Multiple forms of molybdate-stabilized glucocorticoid-receptor complexes from HeLa cell cytosol

Summary The investigation on hydrodynamic parameters of molybdate-stabilized glucocorticoid-receptor complexes from HeLa cell cytosol permitted resolution of four distinct forms. The first one could be detected in concentrated cytosols at low salt concentrations, and had the following properties: sedimentation coefficient = 9 S; R _s = 9.3 nm; M _r = 357,800; f/f _o = 1.83; axial ratio (prolate ellipsoid) = 16. When these cytosol extracts were diluted, a second form could be detected with sedimentation coefficient = 8.3 S; R _s = 9.05 nm; M _r = 320,700;f/f _o = 1.84; axial ratio = 16. Under high salt conditions, glucocorticoid-receptor complexes in concentrated cytosol had the following properties: sedimentation coefficient = 6.4 S; R _s, = 6.7 nm; M _r = 183,100;f/f _o = 1.64; axial ratio = 12. When either these cytosol extracts were diluted, or glucocorticoid-receptor complexes were subjected to repeated analysis, a fourth form was detected with sedimentation coefficient = 3.76 S; R _s = 5.67; M _r = 91,000; f/f _o = 1.75; axial ratio = 14. Besides salt concentration and dilution, the time elapsed between sample dilution and analysis appeared to affect the hydrodynamic properties of glucocorticoid-receptor complexes. On the basis of our findings, it has been concluded that the most likely structure of molybdate-stabilized glucocorticoid-receptor complexes of HeLa cell cytosol can be represented by association of monomers in homodimers, and homotetramers. A homotrimer form could not be deduced from our findings, and the 320,700 glucocorticoid-receptor complex we observed has been suggested to represent an unresolved mixture of trimers and tetramers.     

Biogeographical patterns of endemic diversity and its conservation in Australia's artesian desert springs

Aim

Springs in the Australian arid zone are distinct from other waterways because they house a large number of endemic species. We aimed to assess spatial patterns in endemic diversity at a basin‐wide scale and whether environmental features can help to explain them. In doing so, we take the opportunity to summarize the current state of conservation in the system.

Location

Great Artesian Basin (GAB), arid and semiarid regions of eastern Australia

Methods

We combine data regarding the location of springs with published GIS layers regarding environmental characteristics and a literature review of all species and subspecies documented in the published literature to be endemic to GAB springs.

Results

We found evidence of 96 species and subspecies of fishes, molluscs, crustaceans and plants endemic to these springs. The majority of endemic species are invertebrates with geographical distributions limited to a single spring complex (<61 km²). Endemic taxa are concentrated in 75 of the 326 spring complexes. Spring complexes with a large number of springs, high connectivity via drainage basins and low rainfall were more likely to contain endemic taxa, but environmental models were poor predictors of diversity. Only 24% spring complexes with high conservation value are within conservation reserves, and the majority of endemic species are unassessed under the IUCN and Australian conservation legislation, particularly the invertebrates.

Main conclusions

Diversity in this system is underestimated given the current rate of species discovery and prevailing data deficiency for many taxa. Historical processes and species‐specific environmental requirements may be more important for explaining why diversity is concentrated in particular complexes. Almost a decade after this system was listed as endangered, most complexes of high conservation value remain outside of conservation reserves, and the endangered species status of many taxa, and particularly the invertebrates, remain unassessed.

     

Linkage and association mapping for the slow softening (SwS) trait in peach (<Emphasis Type="Italic">P. persica</Emphasis> L. Batsch) fruit

Fruit texture is a crucial quality factor influencing consumer preference and shelf life. Peach (P. persica L. Batsch) is a highly perishable fruit subjected to a rapid softening after harvest. Improvement of peach shelf life is an important breeding objective, stimulating the characterization and exploitation of texture-related traits. Variants of melting (M) texture have captured an increasing interest, following the economic success of “Big Top” nectarine, one of the most cultivated varieties worldwide. “Big Top” fruit maintains a crispy texture for an extended period before the onset of the melting phase, prolonging its shelf life. Genetic determinants regulating this complex trait, defined as slow softening (SwS), are still unknown, mainly because of limitations in phenotyping methods. In this work, a mechanical approach for measuring SwS fruit texture was used to phenotype offspring derived from a cross between “Rebus028” (SwS texture) and “Max10” (M texture). Mechanical parameters were used in linkage mapping, allowing the identification of a major QTL on chromosome 8 (qSwS8.1). The presence of this QTL was validated by a genome-wide association study (GWAS) in a panel of accessions phenotyped for mechanical properties. Less significant signals were also detected by GWAS in other genomic regions, suggesting that additional loci may regulate the SwS trait, possibly depending on the genetic background. The inheritance pattern of the SwS trait and the presence of additional loci are crucial aspects to be addressed in future studies, along with a better characterization of other important textural attributes.     

Development of microsatellite markers for <Emphasis Type="Italic">Myracrodruon urundeuva</Emphasis> (F.F. &amp; M.F. Allemão), a highly endangered species from tropical forest based on next-generation sequencing

Myracrodruon urundeuva is a tree species of high economic importance due the strength and durability of its wood. Threatened of extinction in Brazil, it is present only in a few forest remnants, mostly in conservation units. Currently, there is little information on the genetic diversity of natural populations in Brazil and even less information about the genome of this species. Here, new species-specific microsatellite loci were developed based on next-generation sequencing (Illumina). More than 100,000 loci were identified in the run, with di- to hexanucleotides motifs. Of these, 20 loci were selected for validation in 30 individuals, with 15 successfully polymorphic loci detected. The number of alleles ranged among loci from 3 to 16, with an average of 7.73, expected (H _e ) and observed (H _o ) heterozygosity ranged from 0.246 to 0.902 and from 0.103 to 0.867, respectively. These results point out that these new set of markers has a great potential for use in population genetic studies for genetic conservation of the species.     

Long‐distance pollen and seed dispersal and inbreeding depression in Hymenaea stigonocarpa (Fabaceae: Caesalpinioideae) in the Brazilian savannah

Hymenaea stigonocarpa is a neotropical tree that is economically important due to its high‐quality wood; however, because it has been exploited extensively, it is currently considered threatened. Microsatellite loci were used to investigate the pollen and seed dispersal, mating patterns, spatial genetic structure (SGS), genetic diversity, and inbreeding depression in H. stigonocarpa adults, juveniles, and open‐pollinated seeds, which were sampled from isolated trees in a pasture and trees within a forest fragment in the Brazilian savannah. We found that the species presented a mixed mating system, with population and individual variations in the outcrossing rate (0.53–1.0). The studied populations were not genetically isolated due to pollen and seed flow between the studied populations and between the populations and individuals located outside of the study area. Pollen and seed dispersal occurred over long distances (>8 km); however, the dispersal patterns were isolated by distance, with a high frequency of mating occurring between near‐neighbor trees and seeds dispersed near the parent trees. The correlated mating for individual seed trees was higher within than among fruits, indicating that fruits present a high proportion of full‐sibs. Genetic diversity and SGS were similar among the populations, but offspring showed evidence of inbreeding, mainly originating from mating among related trees, which suggests inbreeding depression between the seed and adult stages. Selfing resulted in a higher inbreeding depression than mating among relatives, as assessed through survival and height. As the populations are not genetically isolated, both are important targets for in situ conservation to maintain their genetic diversity; for ex situ conservation, seeds can be collected from at least 78 trees in both populations separated by at least 250 m.     

Exosome-mediated delivery of miR-9 induces cancer-associated fibroblast-like properties in human breast fibroblasts

It is established that the interaction between microenvironment and cancer cells has a critical role in tumor development, given the dependence of neoplastic cells on stromal support. However, how this communication promotes the activation of normal (NFs) into cancer-associated fibroblasts (CAFs) is still not well understood. Most microRNA (miRNA) studies focused on tumor cell, but there is increasing evidence of their involvement in reprogramming NFs into CAFs. Here we show that miR-9, upregulated in various breast cancer cell lines and identified as pro-metastatic miRNA, affects the properties of human breast fibroblasts, enhancing the switch to CAF phenotype, thus contributing to tumor growth. Expressed at higher levels in primary triple-negative breast CAFs versus NFs isolated from patients, miR-9 improves indeed migration and invasion capabilities when transfected in immortalized NFs; viceversa, these properties are strongly impaired in CAFs upon miR-9 inhibition. We also demonstrate that tumor-secreted miR-9 can be transferred via exosomes to recipient NFs and this uptake results in enhanced cell motility. Moreover, we observed that this miRNA is also secreted by fibroblasts and in turn able to alter tumor cell behavior, by modulating its direct target E-cadherin, and NFs themselves. Consistently with the biological effects observed, gene expression profiles of NFs upon transient transfection with miR-9 show the modulation of genes mainly involved in cell motility and extracellular matrix remodeling pathways. Finally, we were able to confirm the capability of NFs transiently transfected with miR-9 to promote in vivo tumor growth. Taken together, these data provide new insights into the role of miR-9 as an important player in the cross-talk between cancer cells and stroma.Tumorigenesis is not considered anymore a tumor cell-autonomous mechanism triggered by accumulation of somatic aberrations, but fostered by a two-way interaction between cancer cells and the surrounding microenvironment.Cancer cells are indeed integrated in a biologically complex stroma, composed of different cell types (such as immune system components, endothelial cells, fibroblasts and adipocytes) as well as extracellular matrix (ECM), which originates the heterogeneity of the tumor microenvironment (TME).¹ It is known that a permissive TME has a key role in tumorigenesis.Fibroblasts, which represent the majority of the stromal cells, are very active in the ECM synthesis, regulation of inflammation and wound healing.² Even though the communication between cancer cells and fibroblasts has been extensively described,³ it is still currently unclear how this interaction promotes the activation of quiescent fibroblasts in cancer-associated fibroblasts (CAFs). It has been reported that breast carcinoma-associated stroma differs from its paired normal in deregulated expression of cytokines, ECM molecules and metalloproteinases.^{4, 5}Breast cancer is the leading cause of cancer-related deaths in women.⁶ Clinically, this heterogeneous disease is categorized into four major molecular subtypes: luminal-A, luminal-B, human epidermal growth factor receptor 2 (HER2) overexpressing and triple-negative/basal-like. Triple-negative breast cancer (TNBC) constitutes approximately 15–20% of all breast cancer cases, with the worst outcome of all subtypes.⁷In breast cancer, the biological characteristics and genetic heterogeneity between CAFs and their paired normal fibroblasts (NFs) have been described.^{8, 9} Breast CAFs are characterized by stronger ability in proliferation and cell motility in comparison with NFs and, consistently with this biological behavior, gene expression profiling showed the abnormal regulation of key signaling pathways as cell adhesion and secreting factors in CAFs.¹⁰MicroRNAs (miRNAs) are a class of small non-coding regulatory RNAs that play an important role in various biological processes.¹¹ Their extracellular presence as the major RNA component of exosomes suggests an internalization process by TME cells, thus mediating the cancer–host communication and participating in cancer metastasis by adapting the cell niches.¹² To date, little is known about miRNA expression differences between CAFs and NFs. Array data of primary cultures of CAFs versus their paired NFs from resected breast tumor tissues identified 11 dysregulated miRNAs, and their predicted target genes resulted mainly related to adhesion, migration, secretion and cell–cell interaction.¹³ A set of three miRNAs has been described to be involved in reprogramming NFs to CAFs in ovarian cancer¹⁴ and, very recently, miR-200s were found to contribute to breast cancer cell invasion through CAF activation and ECM remodeling.¹⁵In the present work, our attention focused on miR-9 as a possible player in the cross-talk between breast cancer cells and stroma. Numerous evidence supports this hypothesis: miR-9 has been described as metastamiR in breast cancer and it resulted markedly upregulated in breast cancer cells compared with normal mammary tissues.¹⁶ MiR-9 directly targets E-cadherin (CDH1) leading to increase cancer cell motility and invasiveness.¹⁷ Even more interestingly, miR-9 was found to be secreted by different human tumor cell lines, packaged into microvesicles and directly delivered to endothelial cells where it is able to promote migration and neovascularization activating JACK–STAT pathway. These observations suggest that tumor-secreted miRNAs can be involved in intercellular communication.¹⁸ Moreover, recent data showed that miR-9 overexpression is associated with epithelial–mesenchymal transition and poor prognosis in breast cancer, leading to its possible use as a biomarker for cancer progression and a target for treatment.¹⁹Our data revealed a higher expression of miR-9 in primary triple-negative breast CAFs versus NFs isolated from patients. Cell motility assays of immortalized NFs overexpressing miR-9 and CAFs where the miRNA was inhibited showed miR-9''s ability to affect the fibroblast behavior. Furthermore, tumor-secreted miR-9 can be transferred via exosomes to recipient NFs and this uptake resulted in enhanced cell motility. Gene expression profiles allowed us to identify a subgroup of molecules differentially expressed in NFs overexpressing miR-9 (NFs/miR-9) mainly involved in cell motility pathways and ECM remodeling. Moreover, miR-9-mediated downmodulation of its known target CDH1 in breast cancer cells cultured in conditioned medium from NFs/miR-9 indicated that miR-9 is also released by fibroblasts and transferred to tumor cells, and provided details regarding the biological mechanisms that could explain both the stronger motility and invasiveness of breast cancer cells observed in vitro, and the improved in vivo growth following co-injection with NFs/miR-9.     

Cholinergic antagonism by beta-blockers. II. Sequence of interactions and final steady state effects

On spontaneously beating guinea pig auricles, by assaying acetylcholine and beta-blockers alternate timing, opposite selective initial reactivity had been shown, partially independent of final steady state specific antagonism. Data related to cyclic receptor-sensitivity on acute versus subacute drug combination interaction had been presented.     

The monoclonal antibody-defined CAR-3 antigen is a serological marker associated with pancreatic carcinoma

The monoclonal antibody-defined CAR-3 antigen is a new carcinoma associated marker which is expressed on a mucin-like molecule. Serum concentrations of CAR-3 were assayed in 181 patients with carcinomas of different organs, 20 patients with non-carcinomatous malignancies, 123 patients with inflammatory diseases and 150 healthy controls. Serum levels of CAR-3 were significantly increased in 51% of the patients with pancreatic carcinomas, in 60% of patients with biliary tract carcinomas and in about 15% of the patients with carcinomas of the digestive apparatus. Sera from patients with breast carcinomas were negative, as well as sera from patients with melanomas or sarcomas. CAR-3 values in samples from patients with chronic pancreatitis were constantly negative, as were samples from healthy donors. Significant concentrations of CAR-3 were detected in 20% of the sera from patients with acute pancreatitis and in 15% of the sera from patients with cirrhosis. Because of its high specificity for pancreatic carcinomas compared to chronic pancreatitis, CAR-3 seems a promising marker for distinguishing between neoplastic and chronic inflammatory diseases of the pancreas, whose differential diagnosis is difficult.