Characterization of BMP signaling in breast development and tumorigenesis

Résumé L'influence des hormones reproductives sur le développement du cancer du sein a été établie au travers de nombreuse études épidémiologiques. Nous avons précédemment démontré que le gène Wnt-4 est un médiateur essentiel de la progestérone dans le développement lobulo-alvéolaire de l'épithélium mammaire. De plus, le rôle de la voie de signalisation Wnt dans la tumorigénèse de la glande mammaire mutine est largement établi. Pour comprendre sa fonction dans le cancer du sein, nous avons activée cette voie en surexprimant le gène Wnt-1 dans des cellules épithéliales primaires de sein, au moyen d'un rétrovirus. Ceci a conduit à la transformation oncogénique de ces cellules et à l'obtention d'un modèle de carcinogénèse du sein dénommé Wnt-1 HMEC. L'analyse de l'expression des gènes induits par la surexpression de Wnt-1 dans ces cellules, a permis d'identifier les gènes BMP4 et 7. Alors que des analyses de RT-PCR ont montré leur forte expression dans les cellules Wnt-1-HMECs, la présence d'une grande quantité de la protéine BMP7 a été constatée dans les tumeurs dérivées de ces cellules. L'importante phosphorylation des Smad 1, 5, S dans les Wnt-1 HMECs indique l'activation de la voie BMP, possiblement due à la stimulation ce celle-ci par BMP7. L'activation de la voie Wnt par la ß-Caténine, conduit à la transcription de BMP7, identifiant ainsi ce gène comme un gène cible de la voie canonique. La pertinence de nos observations a par ailleurs été confirmée par le fait que BMP7 est surexprimé dans les tumeurs de seins humains. Afin d'élucider la fonction de la voie BMP dans le sein, nous avons utilisé le modèle mutin. L'expression du gène BMP7 dans les souris transgéniques MMTV Wnt-1 s'est avérée élevée, démontrant qu'il est aussi un gène cible de la voie Wnt in-vivo. L'expression de l'ARN messager .codant pour la protéine BMP7 est induite lors du développement lobulo-alvéolaire, qui se fait sous l'influence de la progestérone et de Wnt-4. Ensemble, ces observations corroborent le fait qu'une stimulation avec de la progestérone suffit à induire la transcription du gène dans les 24h. Nos résultats coïncident d'autre part avec le fait que BMP7 est exprimé dans la couche myoépithéliale de l'épithélium où la voie Wnt est activée. L'analyse de souris reportrices de l'activité de la voie BMP, suggère une activation dans la couche luminale de l'épithélium durant tout le développement de la glande mammaire. Curieusement, cette même voie est active dans le mésenchyme lors de la mammogénèse embryonnaire. Finalement, nos analyses d'immunofluorescence démontrent la capacité de prolifération des cellules ayant activé BMP, ainsi que leur nette ségrégation d'avec les cellules exprimant le récepteur à la progestérone. Nos résultats démontrent que le gène BMP7 est un gène cible de la voie Wnt canonique dans le sein. Son expression dans la couche myoépitheliale est induite par Wnt-4, lui-même sécrété par les cellules luminales sensibles à la progestérone. La sécrétion de la protéine BMP7 conduit finalement à l'activation de la voie BMP dans les cellules négatives pour le récepteur à la progestérone. Abstract Epidemiological studies highlight the repetitive exposure to circulating progesterone as a major risk in the development of breast cancer. Work in our laboratory showed that Wnt-4 is an essential mediator of progesterone-driven side-branch formation, while Wnt signaling has long been established as strongly oncogenic in the mouse mammary gland. To address the role of Wnt in breast tumorigenesis we activated the pathway in primary human breast epithelial cells by means of refroviral Wnt-1 expression. This resulted in a Wnt1-induced breast carcinogenesis model, being referred to as Wnt-1-HMECs. Gene expression profiling revealed the Bone Morphogenetic Protein 4 and 7 (BMP4 and 7) a mong the most upregulated gene by ectopic Wnt-1 expression in primary HMECs. RT-PCR analysis confirmed elevated BMP4 and 7 mRNA levels in Wnt-1-infected HMECs, as well as strong BMP7 expression in the tumors derived from these cells. Smad 1, 5, 8 phosphorylation was high in Wnt-1HMECs whereas below detection limit in primary HMECs suggesting that the increased expression of BMP-7 results in activation of downstream signaling. Ectopic expressíon of a stabilized form of ßcatenin in primary HMECs resulted in increased transcription of BMP-7 suggesting that it is a target of canonical Wnt signaling. The clinical relevance of our observations was confirmed by the finding of BMP7 being upregulated in human breast tumor samples. To elucidate the role of BMP ligands in the breast in-vivo, we made use of the mouse model. Expression of the BMP7 gene was found to be increased in MMTV-Wnt-1 transgenic animals, suggesting that BMP7 may also be a Wnt 1 target gene in vivo. Expression of BMP7 was upregulated in mid-pregnancy which coincides with progesterone/Wnt induced side branching. BMP7 was induced within 24 hours by progesterone. Consistent with it being a target of canonical Wnt signaling, we demonstrated preferential expression of this ligand in the myoepithelial cells, the target cells of Wnt signals. In-vivo analysis of BMP signaling using a reporter mouse revealed the activation of the pathway in the luminal layer of the epithelium throughout postnatal development. Interestingly, during embryonic mammogenesis the pathway was found to be active in the mesenchyme. Immunofluorescence studies demonstrated that cells with BMP activity can proliferate. They also revealed a clear segregation between progesterone receptor positive cells and cells with active BMP signaling. Together our observations suggest that BMP-7 is a canonical Wnt signaling target both in HMECs and in the mouse mammary gland in-vivo. It is expressed in the myoepithelium possibly in response to Wnt-4, which is secreted by steroid receptor positive cells in response to progesterone. BMP-7 in turn may impinge on lumina) epithelial cells and activate BMP signaling in PR negative cells.

Genetic variation in GIPR influences the glucose and insulin responses to an oral glucose challenge.

Glucose levels 2 h after an oral glucose challenge are a clinical measure of glucose tolerance used in the diagnosis of type 2 diabetes. We report a meta-analysis of nine genome-wide association studies (n = 15,234 nondiabetic individuals) and a follow-up of 29 independent loci (n = 6,958-30,620). We identify variants at the GIPR locus associated with 2-h glucose level (rs10423928, beta (s.e.m.) = 0.09 (0.01) mmol/l per A allele, P = 2.0 x 10(-15)). The GIPR A-allele carriers also showed decreased insulin secretion (n = 22,492; insulinogenic index, P = 1.0 x 10(-17); ratio of insulin to glucose area under the curve, P = 1.3 x 10(-16)) and diminished incretin effect (n = 804; P = 4.3 x 10(-4)). We also identified variants at ADCY5 (rs2877716, P = 4.2 x 10(-16)), VPS13C (rs17271305, P = 4.1 x 10(-8)), GCKR (rs1260326, P = 7.1 x 10(-11)) and TCF7L2 (rs7903146, P = 4.2 x 10(-10)) associated with 2-h glucose. Of the three newly implicated loci (GIPR, ADCY5 and VPS13C), only ADCY5 was found to be associated with type 2 diabetes in collaborating studies (n = 35,869 cases, 89,798 controls, OR = 1.12, 95% CI 1.09-1.15, P = 4.8 x 10(-18)).

Efficient targeted transcript discovery via array-based normalization of RACE libraries.

Rapid amplification of cDNA ends (RACE) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. To improve sampling efficiency of human transcripts, we hybridized the products of the RACE reaction onto tiling arrays and used the detected exons to delineate a series of reverse-transcriptase (RT)-PCRs, through which the original RACE transcript population was segregated into simpler transcript populations. We independently cloned the products and sequenced randomly selected clones. This approach, RACEarray, is superior to direct cloning and sequencing of RACE products because it specifically targets new transcripts and often results in overall normalization of transcript abundance. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of new transcripts, and we investigated multiplexing the strategy by pooling RACE reactions from multiple interrogated loci before hybridization.

Molecular features of hepatosplenic T-cell lymphoma unravels potential novel therapeutic targets.

The pathogenesis of hepatosplenic T-cell lymphoma (HSTL), a rare entity mostly derived from γδ T cells and usually with a fatal outcome, remains largely unknown. In this study, HSTL samples (7γδ and 2αβ) and the DERL2 HSTL cell line were subjected to combined gene-expression profiling and array-based comparative genomic hybridization. Compared with other T-cell lymphomas, HSTL had a distinct molecular signature irrespective of TCR cell lineage. Compared with peripheral T-cell lymphoma, not otherwise specified and normal γδ T cells, HSTL overexpressed genes encoding NK-cell-associated molecules, oncogenes (FOS and VAV3), the sphingosine-1-phosphatase receptor 5 involved in cell trafficking, and the tyrosine kinase SYK, whereas the tumor-suppressor gene AIM1 (absent in melanoma 1) was among the most down-expressed. We found highly methylated CpG islands of AIM1 in DERL2 cells, and decitabine treatment induced a significant increase in AIM1 transcripts. Syk was present in HSTL cells and DERL2 cells contained phosphorylated Syk and were sensitive to a Syk inhibitor in vitro. Genomic profiles confirmed recurrent isochromosome 7q (n = 6/9) without alterations at the SYK and AIM1 loci. Our results identify a distinct molecular signature for HSTL and highlight oncogenic pathways that offer rationale for exploring new therapeutic options such as Syk inhibitors and demethylating agents.

Revisiting spatial distribution and biochemical composition of calcium-containing crystals in human osteoarthritic articular cartilage.

INTRODUCTION: Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage. METHODS: We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transform infra-red spectrometry and scanning electron microscopy of 8 to 10 cartilage zones of each knee, including medial and lateral femoral condyles and tibial plateaux and the intercondyle zone. Differential expression of genes involved in the mineralization process between cartilage with and without calcification was assessed in samples from 8 different patients by RT-PCR. Immunohistochemistry and histology studies were performed in 6 different patients. RESULTS: Mean (SEM) age and body mass index of patients at the time of TKR was 74.6 (1.7) years and 28.1 (1.6) kg/m², respectively. Preoperative X-rays showed joint calcifications (chondrocalcinosis) in 4 cases only. The medial femoro-tibial compartment was the most severely affected in all cases, and mean (SEM) Kellgren-Lawrence score was 3.8 (0.1). All 20 OA cartilages showed CaC crystals. The mineral content represented 7.7% (8.1%) of the cartilage weight. All patients showed BCP crystals, which were associated with CPP crystals for 8 joints. CaC crystals were present in all knee joint compartments and in a mean of 4.6 (1.7) of the 8 studied areas. Crystal content was similar between superficial and deep layers and between medial and femoral compartments. BCP samples showed spherical structures, typical of biological apatite, and CPP samples showed rod-shaped or cubic structures. The expression of several genes involved in mineralization, including human homolog of progressive ankylosis, plasma-cell-membrane glycoprotein 1 and tissue-nonspecific alkaline phosphatase, was upregulated in OA chondrocytes isolated from CaC crystal-containing cartilages. CONCLUSIONS: CaC crystal deposition is a widespread phenomenon in human OA articular cartilage involving the entire knee cartilage including macroscopically normal and less weight-bearing zones. Cartilage calcification is associated with altered expression of genes involved in the mineralisation process.

A genome-wide screen for interactions reveals a new locus on 4p15 modifying the effect of waist-to-hip ratio on total cholesterol.

Recent genome-wide association (GWA) studies described 95 loci controlling serum lipid levels. These common variants explain ∼25% of the heritability of the phenotypes. To date, no unbiased screen for gene-environment interactions for circulating lipids has been reported. We screened for variants that modify the relationship between known epidemiological risk factors and circulating lipid levels in a meta-analysis of genome-wide association (GWA) data from 18 population-based cohorts with European ancestry (maximum N = 32,225). We collected 8 further cohorts (N = 17,102) for replication, and rs6448771 on 4p15 demonstrated genome-wide significant interaction with waist-to-hip-ratio (WHR) on total cholesterol (TC) with a combined P-value of 4.79×10(-9). There were two potential candidate genes in the region, PCDH7 and CCKAR, with differential expression levels for rs6448771 genotypes in adipose tissue. The effect of WHR on TC was strongest for individuals carrying two copies of G allele, for whom a one standard deviation (sd) difference in WHR corresponds to 0.19 sd difference in TC concentration, while for A allele homozygous the difference was 0.12 sd. Our findings may open up possibilities for targeted intervention strategies for people characterized by specific genomic profiles. However, more refined measures of both body-fat distribution and metabolic measures are needed to understand how their joint dynamics are modified by the newly found locus.

Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpkCCD(cl4)) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P < 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCCD(cl4) cell line either by Northern blot hybridization or reverse transcription-PCR. The hepatocyte nuclear transcription factor HNF-3-alpha (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

The Wnt receptor FZD1 mediates chemoresistance in neuroblastoma through activation of the Wnt/beta-catenin pathway.

The development of chemoresistance represents a major obstacle in the successful treatment of cancers such as neuroblastoma (NB), a particularly aggressive childhood solid tumour. The mechanisms underlying the chemoresistant phenotype in NB were addressed by gene expression profiling of two doxorubicin (DoxR)-resistant vs sensitive parental cell lines. Not surprisingly, the MDR1 gene was included in the identified upregulated genes, although the highest overexpressed transcript in both cell lines was the frizzled-1 Wnt receptor (FZD1) gene, an essential component of the Wnt/beta-catenin pathway. FZD1 upregulation in resistant variants was shown to mediate sustained activation of the Wnt/beta-catenin pathway as revealed by nuclear beta-catenin translocation and target genes transactivation. Interestingly, specific micro-adapted short hairpin RNA (shRNAmir)-mediated FZD1 silencing induced parallel strong decrease in the expression of MDR1, another beta-catenin target gene, revealing a complex, Wnt/beta-catenin-mediated implication of FZD1 in chemoresistance. The significant restoration of drug sensitivity in FZD1-silenced cells confirmed the FZD1-associated chemoresistance. RNA samples from 21 patient tumours (diagnosis and postchemotherapy), showed a highly significant FZD1 and/or MDR1 overexpression after treatment, underlining a role for FZD1-mediated Wnt/beta-catenin pathway in clinical chemoresistance. Our data represent the first implication of the Wnt/beta-catenin pathway in NB chemoresistance and identify potential new targets to treat aggressive and resistant NB.

Growth promoting signaling by tenascin-C [corrected].

Tenascin-C is an adhesion-modulating extracellular matrix molecule that is highly expressed in tumor stroma and stimulates tumor cell proliferation. Adhesion of T98G glioblastoma cells to a fibronectin substratum is inhibited by tenascin-C. To address the mechanism of action, we performed a RNA expression analysis of T89G cells grown in the presence or absence of tenascin-C and found that tenascin-C down-regulates tropomyosin-1. Upon overexpression of tropomyosin-1, cell spreading on a fibronectin/tenascin-C substratum was restored, indicating that tenascin-C destabilizes actin stress fibers through down-regulation of tropomyosin-1. Tenascin-C also increased the expression of the endothelin receptor type A and stimulated the corresponding mitogen-activated protein kinase signaling pathway, which triggers extracellular signal-regulated kinase 1/2 phosphorylation and c-Fos expression. Tenascin-C additionally caused down-regulation of the Wnt inhibitor Dickkopf 1. In consequence, Wnt signaling was enhanced through stabilization of beta-catenin and stimulated the expression of the beta-catenin target Id2. Finally, our in vivo data derived from astrocytoma tissue arrays link increased tenascin-C and Id2 expression with high malignancy. Because increased endothelin and Wnt signaling, as well as reduced tropomyosin-1 expression, are closely linked to transformation and tumorigenesis, we suggest that tenascin-C specifically modulates these signaling pathways to enhance proliferation of glioma cells.

Identification of cancer/testis-antigen genes by massively parallel signature sequencing.

Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.

The short-rooted phenotype of the brevis radix mutant partly reflects root abscisic acid hypersensitivity.

To gain further insight into abscisic acid (ABA) signaling and its role in growth regulation, we have screened for Arabidopsis (Arabidopsis thaliana) mutants hypersensitive to ABA-mediated root growth inhibition. As a result, we have identified a loss-of-function allele of BREVIS RADIX (BRX) in the Columbia background, named brx-2, which shows enhanced response to ABA-mediated inhibition of root growth. BRX encodes a key regulator of cell proliferation and elongation in the root, which has been implicated in the brassinosteroid (BR) pathway as well as in the regulation of auxin-responsive gene expression. Mutants affected in BR signaling that are not impaired in root growth, such as bes1-D, bzr1-D, and bsu1-D, also showed enhanced sensitivity to ABA-mediated inhibition of root growth. Triple loss-of-function mutants affected in PP2Cs, which act as negative regulators of ABA signaling, showed impaired root growth in the absence of exogenous ABA, indicating that disturbed regulation of ABA sensitivity impairs root growth. In agreement with this result, diminishing ABA sensitivity of brx-2 by crossing it with a 35S:HAB1 ABA-insensitive line allowed significantly higher recovery of root growth after brassinolide treatment. Finally, transcriptomic analysis revealed that ABA treatment negatively affects auxin signaling in wild-type and brx-2 roots and that ABA response is globally altered in brx-2. Taken together, our results reveal an interaction between BRs, auxin, and ABA in the control of root growth and indicate that altered sensitivity to ABA is partly responsible for the brx short-root phenotype.

A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer.

To better understand the relationship between tumor-host interactions and the efficacy of chemotherapy, we have developed an analytical approach to quantify several biological processes observed in gene expression data sets. We tested the approach on tumor biopsies from individuals with estrogen receptor-negative breast cancer treated with chemotherapy. We report that increased stromal gene expression predicts resistance to preoperative chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide (FEC) in subjects in the EORTC 10994/BIG 00-01 trial. The predictive value of the stromal signature was successfully validated in two independent cohorts of subjects who received chemotherapy but not in an untreated control group, indicating that the signature is predictive rather than prognostic. The genes in the signature are expressed in reactive stroma, according to reanalysis of data from microdissected breast tumor samples. These findings identify a previously undescribed resistance mechanism to FEC treatment and suggest that antistromal agents may offer new ways to overcome resistance to chemotherapy.

Isolation and in vitro chondrogenic potential of human foetal spine cells.

Cell therapy for nucleus pulposus (NP) regeneration is an attractive treatment for early disc degeneration as shown by studies using autologous NP cells or stem cells. Another potential source of cells is foetal cells. We investigated the feasibility of isolating foetal cells from human foetal spine tissues and assessed their chondrogenic potential in alginate bead cultures. Histology and immunohistochemistry of foetal tissues showed that the structure and the matrix composition (aggrecan, type I and II collagen) of foetal intervertebral disc (IVD) were similar to adult IVD. Isolated foetal cells were cultured in monolayer in basic media supplemented with 10% Fetal Bovine Serum (FBS) and from each foetal tissue donation, a cell bank of foetal spine cells at passage 2 was established and was composed of around 2000 vials of 5 million cells. Gene expression and immunohistochemistry of foetal spine cells cultured in alginate beads during 28 days showed that cells were able to produce aggrecan and type II collagen and very low level of type I and type X collagen, indicating chondrogenic differentiation. However variability in matrix synthesis was observed between donors. In conclusion, foetal cells could be isolated from human foetal spine tissues and since these cells showed chondrogenic potential, they could be a potential cell source for IVD regeneration.

Exploration of the visual system: Part 1: Dissection of the mouse eye for RNA, protein, and histological analyses

Due to the power of genetics, the mouse has become a widely used animal model in vision research. However, its eyeball has an axial length of only about 2 mm. The present protocol describes how to easily dissect the small rodent eye post mortem. This allows collecting different tissues of the eye, i.e., cornea, lens, iris, retina, optic nerve, retinal pigment epithelium (RPE), and sclera. We further describe in detail how to process these eye samples in order to obtain high‐quality RNA for RNA expression profiling studies. Depending on the eye tissue to be analyzed, we present appropriate lysis buffers to prepare total protein lysates for immunoblot and immuno‐precipitation analyses. Fixation, inclusion, embedding, and cryosectioning of the globe for routine histological analyses (HE staining, DAPI staining, immunohistochemistry, in situ hybridization) is further presented. These basic protocols should allow novice investigators to obtain eye tissue samples rapidly for their experiments.