Rigor, Reproducibility, and Transparency in Shared Research Resources: Follow-Up Survey and Recommendations for Improvements.

Rigor, reproducibility, and transparency (RR&T) are essential components of all scientific pursuits. Shared research resources, also known as core facilities, are on the frontlines of ensuring robust RR&T practices. The Association of Biomolecular Resource Facilities Committee on Core Rigor and Reproducibility conducted a follow-up survey 4 years after the initial 2017 survey to determine if core facilities have seen a positive impact of new RR&T initiatives (including guidance from the National Institutes of Health, new scientific journal requirements on transparency and data provenance, and educational tools from professional organizations). While there were fewer participants in the most recent survey, the respondents' opinions on the role of core facilities and level of best practices adoption remained the same. Overall, the respondents agreed that procedures should be implemented by core facilities to ensure scientific RR&T. They also indicated that there is a strong correlation between institutions that emphasize RR&T and core customers using this expertise in grant applications and publications. The survey also assessed the impact of the COVID-19 pandemic on core operations and RR&T. The answers to these pandemic-related questions revealed that many of the strategies aimed at increasing efficiencies are also best practices related to RR&T, including the development of standard operating procedures, supply chain management, and cross training. Given the consistent and compelling awareness of the importance of RR&T expressed by core directors in 2017 and 2021 contrasted with the lack of apparent improvements over this time period, the authors recommend an adoption of RR&T statements by all core laboratories. Adhering to the RR&T guidelines will result in more efficient training, better compliance, and improved experimental approaches empowering cores to become "rigor champions."

Addressing the Environmental Impact of Science Through a More Rigorous, Reproducible, and Sustainable Conduct of Research.

The pervasiveness of irreproducible research remains a thorny problem for the progress of scientific endeavor, spawning an abundance of opinion, investigation, and proposals for improvement. Irreproducible research has negative consequences beyond the obvious impact on achieving new scientific discoveries that can advance healthcare and enable new technologies. The conduct of science is resource intensive, resulting in a large environmental impact from even the smallest research programs. There is value in making explicit connections between the conduct of more rigorous, reproducible science and commitments to environmental sustainability. Shared research resources (also commonly known as cores) often have an institutional role in supporting researchers in the responsible conduct of research through training, informal mentorship, and services and are particularly well suited to promulgating essential principles of scientific rigor, reproducibility, and transparency. Shared research resources can also play a role in advancing sustainability by virtue of their inherently efficient science model in which singular shared equipment, technology, and expertise resources can serve many different research programs. Programs that elevate shared research resources, scientific rigor, reproducibility, transparency, and environment sustainability in harmony may achieve a unique synergy. Several case studies and quality paradigms are discussed that offer tools and concepts that can be adapted whole or in part by individual shared research resources or research-intensive institutions as part of an overall program of sustainability.

Building a Quality Management System in a Core Facility: A Genomics Core Case Study.

Core facilities are key resources supporting the academic research enterprise, providing access to innovative and essential technologies and expertise. Given the constraints placed on core facilities as recharge centers and the ever-changing research environment, an important competitive differentiator that can support rigorous and reproducible approaches in core labs is the implementation of a quality management system (QMS). This paper describes a systematic approach to building a QMS in a genomics core facility at the University of North Carolina School of Medicine. This model is based on principles of the International Organization for Standardization 9001 system with initiatives focused on process mapping, training (communication, customer service, performance management, development of standard operating procedures, and quality audits), root cause analysis, visual control boards, mock quality audits, and continuous improvement through metrics tracking and "voice of the customer" exercises. The goal of this paper is to share practical step-by-step recommendations and outcomes of this core facility QMS that are generally applicable to academic core facilities, regardless of technical focus. Application of these good laboratory practice principles will foster "competitiveness through compliance" and promote outstanding interdisciplinary research between academic cores and their nonacademic pharmaceutical and federal research partners. Additionally, implementation of the QMS qualified this core to apply for federally funded contracts, thereby diversifying its types of projects and sources of revenue.

Erratum: Publisher's Note: "High yield fabrication of multilayer polydimethylsiloxane devices with freestanding micropillar arrays" [Biomicrofluidics 7, 056503 (2013)].

[This corrects the article on p. 056503 in vol. 7.].

High yield fabrication of multilayer polydimethylsiloxane [corrected] devices with freestanding micropillar arrays.

A versatile method to fabricate a multilayer polydimethylsiloxane (PDMS) device with micropillar arrays within the inner layer is reported. The method includes an inexpensive but repeatable approach for PDMS lamination at high compressive force to achieve high yield of pillar molding and transfer to a temporary carrier. The process also enables micropillar-containing thin films to be used as the inner layer of PDMS devices integrated with polymer membranes. A microfluidic cell culture device was demonstrated which included multiple vertically stacked flow channels and a pillar array serving as a cage for a collagen hydrogel. The functionality of the multilayer device was demonstrated by culturing collagen-embedded fibroblasts under interstitial flow through the three-dimensional scaffold. The fabrication methods described in this paper can find applications in a variety of devices, particularly for organ-on-chip applications.

Clinical trials assessing ototopical agents in the treatment of pain associated with acute otitis media in children.

Otalgia (ear pain) is one of the characteristic symptoms and best predictor of acute otitis media (AOM) in children. Although oral pain medications are the current mainstay for the treatment of AOM-associated otalgia, ototopical agents have been investigated as an alternative treatment strategy. To permit review and assessment of this treatment modality, a systematic literature search was conducted to identify all randomized, controlled trials of ototopical agents. Four trials were identified, including those examining ototopical benzocaine in combination with antipyrine, lidocaine, tetracaine, and herbal extracts. Although the current available evidence suggests ototopical agents may be safe and effective, we conclude that further studies with more rigorous methodology are needed to conclusively demonstrate their utility in this setting.

14-3-3{eta} Amplifies Androgen Receptor Actions in Prostate Cancer.

PURPOSE: Androgen receptor abundance and androgen receptor-regulated gene expression in castration-recurrent prostate cancer are indicative of androgen receptor activation in the absence of testicular androgen. Androgen receptor transactivation of target genes in castration-recurrent prostate cancer occurs in part through mitogen signaling that amplifies the actions of androgen receptor and its coregulators. Herein we report on the role of 14-3-3eta in androgen receptor action. Experimental Design and RESULTS: Androgen receptor and 14-3-3eta colocalized in COS cell nuclei with and without androgen, and 14-3-3eta promoted androgen receptor nuclear localization in the absence of androgen. 14-3-3eta interacted with androgen receptor in cell-free binding and coimmunoprecipitation assays. In the recurrent human prostate cancer cell line, CWR-R1, native endogenous androgen receptor transcriptional activation was stimulated by 14-3-3eta at low dihydrotestosterone concentrations and was increased by epidermal growth factor. Moreover, the dihydrotestosterone- and epidermal growth factor-dependent increase in androgen receptor transactivation was inhibited by a dominant negative 14-3-3eta. In the CWR22 prostate cancer xenograft model, 14-3-3eta expression was increased by androgen, suggesting a feed-forward mechanism that potentiates both 14-3-3eta and androgen receptor actions. 14-3-3eta mRNA and protein decreased following castration of tumor-bearing mice and increased in tumors of castrate mice after treatment with testosterone. CWR22 tumors that recurred 5 months after castration contained 14-3-3eta levels similar to the androgen-stimulated tumors removed before castration. In a human prostate tissue microarray of clinical specimens, 14-3-3eta localized with androgen receptor in nuclei, and the similar amounts expressed in castration-recurrent prostate cancer, androgen-stimulated prostate cancer, and benign prostatic hyperplasia were consistent with androgen receptor activation in recurrent prostate cancer. CONCLUSION: 14-3-3eta enhances androgen- and mitogen-induced androgen receptor transcriptional activity in castration-recurrent prostate cancer. (Clin Cancer Res 2009;15(24):7571-81).

Site-specific androgen receptor serine phosphorylation linked to epidermal growth factor-dependent growth of castration-recurrent prostate cancer.

The androgen receptor (AR) is required for prostate cancer development and contributes to tumor progression after remission in response to androgen deprivation therapy. Epidermal growth factor (EGF) increases AR transcriptional activity at low levels of androgen in the CWR-R1 prostate cancer cell line derived from the castration-recurrent CWR22 prostate cancer xenograft. Here we report that knockdown of AR decreases EGF stimulation of prostate cancer cell growth and demonstrate a mechanistic link between EGF and AR signaling. The EGF-induced increase in AR transcriptional activity is dependent on phosphorylation at mitogen-activated protein kinase consensus site Ser-515 in the AR NH(2)-terminal region and at protein kinase C consensus site Ser-578 in the AR DNA binding domain. Phosphorylation at these sites alters the nuclear-cytoplasmic shuttling of AR and AR interaction with the Ku-70/80 regulatory subunits of DNA-dependent protein kinase. Abolishing AR Ser-578 phosphorylation by introducing an S578A mutation eliminates the AR transcriptional response to EGF and increases both AR binding of Ku-70/80 and nuclear retention of AR in association with hyperphosphorylation of AR Ser-515. The results support a model in which AR transcriptional activity increases castration-recurrent prostate cancer cell growth in response to EGF by site-specific serine phosphorylation that regulates nuclear-cytoplasmic shuttling through interactions with the Ku-70/80 regulatory complex.

Antigonadotropins: a novel strategy to halt Alzheimer's disease progression.

A significant amount of research has been focused on the relationship between hormones and Alzheimer's disease. However, the majority of this work has been on estrogen and more recently testosterone. A serendipitous patient encounter led one of us (RLB) to question whether other hormones of the hypothalamic-pituitary-gonadal axis could be playing a role in the pathogenesis of Alzheimer's disease. The age-related decline in reproductive function results in a dramatic decrease in serum estrogen and testosterone concentrations and an equally dramatic compensatory increase in serum luteinizing hormone concentrations. Indeed, there is growing evidence that the gonadotropin, luteinizing hormone, which regulates serum estrogen and testosterone concentrations, could be an important causative factor in the development of Alzheimer's disease. This review provides information supporting the "gonadotropin hypothesis," puts forth a novel mechanism of how changes in serum luteinizing hormone concentrations could contribute to the pathogenesis of Alzheimer's disease, and discusses potential therapeutic anti-gonadotropin compounds.

Increased expression of delta-catenin/neural plakophilin-related armadillo protein is associated with the down-regulation and redistribution of E-cadherin and p120ctn in human prostate cancer.

delta-Catenin, or neural plakophilin-related armadillo protein, is a unique armadillo domain-containing protein in that it is neural-specific and primarily expressed in the brain. However, our recent analysis of the human genome revealed a consistent association of delta-catenin messenger RNA sequences with malignant cells, although the significance of these findings was unclear. In this study, we report that a number of delta-catenin epitopes were expressed in human prostate cancer cells. Western blot and tissue microarray revealed a close association between increased delta-catenin expression and human primary prostatic adenocarcinomas. The analyses of 90 human prostate cancer and 90 benign prostate tissue samples demonstrated that an estimated 85% of prostatic adenocarcinomas showed enhanced delta-catenin immunoreactivity. delta-Catenin expression increased with prognostically significant increased Gleason scores. By analyzing the same tumor cell clusters using consecutive sections, we showed that an increased delta-catenin immunoreactivity was accompanied by the down-regulation and redistribution of E-cadherin and p120ctn, major cell junction proteins whose inactivation is frequently associated with cancer progression. Furthermore, overexpression of delta-catenin in tumorigenic CWR-R1 cells that are derived from human prostate cancer xenograft resulted in reduced immunoreactivity for E-cadherin and p120ctn at the cell-cell junction. This is the first study comparing overexpression of delta-catenin with the E-cadherin/catenin system in cancer and shows that delta-catenin may be intimately involved in regulating E-cadherin/p120ctn cell-cell adhesion in prostate cancer progression.

Steroid 5alpha-reductase isozymes I and II in recurrent prostate cancer.

PURPOSE: Prostate cancer recurs during androgen deprivation therapy despite reduced circulating androgens. We showed that recurrent prostate cancer tissue has testosterone levels similar to androgen-stimulated benign prostate, whereas dihydrotestosterone levels were reduced 82% to 1.45 nmol/L, sufficient for androgen receptor activation. The altered testosterone/dihydrotestosterone ratio in recurrent prostate cancer suggests loss of 5alpha-reducing capability. The aim of this study was to characterize steroid 5alpha-reductase isozymes I (S5alphaRI) and II (S5alphaRII) in prostate tissues. EXPERIMENTAL DESIGN: A tissue microarray was constructed from 22 recurrent prostate cancer specimens and matched pairs of androgen-stimulated benign prostate and androgen-stimulated prostate cancer from 23 radical prostatectomy specimens. Immunoblots were constructed from eight recurrent prostate cancers, eight androgen-stimulated benign prostate, and eight androgen-stimulated prostate cancer specimens. Isozyme expression was examined in microarray sections and immunoblots using S5alphaRI and S5alphaRII polyclonal antibodies. Isozyme activities were measured in 12 recurrent prostate cancer, 12 androgen-stimulated benign prostate, and 12 androgen-stimulated prostate cancer specimens. RESULTS: Nuclear immunostaining exhibited higher S5alphaRI expression than S5alphaRII in recurrent prostate cancer, androgen-stimulated benign prostate, and androgen-stimulated prostate cancers (P < 0.0001); mean expression was 125, 150, and 115 for S5alphaRI versus 10, 29, and 37 for S5alphaRII, respectively. Cytoplasmic immunostaining was moderate and similar for both isozymes in the three tissue types (P > 0.05). Immunoblots confirmed immunohistochemistry; S5alphaRI was expressed in recurrent prostate cancer specimens and S5alphaRII was not detected. The activity of S5alphaRI (114.4 pmol/mg epithelial protein/minute) was 3.7-fold higher than S5alphaRII (30.7 pmol/mg epithelial protein/minute) in recurrent prostate cancer specimens. CONCLUSIONS: Expression levels and isozyme activity shifts from S5alphaRII toward S5alphaRI in recurrent prostate cancer. Dual inhibition of S5alphaRI and S5alphaRII should reduce dihydrotestosterone biosynthesis and may prevent or delay growth of recurrent prostate cancer.

Protein C inhibitor (plasminogen activator inhibitor-3) expression in the CWR22 prostate cancer xenograft.

The serine protease inhibitor (serpin) protein C inhibitor (PCI) has been found in the prostate and possibly is a marker to distinguish normal prostate, benign prostatic hyperplasia, and prostate cancer. In this study, we assessed PCI expression in normal, hyperplastic, and malignant prostatic tissues, prostate cancer cell lines, and the CWR22 prostate cancer xenograft model that allowed us to study PCI expression and its regulation in response to androgens. By Northern blot, immunohistochemistry, and in situ hybridization, we found that PCI was expressed in both benign and malignant prostate tissues. Protein C inhibitor was expressed in both androgen-independent (PC-3) and androgen-dependent (LNCaP) prostate cancer cell lines. Furthermore, PCI was detected in all CWR22 tumor samples (androgen dependent, 6 days post-castration, 12 days post-castration followed by 72 h of testosterone treatment, and recurrent CWR22 tumor), although expression of the mature forms of both prostate-specific antigen (PSA) and its homolog, kallikrein 2 (hK2), was clearly androgen-dependent. These results suggest that PCI expression is not regulated by androgens and that PCI is unlikely to be a tumor suppressor gene, but also that PCI may be involved in regulating key serine proteases involved in metastatic prostate disease.

Inhibition of HER-2/neu kinase impairs androgen receptor recruitment to the androgen responsive enhancer.

Advanced prostate cancer invariably recurs despite androgen deprivation therapy. The androgen receptor (AR) likely plays a key role in this progression and in the continued survival and proliferation of prostate cancer cells in the low androgen environment. Cross-talk with growth factor receptors, such as epidermal growth factor receptor (EGFR) family, has been postulated as a potential mechanism to activate AR in recurrent prostate cancer. We have investigated the role of HER-2/neu (ErbB-2) tyrosine kinase in AR function by characterizing the effect of inhibiting endogenous HER-2 activity in LNCaP cells. We used two independent methods, expression of intracellular single-chain antibody against HER-2 and treatment with a novel dual EGFR/HER-2 kinase inhibitor GW572016 (lapatinib). Expression of intracellular HER-2 antibody scFv-5R and treatment with GW572016 inhibited HER-2 signaling. This HER-2 inhibition led to impairment of AR-mediated functions, such as androgen-stimulated growth and the induction of endogenous prostate-specific antigen (PSA) mRNA and protein. Androgen-stimulated recruitment of AR and histone acetylation at the androgen responsive enhancer of the PSA gene, detected by chromatin immunoprecipitation analysis, were impaired by HER-2 inhibition. GW572016 was more potent in its ability to inhibit PSA expression and AR recruitment and histone acetylation than the EGFR-selective kinase inhibitor ZD1839 (gefitinib), consistent with the HER-2 kinase playing the major role in AR regulation. These results show that HER-2 signaling is required for optimal transcriptional activity of AR in prostate cancer cells and suggest that HER-2 inhibition may provide a novel strategy to disrupt AR function in prostate cancer.

Heregulin-induced activation of HER2 and HER3 increases androgen receptor transactivation and CWR-R1 human recurrent prostate cancer cell growth.

PURPOSE: The androgen receptor (AR) is a ligand-dependent transcription factor that mediates gene expression and growth of normal and malignant prostate cells. In prostate tumors that recur after androgen withdrawal, the AR is highly expressed and transcriptionally active in the absence of testicular androgens. In these "androgen-independent" tumors, alternative means of AR activation have been invoked, including regulation by growth factors and their receptors in prostate cancer recurrence. EXPERIMENTAL DESIGN AND RESULTS: In this report, we show that HER receptor tyrosine kinases 1 through 4 are expressed in the CWR-R1 recurrent prostate cancer cell line; their stimulation by epidermal growth factor (EGF) and heregulin activates downstream signaling, including mitogen-activated protein kinase and phosphatidylinositol-3 kinase and Akt pathways. We show that heregulin activates HER2 and HER3 and increases androgen-dependent AR transactivation of reporter genes in CWR-R1 cells. Tyrosine phosphorylation of HER2 and HER3, AR transactivation, and cell proliferation induced by heregulin were more potently inhibited by the EGFR/HER2 dual tyrosine kinase inhibitor GW572016 (lapatinib) than the EGFR-specific inhibitor ZD1839 (gefitinib). Basal proliferation in the absence of growth factors was also inhibited by GW572016 to a greater extent than ZD1839, suggesting that low level HER2/HER3 activation perhaps by an autocrine pathway contributes to the proliferation signal. CONCLUSIONS: These data indicate that heregulin signaling through HER2 and HER3 increases AR transactivation and alters growth in a recurrent prostate cancer cell line. Therefore, inhibition of low-level HER2 signaling may be a potential novel therapeutic strategy in prostate cancer.

Neuroendocrine cell differentiation in the CWR22 human prostate cancer xenograft: association with tumor cell proliferation prior to recurrence.

BACKGROUND: Neuroendocrine (NE) cell differentiation is proposed to facilitate prostate cancer (CaP) recurrence following androgen deprivation through secretion by NE cells of growth factors and neuropeptides that support survival and proliferation of CaP cells and vasculature. METHODS: The effect of androgen deprivation on NE differentiation and tumor cell proliferation in the CWR22 model of human CaP was determined by immunohistochemical analysis of the NE cell marker synaptophysin and the cell proliferation marker Ki67. RESULTS: A significant increase in the number of NE cells was observed in CWR22 tumors between 28 and 66 days after castration compared to intact mice, that preceded the increase in tumor cell proliferation that began 70 days after androgen deprivation heralding recurrence. There was a significant positive correlation between the number of tumor-associated NE cells and proliferating CaP cells in tumors from mice after 28-34 days of androgen withdrawal. CONCLUSION: In the CWR22 model, androgen deprivation induces an increase in tumor-associated NE cells prior to increased tumor cell proliferation, with NE cells possibly promoting tumor survival and recurrent disease.

The androgen axis in recurrent prostate cancer.

PURPOSE: Prostate cancer that recurs during androgen deprivation therapy is referred to as androgen-independent. High levels of expression of androgen receptor and androgen receptor-regulated genes in recurrent prostate cancer suggest a role for androgen receptor and its ligands in prostate cancer recurrence. EXPERIMENTAL DESIGN: Recurrent prostate cancer specimens from 22 men whose prostate cancer recurred locally during androgen deprivation therapy and benign prostate specimens from 48 men who had received no prior treatment were studied. Androgen receptor expression was measured using monoclonal antibody and automated digital video image analysis. Tissue androgens were measured using radioimmunoassay. RESULTS: Epithelial nuclei androgen receptor immunostaining in recurrent prostate cancer (mean optical density, 0.284 +/- SD 0.115 and percentage positive nuclei, 83.7 +/- 11.6) was similar to benign prostate (mean optical density, 0.315 +/- 0.044 and percentage positive nuclei, 77.3 +/- 13.0). Tissue levels of testosterone were similar in recurrent prostate cancer (2.78 +/- 2.34 pmol/g tissue) and benign prostate (3.26 +/- 2.66 pmol/g tissue). Tissue levels of dihydrotestosterone, dehydroepiandrosterone, and androstenedione were lower (Wilcoxon, P = 0.0000068, 0.00093, and 0.0089, respectively) in recurrent prostate cancer than in benign prostate, and mean dihydrotestosterone levels, although reduced, remained 1.45 nM. Androgen receptor activation in recurrent prostate cancer was suggested by the androgen-regulated gene product, prostate-specific antigen, at 8.80 +/- 10.80 nmol/g tissue. CONCLUSIONS: Testosterone and dihydrotestosterone occur in recurrent prostate cancer tissue at levels sufficient to activate androgen receptor. Novel therapies for recurrent prostate cancer should target androgen receptor directly and prevent the formation of androgens within prostate cancer tissue.

Epidermal growth factor increases coactivation of the androgen receptor in recurrent prostate cancer.

Growth of normal and neoplastic prostate is mediated by the androgen receptor (AR), a ligand-dependent transcription factor activated by high affinity androgen binding. The AR is highly expressed in recurrent prostate cancer cells that proliferate despite reduced circulating androgen. In this report, we show that epidermal growth factor (EGF) increases androgen-dependent AR transactivation in the recurrent prostate cancer cell line CWR-R1 through a mechanism that involves a post-transcriptional increase in the p160 coactivator transcriptional intermediary factor 2/glucocorticoid receptor interacting protein 1 (TIF2/GRIP1). Site-specific mutagenesis and selective MAPK inhibitors linked the EGF-induced increase in AR transactivation to phosphorylation of TIF2/GRIP1. EGF signaling increased the coimmunoprecipitation of TIF2 and AR. AR transactivation and its stimulation by EGF were reduced by small interfering RNA inhibition of TIF2/GRIP1 expression. The data indicate that EGF signaling through MAPK increases TIF2/GRIP1 coactivation of AR transactivation in recurrent prostate cancer.

Androgen receptor gene amplification and protein expression in recurrent prostate cancer.

PURPOSE: The androgen receptor (AR) is highly expressed in androgen dependent and recurrent prostate cancer, suggesting that it has a role in tumor growth and progression after androgen deprivation. AR amplification may contribute to androgen receptor activation in relative androgen absence. MATERIALS AND METHODS: Formalin fixed and frozen specimens of recurrent prostate cancer were obtained by transurethral resection from men with increasing serum level of prostate specific antigen in whom urinary retention developed. AR amplification and X-chromosome number were determined by 2-color fluorescence in situ hybridization, and AR protein expression was determined by automated image analysis. We compared clinical characteristics and survival of patients with recurrent prostate cancer whose tumors did or did not exhibit AR amplification and X-chromosome polysomy. RESULTS: Thirty-three percent of the 24 recurrent prostate cancer specimens 8 (33%) showed AR amplification. AR was more intensely immunostained in tumors with amplified (AMP) AR (mean optical density 0.36 +/- 0.07) than in tumors lacking amplification (NO AMP) (mean optical density 0.24 +/- 0.09). No differences were found between the 2 groups when comparing serum levels of prostate specific antigen (AMP 11.9, 14.8; NO AMP 26.0, 60.3), Gleason sum (AMP 9.0, 0.5; NO AMP 9.0, 1.0), clinical TNM stage (AMP 4 cases M0, M1 4; NO AMP 8 M0, 8 M1), race (AMP 6 white and 2 black men, NO AMP white and 7 black men) or survival in months (AMP 47.5, 28.5; NO AMP 33.5, 72.0). Three of the recurrent prostate cancer specimens (13%) demonstrated X-chromosome copy number 2 or greater and no differences were found when comparing clinical characteristics between these groups. CONCLUSIONS: AR amplification in recurrent prostate cancer results in higher levels of AR protein expression but does not affect survival.