Esox lucius, Northern Madtom Noturus stigmosus) and a single extinct (Harelip Sucker Moxostoma lacerum) taxa that suggest a combination of late Pleistocene displacement events coupled with more recent changes in habitat that have resulted in modern basin diversity patterns. The Bell Cave locality represents one of the most intact Pleistocene freshwater fish deposits anywhere in North America. Significant preservational, taphonomic, sampling, and identification biases preclude the identification of additional taxa. Overall, this study provides a detailed look into paleo-river ecology, as well as freshwater fish diversity and distribution leading up to the contemporary biodiversity patterns of the Tennessee River Basin and Mississippi River Basin as a whole.The Tennessee River Basin is considered one of the most important regions for freshwater biodiversity anywhere on the globe. The Tennessee River Basin currently includes populations of at least half of the described contemporary diversity of extant North American freshwater fishes, crayfish, mussel, and gastropod species. However, comparatively little is known about the biodiversity of this basin from the Pleistocene Epoch, particularly the late Pleistocene  leading to modern Holocene fish diversity patterns. The objective of this study was to describe the fish assemblages of the Tennessee River Basin from the late Pleistocene using a series of faunas from locales throughout the basin documented from published literature, unpublished reports, and an undocumented fauna from Bell Cave . Herein we discuss 41 unequivocal taxa from 10 late Pleistocene localities within the basin and include a systematic discussion of 11 families, 19 genera, and 24 identifiable species  specific to the Bell Cave locality. Among the described fauna are several extirpated (e.g., Northern Pike  The cave’s locale along the mainstem Tennessee River and position within the basin make it potentially one of the most significant North American locations for documenting Pleistocene freshwater fish biodiversity. To date, excavations of Bell Cave have resulted in the recovery of nearly 4,000 cataloged lots of late Pleistocene faunal remains from four distinct stratigraphic levels within the cave. However, to this point, no formal description of the Bell Cave ichthyofauna has been undertaken.Acipenser, Drum Aplodinotus, Catfish Ictalurus, and Redhorse Moxostoma, but did not cite any cataloged specimens or provide any systematic material or comparisons. Formal descriptions of the fishes from Bell Cave will hopefully help fill a gap in our understanding of regional diversity during this time period.Formal descriptions of other faunas captured from Bell Cave have included amphibians and reptiles , birds , and mamThe purpose of this study is to expand our understanding of late Pleistocene fish diversity in the Tennessee River basin by closely examining existing faunal lists as well as describing the 235 cataloged lots of Bell Cave fish material to develop our understanding of the fish assemblages during this time period. These fish remains represent one of the largest Pleistocene fish faunas reported from North America, the largest from the Tennessee River Basin, and the first published from the state of Alabama. The overarching comparison of the Bell Cave fauna to others within the Tennessee River Basin not only serves to fill a regional gap in terms of knowledge of late Pleistocene fish occurrences, but also provides valuable insights into the biogeography and paleoecology of these fish taxa within the basin during the late Pleistocene.Late Pleistocene faunal lists from localities located within the boundaries of the Tennessee River Basin were recorded based on both published and unpublished accounts. All specimens reported from unpublished localities were directly examined by one or more of the authors. While the primary focus of this manuscript is on the formal description of the Bell Cave fish fauna, all known  late Pleistocene localities are reviewed in this study as a means to compare with the Bell Cave fauna and to provide a larger view of the late Pleistocene fishes within the Tennessee Basin. These late Pleistocene localities were chosen based on the following criteria: (1) locality had a described late Pleistocene fish fauna (published or unpublished); (2) the locality was within the recognized boundary of the Tennessee River Basin; and (3) the site had described faunal remains that were late Wisconsinan in age or fall within the age range of 10,000–30,000 years before present. The fish faunas reviewed herein are the result of a search for sites meeting these criteria and derive localities that span the eastern to western extents of Tennessee River Basin, thus providing a representative overview of the late Pleistocene fishes known from the entirety of the Basin. Localities that fit these criteria are as follows, with general locations presented in 1.Site ACb-2 Bell Cave, Colbert County, AL—Source: This study.2.Site ACb-3, Colbert County, AL—Source: This study.3.Site ACb-5, Colbert County, AL—Source: This study.4.Appalachian Caverns, Sullivan County, TN—Source: This study.5.Baker Bluff Cave, Sullivan County, TN—Source: 6.Beartown Cave, Sullivan County, TN—Source: 7.Cheek Bend Cave, Maury County, TN—Source: 8.Dust Cave, Lauderdale County, AL—Source: 9.Guy Wilson Cave, Sullivan County, TN—Source: This study.10.Saltville, Smythe County, VA—Source: 2 units were excavated within the main room of Bell Cave with matrix removed one stratum at a time. Bags and buckets of removed matrix and bone were carefully dried and later wet-screened through c. 17 mesh screens either onsite or at the Red Mountain Museum laboratory  and recorded in MS Excel. In the event a cataloged lot contained multiple identified taxa, the original catalog number was retained, but sub-numbers were assigned . Multiple elements associated with a single individual were given a single catalog number. All figured specimens were photographed with a tripod mounted Nikon d3000 camera with Nikkor 60 mm lens, and broken specimens were repaired (when possible) with B-76 butvar. Specimen photographs were rendered in Adobe Photoshop CS2 software as part of the production of the presented figures. Due to the number of specimens/fragments it was not feasible to photograph all materials. However, all lots and specimens are available for study upon request.The Fishes of Tennessee (1993) and Boschung and Mayden’s Fishes of Alabama (2004).Bell Cave fish taxa identified as part of this study are provided in Bell Cave is located along the south bank of the Tennessee River in the northwest corner of Alabama in Colbert County . SituateMuseum excavations within Bell Cave revealed an accumulation of approximately 70 cm of sterile clay overlain by 40 cm of bone packed mud . VertebrZones 1, 2, and 3 consist of bone-filled reddish textured clays that were deposited as independent mud flow events from fissures at the southwest end and in the ceiling of the main cave room . Zones 1Zone 1–2 (0–15 cm)—11,820 + 480/−500 B.P. Dated from an Ursus americanus femur collected in the upper part of Zone 2 and the lower part of Zone 1. Date determined by Dicard Radioisotope Company —sample DIC 2929, 1(222-02-3).Zone 3 (15–30 cm)—Not enough large bone was present for dating, but this zone has a similar sedimentary profile and is faunistically identical to Zone 1–2.Zone 4 (30–40 cm)—26,500 + 870/−990 B.P. Dated from large longbone fragments collected at the base of Zone 4, just above the sterile layer. Date determined by Dicard Radioisotope Company —sample DIC 3117, 236-61. As a specific caveat to prior literature on the dating of these zones, in a study of the Bell Cave herpetofauna, Overall, Zones 1–2 and 3 represent secondary depositional events in which consolidation and mixing within each zone may have occurred , Boschung and Mayden’s Fishes of Alabama (2004), and Page and Burr’s Field Guide to Freshwater Fishes (2011). References to current mainstem taxa occurrences and abundances follow recent (1993–2014) Tennessee River mainstem electrofishing and gillnet surveys undertaken by Tennessee Valley Authority (TVA) Biologists . The systematic arrangement of higher taxonomic rankings follows that of Referred Material—see MaterialFin rays, vertebrae, dentaries, premaxillae, teeth, operculi, spines, unknown fragments.OccurrencesZones 1–2, 3, 4, Mixed.Remarksin situ. This element could represent a possible Hiodontidae member. Additionally, lot RMM 4950 contains a few cranial elements that could represent a single Bowfin Amia calva (Amiidae) individual. Other unidentified fragments may actually be smaller non-fish vertebrate fragments such as mammal or bird remains mistakenly apportioned with fishes. Other fragments may be too eroded or amorphic for any identification attempts and remain cataloged as such. Regardless, additional matrix from future cave excavations may result in further taxonomic detail and resolution.Although much of the Bell Cave material could be identified at least to the family level (450 unique identifications arranged in lots), 114 collections of material still remain problematic for definitive identification. The majority of this material is comprised of unidentifiable vertebrae and fin rays; however, several other fragments carry notes in A caveat to any discussion of late Pleistocene fish remains is that these collections almost undoubtedly represent a subset of the actual living assemblage and that numerous taxa are likely missing due to some form of taphonomic bias occurring between decomposition, transport, deposition, preservation, and collection processing. For example, the complete absence of otoliths among the examined material or within the >100 kg of recovered bulk matrix from Bell Cave leads us to believe this absence is preservational. The chemical composition of nearly all Actinopterygiian otoliths is aragonite , Spotted Gar (L. oculatus), and Shortnose Gar (L. platostomus), once considered extirpated from this section of the basin (Atractosteus spatula). Together, the three Lepisosteus taxa comprise 1,809 recorded individuals, with Spotted Gar representing the most common. In the Bell Cave samples, their characteristic ganoid scales coated with ganoin enamel were the most numerous elements identified from Gar taxa. The presence of fine needle-like striated teeth of varying position rooted with a circular base in a shallow socket were also used to delineate Lepisosteidae. Only one dentary element associated with the recovered material was diagnostic beyond the generic level. This particularly long and narrow dentary bone with teeth in situ strongly suggests the presence of Longnose Gar in the samples, while conversely, the presence of more compressed dentary bones with teeth in situ likely indicate the presence of Spotted and/or Shortnose Gar. Several extremely large Lepisosteidae fragments, including comparatively thick scales, may also indicate the presence of Alligator Gar in the Bell Cave samples. However, no Alligator Gar are known to have ever been collected from the mainstem Tennessee and there is not enough intact diagnostic material to confirm this taxon. In addition to the Bell Cave specimens, remains identified as Lepisosteus sp. have been identified from Baker Bluff  and the Redfin Pickeral (E. americanus), while The presence of these remains indicate a range extension into Alabama for two of these taxa. Esox tooth samples. The large size of the teeth were used to rule out either the ‘native’ Redfin or Chain Pickerel. One example of a large, intact palatine with tooth socket combinations was also used to definitively identify the presence of Muskellunge. When present, neural arches were also used to distinguish between Northern Pike and Muskellunge vertebrae. A near complete Muskellunge dentary was identified on the basis of submandibular pore counts , and Central Stoneroller , were identified in recent mainstem surveys in Alabama; however, only the Central Stoneroller is considered native to the basin by C. pauciradii). However, the Bluefin Stoneroller is typically only associated with the Georgia portion of the Tennessee River Drainage Basin, well outside the vicinity of Bell Cave  and River Chub, are noted to occur in the basin, although the former is not always noted as present in the drainage basin , and although remains belonging to the River Chub have been identified from both Bell Cave and Cheek Bend Cave, the latter locality also produced remains belonging to Hornyhead Chub N. biguttatus  and Baker Bluff  in Tenneennessee . Remainsguttatus . Other CVirginia ; Bluntnoend Cave .MaterialEpihyal, hypohyal, basipterygium, hyomandibular, basihyal, scapula, pharyngeal teeth, supracleithrum, fin ray, quadrate, cleithrum, vertebrae, operculum, maxilla, orbitosphenoid, parietal, basioccipital, pharyngeal, angular, ethmoid.OccurrencesZones 1–2, 3, 4, Mixed.MaterialDentary.OccurrencesZones 1–2, 3.MaterialOperculum, hyomandibular.OccurrencesZone 3.MaterialAngular, operculum, rostral, epihyal, angular, parasphenoid, pharyngeal.OccurrencesZones 1–2, 3.MaterialPharyngeal teeth, rostral, cleithrum, epihyal, maxillary, dentary, neural process, basioccipital, parietal, orbitosphenoid, scapula, urohyal.OccurrencesZones 1–2, 3, 4, Mixed.MaterialPremaxillary, palatine.OccurrencesZone 3.MaterialDentary, maxillary.OccurrencesZones 1–2, 3.RemarksMinytrema melanops), Northern Hogsucker (Hypentelium nigricans), and the Blue Sucker (Cycleptus elongatus). Genera represented include Moxostoma, Ictiobus, and Catostomus. All definitive species identified still occur in the mainstem, with one notable exception. Represented by only two cranial fragments, the remains of the extinct Harelip Sucker, are perhaps the most significant among the Bell Cave Catostomidae remains. Little is known about the Harelip Sucker’s niche, distribution, or eventual decline, except that the species is thought to have preferred medium to large warm-water lotic systems with low rates of sedimentation and that population declines occurred concurrently with these habitats becoming increasingly degraded as a result of agricultural sedimentation  of a damaged or weathered cleithrum may be from a Silver Redhorse Moxostoma are widely represented within sites in our study area with specimens identified as Moxostoma sp. reported from Dust Cave, Appalachian Caverns, Baker Bluff Cave. Other Redhorse species found in Bell Cave have also been reported at other localities including River Redhorse from Appalachian Caverns, Baker Bluff Cave, and Cheek Bend Cave as well as Shorthead Redhorse from Cheek Bend Cave. Cheek Bend Cave has also produced three species of Redhorse not identified from Bell Cave, Silver Redhorse, Black Redhorse M. dusquesnei , and Golden Redhorse M. erythrurum . Appalachian Caverns, Baker Bluff, Cheek Bend, and Guy Wilson caves also report the presence of Northern Hogsucker Hypentellum nigricans, another taxon not identified from Bell Cave. Of the localities within our study area, remains belonging to Buffalo are only known from Bell Cave  as well as potential evidence for range expansions for several of these taxa. Madtom spines are often noted by their ornate serrae and curvature patterns coupled with their relatively small size. However, Stonecat Madtoms were distinguished primarily via a general lack of these ornamentations, including any anterior or posterior serrae, but with several posterior notches on the posterior distal end of the pectoral spine. Stonecat Madtom remains also included characteristic premaxillaries with elongated processes extending from functional tooth pads  on the basis of their more prominent, recurved, and unique spacing of anterior serrae  and Cheek Bend Cave in Tennessee . Specimeer Bluff  and Bearer Bluff  in Tenneer Bluff . Remainser Bluff .MaterialPremaxilla, dentary.OccurrencesZones 1–2, 3.RemarksCottus bairdi) and Banded Sculpin (Cottus carolinae). Both of these taxa are still recovered in mainstem fisheries surveys, albeit in relatively low numbers. The Sculpin fragments identified from Bell Cave lots were also some of the most uncommon of the taxa identified. Sculpin fragments were described based on characters outlined in Two Cottidae taxa are recognized natives of the Tennessee River Basin , the MotCottus sp. specimens identified from Bell Cave, this taxon has also been identified from Appalachian Caverns in Tennessee (this study). Both Carolina Sculpin and Mottled Sculpin have been reported from Cheek Bend Cave in Tennessee  and Largemouth Bass , neither of which were definitely identified from Bell Cave samples. The absence of Largemouth Bass and Bluegill is not particularly surprising given their habitat affinities for the more recent impoundment conditions of the Tennessee River mainstem that were nonexistent in the Pleistocene, coupled with decades of introduction efforts throughout the watershed. However, the absence of any Sunfish Lepomis taxa is surprising and should be further explored in future studies as several of these  of these taxa as common native members of the modern Tennessee River fish assemblage. Interestingly, the most common Centrarchidae in recent mainstem collections are Bluegill  was used as a diagnostic feature. Differentiation between the two species was often a function of size inference . Specific to the Pleistocene samples, the lack of any Darter remains is likely a function of preservation, size, collection, recovery and processing biases inherent in the Bell Cave samples.Both of these taxa are native to the Tennessee River drainage and have been surveyed in recent mainstem samples. Relative to modern mainstem TVA collections, the Sauger is a more numerically dominant member of the assemblage than the Walleye. If this trend has historical continuity it could mean that the majority of the unidentified remains should be assigned to Sauger . The prind Perca , severalE. blennioides, Yellow Perch Perca flacescens, and Logperch Percina caprodes have been identified among the remains at Cheek Bend Cave in Tennessee  in the assemblage and likely higher in biomass than the majority of other taxa. This commentary on contemporary abundance appears to be consistent with the assemblages captured in the Bell Cave Pleistocene deposits as Drum samples were very well represented across all zones. Freshwater Drum remains, pharyngeal bones and teeth in particular, were identified from a variety of size classes and comprised the most prevalent materials in the Bell Cave samples. The pharyngeal bone was identified through robust thickness, triangular shape, and rounded tooth sockets of increasing circumference proximal to pharyngeal edges. Corresponding pharyngeal teeth are molariform in shape with rounded edges and a dome shaped crushing functional surface . Freshwater Drum pharyngeal bones and teeth are well-known in the Pleistocene fish literature; however, this represents the first associated museum records with collection information but see  for AlabThe Tennessee River Basin fish assemblage preserved in the late Pleistocene deposits provide a detailed look into the paleoecology of one of the most species rich regions in North America. In total, 41 unequivocal taxa from 10 sites are reported from across the basin. Most significantly, excavations of Bell Cave deposits yielded the largest taxa list with a total of 11 families, 19 genera, and 24 identifiable fish species. Given that numerous uncommon fragment types could not be identified beyond family or genus level, this combined to indicate the presence of at least 28 unequivocal taxa in the assemblage.The Bell Cave assemblage was captured in four snapshots denoted by ‘zones’ which ranged in age from approximately 10,000 to 30,000 years before present. None of the zones exhibited complete turnover in assemblages; however, during this 20,000 year range, Zone 3 (intermediate to Zones 1–2 and 4) was found to comprise the greatest diversity of unequivocal taxa. When grouped according to genera, diversity from Zones 1–2, 3, and 4 included 15, 15, and 8 distinct genera, respectively. Family diversity of Zones 1–2, 3, and 4 included 10, 11, and 8 distinct families, respectively. Of these families, 8 were common to each zone , with the presence of 3 families  driving the differences between zones. Although these zones are similar, differences between them may be attributable to habitat at the time of subaqueous deposition of Zone 4 and viscous mudflows resulting in Zones 1–2 and 3. This indicates full submersion of the cave habitat during the time period of Zone 4 with fish being stranded as mainstem water levels dropped compared with marginal riparian habitat flooding resulting in individuals washing and mixing with mudflows passing through fissures in the cave ceiling.Not surprisingly, much of the contemporary basin wide fish diversity is not represented in the identified fragments or literature review—although the full complement of Tennessee River or basin wide fish diversity would not be expected to occur at a single site. This taxa diversity differential likely represents a taphonomic bias as a result of decomposition, transport, deposition, differential bone preservation, collection processing, or a combination of these factors. From what is preserved, the reconstructed Bell Cave assemblage resembles a typical medium-to-large river fish assemblage of the major contemporary Ohio River Basin tributaries . In comparison of the faunas from all of the late Pleistocene sites with modern diversity, more than 75% of the represented taxa are listed as native and present within the Tennessee River Basin today see .Ectopistes migratorius, an extinction resultant of human impact in historic times while four of the identified bird taxa from Bell Cave are not current residents in Alabama. Range changes in these bird taxa, including the Ruffed Grouse Bonasa umbellus, Greater Prairie Chicken Tympanuchus cupido, Common Raven Corvus corax, and Eurasian Magpie Pica pica are the result of Pleistocene dispersal , and those with boreal affinities that experienced northern dispersal patterns, such as the Red-backed Vole Clethrionomys gapperi, Least Weasel Mustela nivalis, Caribou Rangifer tarandus, and American Red Squirrel Tamiasciurus hudsonicus.These largely similar comparisons between Pleistocene and modern fauna are also paralleled in other taxa. Previous investigations of the Bell Cave (ACb-2) faunas have described the known birds , herpetoispersal . EbersolTaken together as a combined terrestrial and aquatic ecosystem, the presence of many of these mammals, birds, herpetofauna, and fishes suggest a more northern climate and habitat  was present in this region during at least the late Pleistocene. This is further evidenced by the presence of extant stands of boreal trees in northern Alabama, such as hemlocks . The bioOver 50 years ago, However, the presence of 41 unequivocal taxa reported here from late Pleistocene localities within the Tennessee River Basin compared with modern diversity records suggests the incidence of significant biases as a result of any or all of the following: preservational, taphonomic, collection, and identification biases. Nevertheless, the assemblages discussed herein  represent the most intact Pleistocene freshwater fish deposits in North America. Future investigation can hopefully reduce these biases and add to the work currently presented here. In particular, as molecular techniques continue to improve, it may be possible to amplify ultrashort DNA fragments that could remain in the preserved bones . Molecul10.7717/peerj.1648/supp-1Supplemental Information 1Raw data.Click here for additional data file.10.7717/peerj.1648/supp-2Supplemental Information 2Click here for additional data file.


AR gene produces a diverse range of AR-V transcripts in primary breast tumors. The most frequently and highly expressed variant was AR-V7 (exons 1/2/3/CE3), which was detectable at the mRNA level in > 50% of all breast cancers and at the protein level in a subset of ERα-negative tumors. Functionally, AR-V7 is a constitutively active and ADT-resistant transcription factor that promotes growth and regulates a transcriptional program distinct from AR in ERα-negative breast cancer cells. Importantly, we provide ex vivo evidence that AR-V7 is upregulated by the AR antagonist enzalutamide in primary breast tumors. These findings have implications for treatment response in the ongoing clinical trials of ADT in breast cancer.The importance of androgen receptor (AR) signaling is increasingly being recognized in breast cancer, which has elicited clinical trials aimed at assessing the efficacy of androgen deprivation therapy (ADT) for metastatic disease. In prostate cancer, resistance to ADT is frequently associated with the emergence of androgen-independent splice variants of the AR  that lack the LBD and are constitutively active. Women with breast cancer may be prone to a similar phenomenon. Herein, we show that in addition to the prototypical transcript, the  Estrogen signaling, mediated via the estrogen receptor alpha (ERα), is a key determinant of the growth and survival of normal and the majority of malignant breast epithelial cells. As such, inhibition of ERα signaling by ERα antagonists or drugs that block the biosynthesis of estrogens (i.e. aromatase inhibitors) are the mainstay adjuvant treatments for estrogen-sensitive breast cancer, which encompasses approximately 70% of all cases. The remaining breast cancers lack ERα and women with this type of disease do not gain benefit from current adjuvant hormone therapies. A subset of ERα-negative cancers are characterized by amplification or overexpression of human epidermal growth factor receptor 2 (HER2) and can be treated with HER2-targeting agents. However, 15–20% of breast tumors lack ERα, HER2 and a clinically relevant biomarker of ERα signaling, progesterone receptor (PGR); this subtype is termed triple-negative breast cancer (TNBC) [The androgen receptor (AR) is a steroid hormone receptor structurally related to ERα that mediates the action of androgen hormones, is critical for development of the male phenotype and has a role in modulating the female phenotype. AR signaling has primarily been studied in prostate cancer, where it plays a central role in both the initiation and progression of disease , but morThe concept of AR as a breast cancer oncogene, and the availability of effective AR-targeting agents used to treat prostate cancer, has elicited clinical trials assessing the efficacy of AR antagonists and androgen biosynthesis inhibitors in preventing disease progression in women with advanced, metastatic breast cancer. These agents, along with gonadotropin-releasing hormone (GnRH) agonists/antagonists, are components of androgen deprivation therapy (ADT), the current mainstay of treatment for men with metastatic prostate cancer. ADT is initially effective in most men but inevitably fails and the resultant disease, termed castration-resistant prostate cancer (CRPC), is incurable and the primary cause of prostate cancer mortality. The totality of research over the past decade has revealed that the most common event associated with failure of ADT is the inappropriate activation or maintenance of AR signaling . Based oAR gene [AR gene, have variable structures but each lacks all or a portion of the ligand-binding domain (LBD) [Androgen signaling in the “castrate” environment is often mediated by direct changes to AR, including upregulation of its expression , amplifiAR gene , 19 and AR gene , 20–25. AR gene –28. Thesin (LBD) . This cain (LBD) . It is nin (LBD) , 31–34.Given the arrival of AR-targeting agents to the breast cancer clinical arena, the heterogeneous nature of this disease and the dichotomous actions of AR in different breast cancer contexts, the question of whether AR-Vs are expressed in breast malignancies is critically important. Herein, we demonstrate that the AR-V7 variant is commonly expressed in primary breast cancers and breast cancer cell lines and provide evidence that this factor could promote growth and mediate resistance to ADT in a subset of breast tumors.AR gene. For data presentation, the tumors are classified into five subgroups based on the PAM50 classification system. In this cohort, the prototypical, full length AR transcript (AR-FL) was expressed at the highest average levels in Luminal A and lowest levels in Basal tumor sub-groups , 3-CE1 , 2-CE4 , 3-CE5  and 6–9 . In general, the number of reads for predicted ch other , indicatsplicing –38.AR-V7 transcripts compared to other AR splice variants in the TCGA dataset, we validated its expression in an independent cohort of prospectively collected primary breast cancers (n = 54), comprised of the expected proportion of ERα-positive (64.8%) and ERα-negative (35.2%) cases (AR-FL were detected in 53 tumors (98.1%) and transcripts encoding AR-V7 were detected in 29 tumors (53.7%), which closely matched the frequency of occurrence in the TCGA cohort. Most of the tumors had relatively low levels of AR-V7 transcript, with the exception of 3 ERα-negative tumors  and one with intermediate AR-V7 expression (black dot), had roughly equivalent levels of the two transcripts  cases . Transcrs Figure . Copy nus Figure . Similard Figure .AR-V7 transcript copy numbers had readily detectable AR-V7 protein by IHC  to 1.1% (ZR-75–1) .To identify an appropriate model to investigate AR-V7 function in breast cancer, qRT-PCR was used to quantify its expression in a panel of breast cancer cell lines. Prostate cancer cell lines 22Rv1 and VCaP, which express high and medium levels of AR-V7, respectively , were in1 Figure , which e1 Figure . As a pr) Figure . SimilarAR-V7 and AR-FL transcripts  was observed in the two ERα-negative breast cancer lines that had the highest levels of AR-V7 mRNA, MDA-MD-453 and MFM-223, but was not apparent in lines with lower  or no (MDA-MB-231) expression of s Figure . Transfes Figure , confirmFunctional studies were undertaken to assess the potential biological relevance of AR-V7 in an ERα-negative breast cancer context. The MDA-MB-453 cell line was chosen as the primary model for further investigations based on our finding that it expresses detectable levels of AR-V7 protein. Moreover, AR-FL promotes the growth of MDA-MB-453 cells and research in this model represents a key component of the basis for targeting AR signaling in breast cancer , 14, 39.The functionality of AR-V7 in the MDA-MB-453 cell line was further assessed by analyzing its subcellular localization. In prostate cells, AR-FL localizes to the cytoplasm in the absence of androgen stimulation and undergoes rapid nuclear translocation upon hormone treatment. This feature was also characteristic of the prototypical AR in MDA-MB-453 breast cancer cells Figure . By contTo investigate the target genes of AR-V7 in MDA-MB-453 breast cancer cells, we undertook microarray profiling following transient over-expression of AR-V7 in the presence or absence of DHT . In the Interestingly, comparison of genes regulated by forced over-expression of AR-V7 in MDA-MB-453 cells (this study) versus LNCaP prostate cancer cells  revealedAR-V7-regulated genes were examined in a large clinical breast cancer dataset recently published by Ringner and colleagues, which is comprised of multiple different cohorts with long-term outcome data . The corPrevious studies have shown that AR-FL is required for optimal growth of the MDA-MB-453 cell line , 14, whiTo assess whether AR-V7 could mediate resistance to an AR antagonist in breast cancer, we transiently transduced MDA-MB-453 cells with a lentivirus designed to over-express AR-V7  and asseAR-FL and AR-V7 transcripts, whereas treatment with DHT inhibited transcription of the AR gene  in response to treatment with enzalutamide (one sided t test p = 0.004)   identified herein, as potential predictive biomarkers of treatment response.Clinical trials are underway that repurpose ADT agents approved for prostate cancer to different breast cancer subtypes, particularly TNBC . These trials arose from the recognition that a subset of TNBCs that lack viable targets for therapy are positive for AR, combined with pre-clinical studies suggesting that AR-FL signalling promotes growth in this context. There are two caveats to this approach: first, AR expression is used as a biomarker for patient selection but AR-FL activity markedly varies depending on ERα status and molecular subtype [e cancer , 34. MorAR-V7 transcript was expressed at low levels in about half of all breast cancers, with highest expression in ERα-negative cases that were HER2-enriched. It is noteworthy that the ratio of AR-V7 to AR-FL mRNA ranged from 0–100% in the TCGA cohort and 0–86% in our prospective cohort, a greater range than that observed in a collection of CRPC bone metastases that would be expected to have high levels of this variant [AR-V7 occurred in the PAM50 HER2-enriched molecular subgroup. In our prospectively-collected validation cohort, we detected high levels of AR-V7 transcript and protein in TNBCs as well as HER2-enriched cases. Importantly, these are the subtypes in which AR signaling is thought to be oncogenic in certain contexts, further highlighting the potential biological relevance of constitutively active AR-Vs. variant . High le variant –15, 43. AR gene regulation identified herein recapitulate findings in the prostate cancer context, which is not surprising given the known similarities between these two malignancies, particularly in terms of endocrine signaling [AR-V transcripts in breast cancer generally tracked with that of AR-FL, a feature observed in prostate cancer cells and tissues [AR splicing is ubiquitous, such that increased AR gene transcription results in increased levels of AR-V transcripts [AR gene transcription. These similarities suggest that AR-V-targeted agents, which are under clinical development for treatment of prostate cancer [Many aspects of AR-V7 functionality and ignaling . First,  tissues . Mechaninscripts . Third, nscripts  was funce cancer , could bAlthough we report many similarities between AR-V7 in breast and prostate contexts, a striking difference was that this factor governed a gene expression program highly disparate from that of the prototypical receptor. In prostate cancer cell lines, studies aimed at elucidating the AR-V7-driven transcriptome have been equivocal: while some have suggested that this variant primarily regulates a classical androgen-regulated gene set , 30, othRecent studies exploiting the power of deep RNA sequencing have demonstrated that most genes are characterized by frequent and complex alternative splicing, which may represent a ‘readiness’ for adaptation to changing environmental conditions , 37. We In summary, we demonstrate that AR-V7 is expressed in a subset of breast cancers and present evidence that this factor plays a role in regulating response to ADT. This concept suggests that testing for AR-V7 in tumors from women in current clinical trials employing ADT therapy, which to date have demonstrated significant variability in patient response, is warranted.Unprocessed RNA-seq data representing tumors and matched normal samples within the TCGA breast cancer cohort were downloaded from CGHub (July 2013 to Sep 2014). For samples where the data was available as BAM files (generated by MapSplice) and not in FASTQ format, the raw reads were extracted using sam2fastq v1.2 from UNC Bioinformatics Utilities.RNA transcript annotation was combined using isoforms from: 1) MISO v2.0 ; 2) UCSCThe raw RNA-seq reads were processed using the following pipeline: 1) Map all reads to the UCSC hg19 (NCBI GRCh37) human genome assembly using Bowtie v1.0.0 with the -v 2 parameter  and RSEMRNA transcript levels were normalized with the trimmed mean of M values (TMM) method  using a All reads mapping within the AR locus were extracted directly from the BAM files and compared to the manually curated list of AR splice variants. Reads mapping within exons were discarded and only gapped reads spanning splice junctions were used as a measure of the presence of processed RNA variants, to exclude reads possibly originating from unprocessed transcripts or DNA contamination. AR variants containing cryptic exons were considered ‘detected’ in a given sample if there was at least one read spanning the 3′ end of the upstream exon and the 5′ end of the cryptic exon. Exon-skipping AR variants were ‘detected’ if there was one or more reads spanning the splice junction between the 3′ end of the upstream exon and the 5′ site of the exon downstream of the skipped exon(s). There are no junctions that are unique to the full-length AR transcript, so to normalize 3-CE3 reads by AR expression Figure  the numbBicalutamide was obtained from AstraZeneca  and dissolved in ethanol. Enzalutamide (MDV3100) was obtained from SelleckChem  and dissolved in dimethyl sulfoxide (DMSO).pcDNA-AR and pcDNA AR-V7 expression plasmids have been described . pEGFP-AFresh breast cancer specimens were obtained with written informed consent from women undergoing breast surgery at Burnside War Memorial Hospital or Flinders Medical Centre . Ethical approval for this study was granted by the University of Adelaide Human Research Ethics Committee and the individual hospitals . Excised tissue samples were delivered to the laboratory on ice in a sterile container within one hour following surgery and washed in culture medium comprised of phenol red-free RPMI. Representative pieces of tissue were fixed in 4% formalin in phosphate-buffered saline at 4°C overnight and subsequently processed into paraffin blocks. Sections (2 μm) were stained with hematoxylin and eosin and examined by a pathologist to assess histology and pathology.MDA-MB-453, T-47D, 22Rv1, VCaP, ZR75–1, MDA-MB-231, MCF7 and HEK293T/17 cells were obtained from the American Type Culture Collection . MFM-223, Cal-51 and Cal-148 cells were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures . LNCaP-95 cells were a kind gift from Dr. Alan K. Meeker (Johns Hopkins University). MDA-MB-453, T-47D, ZR75–1 and 22Rv1 cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS); VCaP cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS, 1% sodium pyruvate, 1% MEM non-essential amino acids and 0.1 nM 5α-dihydrotestosterone ; MDA-MB-231 cells were maintained in RPMI-1640 medium containing 5% FBS; MCF7 cells were maintained in Eagle's Minimum Essential Medium (EMEM) containing 10% FBS, 2 mM L-glutamine, sodium pyruvate and 0.2 U/mL insulin; MFM-223 cells were maintained in EMEM containing 10% FBS, 2 mM L-glutamine, and insulin-transferrin-sodium selenite (ITS) supplement; Cal-51 cells were maintained in DMEM containing 10% FBS; Cal-148 cells were maintained in DMEM containing 10%FBS, 2 mM L-Glutamine and EGF (1ug/100mL); HEK293T/17 cells were maintained in DMEM containing 10% FBS and 20 mM HEPES.Extraction of RNA from cells was done as described . RNA wasPreparation of whole cell lysates from breast and prostate cancer cell lines and Western blotting were done as described previously . PrimaryImmunohistochemical staining for AR-V7 was done on serial 4 μm breast tissue sections as described previously  using thPreparation of formalin-fixed, paraffin-embedded tissue sections for immunofluorescence was done as described previously . For all5 cells/well in 6-well plates) were transfected in suspension with 100 nM siRNA using RNAiMax  and protein lysates were collected 72 h post-transfection and analyzed by immunoblotting.Small interfering RNAs were purchased from Ambion (Life Technologies) and had the following target sequences: AR (GGAACUCGAUCGUAUCAUU) and AR-V7 (GUAGUUGUGAGUAUCAUGA) . An addiTransactivation assays were performed as described .4 cells/well onto glass coverslips in 12-well plates. Once seeded, cells were transfected with 100 ng/well of plasmids designed to express GFP, GFP-AR or GFP-AR-V7 by mixing plasmid DNA and polyethylenimine at a ratio of 1:3.1 in 0.9% sodium chloride. Cells were subsequently treated with 10 nM DHT or ethanol (vehicle control) and left overnight. The cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Alexa-Fluor 568 phalloidin  was used according to the manufacturer's protocol to visualize actin. ProLong Gold Antifade Reagent with DAPI (Life Technologies) was used as mounting media and to detect cell nuclei. Cells were viewed using Zeiss 700 confocal microscope with a pinhole aperture of 1.5 airy units.MDA-MB-453 cells were seeded at a density of 6.0 × 105 cells/well in a 6-well plate) were transiently transfected with 1 μg of plasmid DNA designed to express AR or AR-V7 (or an empty vector control) using Lipofectamine-2000. After 4 h, cells were treated with 1 nM DHT or ethanol (vehicle control). After 24 h, RNA was extracted using TRIzol  and purified using an RNeasy Mini Kit (Qiagen). Prior to microarray analysis, AR and AR-V7 overexpression and evidence of efficacy of DHT treatment (i.e. FKBP5 induction) was confirmed by qRT-PCR. Subsequently, RNA was analyzed using Affymetrix Human Gene 2.0 ST arrays by the Adelaide Microarray Centre, as described previously [p-value adjusted using a step-up multiple test correction to control the false discovery rate. Adjusted p-values < 0.05 were considered to be significant. Raw and normalized data have been deposited in the Gene Expression Omnibus database (accession number GSE65738).MDA-MB-453 cells (6.6 × 10eviously . Differe3 cells/well) and left to adhere overnight. The following day, day zero and standard curve plates were assayed by adding 10 μl of 10 mg/mL MTT (Sigma) per well and incubated at 37°C for 4 hours. 100 μl of 20% SDS (in 0.2 M HCl) was then added to each well and left to solubilize overnight. The next day the absorbance at 570 nm in each well was measured using a FluoStar Omega plate reader . All experimental plates were treated with 10 nM DHT at day zero and assayed using the procedure above at the indicated time points.MTT -2,5-Diphenyl Tetrazolium Bromide) assays were performed on MDA-MB-453 cells treated with siRNAs targeting AR or AR-V7. MDA-MB-453 cells in RPMI-1640 medium containing 10% charcoal stripped serum (CSS) were transfected in suspension with 100 nM siRNA using RNAiMax (Life Technologies), seeded in 96 well plates (6 × 104 cells/well in RPMI-1640 medium containing 10% CSSS in 24-well plates and transfected in suspension with 100 nM siRNA using RNAiMax (Life Technologies). 72 h post-transfection, day zero counts were taken and cells were treated with 10 nM DHT (vehicle control). Live and dead cells were quantified at indicated time points using Trypan blue.Trypan blue exclusion counts were performed on MDA-MB-453 cells treated with siRNAs targeting AR or AR-V7. Cells were seeded at 4 × 106 cells per T25 flask and left overnight to adhere. The next day, cells were transduced with concentrated lentivirus using a MOI of 1 and 6 μg/mL Polybrene (Sigma) in normal growth media.Lentivirus particles designed to express GFP and AR-V7 were prepared using a standard third generation packaging system in HEK293T/17 cells. For viral transduction experiments, MDA-MB-453 cells were seeded at 1.65 × 105 cells/well in 24-well plates and left to adhere overnight. Cells were treated the next day with vehicle control (DMSO) or 10 μM enzalutamide. At the indicated time points, cells were fixed and stained with Crystal Violet.Cell growth of lentivirus-transduced MDA-MB-453 cells expressing AR and AR-V7 was assessed by Crystal Violet assay, essentially as described . Briefly5 cells/well in 12-well plate) were seeded in phenol red-free RPMI containing 10% charcoal-stripped FBS. After 24 h, the media was removed and replaced with media containing 10 nM DHT, 10 μM bicalutamide, 10 μM enzalutamide or ethanol/DMSO (vehicle control) for 24 h. RNA and protein samples were analyzed by qRT-PCR or Western blotting, respectively.MDA-MB-453 cells .Raw and normalized microarray data from this study have been deposited in the Gene Expression Omnibus database (accession number GSE65738).


It has been well known that pediatric allergic rhinitis was associated with poor performance at school due to attention deficit. However, there were no cohort studies for the effect of treatment of allergic rhinitis on attention performance in pediatric population. Thus, the aim of this study was to investigate whether attention performance was improved after treatment in children with allergic rhinitis.In this ARCO-Kids (Allergic Rhinitis Cohort Study for Kids), consecutive pediatric patients with rhinitis symptoms underwent a skin prick test and computerized comprehensive attention test. According to the skin prick test results, the children were diagnosed as allergic rhinitis or non- allergic rhinitis. All of the patients were regularly followed up and treated with oral medication or intranasal corticosteroid sprays. The comprehensive attention tests consisted of sustained and divided attention tasks. Each of the tasks was assessed by the attention score which was calculated by the number of omission and commission errors. The comprehension attention test was repeated after 1 year.vs 97.0±16.6) and divided attention task (99.1±15.8 vs 91.8±23.5). Meanwhile, there was no significant difference of attention scores in children with non-allergic rhinitis.A total of 797 children with allergic rhinitis and 239 children with non-allergic rhinitis were included. Initially, the attention scores of omission and commission errors on divided attention task were significantly lower in children with allergic rhinitis than in children with non-allergic rhinitis. After 1 year of treatment, children with allergic rhinitis showed improvement in attention: commission error of sustained (95.6±17.0 Our study showed that management of allergic rhinitis might be associated with improvement of attention. Allergic rhinitis (AR) is one of the most common chronic diseases and a global health issue that represents a significant healthcare burden with serious adverse effects on quality of life. The prevalence of AR in adults is estimated to be between 10% and 30%, whereas several studies have estimated the prevalence of AR in children to be closer to 40% Although AR is still considered by many to be a bothersome condition of childhood Thus, we hypothesized that regular treatment of AR would improve attention. The aim of this prospective study was to investigate whether appropriate treatments were able to improve the attention performance in children with AR by analyzing data collected from children of a large Korean AR cohort.The present study was a longitudinal prospective cohort analysis conducted from February, 2009 through June, 2013, using a database of ARCO-Kids (Allergic Rhinitis Cohort Study for Kids), which is a prospective hospital-based cohort of children with allergic or non-allergic rhinitis (NAR) in Korea. All of the enrolled children received an endoscopic examination and a skin prick test (SPT). Exclusion criteria were as follows: (1) children younger than 3 years or older than 16 years of age; (2) children who had previously been diagnosed with attention deficit hyperactivity disorder or other psychotic disorders; (3) children who had hearing or visual problem; (4) children with adenotonsillar hypertrophy, craniofacial syndromes, or neuromuscular diseases. A written informed consent was obtained from participants or their parents. The study protocol was approved by the Institutional Review Board of Seoul National University Bundang Hospital and Seoul National University Hospital.Dermatophagoides pteronyssinus, Dermatophagoides farinae), molds , animal danders , pollens  and cockroach (Blattella germanica). Histamine and isotonic saline were used as positive and negative controls, respectively. The positive result of SPT was diagnosed when the ratio of an allergen to histamine wheal diameter was equal to 1 or greater and the mean wheal diameter of the allergen was 3 mm larger than that of negative  control. Then, the children were categorized into AR and non-AR groups. For children with AR, oral medication and/or intranasal steroid sprays were used regularly for 1 year, whereas for those with non-AR, symptomatic treatment was performed on demand.All of the children underwent SPT for evaluating their atopic status. The SPT was performed by using standardized extracts  for 13 common aeroallergens, including house dust mites , which was made by the Korean Academy of Child and Adolescent Psychiatry In addition, there were two dependent variables (omission and commission errors) on each of the 2 attention tasks. Omission errors were measures of inattention and were defined as failures to respond to the target. Commission errors were measures of impulsivity and were defined as inappropriate responses to the non-target. For removal of potential confounding factors, the resulting values of computerized CAT were presented as the attention score that is an adjusted value based on the standard population who matched gender and age. The standard population showed scores of attention task with mean of 100 and standard deviation of 15.t-test was used to determine whether there were associations between allergic rhinitis and inattention. In addition, the paired t-test was performed to compare the repeated computerized CAT results. The SPSS statistical software version 18.0  was used for statistical analyses. A P-value less than 0.05 were considered statistically significant.The values are presented as mean ± standard deviation (SD) unless otherwise specified. The independent A total of 1070 consecutive children were enrolled initially in our cohort. Among them, 1036 children (96.8%) who completed CAT at baseline and after 12 months were eligible for analyses in our study. Their mean age was 10.8 years , and 704 participants (67.9%) were males. Among them, 797 children (76.9%) were classified as having AR and 239 children (23.1%) as having non-AR. The general characteristics of the participants are presented in According to the presence of AR, the initial attention status was evaluated using the CAT. On the sustained attention task of non-AR group, the mean attention score of omission errors and commission errors was 98.3±17.8 and 98.2±17.3, respectively. Meanwhile, AR group had a 99.9±19.3 of omission errors and a 95.6±17.0 of commission errors on the sustained attention task. Although AR group children made more commission errors than non-AR group children on the sustained attention task, the differences were not statistically significant (p = 0.056).On the divided attention task, the mean attention score of omission errors and commission errors in non-AR group was 100.4±23.1 and 99.1±15.8, respectively, whereas AR group showed a 93.9±16.4 of omission errors and a 91.8±23.5 of commission errors. AR group children made significantly lesser omission errors (p = 0.022) and commission errors (p = 0.046) on the divided attention task than non-AR group children.To investigate the treatment effect on attention status in each group, we compared the mean attention score of CAT between baseline and after 1 year. Compared to pre-treatment attention performance, AR group showed significantly higher attention score of commission errors in both sustained attention task (p = 0.003) and divided attention task (p = 0.011) , but thePediatric AR is a common debilitating disorder that can adversely affect the quality of life and the academic performance of school-age children. The overall socioeconomic burden of AR is a great expense, with direct expenditures, such as costs of physician visits and medications. Moreover, indirect costs of this disease including missed school days, decreased learning capacity, reduced school performance, impaired sleep, and diminished quality of life are substantial In the present study, we found that children with AR showed a worse performance on divided attention compared to children with non-AR. In addition, in terms of commission errors of sustained/divided attention, the partial improvement of attention status was observed by treatment in pediatric AR. Our findings could be supported by previous studies showing that treatment of pediatric AR improved learning ability Sustained attention means the ability to direct and focus cognitive activity on specific stimuli Because attention deficit is known to be multifactorial, the present study was designed to examine independent therapeutic effects of AR on attention by using the attention score, adjusting for potential confounding factors, such as gender and age. Based on our results, the appropriate treatment of AR is beneficial for improvement of attention, especially on commission errors of sustained or divided attention tasks. Meanwhile, the therapeutic effect did not appear on omission errors. Our different findings may be explained by prior several studies Non-AR has similar symptoms of AR but no identified allergic reaction However, our study has some limitations. Firstly, the present study has no healthy control group. Therefore, it is hard to differentiate whether the improvement of attention was the effect of AR treatment or natural improvement by aging. However, non-AR patients who were set as a disease control group did not show improvement. Secondly, there may be a selection bias because our cohort was a hospital-based one. However, it may have been partially overcome because the present study was prospectively performed. Thirdly, our study lacked some important factors such as socioeconomic status and parental education levels that may influence the attention status of children. In spite of these limitations, the advantages of the present study may be its large sample size, the use of objective computerized attention test and a longitudinal design.The present study suggests that AR be associated with attention problems compared to non-AR in children. The commission errors of sustained and divided attention tasks could be improved by treatment of AR. Therefore, regular treatment of AR might also be warranted, in particular, in terms of improvement of attention problems in children with AR.


Structural determinants for CXCR4 heteromerization and functional consequences of CXCR4:α1A/B-AR heteromerization in intact arteries, however, remain unknown. Utilizing proximity ligation assays (PLA) to visualize receptor interactions in VSMC, we show that peptide analogs of transmembrane-domain (TM) 2 and TM4 of CXCR4 selectively reduce PLA signals for CXCR4:α1A-AR and CXCR4:ACKR3 interactions, respectively. While both peptides inhibit CXCL12-induced chemotaxis, only the TM2 peptide inhibits phenylephrine-induced Ca2+-fluxes, contraction of VSMC and reduces efficacy of phenylephrine to constrict isolated arteries. In a Cre-loxP mouse model to delete CXCR4 in VSMC, we observed 60% knockdown of CXCR4. PLA signals for CXCR4:α1A/B-AR and CXCR4:ACKR3 interactions in VSMC, however, remained constant. Our observations point towards TM2/4 of CXCR4 as possible contact sites for heteromerization and suggest that TM-derived peptide analogs permit selective targeting of CXCR4 heteromers. A molecular dynamics simulation of a receptor complex in which the CXCR4 homodimer interacts with α1A-AR via TM2 and with ACKR3 via TM4 is presented. Our findings further imply that CXCR4:α1A-AR heteromers are important for intrinsic α1-AR function in intact arteries and provide initial and unexpected insights into the regulation of CXCR4 heteromerization in VSMC.Recent evidence suggests that C-X-C chemokine receptor type 4 (CXCR4) heteromerizes with α G protein-coupled receptors (GPCR) comprise the largest group of cell surface receptors and mediate a multitude of important cell functions. It is well established that the physiological range of GPCR-mediated signaling events and functions is amplified through cross-talk between GPCRs ,3,4,5,6.1A/B-adrenergic receptors (AR) on the cell surface of vascular smooth muscle cells (VSMC). As interference with CXCR4:α1A/B-AR heteromerization inhibited α1-AR mediated responses of VSMC, these data suggested that CXCR4:α1A/B-AR heteromerization is important for normal α1-AR function. Furthermore, we have previously reported that a peptide analog of transmembrane helix (TM) 2 of CXCR4 disturbs heteromeric complex formation between CXCR4 and α1A/B-AR, but does not influence CXCR4:ACKR3 heteromerization [1A/B-AR heteromers, however, is unknown and the effects of CXCR4:α1A/B-AR heteromerization in VSMC on the function of intact arteries or on blood pressure regulation remain to be determined.Multiple lines of evidence suggest that chemokine receptors and adrenergic receptors (AR) may form heteromeric receptor complexes with receptors of the same family and that chemokine receptors may also form heteromeric complexes with adrenergic receptors ,27,28,29rization . Whether1A-AR and CXCR4:ACKR3 can be selectively targeted utilizing peptide analogs of transmembrane domain (TM) 2 and 4 of CXCR4, respectively, suggesting TM2 and TM4 of CXCR4 as possible contact sites in heteromeric receptor complexes. Accordingly, we modeled a hetero-hexameric receptor complex in which the CXCR4 homodimer interacts with α1A-AR via TM2 and with ACKR3 via TM4. Furthermore, we provide evidence that CXCR4:α1A/B-AR heteromers regulate intrinsic α1-AR function in intact arteries and new insights into the regulation of CXCR4 heteromerization in vascular smooth muscle from observations in a Cre-loxP mouse model.Here we show that CXCR4:α1A-AR and CXCR4:ACKR3 interactions in human VSMC (hVSMC) incubated with or without the TM peptide analogs and 1A-AR complexes, but did not affect PLA signals for CXCR4:ACKR3 interactions .To address the possibility that the observed changes in PLA signals are caused by TM peptide analog-induced receptor internalization, we then studied receptor expression levels on hVSMC by FACS analyses. As shown in n levels ,35, we tNext, we studied how interference with receptor heteromerization by the TM-derived peptides influences receptor function. To assess the effects of the TM peptide analogs on CXCR4-mediated effects, we first measured CXCL12-induced chemotaxis as a functional read-out. As shown in 1-AR function, we then tested whether the TM peptide analogs affect signaling and function of VSMC upon exposure to the selective α1-AR agonist phenylephrine. The TM2 peptide analog significantly inhibited phenylephrine-induced Ca2+ mobilization, whereas the TM4 peptide analog did not (p < 0.05 vs. control); TM4: 60% ± 7%; To assess the effects on α did not B. Simila50 for phenylephrine to induce vasoconstriction was 1.3 ± 0.04 μM and maximal constriction in response to phenylephrine was 54% ± 3% of the outer diameter (o.d.) in the absence of TM peptide analogs. In the presence of the TM2 peptide analog, the EC50 for phenylephrine was 2.3 ± 0.1 μM and maximal constriction 43% ± 3% of the o.d. , whose dimerization interface is on the opposite side compared to the CXCR4 dimer, as a reference structure. We aligned the β1-AR subunit A (not shown) with the CXCR4 subunit A  (not shown), whose TM4 is in its dimerization interface, with the CXCR4 subunit A so the histamine H1 receptor subunit B could be used for the arrangement of the ACKR3 model  as the core of a hetero-hexameric receptor cluster. We assumed TM2 of CXCR4 as a likely contact site for αubunit A A so the R3 model A. Likewiincluded B.1A-AR and ACKR3, respectively. The final structural coordinates of the receptor cluster  signals in a HEK293T expression system . The TM nd ACKR3 ,33. Whetr α1B-AR ,30,39,401A/B-AR heteromers on vascular function, we then utilized a Cre-loxP mouse model to delete CXCR4 in smooth muscle. CreERT2 positive mice, in which Cre-mediated recombination occurs exclusively in smooth muscle cells, have been used previously to study various aspects of vascular smooth muscle function [T2 positive mice homozygous for the CXCR4 loxP allele, as compared with mice after injection of vehicle. Quantification of the degree of knockdown revealed 60% reduction of CXCR4 expression on VSMC. This indicates incomplete excision of the loxP-flanked gene after Cre induction and is in agreement with incomplete deletion of loxP-flanked genes that have been observed in other transgenic mouse models utilizing smooth muscle myosin heavy chain CreERT2 [To gain initial information on the physiological role of CXCR4:αfunction . As expe CreERT2 ,43,44,451A/B-AR and CXCR4:ACKR3 heteromeric complexes in VSMC were not reduced, despite significant reduction of PLA signals for CXCR4, was unexpected. We previously showed that CXCR4 gene silencing in cultured human VSMC resulted in reductions of PLA signals for CXCR4 heteromers, in proportion to the degree of CXCR4 knockdown [CXCR4 gene silencing reduced PLA signals for CXCR4 heteromers in cultured human VSMC may not be contradictory because receptor expression levels in freshly isolated mouse VSMC that we used in the present study, and in cultured human VSMC that we utilized previously, could be substantially different [The finding that CXCR4:αnockdown . Our obsifferent .1A/B-AR heteromer expression levels in VSMC in this transgenic mouse model.Alternatively, it could be speculated that regulatory mechanisms occur in our genetic model, which promote CXCR4 heteromerization, and that such regulation is absent in cell cultures. Nevertheless, our finding that phenylephrine-reactivity of isolated arteries in pressure myography experiments and blood pressures of conscious mice were not affected after reduction of CXCR4 in VSMC is consistent with the maintenance of CXCR4: α1A/B-AR heteromers in VSMC as those were maintained after CXCR4 knockdown. This documents an unexpected outcome of a genetic animal model despite a straight forward experimental approach that was based on findings from cell culture experiments. Thus, genetic models that permit complete CXCR4 knockdown to abolish CXCR4:α1A/B-AR heteromers or in which mutated CXCR4 that is unable to interact with α1A/B-AR is expressed in VSMC will be required to answer this question in the future.Although our observations provide initial insights into the regulation of receptor heteromerization, our experimental approach was unable to evaluate the physiological function of CXCR4:α1A/B-AR and ACKR3, respectively, and suggest that TM-derived peptides permit selective targeting of CXCR4 heteromers. We anticipate that further development and refinement of TM-derived peptide analogs or small molecules that target specific receptor–receptor interaction sites can serve as useful tools to clarify the roles and mechanisms of GPCR heteromerization and may lead to the development of new compounds with therapeutic applicability. Our findings further support the notion that CXCR4:α1A/B-AR heteromers are important for intrinsic α1-AR function in intact arteries and provide initial and unexpected insights into the regulation of CXCR4 heteromerization in VSMC.In conclusion, our findings point towards TM2 and TM4 of CXCR4 as possible contact sites for heteromerization with αAll procedures involving healthy blood donors were conducted in accordance with the Declaration of Helsinki-Ethical Principles for Medical Research Involving Human Subjects and the National Institutes of Health human subjects policies and guidance and were approved by the Loyola University Chicago Health Sciences Division Institutional Review Board for the Protection of Human Subjects. Informed consent was obtained from all blood donors. All procedures involving animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, 8th Edition and were approved by the Institutional Animal Care and Use Committee of Loyola University Chicago . 2) and TM4 (VYVGVWIPALLLTIPDFIFANDD-OH) of CXCR4 were generated using the Fmoc protected amino acids in a solid-phase synthesis on a 433A Applied Biosciences Peptide Synthesizer and Liberty Blue Microwave peptide synthesizer  using Fmoc chemistry. The peptides were cleaved from the resin and deprotected with a mixture of 90.0% (v/v) trifluoroacetic acid (TFA) with 2.5% water, 2.5% triisopropyl-silane, 2.5% 2,2′-(ethylenedioxy)diethanethiol and 5% thioanisol. Peptides were purified on a preparative (25 mm × 250 mm) Atlantis C3 reverse phase column  in a 90 min gradient of 0.1% (v/v) trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile, with a 10 mL/min flow rate. The fractions containing peptides were analyzed on Agilent 6100 LC/MS spectrometer  with the use of a Zorbax 300SB-C3 PoroShell column and a gradient of 5% acetic acid in water and acetonitrile. Fractions that were more than 95% pure were combined and freeze dried.Phenylephrine was purchased from Sigma-Aldrich  and CXCL12 from Protein Foundry, Milwaukee, WI, USA. siRNA reagents were purchased from GE Dharmacon . The peptide analogs of transmembrane helix 2 (TM2)  and the A7r5 rat aortic smooth muscle cell line were obtained from American Type Culture Collection  and cultured as described . MesenteHuman monocytes were isolated from whole blood from healthy volunteers. In brief, whole blood was drawn and peripheral blood mononuclear cells were isolated by standard density gradient centrifugation. Monocytes were then positively selected using the EasySepTM Human CD14 positive selection kit  and suspended in RPMI supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin.tm2Yzo/J mice and male B6.FVB-Tg(Myh11-cre/ERT2)1Soff/J were purchased form Jackson Laboratory  and mated. B6.129P2-Cxcr4tm2Yzo/J mice possess loxP sites on either side of exon 2 of the CXCR4 gene [T2 expression, a fusion protein of the Cre recombinase with the modified estrogen receptor binding domain, is under control of the mouse smooth muscle myosin, heavy polypeptide 11, smooth muscle (Myh11) promoter/enhancer regions on the bacterial artificial chromosome (BAC) transgene, which is inserted on the Y chromosome [tm2Yzo/J mice. Male progeny carrying the CreERT2 transgene and homozygous for the loxP allele were then mated to female B6.129P2-Cxcr4tm2Yzo/J mice and male progeny selected as experimental animals. For Cre-induction, mice ≥12 weeks-old were injected intraperitoneally with 1 mg/kg body weight tamoxifen or vehicle (corn oil) for five consecutive days. Tissues were harvested not earlier than 7 days after the final tamoxifen injection.Female B6.129P2-Cxcr4CR4 gene . In B6.Fromosome . Male oftm2Yzo  and Tg(Myh11-cre/ERT2)1Soff .Genotyping of transgenic mice with DNA extracted from tail-snips was performed using standard PCR according to the Jackson Laboratory protocols Cxcr4Direct blood pressure measurements in conscious mice were performed utilizing implantable radio telemetry devices, as described . In brie2PO4, 1.17 mM MgSO4·7H2O, 14.9 mM NaHCO3, 5.5 mM d-Glucose, 0.026 mM EDTA, 1.16 mM CaCl2 aerated with 95% O2, 5% CO2 at 37 °C. The animal was than euthanized by cardiectomy and bilateral decompression of the lungs. Third or 4th order mesenteric arteries were dissected free from the mesentery, mounted onto two glass cannulae with United States Pharmacopeia (USP) scale 11-0 sutures and pressurized as required in a DMT 110P Pressure myograph system . The intraluminal solution and the vessel bath solution were the same as described before. The vessel bath solution was continuously aerated with 95% O2, 5% CO2 throughout the experiment. The outer o.d. of the pressurized vessel was then continuously measured and recorded via digital video-edge detection. For each condition or drug treatment, the vessel was observed for 7 min and the mean o.d. during the final minute of the dosing period calculated as percent of the o.d. before drug treatment.Pressure myography was performed as described in detail previously with slight modifications . Animals1A-AR siRNA gene silencing were performed as described previously [1A-AR siRNA were reconstituted with siRNA buffer to a stock concentration of 100 µM. Cells were then transfected with 10 nmol CXCR4, ACKR3 or α1A-AR siRNA and incubated for 72 h at 37 °C, 5% CO2. Accell non-targeting (NT) siRNA pool was used as negative control. After 72 h, cells were assayed for receptor cell surface expression.CXCR4, ACKR3 and αeviously ,52. In b2, 2 mM CaCl2, 10 mM HEPES, and 10 mM d-glucose, pH 7.3, 298 mOsm/L. Images were acquired using C-Imaging System  with an Olympus 1X71 inverted epifluorescence microscope  and Simple PCI software  every 2 s for 1 min prior to the application of 10 µM phenylephrine and for additional 3 min after phenylephrine application. The total number of the cells and the number of the cells that contracted in response to phenylephrine were counted in each field. For each experiment, cells from a single animal were tested in triplicate or quadruplicate.Contraction assays were performed as described previously . In brie1A-AR  (1:400) antibodies at 37 °C for 2 h in a humidifying chamber to visualize receptor–receptor interaction. Mouse VSMC were incubated either with rabbit anti-CXCR4 (AbCam ab2074) (1:500), mouse anti-ACKR3  (1:500), mouse anti-α1A-AR (AbCam ab87990) (1:500) or goat anti-α1B-AR  for detection of individual receptors or with the following antibody combinations  for the detection of receptor–receptor interactions: rabbit anti-CXCR4 (AbCam ab2074)/mouse anti-ACKR3 (R&D Systems MAB42273), rabbit anti-CXCR4 (AbCam ab2074)/mouse anti-α1A-AR (AbCam ab87990) and rabbit anti-CXCR4 (AbCam ab2074)/goat anti-α1B-AR (Santa Cruz sc27136). Slides were then incubated with secondary anti-species specific antibodies conjugated with plus and minus Duolink II PLA probes (1:5), as appropriate. As controls, slides were incubated with PBS instead of primary antibody or with rabbit anti-CXCR4 (AbCam ab2074) in combination with a single secondary probe. After washing, slides were then incubated with ligation-ligase solution  followed by incubation with amplification-polymerase solution . Slides were then mounted with a minimal volume of Duolink II Mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) for 15–30 min and PLA signals (Duolink In Situ Detection Reagents Green (λexcitation/emission 495/527 nm) or Red (λexcitation/emission 598/634 nm)) were identified as fluorescent spots under a fluorescence microscope using 40× or 100× objectives. The number of PLA signals per cell were analyzed using the Duolink Image Tool software (Sigma-Aldrich). Images were imported in merged.tiff formats containing both signal and nuclei channels. Merged images were checked visually and verified for analytical quality. Comparisons and statistical analyses were performed only when PLA assays were performed on the same day in parallel experiments and fluorescence microscopy was performed with identical settings. For hVSMC, 10 vision fields were analyzed for each condition and experiment. For mouse VSMC, 6–15 vision fields were analyzed for each condition and animal (n = 7–8).The Duolink proximity ligation assay was purchased from Sigma-Aldrich and performed in a format suitable for the detection of single proteins and protein-protein interactions, as described in detail previously ,53,54. I1A-AR  and polyclonal rabbit anti-ACKR3  in combination either with anti-rabbit FITC conjugated goat IgG  or anti-mouse FITC conjugated goat IgG (ab6785). Rabbit IgG  in combination with FITC conjugated anti-rabbit goat IgG  was used as a negative control. The geometric fluorescence intensities of at least 3 × 104 cells were recorded and analyzed using the FlowJo software .FACS analyses were performed as described previously ,53. Cell2+ in A7r5 cells was measured using the Fluo-4 NW Calcium Assay Kit , as described [Intracellular Caescribed ,52,55,565 cells were loaded onto the ChemoTX filter over each well and incubated for 2 h at 37 °C, 5% CO2. After incubation, transmigrated cells were washed, fixed and stained with Accustain Wright-Giemsa stain (Sigma-Aldrich). Stained cells were counted under a light microscope (400×) and average cell numbers were determined by counting the cells in three random non-overlapping vision fields. The chemotactic index was calculated as the ratio of cells that transmigrated through the filter in the presence versus absence (=PBS/control) of the test solutions.Cell migration of hVSMC and monocytes towards CXCL12 was assessed using the ChemoTx96-well cell migration system . ChemoTX filters were pre-coated with 100 µg/mL Collagen IV for 48 h at 25 °C when hVSMC were tested. The bottom wells were loaded with 30 µL of test solutions (100 ng/mL CXCL12). Twenty-five microliters of cell suspension containing 10Tissue biopsies  were fixed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). Slides were examined under light microscopy by a pathologist who was blinded as to the origin of the tissues.http://gpcrdb.org/), where sequence similarity is defined as the percentage of similar amino acids and similar is defined as a BLOSUM62 score >0 [Structure-based sequence alignments were performed with the web-based GPCRdb similarity search tool  server [Structural modeling of ACKR3 and α) server . Experimn independent experiments that were performed on different days. Data were analyzed with non-linear regression analyses and with one-way analyses of variance (ANOVA) with Bonferroni’s multiple comparison post-hoc test or with two-way ANOVA with Sidak’s multiple comparisons test for multiple comparisons, as appropriate. Best-fit values were compared with the extra sum-of-squares F test. All analyses were calculated with the GraphPad-Prism 6 software . A two-tailed p < 0.05 was considered significant.Data are expressed as mean ± standard error of the mean (SEM) from


To determine whether the public and scientists consider common arguments (and counterarguments) in support (or not) of animal research (AR) convincing.After validation, the survey was sent to samples of public , Amazon Mechanical Turk , a Canadian city festival and children’s hospital), medical students (two second-year classes), and scientists . We presented questions about common arguments (with their counterarguments) to justify the moral permissibility (or not) of AR. Responses were compared using Chi-square with Bonferonni correction.n = 586; AMT, n = 439; Festival, n = 195; Hospital n = 107], 194/331 (59 %) medical student, and 19/319 (6 %) scientist [too few to report] responses. Most public respondents were <45 years (65 %), had some College/University education (83 %), and had never done AR (92 %). Most public and medical student respondents considered ‘benefits arguments’ sufficient to justify AR; however, most acknowledged that counterarguments suggesting alternative research methods may be available, or that it is unclear why the same ‘benefits arguments’ do not apply to using humans in research, significantly weakened ‘benefits arguments’. Almost all were not convinced of the moral permissibility of AR by ‘characteristics of non-human-animals arguments’, including that non-human-animals are not sentient, or are property. Most were not convinced of the moral permissibility of AR by ‘human exceptionalism’ arguments, including that humans have more advanced mental abilities, are of a special ‘kind’, can enter social contracts, or face a ‘lifeboat situation’. Counterarguments explained much of this, including that not all humans have these more advanced abilities [‘argument from species overlap’], and that the notion of ‘kind’ is arbitrary . Medical students were more supportive (80 %) of AR at the end of the survey (p < 0.05).There were 1220 public [SSI, Responses suggest that support for AR may not be based on cogent philosophical rationales, and more open debate is warranted.The online version of this article (doi:10.1186/s12910-016-0100-x) contains supplementary material, which is available to authorized users. Given the massive public funding of animal research (AR) in democratic societies, it might be expected that the arguments for and against AR are well settled , 2. HoweThe question to debate is this: is any or all AR that involves seriously harming animals morally permissible? The moral justification is usually given by one of three types of arguments –7. “Benen = 4 experts), face and content validation by pilot testing using a clinical sensibility tool and informal semi-structured interviews   [The development and reporting of the questionnaire followed published recommendations . As descviduals) . Not allWe surveyed the public (4 groups), adults with biomedical science training (2 groups), and animal researchers (2 groups), the details of which are as follows:n = 195). These adults were asked if they would fill out a paper survey about AR in return for $5 food ticket as an incentive. We did not track the number of people asked to participate, but our impression was that most adults approached completed the survey. ii) A random sample of Canadian adults accessed using the marketing firm Survey Sampling International (SSI) in November 2014 (n = 586). The sample “was selected to be reflective of the Canadian population over age 18 years with at least a high school education, by age, gender, race/ethnicity, income, and geographic region.” Of those invited to participate, 1 terminated, 85 submitted partial responses, and 501 submitted complete responses. iii) A sample of US adults using Amazon Mechanical Turk (AMT) in February 2015 (n = 439). For this sample, we limited potential responders to those living in the US, with a Human Intelligence Task approval rate >90 %, and we paid $1.60 on completion. The survey was on the AMT site for <4.5 h, and the average time spent per survey was 31 min. Crowdsourcing on AMT has found results to be psychometrically valid, with high test-retest reliability, attentiveness, and truthfulness, even on complex cognitive and decision-making tasks [n = 107). We did not track the number of people asked to participate, but our impression was that most of those approached completed the paper survey.1. Public: we chose 4 groups that represented a range of situations that were conveniently accessible for survey distribution: i) A convenience sample of adults approached while in various line-ups at the Heritage Festival in Edmonton, Alberta, Canada in August 2013  The second year University of Alberta Medical School in September 2013, and ii) The next second year class in November 2014. Non-respondents were sent three reminders at about 2–3 week intervals.3. Animal researchers: i) The corresponding authors of AR papers published during the 6 months from October 2012 to March 2013 in the high impact journals Nature, Science, and Critical Care Medicine ; and ii) All academic faculty pediatricians at The Hospital for Sick Children in Toronto, Ontario, Canada, as listed by email on the University of Toronto website. We assumed these pediatricians would have many ties to research activities, including knowledge of, if not participation in, AR. Non-responders were sent three reminders at about 2–3 week intervals.The surveys were done using the web-based tool REDCap, which allows anonymous survey responses to be collected or entered, and later downloaded into statistical software for analysis . The papThe background section stated “In this survey, ‘animals’ means: mammals, such as mice, rats, dogs, and cats. It has been estimated that over 100 million animals are used in the world for research each year. There are many good reasons to justify animal research, which is the topic of this survey. Nevertheless, some people argue that these animals are harmed in experimentation, because their welfare is worsened. In this survey, ‘harmful’ means such things as: pain, suffering , and early death. We value your opinion on the very important issue of the ethical dimension of animal research.” We chose mammals to represent a group of sentient animals that are thus capable of being harmed.We presented demographic questions, 3 questions about support for AR, and 12 arguments with their counterarguments to consider. The survey stated: “a) First, we give you an argument to justify harmful animal research, and we ask if you agree with that argument; b) Then we give some responses to the argument, and we ask if you think each response would make it harder for someone to justify harmful animal research using the initial argument . All the arguments and responses in this survey are those commonly made in the literature on animal research.” When each argument was presented, it was followed with the question “Is this a good enough reason to justify using animals in medical research?” When the responses to an argument were presented, they were followed with the question: “Do any of the following responses make it harder for someone to justify animal research using Argument x ?”P < 0.05 after Bonferroni correction for multiple comparisons considered statistically significant. Prior to statistical analysis, we defined a clinically significant difference between groups as one where the comparison is statistically significant, in addition to having a clear majority (at least 60 %) on different sides of the yes/no response option. This was done because we are interested in whether the groups have opinions that could result in different practical consequences both for the animals, and for the researchers involved. For example, if many respondents have very different opinions about the moral permissibility of AR, this might lead to very different levels of support of AR, and thus have different implications for the actual practice of AR.We used REDCap as our web-based survey management tool, allowing anonymous survey responses to be collected, and later downloaded into an SPSS database for analysis . ResponsThere were 195 respondents from the local festival, 586 from SSI, 439 from AMT, and 107 from the children’s hospital samples. Most respondents were under 45 years old, had at least some college or university training, and the vast majority had never done AR  and 82/167 (49 %) in the two medical school classes. Most respondents were under 35 years, and 60–62 % had never done AR  were invited to participate by e-mail. The response rate was 5/178 (3 %) after 4 mailings; therefore, no useful results can be reported here. Academic pediatricians at the Hospital for Sick Children (n = 141) were invited to participate by e-mail. The response rate to the demographics questions was 18/141 (13 %); however, before all the benefits arguments were presented the response rate dropped to only 14/141 (10 %), and therefore no useful results can be reported here. The difference in response rates between medical students and animal researchers was statistically significant (Chi-square p < 0.001).Corresponding authors  and medical students (80 %) are supportive of AR. Second, ‘benefits arguments’ were usually thought sufficient to justify AR. However, when confronted with counterarguments pointing out that alternative research methods may be available, most were not as convinced of the initial argument. In addition, counterarguments suggesting that it is unclear why the same ‘benefits arguments’ do not apply to using humans in the same medical experiments were convincing for about half of the public and one third of medical students. Third, almost all respondents were not convinced by “characteristics of NHA arguments”, including that NHA may not be sentient or are simply property. Fourth, most respondents were not convinced by ‘human exceptionalism’ arguments; these are the arguments that justify AR due to benefits but claim the same benefits do not justify human research. These include arguments that humans have more advanced mental abilities than NHAs, are of a special ‘kind’, can enter into social contracts, or face a lifeboat situation where human interests trump NHA interests. Fifth, common counterarguments explained much of the respondents’ lack of acceptance of ‘human exceptionalism’ arguments: the ‘argument from species overlap’  –31; the Previous surveys of the public and scientists have generally asked only whether people support AR for human benefit, and not asked people to evaluate their reasons for supporting (or not) AR Table  16–20, , 20, 34.This study has several limitations. Response rates for medical school classes were 68 % and 49 %; thus we cannot rule out biased participation in the survey. In addition, we do not know the true response rate for our public festival, hospital wards, and AMT samples. Nevertheless, we estimate that most people approached for the paper surveys agreed to participate, the AMT surveys were completed in a very short period of time (<4.5 h on-line), and the SSI surveys were fully completed by 501/587 of those invited (85 %). Arguments and counterarguments presented needed to be short and concise, and this may have left out important details that would have influenced the understanding and response to the text. We did not ask about specific subtypes of AR and thus do not know if opinions will vary by whether research is on toxicology or efficacy, cancer or sepsis, etc. Nevertheless, the questions were framed assuming great benefit to humans from AR, and thus we believe the opinions are reflective of beliefs for types of AR that have such great benefit. We did not involve animal researchers in the development of the survey, and this may have biased the wording of the questions. Finally, our description of AR as harmful may have biased the responses. However, the survey was meant to apply to AR that is harmful to the animals; AR is a moral issue precisely because it is harmful to the animals; and, that AR is said by advocates to be done as ‘humanely’ as possible, with suffering minimized, entails that it is harmful to the animals.Strengths of this study include the rigorous survey development process , and the inclusion of the most common and accepted arguments/counterarguments in the literature. This is the first survey we are aware of that asks the public and medical students to consider not just whether they support AR, but rather to consider the most common arguments in the literature in favor of and against AR. Another strength is the large sample size of the public, obtained from 4 different samples, with strikingly similar responses, enhancing the generalizability of our results. The similarity of findings to our previous sample of pediatric health care workers also enhances the generalizability of the results .When presented with common arguments to justify AR, most respondents accepted ‘benefits’ arguments, and only a minority found the ‘characteristics of NHA arguments’ and ‘human exceptionalism’ arguments convincing. Most found the argument to justify AR significantly weakened by common counterarguments, including those who initially found the ‘benefits’ arguments convincing. These responses to all the common arguments/counterarguments on offer in the literature regarding the moral permissibility of AR suggest that the public may want to re-consider whether public funds ought to support AR. Engagement with and serious discussion of the arguments on both sides of the AR debate by the public and scientists ; and deliberate extensive investigation of alternative research methods, and the return on investment from using them  The study was approved by the health research ethics board of the University of Alberta (Pro00039590), and return of the survey was considered consent to participate.Not applicable.The survey instrument, and all aggregated data is reported in the manuscript. The database can be made available on request to the authors.


CHANCE  is a standalone package for ChIP-seq quality control and protocol optimization. Our user-friendly graphical software quickly estimates the strength and quality of immunoprecipitations, identifies biases, compares the user's data with ENCODE's large collection of published datasets, performs multi-sample normalization, checks against quantitative PCR-validated control regions, and produces informative graphical reports. CHANCE is available at  The foremost question that challenges an experimentalist about their ChIP-seq is, 'did my experiment work?' CHANCE  is a software package that provides quantitatively rigorous yet intuitive answers to this fundamental question in the following ways:1. CHANCE assesses the strength of immunoprecipitation (IP) enrichment to identify potentially failed experiments. CHANCE identifies insufficient sequencing depth, PCR amplification bias in library preparation, and batch effects.2. CHANCE identifies biases in sequence content and quality, as well as cell-type and laboratory-dependent biases in read density. Read-density bias reduces the statistical power to distinguish subtle but real enrichment from background noise -3. CHANC3. CHANCE cross-validates enrichment with previous ChIP-qPCR results. Experimentalists frequently use ChIP-qPCR to check the enrichment of positive control regions and the background level of negative control regions in their immunoprecipitation DNA (IP) relative to input DNA (Input). It is thus important to verify whether those select regions originally checked with PCR are captured correctly in the sequencing data. CHANCE's spot-validation tool provides a fast way to perform this verification. CHANCE also compares enrichment in the user's experiment with enrichment in a large collection of experiments from public ChIP-seq databases.Despite having different goals, some software packages partially overlap with CHANCE in functionality: htSeqTools  is an R While Python scripts and Java applications are available for correcting read density for mappability and GC content biases , to our CHANCE works with reads mapped to a reference genome from IP and control (Input) samples. It can import reads in BED, tagAlign , SAM, anq-value for the IP enrichment level based on this training data and uses the q-value to identify potentially failed experiments.IP enrichment strength is important for calling robust peaks that correspond to transcription factor (TF) binding sites or epigenetic modification sites. To estimate the IP strength, CHANCE attempts to decompose the population of IP reads into two distinct components: those pulled down by the antibody, and background. To accomplish this task, CHANCE uses signal extraction scaling (SES), which is based on order statistics . SES estCARM1 in human embryonic stem cells (GEO accession GSM801064), where the IP sample is statistically indistinguishable from Input.In addition to assessing the strength of IP, it is also important to monitor the levels and sources of different biases present in the data. Identifying these biases is useful for optimizing experimental protocols. During the estimation of IP strength, CHANCE thus also detects several forms of bias. Figure CHANCE can also compare two or more IP samples  by constructing a consensus profile based on signal processing techniques designed to identify regions of mutual enrichment -19  for a given value of the divergence test statistic as the fraction of Input-Input pairs in the set all samples with divergence test values greater than or equal to the user's divergence test value. The q-value is thus interpreted as the fraction of comparisons from ENCODE that show differential enrichment at the level of the user's data, but turn out to be technical replicates of the Input channel.In order to ascertain the precision and recall of the divergence test as a classifier of successful experiments, we calibrated CHANCE on a data set obtained from the ENCODE repository. We downloaded all ENCODE ChIP-seq data sets with replicate inputs  histone mark in normal cells, (b) histone mark in cancer cells, (c) TF in normal cells, (d) TF in cancer cells, and (e) all samples. Figure q-values are above 5%, but the user may also compare their experiment by category.While the majority of histone mark ChIP-seq enrichment profiles tend to be spread out, profiles for TFs tend to be more punctate. This spreading can result in a reduction in ChIP signal and lead to a stronger mixing between the distributions of Input versus Input and IP versus Input divergence test statistics for histone mark ChIP-seq, whereas the distributions are more separable for TF ChIP-seq, as shown in Figure p(α) denote the percentage of reads in the IP channel contained in the first α percent of 1 kb non-overlapping bins sorted in an increasing order of read density. Similarly, let q(α) denote the percentage of the matching tag counts in Input, reordered by the sorting induced by the sorting of the IP channel. If IP had sufficient enrichment, then we must have p(α) ≤ q(α), since reads accumulate significantly in a small genomic subset targeted by IP, while the majority of sequences in the Input channel are more uniformly distributed throughout the genome. On the other hand, if there is insufficient sequencing depth in the Input channel, then there will be abundant zero counts in Input tag bins; and for α sufficiently small, we will have q(α) ≤ p(α). If CHANCE detects this crossing of p(α) from below by q(α), it reports a warning of potential low coverage in the Input channel.As in , let p(αp(α) will be zero for α ≤ α0 for some α0 > 0, α0 therefore being the percentage of the genome with zero coverage. In some extreme cases, the maximal percentage differential enrichment of IP over Input occurs at α0  and distribution of coverage (the same genome-wide spread in tag count). The spectral energy landscape of a sample with minimal bias will be similar to that of the simulation /q/(1 - q). We then compute the same odds ratio for each IP-Input pair, in ENCODE, for the same TF or histone mark. The distribution of odds ratios gives the user a sense of how cell type-specific enrichment for that particular mark is. If the user's odds ratio is much less than one, this indicates that the user's data set is somewhat of an outlier, compared to ENCODE. We compute the log of the odds ratio, since the log odds is approximately normal. This allows us to fit a normal curve to the distribution of ENCODE log odds ratios. The cumulative distribution at the log odds of the user's data then gives a probability indicating how much of an outlier the user's data set is. Although not definitive of a failed experiment on its own, a small odds ratio provides additional evidence of a potentially failed experiment.The ENCODE project provides representative transcriptional and epigenetic maps of the mammalian genomes. We thus reasoned that the ENCODE data can provide a rough landscape of TF binding and epigenetic modification sites that are applicable to multiple cell types. The 'Comparison with ENCODE' module thus allows one to compare one's own dataset with corresponding ENCODE datasets to determine if the user's data show an accumulation of reads within ENCODE peaks. For each TF or epigenetic mark for which ENCODE has called peaks (Additional file https://github.com/songlab/chance.CHANCE is open source, published under the GNU General Public License. The Matlab source code, User Guide, examples, and executables for Mac OS, Windows, and Linux are available at ChIP: chromatin immunoprecipitation; CHANCE: CHip-seq ANalytics and Confidence Estimation; GEO: Gene Expression Omnibus; GUI: graphical user interface; HESC: human embryonic stem cell; IP: immunoprecipitation; NSC: neural stem cell; qPCR: quantitative polymerase chain reaction; SES: signal extraction scaling; TF: transcription factor; UCSF: University of California: San Francisco.AD and JSS designed the project and developed the underlying algorithms. AD designed and developed the software package. AN implemented the BAM/SAM file reader interface. AD and AN wrote the User Guide, and all authors together wrote the paper.CHANCE feature comparison table.Click here for fileCHANCE training data table. This file enumerates the experiment information and url for each ENCODE sample used in either the training data set for the false discovery rate computation in the "IP enrichment" module or the "Comparison with ENCODE module".Click here for file