approach, we showed that the immunopurified IP3R-3 was efficiently phosphorylated by exogenous PKC. With a direct in cellulo approach and an indirect in cellulo back-phosphorylation approach we showed that phorbol-12-myristate-13-acetate (PMA) causes the phosphorylation of IP3R-3 in intact RINm5F cells. In saponin-permeabilized RINm5F cells, 3-induced Ca2+ release was reduced after a pre-treatment with PMA. PMA also reduced the Ca2+ response of intact RINm5F cells stimulated with carbachol and EGF, two agonists that use different receptor types to activate phospholipase C. These results suggest the existence of a negative feedback mechanism involving two components of the Ca2+ signalling cascade, whereby activated PKC dampens IP3R-3 activity.     

The exceptional stem cell system of Macrostomum lignano: Screening for gene expression and studying cell proliferation by hydroxyurea treatment and irradiation

Background

Flatworms are characterized by an outstanding stem cell system. These stem cells (neoblasts) can give rise to all cell types including germ cells and power the exceptional regenerative capacity of many flatworm species. Macrostomum lignano is an emerging model system to study stem cell biology of flatworms. It is complementary to the well-studied planarians because of its small size, transparency, simple culture maintenance, the basal taxonomic position and its less derived embryogenesis that is more closely related to spiralians. The development of cell-, tissue- and organ specific markers is necessary to further characterize the differentiation potential of flatworm stem cells. Large scale in situ hybridization is a suitable tool to identify possible markers. Distinguished genes identified in a large scale screen in combination with manipulation of neoblasts by hydroxyurea or irradiation will advance our understanding of differentiation and regulation of the flatworm stem cell system.

Results

We have set up a protocol for high throughput large scale whole mount in situ hybridization for the flatworm Macrostomum lignano. In the pilot screen, a number of cell-, tissue- or organ specific expression patterns were identified. We have selected two stem cell- and germ cell related genes – macvasa and macpiwi – and studied effects of hydroxyurea (HU) treatment or irradiation on gene expression. In addition, we have followed cell proliferation using a mitosis marker and bromodeoxyuridine labeling of S-phase cells after various periods of HU exposure or different irradiation levels. HU mediated depletion of cell proliferation and HU induced reduction of gene expression was used to generate a cDNA library by suppressive subtractive hybridization. 147 differentially expressed genes were sequenced and assigned to different categories.

Conclusion

We show that Macrostomum lignano is a suitable organism to perform high throughput large scale whole mount in situ hybridization. Genes identified in such screens – together with BrdU/H3 labeling – can be used to obtain information on flatworm neoblasts.     

Revisiting the trail pheromone components of the red imported fire ant,Solenopsis invicta Buren

Ants use species-specific trail pheromones to coordinate their sophisticated foraging behavior. During the past decades, many trail pheromone components with various structures have been identified in ants, including the red imported fire ant, Solenopsis invicta, a notorious invasive species worldwide. Four compounds, Z,E- (ZEF) and E,E-α-farnesene (EEF), Z,E- (ZEHF) and E,E-α-homofarnesene (EEHF), have been reported as components of S. invicta trail pheromone. However, another study reported an analog of α-farnesene, Z,Z,Z-allofarnesene, as a key trail pheromone component. These contrasting results caused some uncertainty about the trail pheromone composition in S. invicta. In this study, we synthesized ZEF and EEF, ZEHF and EEHF, and reanalyzed the chemicals in the Dufour gland extract and in the trail pheromone fraction of S. invicta worker body extract. The reported isomers of farnesene and homofarnesene were detected and showed trail-following activity, with ZEF as the major compound, while no allofarnesene was found, neither in the Dufour gland extract nor in the whole-body extract. Our results confirm ZEF and EEF, ZEHF and EEHF as trail pheromone components of S. invicta.     

Cas9 Cuts and Consequences; Detecting,Predicting, and Mitigating CRISPR/Cas9 On- and Off-Target Damage

Large deletions and genomic re-arrangements are increasingly recognized as common products of double-strand break repair at Clustered Regularly Interspaced, Short Palindromic Repeats - CRISPR associated protein 9 (CRISPR/Cas9) on-target sites. Together with well-known off-target editing products from Cas9 target misrecognition, these are important limitations, that need to be addressed. Rigorous assessment of Cas9-editing is necessary to ensure validity of observed phenotypes in Cas9-edited cell-lines and model organisms. Here the mechanisms of Cas9 specificity, and strategies to assess and mitigate unwanted effects of Cas9 editing are reviewed; covering guide-RNA design, RNA modifications, Cas9 modifications, control of Cas9 activity; computational prediction for off-targets, and experimental methods for detecting Cas9 cleavage. Although recognition of the prevalence of on- and off-target effects of Cas9 editing has increased in recent years, broader uptake across the gene editing community will be important in determining the specificity of Cas9 across diverse applications and organisms.     

Dryoptkirbioside,A New Fructofuranoside Glycerol,and Other Constituents from Dryopteris kirbi Hook et Grav Rhizomes

A new fructofuranoside glycerol, dryoptkirbioside ( 1 ), along with thirteen known compounds ( 2 - 14 ), was isolated from the MeOH extract of Dryopteris kirbi rhizomes by silica gel column chromatography, Sephadex LH-20 column chromatography, and semipreparative HPLC. The structure of the new compound was determined by analyses of its spectroscopic data including nuclear magnetic resonance (NMR), and high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS) and chemical conversions. The hexane-soluble portion and the EAF_A fraction showed strong activities against lung (A549), breast (MCF-7), and cervical (HeLa) human cancer cell lines (IC₅₀ values ranging from 4.0 to 8.8 μg/mL). Aspidinol P ( 5 ) and aspidinol B ( 6 ) exhibited moderate to low cytotoxicity on the three cell lines (IC₅₀ values ranging from 20.4 to 58.7 μM). The MeOH extract and hexane-soluble portion had excellent activities against Staphylococcus aureus and Bacillus subtilis (MICs 11.7 and 23.4 μg/mL), whereas the AcOEt- and BuOH-soluble portions were significantly active on S. aureus (MICs 46.9 and 93.8 μg/mL). The main fractions EAF_B, EAF_C and nBF_B displayed excellent activity against S. aureus (MICs 11.7 and 23.4 μg/mL). Aspidinol B ( 6 ) had significant activity, while aspidinol P ( 5 ) was moderately active against S. aureus and B. subtilis (MICs 42.0 and 89.5 μM).     

Expression of a truncated form of inositol 1,4,5-trisphosphate receptor type III in the cytosol of DT40 triple inositol 1,4,5-trisphosphate receptor-knockout cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel playing a major role in Ca2+ signaling. Three isoforms of IP3R have been identified and most cell types express different proportions of each isoform. The DT40 B lymphocyte cell line lacking all three IP3R isoforms (DT40IP3R-KO cells) represents an excellent model to re-express any recombinant IP3R and analyze its specific properties. In the study presented here, we confirmed that DT40IP3R-KO cells do not express any IP3-sensitive Ca2+ release channel. However, with an immunoblot approach and a [3H]IP3 binding approach we demonstrated the presence of a C-terminally truncated form of IP3R type III in the cytosolic fraction of DT40IP3R-KO cells. We further showed that this truncated IP3R retained the ability to couple to the Ca2+ entry channel TRPC6. Therefore, a word of caution is offered about the interpretation of results obtained in using DT40IP3R-KO cells to study the cellular mechanisms of Ca2+ entry.     

Effects of Hybridization and Evolutionary Constraints on Secondary Metabolites: The Genetic Architecture of Phenylpropanoids in European Populus Species

The mechanisms responsible for the origin, maintenance and evolution of plant secondary metabolite diversity remain largely unknown. Decades of phenotypic studies suggest hybridization as a key player in generating chemical diversity in plants. Knowledge of the genetic architecture and selective constraints of phytochemical traits is key to understanding the effects of hybridization on plant chemical diversity and ecological interactions. Using the European Populus species P. alba (White poplar) and P. tremula (European aspen) and their hybrids as a model, we examined levels of inter- and intraspecific variation, heritabilities, phenotypic correlations, and the genetic architecture of 38 compounds of the phenylpropanoid pathway measured by liquid chromatography and mass spectrometry (UHPLC-MS). We detected 41 quantitative trait loci (QTL) for chlorogenic acids, salicinoids and flavonoids by genetic mapping in natural hybrid crosses. We show that these three branches of the phenylpropanoid pathway exhibit different geographic patterns of variation, heritabilities, and genetic architectures, and that they are affected differently by hybridization and evolutionary constraints. Flavonoid abundances present high species specificity, clear geographic structure, and strong genetic determination, contrary to salicinoids and chlorogenic acids. Salicinoids, which represent important defence compounds in Salicaceae, exhibited pronounced genetic correlations on the QTL map. Our results suggest that interspecific phytochemical differentiation is concentrated in downstream sections of the phenylpropanoid pathway. In particular, our data point to glycosyltransferase enzymes as likely targets of rapid evolution and interspecific differentiation in the ‘model forest tree’ Populus.