TWEAK/Fn14 signalling driven super-enhancer reprogramming promotes pro-metastatic metabolic rewiring in triple-negative breast cancer.

Triple Negative Breast Cancer (TNBC) is the most aggressive breast cancer subtype suffering from limited targeted treatment options. Following recent reports correlating Fibroblast growth factor-inducible 14 (Fn14) receptor overexpression in Estrogen Receptor (ER)-negative breast cancers with metastatic events, we show that Fn14 is specifically overexpressed in TNBC patients and associated with poor survival. We demonstrate that constitutive Fn14 signalling rewires the transcriptomic and epigenomic landscape of TNBC, leading to enhanced tumour growth and metastasis. We further illustrate that such mechanisms activate TNBC-specific super enhancers (SE) to drive the transcriptional activation of cancer dependency genes via chromatin looping. In particular, we uncover the SE-driven upregulation of Nicotinamide phosphoribosyltransferase (NAMPT), which promotes NAD+ and ATP metabolic reprogramming critical for filopodia formation and metastasis. Collectively, our study details the complex mechanistic link between TWEAK/Fn14 signalling and TNBC metastasis, which reveals several vulnerabilities which could be pursued for the targeted treatment of TNBC patients.

Aberrant non-canonical NF-κB signalling reprograms the epigenome landscape to drive oncogenic transcriptomes in multiple myeloma.

In multiple myeloma, abnormal plasma cells establish oncogenic niches within the bone marrow by engaging the NF-κB pathway to nurture their survival while they accumulate pro-proliferative mutations. Under these conditions, many cases eventually develop genetic abnormalities endowing them with constitutive NF-κB activation. Here, we find that sustained NF-κB/p52 levels resulting from such mutations favours the recruitment of enhancers beyond the normal B-cell repertoire. Furthermore, through targeted disruption of p52, we characterise how such enhancers are complicit in the formation of super-enhancers and the establishment of cis-regulatory interactions with myeloma dependencies during constitutive activation of p52. Finally, we functionally validate the pathological impact of these cis-regulatory modules on cell and tumour phenotypes using in vitro and in vivo models, confirming RGS1 as a p52-dependent myeloma driver. We conclude that the divergent epigenomic reprogramming enforced by aberrant non-canonical NF-κB signalling potentiates transcriptional programs beneficial for multiple myeloma progression.

A Description of NAPLEX and MPJE Preparation Strategies Among US Colleges and Schools of Pharmacy.

OBJECTIVE: The purpose of this study was to describe the different strategies used to supplement North American Pharmacist Licensure Examination (NAPLEX) and Multistate Pharmacy Jurisprudence Examination (MPJE) preparation in the US pharmacy programs. METHODS: An online survey was developed to gather information from 141 accredited schools/colleges of pharmacy about the preparation methods used during the 2021-22 academic year. The questionnaire contained 19 NAPLEX- and 10 MPJE-specific questions related to timing, content, use of commercial products and programs, faculty involvement, and whether these activities were required or recommended. Characteristics of schools/colleges were compared based on the presence or absence of preparation programs; preparation programs were descriptively reported. RESULTS: The response rate was 71%. Most schools (87/100 respondents) provided NAPLEX preparation programs starting in the advanced pharmacy practice experiential year, required students to participate, and focused on reviewing the content instead of assessing students' examination readiness. Similar elements were reported among 61 schools providing MPJE preparation programs. Schools used a variety of resources including access to vendor-based question banks or review materials, and completing live, proctored, NAPLEX-like examinations. Characteristics of schools or colleges did not differ significantly based on presence or absence of a preparation program. CONCLUSION: Schools/colleges of pharmacy use a variety of strategies to prepare students for licensing examinations. Many require student participation in vendor-based preparation programs for NAPLEX, and homegrown programs for MPJE preparation. The next step will be to determine the effectiveness of various approaches used by the schools/colleges on first-time licensure examination attempts.

An Updated National Profile of Curriculum Committees at US Schools and Colleges of Pharmacy.

Objective. To assess how curriculum committees at US schools and colleges of pharmacy have evolved since 2011 regarding their responsibilities, structures, functions, charges, and activities.Methods. A total of 133 fully accredited schools and colleges of pharmacy were included in the survey. Data collection occurred between March and September 2020, and survey questions pertained to academic year 2019-2020. Data were collected on committee membership, leadership, functions, and charges. New questions explored ties to assessments and Standards 2016. Analysis included descriptive statistics and comparisons to the 2011 survey results.Results. The response rate was 80%; one partial response was excluded from analysis. Most schools and colleges (93%) rely on a curriculum committee to provide curriculum oversight. Faculty and students remain the most frequent types of members, but increases have occurred in the number of committees with members from other areas, including experiential programs, staff, directors, librarians, and pharmacy residents. Committee charges have increased beyond the traditional activities of curriculum planning, mapping, and review to include newer tasks. In one-third of the institutions, the primary responsibility for various assessment activities is shared by both committees.Conclusion. Curriculum committees remain a key part of pharmacy education but continue to evolve to meet their responsibilities related to new and increasing numbers of charges and to find ways to communicate and share duties with their assessment counterparts. Based on these findings, recommendations include having clear guidance for curriculum committees and reducing the frequency of their scheduled work to ensure they will be able to address new challenges as they emerge.

Approaches to Identify and Characterise the Post-Transcriptional Roles of lncRNAs in Cancer.

It is becoming increasingly evident that the non-coding genome and transcriptome exert great influence over their coding counterparts through complex molecular interactions. Among non-coding RNAs (ncRNA), long non-coding RNAs (lncRNAs) in particular present increased potential to participate in dysregulation of post-transcriptional processes through both RNA and protein interactions. Since such processes can play key roles in contributing to cancer progression, it is desirable to continue expanding the search for lncRNAs impacting cancer through post-transcriptional mechanisms. The sheer diversity of mechanisms requires diverse resources and methods that have been developed and refined over the past decade. We provide an overview of computational resources as well as proven low-to-high throughput techniques to enable identification and characterisation of lncRNAs in their complex interactive contexts. As more cancer research strategies evolve to explore the non-coding genome and transcriptome, we anticipate this will provide a valuable primer and perspective of how these technologies have matured and will continue to evolve to assist researchers in elucidating post-transcriptional roles of lncRNAs in cancer.

Sequence- and structure-specific cytosine-5 mRNA methylation by NSUN6.

The highly abundant N6-methyladenosine (m6A) RNA modification affects most aspects of mRNA function, yet the precise function of the rarer 5-methylcytidine (m5C) remains largely unknown. Here, we map m5C in the human transcriptome using methylation-dependent individual-nucleotide resolution cross-linking and immunoprecipitation (miCLIP) combined with RNA bisulfite sequencing. We identify NSUN6 as a methyltransferase with strong substrate specificity towards mRNA. NSUN6 primarily targeted three prime untranslated regions (3'UTR) at the consensus sequence motif CTCCA, located in loops of hairpin structures. Knockout and rescue experiments revealed enhanced mRNA and translation levels when NSUN6-targeted mRNAs were methylated. Ribosome profiling further demonstrated that NSUN6-specific methylation correlated with translation termination. While NSUN6 was dispensable for mouse embryonic development, it was down-regulated in human tumours and high expression of NSUN6 indicated better patient outcome of certain cancer types. In summary, our study identifies NSUN6 as a methyltransferase targeting mRNA, potentially as part of a quality control mechanism involved in translation termination fidelity.

Recommendations for integration of foundational and clinical sciences throughout the pharmacy curriculum.

INTRODUCTION: Pharmacy faculty have the often difficult task of translating and incorporating existing concepts and advances from the foundational sciences into the clinical sciences and practice. This commentary focuses on content integration as a curricular and educational strategy, outcomes data from integration, and recommendations for programs employing or considering curricular integration. COMMENTARY: Integration of foundational and clinical sciences across the curriculum has been emphasized in accreditation standards but met with mixed reactions by faculty across different disciplines in the academy. Many pharmacy programs have already incorporated some level of integration in didactic courses. However, most report coordination of curricular delivery rather than higher levels of integration in which different disciplines work together to design and deliver instructional materials across the entire curriculum. IMPLICATIONS: Curricular integration models should be optimized to minimize or eliminate the risks of marginalization of foundational sciences in pharmacy curricula. A significant problem in implementing curricular integration is determining the appropriate balance between foundational and clinical sciences. Well-designed curricular integration with ongoing reinforcement that builds in complexity over time could enhance knowledge retention, critical thinking abilities, and clinical decision making. Further research is needed into the outcomes achieved from various integrated curricular approaches in pharmacy education.

FICC-Seq: a method for enzyme-specified profiling of methyl-5-uridine in cellular RNA.

Methyl-5-uridine (m5U) is one the most abundant non-canonical bases present in cellular RNA, and in yeast is found at position U54 of tRNAs where modification is catalysed by the methyltransferase Trm2. Although the mammalian enzymes that catalyse m5U formation are yet to be identified via experimental evidence, based on sequence homology to Trm2, two candidates currently exist, TRMT2A and TRMT2B. Here we developed a genome-wide single-nucleotide resolution mapping method, Fluorouracil-Induced-Catalytic-Crosslinking-Sequencing (FICC-Seq), in order to identify the relevant enzymatic targets. We demonstrate that TRMT2A is responsible for the majority of m5U present in human RNA, and that it commonly targets U54 of cytosolic tRNAs. By comparison to current methods, we show that FICC-Seq is a particularly robust method for accurate and reliable detection of relevant enzymatic target sites. Our associated finding of extensive irreversible TRMT2A-tRNA crosslinking in vivo following 5-Fluorouracil exposure is also intriguing, as it suggests a tangible mechanism for a previously suspected RNA-dependent route of Fluorouracil-mediated cytotoxicity.

Revisiting Faculty Citizenship.

This commentary describes the significance of faculty citizenship in the broader context of institutional culture and defines faculty citizenship for use across all aspects of faculty roles in the Academy. The definition includes two key components (engagement and collegiality) that can be used to measure citizenship behaviors. Continued discussion and study of faculty citizenship will further the Academy's understanding and use of the concept.

Pilot study of rapid MR pancreas screening for patients with BRCA mutation.

PURPOSE: To develop and optimize a rapid magnetic resonance imaging (MRI) screening protocol for pancreatic cancer to be performed in conjunction with breast MRI screening in breast cancer susceptibility gene (BRCA)-positive individuals. METHODS: An IRB-approved prospective study was conducted. The rapid screening pancreatic MR protocol was designed to be less than 10 min to be performed after a standard breast MRI protocol. Protocol consisted of coronal NT T2 SSFSE, axial NT T2 SSFSE and axial NT rFOV FOCUS DWI, and axial T1. Images were acquired with the patient in the same prone position of breast MRI using the built-in body coil. Image quality was qualitatively assessed by two radiologists with 12 and 13 years of MRI experience, respectively. The imaging protocol was modified until an endpoint of five consecutive patients with high-quality diagnostic images were achieved. Signal-to-noise ratio and contrast-to-noise ratio were assessed. RESULTS: The rapid pancreas MR protocol was successfully completed in all patients. Diagnostic image quality was achieved for all patients. Excellent image quality was achieved for low b values; however, image quality at higher b values was more variable. In one patient, a pancreatic neuroendocrine tumor was found and the patient was treated surgically. In four patients, small pancreatic cystic lesions were detected. In one subject, a hepatic mass was identified and confirmed as adenoma by liver MRI. CONCLUSION: Rapid MR protocol for pancreatic cancer screening is feasible and has the potential to play a role in screening BRCA patients undergoing breast MRI. KEY POINT: • Develop and optimize a rapid magnetic resonance imaging (MRI) screening protocol for pancreatic cancer to be performed in conjunction with breast MRI screening in BRCA mutation positive individuals.

Formalization of an antimicrobial stewardship program in a small community hospital.

PURPOSE: Results of a study to formalize an antimicrobial stewardship program (ASP) in a small community hospital are presented. METHODS: The formalization process began with a gap analysis of the hospital's antimicrobial services, followed by the development of a fully integrated, multipharmacist ASP. The impact was studied with an institutional review board-approved study design. Retrospective pre-ASP data were pulled from March 1 to June 30, 2012 and 2013 patient records; prospective post-ASP data were collected for March 1 to June 30, 2015. Analyses included descriptive and inferential statistics. RESULTS: No significant differences in age, percent of patients on antimicrobials, or length of stay were found between the 2 groups. The post-ASP period showed a 30.2% decrease in defined daily dose (DDD) per 1,000 patient-days for the 18 most frequently used parenteral antimicrobial agents (p < 0.001). For all nursing units except nursery, the vancomycin and piperacillin-tazobactam DDD per 1,000 patient-days decreased by 63% (p < 0.001) and 36% (p < 0.001), respectively. Mean antibiotic charges per patient-day decreased from $10.44 to $3.09 (p < 0.001) and from $18.04 to $11.29 (p < 0.001) for vancomycin and piperacillin-tazobactam, respectively. Pharmacist interventions increased from 19.3 per 1,000 patients to 104.3 per 1,000 patients. Deescalation of therapy was the most common intervention (46% and 29%) in both time periods. CONCLUSION: In a small community hospital, a new formalized ASP with pharmacists showed a decrease in the DDD per 1,000 patient-days and average antibiotic charges per patient-day for vancomycin and piperacillin-tazobactam within 4 months of implementation. The approach used to develop a formalized ASP could be used as an example for development in small community hospitals with similar resources.

Robust long-read native DNA sequencing using the ONT CsgG Nanopore system.

Background: The ability to obtain long read lengths during DNA sequencing has several potentially important practical applications. Especially long read lengths have been reported using the Nanopore sequencing method, currently commercially available from Oxford Nanopore Technologies (ONT). However, early reports have demonstrated only limited levels of combined throughput and sequence accuracy. Recently, ONT released a new CsgG pore sequencing system as well as a 250b/s translocation chemistry with potential for improvements. Methods: We made use of such components on ONTs miniature 'MinION' device and sequenced native genomic DNA obtained from the near haploid cancer cell line HAP1. Analysis of our data was performed utilising recently described computational tools tailored for nanopore/long-read sequencing outputs, and here we present our key findings. Results: From a single sequencing run, we obtained ~240,000 high-quality mapped reads, comprising a total of ~2.3 billion bases. A mean read length of 9.6kb and an N50 of ~17kb was achieved, while sequences mapped to reference with a mean identity of 85%. Notably, we obtained ~68X coverage of the mitochondrial genome and were able to achieve a mean consensus identity of 99.8% for sequenced mtDNA reads. Conclusions: With improved sequencing chemistries already released and higher-throughput instruments in the pipeline, this early study suggests that ONT CsgG-based sequencing may be a useful option for potential practical long-read applications.

Studying Oogenesis in a Non-model Organism Using Transcriptomics: Assembling, Annotating, and Analyzing Your Data.

This chapter provides a guide to processing and analyzing RNA-Seq data in a non-model organism. This approach was implemented for studying oogenesis in the Speckled Wood Butterfly Pararge aegeria. We focus in particular on how to perform a more informative primary annotation of your non-model organism by implementing our multi-BLAST annotation strategy. We also provide a general guide to other essential steps in the next-generation sequencing analysis workflow. Before undertaking these methods, we recommend you familiarize yourself with command line usage and fundamental concepts of database handling. Most of the operations in the primary annotation pipeline can be performed in Galaxy (or equivalent standalone versions of the tools) and through the use of common database operations (e.g. to remove duplicates) but other equivalent programs and/or custom scripts can be implemented for further automation.

Comparison of Intraoperative Periarticular Injections Versus Liposomal Bupivacaine as Part of a Multimodal Approach to Pain Management in Total Knee Arthroplasty.

BACKGROUND: Total knee arthroplasty (TKA) has been shown to restore mobility, return an individual to activities of daily living, and improve quality of life. Nearly 80% of patients undergoing TKA report moderate to severe pain in the first 2 weeks following surgery. METHODS: A retrospective study was conducted in 103 patients who underwent TKA between October 12, 2014 and May 30, 2015 by a single surgeon at a small community hospital. During this period, data were analyzed for differences in outcomes with a change from intraoperative periarticular (IOPA) injections containing an anesthetic/analgesic mixture of ropivacaine, epinephrine, ketorolac, and clonidine to liposomal bupivacaine. Patient records were reviewed to extract study data including postoperative opioid use, length of stay (LOS), opioid-associated adverse events, and non-opioid analgesic use. RESULTS: No statistical differences were determined between groups for mean postoperative opiate usage in morphine equivalences during any time frame or for total opiate usage (79.4 vs 89.2 mg; P = .259) during the first 72 postoperative hours. Patients who received a liposomal bupivacaine injection did have a statistically significant increase in hospital LOS (70.0 vs 75.5 hours; P = .013) when compared to patients who received an IOPA injection. The incidence of nausea or vomiting, pruritus, or oversedation did not differ between groups. CONCLUSION: Pain control in TKA with a multimodal pain management protocol was not improved with the addition of liposomal bupivacaine compared to the IOPA injection at a community hospital.

Divergent RNA Localisation Patterns of Maternal Genes Regulating Embryonic Patterning in the Butterfly Pararge aegeria.

The maternal effect genes responsible for patterning the embryo along the antero-posterior (AP) axis are broadly conserved in insects. The precise function of these maternal effect genes is the result of the localisation of their mRNA in the oocyte. The main developmental mechanisms involved have been elucidated in Drosophila melanogaster, but recent studies have shown that other insect orders often diverge in RNA localisation patterns. A recent study has shown that in the butterfly Pararge aegeria the distinction between blastodermal embryonic (i.e. germ band) and extra-embryonic tissue (i.e. serosa) is already specified in the oocyte during oogenesis in the ovariole, long before blastoderm cellularisation. To examine the extent by which a female butterfly specifies and patterns the AP axis within the region fated to be the germ band, and whether she specifies a germ plasm, we performed in situ hybridisation experiments on oocytes in P. aegeria ovarioles and on early embryos. RNA localisation of the following key maternal effect genes were investigated: caudal (cad), orthodenticle (otd), hunchback (hb) and four nanos (nos) paralogs, as well as TDRD7 a gene containing a key functional domain (OST-HTH/LOTUS) shared with oskar. TDRD7 was mainly confined to the follicle cells, whilst hb was exclusively zygotically transcribed. RNA of some of the nos paralogs, otd and cad revealed complex localisation patterns within the cortical region prefiguring the germ band (i.e. germ cortex). Rather interestingly, otd was localised within and outside the anterior of the germ cortex. Transcripts of nos-O formed a distinct granular ring in the middle of the germ cortex possibly prefiguring the region where germline stem cells form. These butterfly RNA localisation patterns are highly divergent with respect to other insects, highlighting the diverse ways in which different insect orders maternally regulate early embryogenesis of their offspring.

JNK signalling is necessary for a Wnt- and stem cell-dependent regeneration programme.

Regeneration involves the integration of new and old tissues in the context of an adult life history. It is clear that the core conserved signalling pathways that orchestrate development also play central roles in regeneration, and further study of conserved signalling pathways is required. Here we have studied the role of the conserved JNK signalling cascade during planarian regeneration. Abrogation of JNK signalling by RNAi or pharmacological inhibition blocks posterior regeneration and animals fail to express posterior markers. While the early injury-induced expression of polarity markers is unaffected, the later stem cell-dependent phase of posterior Wnt expression is not established. This defect can be rescued by overactivation of the Hh or Wnt signalling pathway to promote posterior Wnt activity. Together, our data suggest that JNK signalling is required to establish stem cell-dependent Wnt expression after posterior injury. Given that Jun is known to be required in vertebrates for the expression of Wnt and Wnt target genes, we propose that this interaction may be conserved and is an instructive part of planarian posterior regeneration.

Ancient expansion of the hox cluster in lepidoptera generated four homeobox genes implicated in extra-embryonic tissue formation.

Gene duplications within the conserved Hox cluster are rare in animal evolution, but in Lepidoptera an array of divergent Hox-related genes (Shx genes) has been reported between pb and zen. Here, we use genome sequencing of five lepidopteran species (Polygonia c-album, Pararge aegeria, Callimorpha dominula, Cameraria ohridella, Hepialus sylvina) plus a caddisfly outgroup (Glyphotaelius pellucidus) to trace the evolution of the lepidopteran Shx genes. We demonstrate that Shx genes originated by tandem duplication of zen early in the evolution of large clade Ditrysia; Shx are not found in a caddisfly and a member of the basally diverging Hepialidae (swift moths). Four distinct Shx genes were generated early in ditrysian evolution, and were stably retained in all descendent Lepidoptera except the silkmoth which has additional duplications. Despite extensive sequence divergence, molecular modelling indicates that all four Shx genes have the potential to encode stable homeodomains. The four Shx genes have distinct spatiotemporal expression patterns in early development of the Speckled Wood butterfly (Pararge aegeria), with ShxC demarcating the future sites of extraembryonic tissue formation via strikingly localised maternal RNA in the oocyte. All four genes are also expressed in presumptive serosal cells, prior to the onset of zen expression. Lepidopteran Shx genes represent an unusual example of Hox cluster expansion and integration of novel genes into ancient developmental regulatory networks.

Factors predicting development of opioid use disorders among individuals who receive an initial opioid prescription: mathematical modeling using a database of commercially-insured individuals.

BACKGROUND: Prescription drug abuse in the United States and elsewhere in the world is increasing at an alarming rate with non-medical opioid use, in particular, increasing to epidemic proportions over the past two decades. It is imperative to identify individuals most likely to develop opioid abuse or dependence to inform large-scale, targeted prevention efforts. METHODS: The present investigation utilized a large commercial insurance claims database to identify demographic, mental health, physical health, and healthcare service utilization variables that differentiate persons who receive an opioid abuse or dependence diagnosis within two years of filling an opioid prescription (OUDs) from those who do not receive such a diagnosis within the same time frame (non-OUDs). RESULTS: When compared to non-OUDs, OUDs were more likely to: (1) be male (59.9% vs. 44.2% for non-OUDs) and younger (M=37.9 vs. 47.7); (2) have a prescription history of more opioids (1.7 vs. 1.2), and more days supply of opioids (M=272.5, vs. M=33.2; (3) have prescriptions filled at more pharmacies (M=3.3 per year vs. M=1.3); (4) have greater rates of psychiatric disorders; (5) utilize more medical and psychiatric services; and (6) be prescribed more concomitant medications. A predictive model incorporating these findings was 79.5% concordant with actual OUDs in the data set. CONCLUSIONS: Understanding correlates of OUD development can help to predict risk and inform prevention efforts.

Unscrambling butterfly oogenesis.

BACKGROUND: Butterflies are popular model organisms to study physiological mechanisms underlying variability in oogenesis and egg provisioning in response to environmental conditions. Nothing is known, however, about; the developmental mechanisms governing butterfly oogenesis, how polarity in the oocyte is established, or which particular maternal effect genes regulate early embryogenesis. To gain insights into these developmental mechanisms and to identify the conserved and divergent aspects of butterfly oogenesis, we analysed a de novo ovarian transcriptome of the Speckled Wood butterfly Pararge aegeria (L.), and compared the results with known model organisms such as Drosophila melanogaster and Bombyx mori. RESULTS: A total of 17306 contigs were annotated, with 30% possibly novel or highly divergent sequences observed. Pararge aegeria females expressed 74.5% of the genes that are known to be essential for D. melanogaster oogenesis. We discuss the genes involved in all aspects of oogenesis, including vitellogenesis and choriogenesis, plus those implicated in hormonal control of oogenesis and transgenerational hormonal effects in great detail. Compared to other insects, a number of significant differences were observed in; the genes involved in stem cell maintenance and differentiation in the germarium, establishment of oocyte polarity, and in several aspects of maternal regulation of zygotic development. CONCLUSIONS: This study provides valuable resources to investigate a number of divergent aspects of butterfly oogenesis requiring further research. In order to fully unscramble butterfly oogenesis, we also now also have the resources to investigate expression patterns of oogenesis genes under a range of environmental conditions, and to establish their function.

The role of curriculum committees in pharmacy education.

OBJECTIVE: To conduct a follow-up survey of curriculum committee chairs in US colleges and schools of pharmacy to describe current committee structures and functions and determine whether changes have occurred over time. METHODS: A descriptive cross-sectional study design using a 30-item survey instrument regarding the structure, function, and charges of curriculum committees was sent to 100 curriculum committee chairs. Several new variables were added to the questionnaire to explore the use of systematic reviews, oversight of experiential education, and the impact of accreditation standards on work focus. RESULTS: Eighty-five chairs responded. Curriculum committees are on average 1 person larger, less likely to have a student vote, more likely to have formal charges, and more likely to be involved in implementing an outcomes-based curriculum compared with 1994. Committees have shifted their work focus from review of curricular content to curricular revision. CONCLUSIONS: Curriculum committees continue to evolve as they respond to changes in pharmacy education and accreditation standards.