A Deeper Reflection on the Integration of the Pharmacists' Patient Care Process.

OBJECTIVE: Given the importance of developing student understanding and application of the Pharmacists' Patient Care Process (PPCP), programs may be able to use successful approaches from other institutions to enhance their curricular and experiential learning and assessment of student outcomes. The study objective was to explore successful methods of integrating the PPCP and outline areas of challenge. METHODS: This study used a qualitative study design with semistructured interviews to gain insight from participants' lived experiences. Pharmacy faculty members participating in a national survey or who were authors of articles about PPCP initiatives were recruited to provide greater detail about building successful and innovative curricula. Thematic analysis identified commonalities and differences among the interviewed participants. RESULTS: A total of 10 interviews were conducted. The following 4 overarching themes arose from the data: discussions around intentional integration of the PPCP across multiple core courses may foster innovations in teaching strategies; intentional integration alone does not equate to PPCP integration across the curriculum; intentional integration may enhance program assessment; and PPCP data from experiential coursework may not be widely used in curricular continuous quality improvement. CONCLUSION: Pharmacy programs will ideally involve the entire faculty, including experiential and basic and social/administrative science members, in weaving the PPCP throughout the curriculum. Rigorous assessment can better inform interventions related to student competency in various steps of the PPCP. Pharmacy programs should also clarify how data obtained from preceptors observing student performance in each of the PPCP steps are used to assess student mastery of this critical skill.

How Preceptors Support Pharmacy Learner Professional Identity Formation.

OBJECTIVE: Pharmacy preceptors play a role in helping learners form professional identities during experiential education. However, it is not clear what specific roles and precepting strategies best foster professional identity formation (PIF). The objective of this study was to explore how preceptors support pharmacy learner PIF. METHODS: This qualitative study used an interpretative descriptive approach. Preceptors from 5 experiential education programs were recruited using purposive sampling for individual semistructured interviews. Interviews were recorded, transcribed, coded, and analyzed by thematic analysis. Team members used a reflective and iterative approach for data analysis and generation of themes. RESULTS: A total of 22 participants were interviewed from various pharmacy practice settings and precept a range of learners, including introductory pharmacy practice experiences, advanced pharmacy practice experiences, and residents. Four main themes were identified to support pharmacy leaner PIF: making learners part of the practice and team, preparing learners to assume the role of a pharmacist, helping learners navigate emotions during practice experiences, and supporting learners in finding the right fit within the profession. Specific precepting strategies associated with each theme were identified. CONCLUSION: Preceptors play an important role in supporting learners in thinking and acting as professionals while also helping navigate emotional experiences that may impact PIF and having conversations to help define learner's future aspirations of the pharmacist they want to become. Strategies identified can inform curricular approaches and preceptor development that intentionally supports PIF.

Spatiotemporal modulation of growth factors directs the generation of multilineage mouse embryonic stem cell-derived mammary organoids.

Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG in vivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling.

Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9.

Here, we present a multiplexed assay for variant effect protocol to assess the functional impact of all possible genetic variations within a particular genomic region. We describe steps for saturation genome editing by designing and cloning of single-guide RNA (sgRNA). We then detail steps for nucleofection of sgRNA, testing drug response on variants, and amplification of genomic DNA for next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Sahu et al.1.

Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs.

Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 17,000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUSs). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. We now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants using pools of mESCs expressing 10-25 BRCA2 variants from a given exon. We use this approach for functional evaluation of 223 variants listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or those reported by other orthogonal assays.

Characterization of BRCA2 R3052Q variant in mice supports its functional impact as a low-risk variant.

Pathogenic variants in BRCA2 are known to significantly increase the lifetime risk of developing breast and ovarian cancers. Sequencing-based genetic testing has resulted in the identification of thousands of BRCA2 variants that are considered to be variants of uncertain significance (VUS) because the disease risk associated with them is unknown. One such variant is p.Arg3052Gln, which has conflicting interpretations of pathogenicity in the ClinVar variant database. Arginine at position 3052 in BRCA2 plays an important role in stabilizing its C-terminal DNA binding domain. We have generated a knock-in mouse model expressing this variant to examine its role on growth and survival in vivo. Homozygous as well as hemizygous mutant mice are viable, fertile and exhibit no overt phenotype. While we did not observe any hematopoietic defects in adults, we did observe a marked reduction in the in vitro proliferative ability of fetal liver cells that were also hypersensitive to PARP inhibitor, olaparib. In vitro studies performed on embryonic and adult fibroblasts derived from the mutant mice showed significant reduction in radiation induced RAD51 foci formation as well as increased genomic instability after mitomycin C treatment. We observed mis-localization of a fraction of R3052Q BRCA2 protein to the cytoplasm which may explain the observed in vitro phenotypes. Our findings suggest that BRCA2 R3052Q should be considered as a hypomorphic variant.

A Qualitative Study of Experiences Contributing to Professional Identity Formation in Recent Pharmacy Graduates.

OBJECTIVE: A professional identity has been described as "an individual thinking, acting, and feeling" like a person within the profession. The objective of this study was to learn about professional identity formation (PIF) in recent graduates of a pharmacy program. METHODS: In-depth interviews were conducted with students graduating from a doctor of pharmacy degree program. Investigators performed a thematic content analysis of interview transcripts. RESULTS: Participants were from community pharmacy practice (4), residencies (4), industry (1), and ambulatory care (1). At the time of the interview, participants were a range of 5-13 months out from graduation. Analysis of the data revealed 4 thematic findings. First, thinking and acting like a pharmacist occurred frequently while in school but feeling like a pharmacist occurred mostly after graduation. Second, feeling like a pharmacist meant participants felt confident in their knowledge base and ability to independently make decisions. Third, real-world practice is critical to PIF, particularly through interactions with patients. Finally, feedback, mentoring, and reflection support PIF and can aid in reconciling the tensions between concepts taught in school and experiences in practice. CONCLUSIONS: In this qualitative analysis of data about PIF obtained from recent graduates from a pharmacy school, we found that thinking and acting like a pharmacist preceded feeling like a pharmacist; feeling like a pharmacist involved confidence in the ability to work autonomously; feedback, mentoring, and reflection on experiences supported PIF; and real-world experiences were critical to PIF.

Saturation genome editing of 11 codons and exon 13 of BRCA2 coupled with chemotherapeutic drug response accurately determines pathogenicity of variants.

The unknown pathogenicity of a significant number of variants found in cancer-related genes is attributed to limited epidemiological data, resulting in their classification as variant of uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate the response of these variants to the DNA-damaging agents, cisplatin and olaparib, allowing us to use cellular survival and drug response as parameters for variant classification. Using this approach, we have categorized 599 BRCA2 variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional and 60 non-functional variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS.

A translational blueprint for developing intraoperative imaging agents via radiopharmaceutical-guided drug design.

Cancer imaging is a rapidly evolving field due to the discovery of novel molecular targets and the availability of corresponding techniques to detect them with high precision, accuracy, and sensitivity. Nuclear medicine is the most widely used molecular imaging modality and has a growing toolkit of clinically used radiopharmaceuticals that enable whole-body tumor visualization, staging, and treatment monitoring for a variety of tumors in a non-invasive manner. The need for similar imaging capabilities in the operating room has led to the emergence of fluorescence-guided surgery (FGS) as a powerful technique that gives surgeons unprecedented ability to distinguish tumors from healthy tissues. While a variety of strategies have been used to develop contrast agents for FGS, the use of radiopharmaceuticals as models brings exceptional translational potential and has increasingly been explored. Here, we review strategies used to convert clinically used radiopharmaceuticals into fluorescent and multimodal counterparts. Unique preclinical and clinical capabilities stemming from radiopharmaceutical-based agent design are also discussed to illustrate the advantages of this approach.

Abrogation of Rb Tumor Suppression Initiates GBM in Differentiated Astrocytes by Driving a Progenitor Cell Program.

Glioblastoma (GBM) remains lethal with no effective treatments. Despite the comprehensive identification of commonly perturbed molecular pathways, little is known about the disease's etiology, particularly in early stages. Several studies indicate that GBM is initiated in neural progenitor and/or stem cells. Here, we report that differentiated astrocytes are susceptible to GBM development when initiated by perturbation of the RB pathway, which induces a progenitor phenotype. In vitro and in vivo inactivation of Rb tumor suppression (TS) induces cortical astrocytes to proliferate rapidly, express progenitor markers, repress differentiation markers, and form self-renewing neurospheres that are susceptible to multi-lineage differentiation. This phenotype is sufficient to cause grade II astrocytomas which stochastically progress to GBM. Together with previous findings, these results demonstrate that cell susceptibility to GBM depends on the initiating driver.

Complete Genome Sequence of Stenotrophomonas maltophilia Siphophage Suzuki.

Stenotrophomonas maltophilia is a Gram-negative bacterium known to cause respiratory tract infections and other diseases in humans. Here, we describe the isolation and genome annotation of S. maltophilia siphophage Suzuki. Its 56,042-bp genome has 83 predicted protein-coding genes and demonstrates similarity with Xylella phages Sano and Salvo.

Sudden Unexpected Death in Infancy [SUDI]: What the clinician, pathologist, coroner and researchers want to know.

The loss of an apparently healthy infant is confronting for any family, puzzling for a clinician and challenging for the pathologist charged with the task of demonstrating a cause for death. The term "cot death" evolved to "sudden infant death syndrome" [SIDS] and now "sudden unexpected death in infancy [SUDI]" as the epidemiology and pathology of infant death changed. Community interventions were successful in changing sleep practices for young babies. The current research focus is on understanding genetic predispositions to unexpected death in early childhood. Whilst much has been achieved in reducing the infant mortality rate from SUDI by between 50%, and 80% in some countries, over the last 30 years, there remain challenges for improving rates of accurate diagnosis and reaching out to more vulnerable families with clearly modifiable risk factors for SUDI. These challenges directly involve the clinician through taking a systematic and detailed history and better standardised death scene evaluations with specifically accredited assessors. Better knowledge regarding circumstances of SUDI cases will help Coroners and researchers provide answers for grieving families now, and in the future contribute to further reductions in the rate of SUDI in communities across the world.

Inclusion of the Pharmacist Patient Care Process in Doctor of Pharmacy Curricula.

Objective. With the inclusion of the Pharmacists' Patient Care Process (PPCP) in the most recent Accreditation Council for Pharmacy Education standards, institutions must determine how best to vertically and horizontally integrate and assess the PPCP in the curriculum. The objective of this study was to identify the breadth and depth of PPCP implementation as well as faculty involvement in teaching the PPCP at ACPE-accredited institutions.Methods. A survey to address the study objectives was developed, piloted, and distributed electronically to all US pharmacy institutions in candidate or accredited status. Electronic reminders were implemented to improve response rates. The data were analyzed descriptively.Results. Approximately 70% of institutions responded to the survey. Integration of the PPCP was most often championed by an individual faculty member and/or a committee. Practice faculty taught PPCP at nearly all institutions, while only a third of survey respondents reported that foundational and social administrative faculty taught the PPCP. Development related to PPCP curricular integration mainly focused on preceptors. Most institutions integrated the PPCP through the didactic and experiential curriculum in an approach that allowed for reinforcement or mastery of concepts. There were limited integration efforts into interprofessional education. Institutions had a plan for assessing the effectiveness of the integration, but were varied in their approach.Conclusion. Institutions have embraced integrating the PPCP into their curricula, didactically and experientially. Progress still needs to be made regarding inclusion of all faculty in teaching the PPCP as well as integrating the PPCP into other key curricular areas, such as interprofessional learning. Faculty development efforts may be beneficial to address these aspects.

A Qualitative Pilot Study to Investigate Caregiver Attitudes on Healthy Lifestyle Discussions During Dental Visits for Children Younger Than Six Years.

Purpose: The purpose of this qualitative pilot study was to investigate caregivers' attitudes about healthy lifestyles and weight-related discussions during dental visits. Methods: Twenty-one caregivers of children younger than six years old at two community dental clinics in Washington State-a rural community clinic serving children of seasonal farmworkers and an urban clinic primarily serving children with special health care needs-were interviewed using a semi-structured guide. Interview data were analyzed inductively via thematic content analysis. Results: Three themes emerged from the data: (1) supporting conversations about healthy lifestyles in the dental office; (2) crafting the conversation and identifying next steps; and (3) ensuring that the dentist is perceived as a caregiver ally. Caregivers were supportive of healthy lifestyle conversations with dentists. Concerns about weight-specific discussions were expressed. Conclusion: Caregivers' attitudes indicated support for conversations on healthy lifestyles. A future workaround incorporating healthy lifestyle discussion into pediatric dental visits is warranted.

A Pathway to Professional Identity Formation: Report of the 2020-2021 AACP Student Affairs Standing Committee.

EXECUTIVE SUMMARY Professional identity formation (PIF) involves internalizing and demonstrating the behavioral norms, standards, and values of a professional community, such that one comes to "think, act and feel" like a member of that community. Professional identity influences how a professional perceives, explains, presents and conducts themselves. This report of the 2020-2021 AACP Student Affairs Standing Committee (SAC) describes the benefits of a strong professional identity, including its importance in advancing practice transformation. Responding to a recommendation from the 2019-2020 SAC, this report presents an illustrative and interpretative schema as an initial step towards describing a pharmacist's identity. However, the profession must further elucidate a universal and distinctive pharmacist identity, in order to better support pharmacists and learners in explaining and presenting the pharmacist's scope of practice and opportunities for practice change. Additionally, the report outlines recommendations for integrating intentional professional identity formation within professional curricula at colleges and schools of pharmacy. Although there is no standardized, single way to facilitate PIF in students, the report explores possibilities for meeting the student support and faculty development needs of an emerging new emphasis on PIF within the Academy.

Epidemiological and ES cell-based functional evaluation of BRCA2 variants identified in families with breast cancer.

The discovery of high-risk breast cancer susceptibility genes, such as Breast cancer associated gene 1 (BRCA1) and Breast cancer associated gene 2 (BRCA2) has led to accurate identification of individuals for risk management and targeted therapy. The rapid decline in sequencing costs has tremendously increased the number of individuals who are undergoing genetic testing world-wide. However, given the significant differences in population-specific variants, interpreting the results of these tests can be challenging especially for novel genetic variants in understudied populations. Here we report the characterization of novel variants in the Malaysian and Singaporean population that consist of different ethnic groups (Malays, Chinese, Indian, and other indigenous groups). We have evaluated the functional significance of 14 BRCA2 variants of uncertain clinical significance by using multiple in silico prediction tools and examined their frequency in a cohort of 7840 breast cancer cases and 7928 healthy controls. In addition, we have used a mouse embryonic stem cell (mESC)-based functional assay to assess the impact of these variants on BRCA2 function. We found these variants to be functionally indistinguishable from wild-type BRCA2. These variants could fully rescue the lethality of Brca2-null mESCs and exhibited no sensitivity to six different DNA damaging agents including a poly ADP ribose polymerase inhibitor. Our findings strongly suggest that all 14 evaluated variants are functionally neutral. Our findings should be valuable in risk assessment of individuals carrying these variants.

A computational model for classification of BRCA2 variants using mouse embryonic stem cell-based functional assays.

Sequencing-based genetic tests to identify individuals at increased risk of hereditary breast and ovarian cancers have resulted in the identification of more than 40,000 sequence variants of BRCA1 and BRCA2. A majority of these variants are considered to be variants of uncertain significance (VUS) because their impact on disease risk remains unknown, largely due to lack of sufficient familial linkage and epidemiological data. Several assays have been developed to examine the effect of VUS on protein function, which can be used to assess their impact on cancer susceptibility. In this study, we report the functional characterization of 88 BRCA2 variants, including several previously uncharacterized variants, using a well-established mouse embryonic stem cell (mESC)-based assay. We have examined their ability to rescue the lethality of Brca2 null mESC as well as sensitivity to six DNA damaging agents including ionizing radiation and a PARP inhibitor. We have also examined the impact of BRCA2 variants on splicing. In addition, we have developed a computational model to determine the probability of impact on function of the variants that can be used for risk assessment. In contrast to the previous VarCall models that are based on a single functional assay, we have developed a new platform to analyze the data from multiple functional assays separately and in combination. We have validated our VarCall models using 12 known pathogenic and 10 neutral variants and demonstrated their usefulness in determining the pathogenicity of BRCA2 variants that are listed as VUS or as variants with conflicting functional interpretation.

Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System.

BRCA2 is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all BRCA2 VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL BRCA2 transcripts. Of 100 BRCA2e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL BRCA2 transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL BRCA2 with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.

Functional evaluation of five BRCA2 unclassified variants identified in a Sri Lankan cohort with inherited cancer syndromes using a mouse embryonic stem cell-based assay.

Next-generation sequencing of Sri Lankan families with inherited cancer syndromes resulted in the identification of five BRCA2 variants of unknown clinical significance. Interpreting such variants poses significant challenges for both clinicians and patients. Using a mouse embryonic stem cell-based functional assay, we found I785V, N830D, and K2077N to be functionally indistinguishable from wild-type BRCA2. Specific but mild sensitivity to olaparib and reduction in homologous recombination (HR) efficiency suggest partial loss of function of the A262T variant. This variant is located in the N-terminal DNA binding domain of BRCA2 that can facilitate HR by binding to dsDNA/ssDNA junctions. P3039P is clearly pathogenic because of premature protein truncation caused by exon 23 skipping. These findings highlight the value of mouse embryonic stem cell-based assays for determining the functional significance of variants of unknown clinical significance and provide valuable information regarding risk estimation and genetic counseling of families carrying these BRCA2 variants.

A Review of Strategies for Enhancing Clarity and Reader Accessibility of Qualitative Research Results.

Objective. To characterize elements of the results section of qualitative research reports that make findings more accessible to readers. Methods. Two analytical methods were used for this review. First, published reviews and textbooks written by experts outlining how to evaluate qualitative research were retrieved and reviewed to identify common elements that enhance clarity of the results section. In the second analysis, the authors analyzed the results sections of a subset of qualitative studies to identify, from a reader's point of view, aspects that enhanced and detracted from communication of the results. Findings. Four elements improve accessibility of the results section for readers of qualitative research reports. Content, the first element, describes what information the reader should look for in the results section. Style of results, the second element, identifies wording choices that improve reader accessibility and understanding. Narrative flow, the third element, describes a results section that flows smoothly and logically. Structural cohesiveness, the final element, outlines effective organization of the results section. Results. While authors take several approaches to the presentation of results in qualitative research reports, some strategies appear to be more common and effective than others. The efficient presentation of results can impact a reader's assessment of the quality and credibility of a study. Identified content and stylistic elements should be considered by authors hoping to make the results of their qualitative research more accessible and comprehensible to readers.