Detection of MET mRNA in gastric cancer in situ. Comparison with immunohistochemistry and sandwich immunoassays

Determination of predictive biomarkers by immunohistochemistry (IHC) relies on antibodies with high selectivity. RNA in situ hybridization (RNA ISH) may be used to confirm IHC and may potentially replace it if suitable antibodies are not available or are insufficiently selective to discriminate closely related protein isoforms. We validated RNA ISH as specificity control for IHC and as a potential alternative method for selecting patients for treatment with MET inhibitors. MET, the HGF receptor, is encoded by the MET proto-oncogene that may be activated by mutation or amplification. MET expression and activity were tested in a panel of control cell lines. MET could be detected in formalin fixed paraffin, embedded (FFPE) samples by IHC and RNA ISH, and this was confirmed by sandwich immunoassays of fresh frozen samples. Gastric cancer cell lines with high MET expression and phosphorylation of tyrosine-1349 respond to the MET inhibitor, BAY-853474. High expression and phosphorylation of MET is a predictive biomarker for response to MET inhibitors. We then analyzed MET expression and activity in a matched set of FFPE vs. fresh frozen tumor samples consisting of 20 cases of gastric cancer. Two of 20 clinical samples investigated exhibited high MET expression with RNA ISH and IHC. Both cases were shown by sandwich immunoassays to exhibits strong functional activity. Expression levels and functional activity in these two cases were in a range that predicted response to treatment. Our findings indicate that owing to its high selectivity, RNA ISH can be used to confirm findings obtained by IHC and potentially may replace IHC for certain targets if no suitable antibodies are available. RNA ISH is a valid platform for testing predictive biomarkers for patient selection.     

Drivers of beta diversity of macroinvertebrate communities in tropical forest streams

1. There has recently been increasing interest in patterns of beta diversity but we still lack a comprehensive understanding of these patterns in various regions (e.g. the tropics), ecosystems (e.g. streams) and organism groups (e.g. invertebrates). 2. Our aim was to investigate the patterns of beta diversity of stream macroinvertebrates in relation to key environmental (i.e. stream size, pH and habitat degradation) and geographical variables (i.e. latitude, longitude, altitude) in a tropical region. We surveyed a total of 8–10 riffle sites in each of 34 streams (altogether 337 riffle sites were sampled) in Peninsular Malaysia to examine variation in macroinvertebrate community composition at within‐stream and among‐stream scales. 3. Based on test of homogeneity of dispersion, we found that the streams studied differed significantly in within‐stream variation in community composition (i.e. among‐site variation of within stream beta diversity). The patterns were similar based on Bray–Curtis coefficient on abundance data, Sorensen coefficient on presence–absence data and Simpson coefficient on presence–absence data. We also found that within‐stream beta diversity was significantly related to stream size, pH and latitude, with each of these variables individually accounting for around 20% of the variation in beta diversity in simple regressions, while the total variation explained by the three significant variables amounted to around 50% in multiple regressions. By contrast, habitat degradation, longitude and altitude were not significantly related to beta diversity. We also found that the factor drainage basin accounted for much of the variation in beta diversity in general linear models, suppressing the effects of environmental variables. 4. We concluded that within‐stream beta diversity is mainly related to a combination of the identity of a drainage basin and stream environmental factors. Our findings provide important background for stream environmental assessment and conservation planning by emphasising that (i) macroinvertebrate communities within streams are not homogeneous, but show considerable beta diversity, (ii) streams differ in their degree of within‐stream beta diversity, (iii) stream size and water pH should be considered in applied contexts related to within‐stream beta diversity and (iv) historical effects may be different in different drainage basins and may affect present‐day patterns of within‐stream beta diversity.     

Housing with Females Increases Testosterone and Cortisol Levels in Captive Groups of Black-Handed Spider Monkeys (Ateles geoffroyi)

The black‐handed spider monkey (Ateles geoffroyi) is a seasonal reproducer that requires a seclusiveness to copulate and has a fusion–fission social system. These features impose important restrictions to achieve reproduction of captive animals. We investigated if group composition in captive spider monkeys has any endocrine effects. We compared testosterone and cortisol concentrations during the mating season in all‐male and multifemale–multimale groups to study if the former condition impairs reproductive potential and increases stress. Concentrations of testosterone and cortisol of males living with females were higher than those of all‐male groups. In the multifemale–multimale condition, dominant males had the highest levels of testosterone, while the youngest males showed the highest concentrations of cortisol. Results show that males adjust well to isosexual grouping, this being an appropriate condition to keep animals when controlled reproduction is sought. Zoo Biol 31:490;–497, 2012. © 2012 Wiley Periodicals, Inc.     

Isolation,Identification, Biocontrol Activity,and Plant Growth Promoting Capability of a Superior Streptomyces tricolor Strain HM10

Streptomyces is a genus with known biocontrol activity, producing a broad range of biologically active substances. Our goal was to isolate local Streptomyces species, evaluate their capacity to biocontrol the selected phytopathogens, and promote the plant growth via siderophore and indole acetic acid (IAA) production and phosphate solubilization. Eleven isolates were obtained from local soil samples in Saudi Arabia via the standard serial dilution method and identified morphologically by scanning electron microscope (SEM) and 16S rRNA amplicon sequencing. The biocontrol of phytopathogens was screened against known soil-borne fungi and bacteria. Plant growth promotion capacity was evaluated based on siderophore and IAA production and phosphate solubilization capacity. From eleven isolates obtained, one showed 99.77% homology with the type strain Streptomyces tricolor AS 4.1867, and was designated S. tricolor strain HM10. It showed aerial hyphae in SEM, growth inhibition of ten known phytopathogens in in vitro experiments, and the production of plant growth promoting compounds such as siderophores, IAA, and phosphate solubilization capacity. S. tricolor strain HM10 exhibited high antagonism against the fungi tested (i.e., Colletotrichum gloeosporides with an inhibition zone exceeding 18 mm), whereas the lowest antagonistic effect was against Alternaria solani (an inhibition zone equal to 8 mm). Furthermore, the most efficient siderophore production was recorded to strain HM8, followed by strain HM10 with 64 and 22.56 h/c (halo zone area/colony area), respectively. Concerning IAA production, Streptomyces strain HM10 was the most effective producer with a value of 273.02 μg/ml. An autochthonous strain S. tricolor HM10 should be an important biological agent to control phytopathogens and promote plant growth.     

Phylogeny of Six Genera of the Subclass Haptoria (Ciliophora,Litostomatea) Inferred from Sequences of the Gene Coding for Small Subunit Ribosomal RNA

ABSTRACT. The small subunit ribosomal RNA genes of nine species belonging to six genera of litostome ciliates, namely Amphileptus aeschtae, Chaenea teres, Chaenea vorax, Lacrymaria marina, Litonotus paracygnus, Loxophyllum sp.‐GD‐070419, Loxophyllum jini, Loxophyllum rostratum, and Phialina salinarum, were sequenced for the first time. Phylogenetic trees were constructed using different methods to assess the inter‐ and intra‐generic relationships of haptorians, of which Chaenea, Lacrymaria, Litonotus, and Phialina were analyzed for the first time based on molecular data. Monophyly of the order Pleurostomatida was strongly confirmed, and the two existing families of pleurostomatids, created on the basis of morphology, were confirmed by molecular evidence. Within the Pleurostomatida, Siroloxophyllum utriculariae occupied a well‐supported position basal to the Loxophyllum clade, supporting the separation of these genera from one another. Both the subclass Haptoria and the order Haptorida were partially unresolved, possibly paraphyletic assemblages of taxa in all analyses, creating doubts about the traditional placement of some haptorid taxa. The existing sequence of L. rostratum in GenBank (DQ411864) was conspicuously different from that of the isolate from Qingdao, China sequenced in the present work, indicating that they are different species. The isolate from Qingdao was verified as L. rostratum by morphological analysis, and the published morphology of existing GenBank record of L. rostratum is different from it. Based on both morphological and molecular evidence, the latter may be congeneric with an undescribed species of Loxophyllum from Guangdong Province, China.     

Re‐targeting of a plant defense protease by a cyst nematode effector

Plants mount defense responses during pathogen attacks, and robust host defense suppression by pathogen effector proteins is essential for infection success. 4E02 is an effector of the sugar beet cyst nematode Heterodera schachtii. Arabidopsis thaliana lines expressing the effector‐coding sequence showed altered expression levels of defense response genes, as well as higher susceptibility to both the biotroph H. schachtii and the necrotroph Botrytis cinerea, indicating a potential suppression of defenses by 4E02. Yeast two‐hybrid analyses showed that 4E02 targets A. thaliana vacuolar papain‐like cysteine protease (PLCP) ‘Responsive to Dehydration 21A’ (RD21A), which has been shown to function in the plant defense response. Activity‐based protein profiling analyses documented that the in planta presence of 4E02 does not impede enzymatic activity of RD21A. Instead, 4E02 mediates a re‐localization of this protease from the vacuole to the nucleus and cytoplasm, which is likely to prevent the protease from performing its defense function and at the same time, brings it in contact with novel substrates. Yeast two‐hybrid analyses showed that RD21A interacts with multiple host proteins including enzymes involved in defense responses as well as carbohydrate metabolism. In support of a role in carbohydrate metabolism of RD21A after its effector‐mediated re‐localization, we observed cell wall compositional changes in 4E02 expressing A. thaliana lines. Collectively, our study shows that 4E02 removes RD21A from its defense‐inducing pathway and repurposes this enzyme by targeting the active protease to different cell compartments.     

Molecular phylogeny of Solms‐laubachia (Brassicaceae) s.l., based on multiple nuclear and plastid DNA sequences,and its biogeographic implications

Abstract The Hengduan Mountains region of south‐west China is a noted biodiversity hotspot, but the geographic origins and historical assembly of its rich endemic flora, including the sky‐island species of Solms‐laubachia Muschl. (Brassicaceae), have been little studied. Previous molecular studies on the phylogeny of Solms‐laubachia showed it to be paraphyletic, leading to considerable expansion not only of its taxonomic limits, but also its geographic range, with the inclusion of taxa from outside the Hengduan region. However, these studies provided little resolution of interspecific relationships, preventing inferences about historical biogeography within the clade. In the present study, new sequence data from two nuclear genes (LEAFY and G3pdh) and two chloroplast intergenic spacers (petN–psbM and psbM–trnD) were combined with existing markers to increase phylogenetic signals. Phaeonychium villosum (Maxim.) Al‐Shehbaz was found to be nested within Solms‐laubachia s.l. In general, phylogenetic relationships appear to be a good predictor of geography, with the Hengduan Mountain endemics embedded in a paraphyletic grade of species from the western Himalayas and central Asia, but they also imply morphological homoplasy. Incongruence was detected between the nuclear and chloroplast gene trees, perhaps resulting from incomplete lineage sorting of ancestral polymorphisms. The crown age of Solms‐laubachia s.l. was estimated to be approximately 1.42–3.68 mya, using Bayesian relaxed molecular clock analysis. Historical biogeographic analysis using a parametric dispersal–extinction–cladogenesis model inferred central Asia and the western Himalayas as most probable ancestral range of Solms‐laubachia s.l., and estimated higher rates of eastward expansion than westward during the diversification of descendant lineages. In summary, our results suggest that Solms‐laubachia s.l. originated during the Pliocene in central Asia, and subsequently migrated eastward into the Hengduan Mountains, colonizing sky‐island, alpine scree‐slope habitats that may have provided novel ecological opportunity and accelerated speciation, ultimately establishing this region as the present center of diversity of the genus.     

Age-dependent changes in fecal 17β-estradiol and progesterone concentrations in female spider monkeys (Ateles geoffroyi)

The objective of this study was to investigate whether sex steroids decreased with age in female black-handed spider monkeys (Ateles geoffroyi). Fecal concentrations of 17β-estradiol and progesterone (five samples/wk) and the number of ovulatory and anovulatory cycles were compared between adult (n = 3) and aged females (n = 2). All animals (regardless of age) had higher 17β-estradiol concentrations during the fertile than the nonfertile phases. However, during the fertile phase, concentrations of this hormone were significantly higher in adult females. Conversely, progesterone concentrations varied normally throughout the menstrual cycle in both adult and aged animals, with no significant difference between age classes. Similarly, there was no significant effect of age on the number of ovulatory and anovulatory cycles. In conclusion, we inferred that the aged female spider monkeys did not reach menopause, instead they remained in a perimenopausal period characterized by changes in fecal concentrations of ovarian steroids and hypothalamus-hypophysis-ovary axis activity, as well as irregular menstrual flows, for prolonged intervals.