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BackgroundIn the context of the SARS-CoV-2 pandemic, reuse of personal protective equipment, specifically that of medical face coverings, has been recommended. The reuse of these typically single-use only items necessitates procedures to inactivate contaminating human respiratory and gastrointestinal pathogens. We previously demonstrated decontamination of surgical masks and respirators contaminated with infectious SARS-CoV-2 and various animal coronaviruses via low concentration- and short exposure methylene blue photochemical treatment (10 µM methylene blue, 30 minutes of 12,500-lux red light or 50,000 lux white light exposure).MethodsHere, we describe the adaptation of this protocol to the decontamination of a more resistant, non-enveloped gastrointestinal virus and demonstrate efficient photodynamic inactivation of murine norovirus, a human norovirus surrogate.ResultsMethylene blue photochemical treatment (100 µM methylene blue, 30 minutes of 12,500-lux red light exposure) of murine norovirus-contaminated masks reduced infectious viral titres by over four orders of magnitude on surgical mask surfaces.Discussion and ConclusionsInactivation of a norovirus, the most difficult to inactivate of the respiratory and gastrointestinal human viruses, can predict the inactivation of any less resistant viral mask contaminant. The protocol developed here thus solidifies the position of methylene blue photochemical decontamination as an important tool in the package of practical pandemic preparedness.

Human noroviruses are recognized as the leading worldwide cause of sporadic and epidemic viral gastroenteritis, causing morbidity and mortality in impoverished developing countries and engendering enormous economic losses in developed countries. Transmitted faecal-orally, either via person-to-person contact, or by consumption of contaminated foods or water, norovirus outbreaks are often reported in institutional settings or in the context of communal dining. Bivalve molluscs, which accumulate noroviruses via filter feeding and are often eaten raw or insufficiently cooked, are a common food vehicle implicated in gastroenteritis outbreaks. The involvement of bivalve molluscs in norovirus outbreaks and epidemiology over the past two decades are reviewed. The authors describe how their physiology of filter feeding can render them concentrated vehicles of norovirus contamination in polluted environments and how high viral loads persist in molluscs even after application of depuration practices and typical food preparation steps. The global prevalence of noroviruses in bivalve molluscs as detected by different monitoring efforts is determined and the various methods currently utilized for norovirus extraction and detection from bivalve matrices described. An overview of gastroenteritis outbreaks affirmatively associated with norovirus-contaminated bivalve molluscs as reported in the past 18 years is also provided. Strategies for risk reduction in shellfish contamination and subsequent human infection are discussed.

Human noroviruses impose a considerable health burden globally. Here, a flow cytometry approach designed for their detection in biological waste and food samples was developed using antibody-coated magnetic beads. Antipeptide antibodies against murine norovirus and various human norovirus genotypes were generated for capture and coated onto magnetic beads. A flow cytometry assay was then implemented to detect bead-bound human norovirus GI.3 in patient stool samples and in norovirus-spiked mussel digestive tissues. The detection limit for stool samples was 105 gc/mL, thus bettering detection limits of commercially available norovirus diagnosis quick kits of 100-fold; the detection limit in spiked mussels however was ten-fold higher than in stool samples. Further assays showed a decrease in fluorescence intensity for heat- or UV-inactivated virus particles. Overall, we demonstrate the application of a flow cytometry approach for direct detection of small non-enveloped virus particles such as noroviruses. An adaptation of the technology to routine diagnostics has the potential to contribute a rapid and sensitive tool to norovirus outbreak investigations. Further improvements to the method, notably decreasing the detection limit of the approach, may allow the analysis of naturally contaminated food and environmental samples.

Human noroviruses are a major cause for gastroenteritis outbreaks. Filter-feeding bivalve molluscs, which accumulate noroviruses in their digestive tissues, are a typical vector for human infection. RT-qPCR, the established method for human norovirus detection in food, does not allow discrimination between infectious and non-infectious viruses and can overestimate potentially infectious viral loads. To develop a more accurate method of infectious norovirus load estimation, we combined intercalating agent propidium monoazide (PMAxx™)-pre-treatment with RT-qPCR assay using in vitro-cultivable murine norovirus. Three primer sets targeting different genome regions and diverse amplicon sizes were used to compare one-step amplification of a short genome fragment to three two-step long-range RT-qPCRs (7 kbp, 3.6 kbp and 2.3 kbp amplicons). Following initial assays performed on untreated infectious, heat-, or ultraviolet-inactivated murine noroviruses in PBS suspension, PMAxx™ RT-qPCRs were implemented to detect murine noroviruses subsequent to their extraction from mussel digestive tissues; virus extraction via anionic polymer-coated magnetic beads was compared with the proteinase K-dependent ISO norm. The long-range RT-qPCR process detecting fragments of more than 2.3 kbp allowed accurate estimation of the infectivity of UV-damaged murine noroviruses. While proteinase K extraction limited later estimation of PMAxx™ pre-treatment effects and was found to be unsuited to the assay, magnetic bead-captured murine noroviruses retained their infectivity. Genome copies of heat-inactivated murine noroviruses differed by 2.3 log10 between RT-qPCR and PMAxx™-RT-qPCR analysis in bivalve molluscs, the PMAxx™ pre-treatment allowing a closer approximation of infectious titres. The combination of bead-based virus extraction and PMAxx™ RT-qPCR thus provides a more accurate model for the estimation of noroviral bivalve mollusc contamination than the conjunction of proteinase K extraction and RT-qPCR and has the potential (once validated utilising infectious human norovirus) to provide an added measure of security to food safety authorities in the hazard assessment of potential bivalve mollusc contamination.

BackgroundAs the SARS-CoV-2 pandemic accelerates, the supply of personal protective equipment remains under strain. To combat shortages, re-use of surgical masks and filtering facepiece respirators has been recommended. Prior decontamination is paramount to the re-use of these typically single-use only items and, without compromising their integrity, must guarantee inactivation of SARS-CoV-2 and other contaminating pathogens.AimWe provide information on the effect of time-dependent passive decontamination at room temperature and evaluate inactivation of a SARS-CoV-2 surrogate and a non-enveloped model virus as well as mask and respirator integrity following active multiple-cycle vaporised hydrogen peroxide (VHP), ultraviolet germicidal irradiation (UVGI), and dry heat (DH) decontamination.MethodsMasks and respirators, inoculated with infectious porcine respiratory coronavirus or murine norovirus, were submitted to passive decontamination or single or multiple active decontamination cycles; viruses were recovered from sample materials and viral titres were measured via TCID50 assay. In parallel, filtration efficiency tests and breathability tests were performed according to EN standard 14683 and NIOSH regulations.Results and DiscussionInfectious porcine respiratory coronavirus and murine norovirus remained detectable on masks and respirators up to five and seven days of passive decontamination. Single and multiple cycles of VHP-, UVGI-, and DH were shown to not adversely affect bacterial filtration efficiency of masks. Single- and multiple UVGI did not adversely affect respirator filtration efficiency, while VHP and DH induced a decrease in filtration efficiency after one or three decontamination cycles. Multiple cycles of VHP-, UVGI-, and DH slightly decreased airflow resistance of masks but did not adversely affect respirator breathability. VHP and UVGI efficiently inactivated both viruses after five, DH after three, decontamination cycles, permitting demonstration of a loss of infectivity by more than three orders of magnitude. This multi-disciplinal approach provides important information on how often a given PPE item may be safely reused.

BACKGROUND: As the SARS-CoV-2 pandemic accelerates, the supply of personal protective equipment remains under strain. To combat shortages, re-use of surgical masks and filtering facepiece respirators has been recommended. Prior decontamination is paramount to the re-use of these typically single-use only items and, without compromising their integrity, must guarantee inactivation of SARS-CoV-2 and other contaminating pathogens. AIM: We provide information on the effect of time-dependent passive decontamination (infectivity loss over time during room temperature storage in a breathable bag) and evaluate inactivation of a SARS-CoV-2 surrogate and a non-enveloped model virus as well as mask and respirator integrity following active multiple-cycle vaporised hydrogen peroxide (VHP), ultraviolet germicidal irradiation (UVGI), and dry heat (DH) decontamination. METHODS: Masks and respirators, inoculated with infectious porcine respiratory coronavirus or murine norovirus, were submitted to passive decontamination or single or multiple active decontamination cycles; viruses were recovered from sample materials and viral titres were measured via TCID50 assay. In parallel, filtration efficiency tests and breathability tests were performed according to EN standard 14683 and NIOSH regulations. RESULTS AND DISCUSSION: Infectious porcine respiratory coronavirus and murine norovirus remained detectable on masks and respirators up to five and seven days of passive decontamination. Single and multiple cycles of VHP-, UVGI-, and DH were shown to not adversely affect bacterial filtration efficiency of masks. Single- and multiple UVGI did not adversely affect respirator filtration efficiency, while VHP and DH induced a decrease in filtration efficiency after one or three decontamination cycles. Multiple cycles of VHP-, UVGI-, and DH slightly decreased airflow resistance of masks but did not adversely affect respirator breathability. VHP and UVGI efficiently inactivated both viruses after five, DH after three, decontamination cycles, permitting demonstration of a loss of infectivity by more than three orders of magnitude. This multi-disciplinal approach provides important information on how often a given PPE item may be safely reused. ispartof: PLOS ONE vol:16 issue:5 ispartof: location:United States status: published

Human noroviruses are a major cause for gastroenteritis outbreaks. Filter-feeding bivalve molluscs, which accumulate noroviruses in their digestive tissues, are a typical vector for human infection. RT-qPCR, the established method for human norovirus detection in food, does not allow discrimination between infectious and non-infectious viruses and can overestimate potentially infectious viral loads. To develop a more accurate method of infectious norovirus load estimation, we combined intercalating agent propidium monoazide (PMAxx™)-pre-treatment with RT-qPCR assay using in vitro-cultivable murine norovirus. Three primer sets targeting different genome regions and diverse amplicon sizes were used to compare one-step amplification of a short genome fragment to three two-step long-range RT-qPCRs (7 kbp, 3.6 kbp and 2.3 kbp amplicons). Following initial assays performed on untreated infectious, heat-, or ultraviolet-inactivated murine noroviruses in PBS suspension, PMAxx™ RT-qPCRs were implemented to detect murine noroviruses subsequent to their extraction from mussel digestive tissues; virus extraction via anionic polymer-coated magnetic beads was compared with the proteinase K-dependent ISO norm. The long-range RT-qPCR process detecting fragments of more than 2.3 kbp allowed accurate estimation of the infectivity of UV-damaged murine noroviruses. While proteinase K extraction limited later estimation of PMAxx™ pre-treatment effects and was found to be unsuited to the assay, magnetic bead-captured murine noroviruses retained their infectivity. Genome copies of heat-inactivated murine noroviruses differed by 2.3 log

Estrogen receptor alpha (ERalpha) is a modular protein of the steroid/nuclear receptor family of transcriptional regulators that upon binding to the hormone undergoes structural changes, resulting in its nuclear translocation and docking to specific chromatin sites. In the nucleus, ERalpha assembles in multiprotein complexes that act as final effectors of estrogen signaling to the genome through chromatin remodeling and epigenetic modifications, leading to dynamic and coordinated regulation of hormone-responsive genes. Identification of the molecular partners of ERalpha and understanding their combinatory interactions within functional complexes is a prerequisite to define the molecular basis of estrogen control of cell functions. To this end, affinity purification was applied to map and characterize the ERalpha interactome in hormone-responsive human breast cancer cell nuclei. MCF-7 cell clones expressing human ERalpha fused to a tandem affinity purification tag were generated and used to purify native nuclear ER-containing complexes by IgG-Sepharose affinity chromatography and glycerol gradient centrifugation. Purified complexes were analyzed by two-dimensional DIGE and mass spectrometry, leading to the identification of a ligand-dependent multiprotein complex comprising beta-actin, myosins, and several proteins involved in actin filament organization and dynamics and/or known to participate in actin-mediated regulation of gene transcription, chromatin dynamics, and ribosome biogenesis. Time course analyses indicated that complexes containing ERalpha and actin are assembled in the nucleus early after receptor activation by ligands, and gene knockdown experiments showed that gelsolin and the nuclear isoform of myosin 1c are key determinants for assembly and/or stability of these complexes. Based on these results, we propose that the actin network plays a role in nuclear ERalpha actions in breast cancer cells, including coordinated regulation of target gene activity, spatial and functional reorganization of chromatin, and ribosome biogenesis.

Background: Microarray experiments enable simultaneous measurement of the expression levels of virtually all transcripts present in cells, thereby providing a 'molecular picture' of the cell state. On the other hand, the genomic responses to a pharmacological or hormonal stimulus are dynamic molecular processes, where time influences gene activity and expression. The potential use of the statistical analysis of microarray data in time series has not been fully exploited so far, due to the fact that only few methods are available which take into proper account temporal relationships between samples. Results: We compared here four different methods to analyze data derived from a time course mRNA expression profiling experiment which consisted in the study of the effects of estrogen on hormone-responsive human breast cancer cells. Gene expression was monitored with the innovative Illumina BeadArray platform, which includes an average of 30-40 replicates for each probe sequence randomly distributed on the chip surface. We present and discuss the results obtained by applying to these datasets different statistical methods for serial gene expression analysis. The influence of the normalization algorithm applied on data and of different parameter or threshold choices for the selection of differentially expressed transcripts has also been evaluated. In most cases, the selection was found fairly robust with respect to changes in parameters and type of normalization. We then identified which genes showed an expression profile significantly affected by the hormonal treatment over time. The final list of differentially expressed genes underwent cluster analysis of functional type, to identify groups of genes with similar regulation dynamics. Conclusions: Several methods for processing time series gene expression data are presented, including evaluation of benefits and drawbacks of the different methods applied. The resulting protocol for data analysis was applied to characterization of the gene expression changes induced by estrogen in human breast cancer ZR-75.1 cells over an entire cell cycle.

Luminal-like breast tumor cells express estrogen receptor alpha (ERalpha), a member of the nuclear receptor family of ligand-activated transcription factors that controls their proliferation, survival, and functional status. To identify the molecular determinants of this hormone-responsive tumor phenotype, a comprehensive genome-wide analysis was performed in estrogen stimulated MCF-7 and ZR-75.1 cells by integrating time-course mRNA expression profiling with global mapping of genomic ERalpha binding sites by chromatin immunoprecipitation coupled to massively parallel sequencing, microRNA expression profiling, and in silico analysis of transcription units and receptor binding regions identified. All 1270 genes that were found to respond to 17beta-estradiol in both cell lines cluster in 33 highly concordant groups, each of which showed defined kinetics of RNA changes. This hormone-responsive gene set includes several direct targets of ERalpha and is organized in a gene regulation cascade, stemming from ligand-activated receptor and reaching a large number of downstream targets via AP-2gamma, B-cell activating transcription factor, E2F1 and 2, E74-like factor 3, GTF2IRD1, hairy and enhancer of split homologue-1, MYB, SMAD3, RARalpha, and RXRalpha transcription factors. MicroRNAs are also integral components of this gene regulation network because miR-107, miR-424, miR-570, miR-618, and miR-760 are regulated by 17beta-estradiol along with other microRNAs that can target a significant number of transcripts belonging to one or more estrogen-responsive gene clusters.

In the context of global warming and increasing wildfire occurrence, this study aims to examine, for the first time, the changes in multi-level biodiversity and key soil features related to soil functioning in a burned Mediterranean beech forest. Two years after the 2017 wildfire, changes between burned and unburned plots of beech forest were analyzed for plant communities (vascular plant and cover, bryophytes diversity, structural, chorological, and ecological variables) and soil features (main chemical properties, microbial biomass and activity, bacterial community composition, and diversity), through a synchronic study. Fire-induced changes in the micro-environmental conditions triggered a secondary succession process with colonization by many native pioneer plant species. Indeed, higher frequency (e.g., Scrophularia vernalis L., Rubus hirtus Waldst. and Kit. group, and Funaria hygrometrica Hedw.) or coverage (e.g., Verbascum thapsus L. subsp. thapsus and Digitalis micrantha Roth ex Schweigg.) of the species was observed in the burned plots, whereas the typical forest species showed a reduction in frequency, but not in cover, except for Fagus sylvatica subsp. sylvatica. Overall, an increase in plant species and family richness was found in the burned plots, mainly in the herbaceous and bryophyte layers, compared to the unburned plots. Burned plots showed an increase in therophytes, chamaephytes, cosmopolites, steno-Mediterranean and Atlantic species, and a decrease in geophytes and Eurasiatic plants. Significant differences were found in burned vs. control soils for 10 phyla, 40 classes, 79 orders, 145 families, 342 genera, and 499 species of bacteria, with about 50% of each taxon over-represented and 50% under-represented in burned than in control. Changes in bacterial richness within several families (reduction in Acidobacteriaceae, Solibacteraceae, Rhodospirillaceae, and Sinobacteraceae, increase in Micrococcaceae, Comamonadaceae, Oxalobacteraceae, Pseudomonadaceae, Hymenobacteraceae, Sphingomonadaceae, Cytophagaceae, Nocardioidaceae, Opitutaceae, Solirubrobacteraceae, and Bacillaceae) in burned soil were related to fire-induced chemical changes of soil (pH, electrical conductivity, and cation exchange capacity). No evident effect of the wildfire was found on organic C content, microbial biomass (total microbial carbon and fungal mycelium) and activity, and microbial indexes (fungal percentage of microbial C, metabolic quotient, and quotient of mineralization), suggesting that soil functions remained unchanged in the burned area. Therefore, we hypothesize that, without an additional disturbance event, a re-establishment of beech forest can be expected but with an unpredictable time of post-fire succession.

Aims Cardiac myxomas usually develop in the atria and consist of an acid-mucopolysaccharide-rich myxoid matrix with polygonal stromal cells scattered throughout. These human benign tumours are a valuable research model because of the rarity of cardiac tumours, their clinical presentation and uncertain origin. Here, we assessed whether multipotent cardiac stem/progenitor cells (CSCs) give rise to atrial myxoma tissue. Methods and results Twenty-three myxomas were collected and analysed for the presence of multipotent CSCs. We detected myxoma cells positive for c-kit (c-kitpos) but very rare Isl-1 positive cells. Most of the c-kitpos cells were blood lineage-committed CD45pos/CD31pos cells. However, c-kitpos/CD45neg/CD31neg cardiac myxoma cells expressed stemness and cardiac progenitor cell transcription factors. Approximately ≤10% of the c-kitpos/CD45neg/CD31neg myxoma cells also expressed calretinin, a characteristic of myxoma stromal cells. In vitro, the c-kitpos/CD45neg/CD31neg myxoma cells secrete chondroitin-6-sulfate and hyaluronic acid, which are the main components of gelatinous myxoma matrix in vivo. In vitro, c-kitpos/CD45neg/CD31neg myxoma cells have stem cell properties being clonogenic, self-renewing, and sphere forming while exhibiting an abortive cardiac differentiation potential. Myxoma-derived CSCs possess a mRNA and microRNA transcriptome overall similar to normal myocardium-derived c-kitpos/CD45neg/CD31negCSCs , yet showing a relatively small and relevant fraction of dysregulated mRNA/miRNAs (miR-126-3p and miR-335-5p, in particular). Importantly, myxoma-derived CSCs but not normal myocardium-derived CSCs, seed human myxoma tumours in xenograft’s in immunodeficient NOD/SCID mice. Conclusion Myxoma-derived c-kitpos/CD45neg/CD31neg CSCs fulfill the criteria expected of atrial myxoma-initiating stem cells. The transcriptome of these cells indicates that they belong to or are derived from the same lineage as the atrial multipotent c-kitpos/CD45neg/CD31neg CSCs. Taken together the data presented here suggest that human myxomas could be the first-described CSC-related human heart disease.

In order to improve the floristic knowledge of the Italian territory, we report the inventory of the taxa collected during the annual field trip of the working group for Floristics, Systematics and Evolution of the Italian Botanical Society held in 2015 in eastern Irpinia and Vulture-Melfese area (South Italy). The investigated territories are located in southern Apennines, along the border between the Campania and Basilicata administrative regions. These areas are scarcely known in terms of vascular flora. The floristic samplings were performed in 19 sites selected as representative of the local environmental diversity as regards to climate, litho-morphology and land-use. The research led to the identification of 4,137 specimens of vascular plants, belonging to 815 species and subspecies, 399 genera, and 85 families. Among these taxa, 42 were endemic to Italy, 38 were included in the IUCN Red List of the Italian Flora, 28 were alien and 5 were cryptogenic in Campania and/or Basilicata administrative regions. Two taxa, Aquilegia coerulea (casual alien, native to North America) and Lolium × boucheanum (native), were found to be new for Italy. On the basis of the available floristic literature the first one is also to be considered new for the European flora. At regional scale, we have found 18 taxa new for the Campania and 15 new for the Basilicata region. Finally, 10 taxa were confirmed for Campania. Data obtained during this study, confirmed the important role of a collaborative approach among botanists and the great relevance of these territories for plant diversity.

Microarray-based gene expression profiling is well suited for parallel quantitative analysis of large numbers of RNAs, but its application to cancer biopsies, formalin-fixed paraffin-embedded (FFPE) archived tissues in particular, is limited by the poor quality of the RNA recovered. This represents a serious drawback, since FFPE tumor tissue banks are available with clinical and prognostic annotations, which could be exploited for molecular profiling studies, provided that reliable analytical technologies are found. We applied and evaluated here a microarray-based cDNA-mediated annealing, selection, extension and ligation (DASL) assay for analysis of 502 mRNAs in highly degraded total RNA extracted from cultured cells or FFPE breast cancer biopsies. The study included quantitative and qualitative comparison of data obtained by analysis of the same RNAs with genome-wide oligonucleotide microarrays vs. DASL arrays and, by DASL, before and after extensive in vitro RNA fragmentation. The DASL-based expression profiling assay applied to RNA extracted from MCF-7 cells before or after 24 hours stimulation with a mitogenic dose of 17β-estradiol consistently allowed to detect hormone-induced gene expression changes also following extensive RNA degradation in vitro. Comparable results where obtained with tumor RNA extracted from FFPE breast cancer biopsies (6 to 19 years old). The method proved itself sensitive, reproducible and accurate, when compared with results obtained by microarray analysis of RNA extracted from snap-frozen tissue of the same tumor.

Oestrogen receptor alpha (ERα) is a ligand-dependent transcription factor that mediates oestrogen effects in hormone-responsive cells. Following oestrogenic activation, ERα directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) represent a class of small noncoding RNAs that function as negative regulators of protein-coding gene expression. They are found aberrantly expressed or mutated in cancer, suggesting their crucial role as either oncogenes or tumour suppressor genes. Here, we analysed changes in miRNA expression in response to oestrogen in hormone-responsive breast cancer MCF-7 and ZR-75.1 cells by microarray-mediated expression profiling. This led to the identification of 172 miRNAs up- or down-regulated by ERα in response to 17β-oestradiol, of which 52 are similarly regulated by the hormone in the two cell models investigated. To identify mechanisms by which ERα exerts its effects on oestrogen-responsive miRNA genes, the oestrogen-dependent miRNA expression profiles were integrated with global in vivo ERα binding site mapping in the genome by ChIP-Seq. In addition, data from miRNA and messenger RNA (mRNA) expression profiles obtained under identical experimental conditions were compared to identify relevant miRNA target transcripts. Results show that miRNAs modulated by ERα represent a novel genomic pathway to impact oestrogen-dependent processes that affect hormone-responsive breast cancer cell behaviour. MiRNome analysis in tumour tissues from breast cancer patients confirmed a strong association between expression of these small RNAs and clinical outcome of the disease, although this appears to involve only marginally the oestrogen-regulated miRNAs identified in this study.

C/EBPs are a family of B-Zip transcription factors -TFs- involved in the regulation of differentiation in several tissues. The two most studied members -C/EBPα and C/EBPβ- play important roles in skin homeostasis and their ablation reveals cells with stem cells signatures. Much less is known about C/EBPδ which is highly expressed in the granular layer of interfollicular epidermis and is a direct target of p63, the master regular of multilayered epithelia. We identified C/EBPδ target genes in human primary keratinocytes by ChIP on chip and profiling of cells functionally inactivated with siRNA. Categorization suggests a role in differentiation and control of cell-cycle, particularly of G2/M genes. Among positively controlled targets are numerous genes involved in barrier function. Functional inactivation of C/EBPδ as well as overexpressions of two TF targets -MafB and SOX2- affect expression of markers of keratinocyte differentiation. We performed IHC on skin tumor tissue arrays: expression of C/EBPδ is lost in Basal Cell Carcinomas, but a majority of Squamous Cell Carcinomas showed elevated levels of the protein. Our data indicate that C/EBPδ plays a role in late stages of keratinocyte differentiation. © 2010 Borrelli et al.

Estrogen receptors α (ER-α) and β (ER-β) play distinct biological roles in onset and progression of hormone-responsive breast cancer, with ER-β exerting a modulatory activity on ER-α-mediated estrogen signaling and stimulation of cell proliferation by mechanisms still not fully understood. We stably expressed human ER-β fused to a tandem affinity purification-tag in estrogen-responsive MCF-7 cells and applied tandem affinity purification and nanoLC-MS/MS to identify the ER-β interactome of this cell type. Functional annotation by bioinformatics analyses of the 303 proteins that co-purify with ER-β from nuclear extracts identify several new molecular partners of this receptor subtype that represents nodal points of a large protein network controlling multiple processes and functions in breast cancer cells.

Estrogen effects on mammary epithelial and breast cancer (BC) cells are mediated by the nuclear receptors ERα and ERβ, transcription factors that display functional antagonism with each other, with ERβ acting as oncosuppressor and interfering with the effects of ERα on cell proliferation, tumor promotion and progression. Indeed, hormone-responsive, ERα+ BC cells often lack ERβ, which when present associates with a less aggressive clinical phenotype of the disease. Recent evidences point to a significant role of microRNAs (miRNAs) in BC, where specific miRNA expression profiles associate with distinct clinical and biological phenotypes of the lesion. Considering the possibility that ERβ might influence BC cell behavior via miRNAs, we compared miRNome expression in ERβ+ vs ERβ- hormone-responsive BC cells and found a widespread effect of this ER subtype on the expression pattern of these non-coding RNAs. More importantly, the expression pattern of 67 miRNAs, including 10 regulated by ERβ in BC cells, clearly distinguishes ERβ+, node-negative, from ERβ-, metastatic, mammary tumors. Molecular dissection of miRNA biogenesis revealed multiple mechanisms for direct regulation of this process by ERβ+ in BC cell nuclei. In particular, ERβ downregulates miR-30a by binding to two specific sites proximal to the gene and thereby inhibiting pri-miR synthesis. On the other hand, the receptor promotes miR-23b, -27b and 24-1 accumulation in the cell by binding in close proximity of the corresponding gene cluster and preventing in situ the inhibitory effects of ERα on pri-miR maturation by the p68/DDX5-Drosha microprocessor complex. These results indicate that cell autonomous regulation of miRNA expression is part of the mechanism of action of ERβ in BC cells and could contribute to establishment or maintenance of a less aggressive tumor phenotype mediated by this nuclear receptor.