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Background/Aim: There exists considerably large interpatient variability in pharmacokinetic exposure of high dose melphalan in multiple myeloma patients with hematopoietic stem-cell transplantation. In this study, we aimed to evaluate the potential impacts of CYP3A4*1B (rs2940574) and CYP3A5*3 (rs776746) variations on pharmacokinetic properties of melphalan and clinical outcomes in multiple myeloma (MM) patients. Patients and Methods: Genotypes of CYP3A4*1B (rs2940574) and CYP3A5*3 (rs776746) were determined by validated gene-specific real-time PCR (RT-PCR) assays using DNA samples from 108 MM patients; plasma concentrations of melphalan at different time points were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/ΜS). Results: CYP3A4*1B/*1B and CYP3A5*3/*3 carriers appeared to have a short median progression-free survival time and a higher maximum melphalan plasma concentration than non-carriers [792 vs. over 950 days, p=0.08; 9.91 (2.67, 34.03) vs. 8.66 (4.46, 17.61) mg/l, p=0.039]. Conclusion: CYP3A4*1B/*1B and CYP3A5*3/*3 variations might influence melphalan therapy in MM patients through yet-to-be-identified mechanisms.

Video 3. N+/E- cells 3D reconstruction animation shows localization of ENT1 (red) and DAPI (blue)

Epithelial–mesenchymal transition (EMT) in cancer cells drives cancer chemoresistance, yet the molecular events of EMT that underpin the acquisition of chemoresistance are poorly understood. Here, we demonstrate a loss of gemcitabine chemosensitivity facilitated by human equilibrative nucleoside transporter 1 (ENT1) during EMT in pancreatic cancer and identify that cadherin switching from the epithelial (E) to neuronal (N) type, a hallmark of EMT, contributes to this loss. Our findings demonstrate that N-cadherin decreases ENT1 expression, membrane localization, and gemcitabine transport, while E-cadherin augments each of these. Besides E- and N-cadherin, another epithelial cell adhesion molecule, EpCAM, played a more prominent role in determining ENT1 membrane localization. Forced expression of EpCAM opposed cadherin switching with restored ENT1 expression, membrane localization, and gemcitabine transport in EMT-committed pancreatic cancer cells. In gemcitabine-treated mice, EpCAM-positive tumors had high ENT1 expression and reduced metastasis, whereas tumors with N-cadherin expression resisted gemcitabine treatment and formed extensive secondary metastatic nodules. Tissue microarray profiling and multiplexed IHC analysis of pancreatic cancer patient-derived primary tumors revealed EpCAM and ENT1 cell surface coexpression is favored, and ENT1 plasma membrane expression positively predicted median overall survival times in patients treated with adjuvant gemcitabine. Together, our findings identify ENT1 as an inadvertent target of EMT signaling mediated by cadherin switching and provide a mechanism by which mesenchymal pancreatic cancer cells evade gemcitabine therapy during EMT.

Video 2. Animation displays 3D reconstruction of PANC-1 cells grown in monolayer immunostained for ENT1 (red) and DAPI (blue)

Video 4. E+/N- cells animation displays 3D reconstruction of ENT1 (red) and DAPI (blue)

Video 5. PANC-1 cells stably overexpressing EpCAM are shown in this animation of a 3D reconstruction of compiled z-stack images. ENT1 (red), EpCAM (green), DAPI (blue).

Video 1. PANC-1 cells stably overexpressing E-cadherin are shown in this animation of a 3D reconstruction of compiled z-stack images. ENT1 (red), E-cadherin (green), DAPI (blue).

An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAAAC→GAG//GTAAGT) in the TOP2α gene’s suboptimal exon 19/intron 19 5′ splice site (E19/I19 5′ SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 mRNA/protein, and increased TOP2α/170 mRNA/protein. Sensitivity to etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition was restored to levels comparable to those in parental K562 cells. Together, the results indicate that our gene-editing strategy for optimizing the TOP2α E19/I19 5′ SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2α-targeted drugs. Significance Statement Results presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 5′ splice site in the DNA topoisomerase IIα (TOP2α) gene results in circumvention of acquired drug resistance to etoposide and other TOP2α-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2α 90 kDa isoform and increasing expression of full-length TOP2α 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2α-targeted drugs.

The introduction of direct oral anticoagulants (DOACs) to the market has expanded anticoagulation options for outpatient use. Routine evaluation by health care professionals is recommended as it is with warfarin, therefore requiring adjustments in practices of anticoagulation management services (AMS). This study aims to describe trends that occurred following the incorporation of DOACs into AMS at a large academic medical center. A retrospective chart review of pharmacist-run AMS was used to compare patients on DOAC therapy versus other types of anticoagulation, including warfarin and parenteral agents. Primary outcomes included trends in the number of unique patients, management encounters, and telephone encounters throughout the study period. Secondary outcomes included trends in new encounters, and changes in patient characteristics, resources utilized, and patient satisfaction scores. A total of 2976 unique patients, 74,582 management encounters, and 13,282 telephone encounters were identified. From study beginning to end, results showed stable numbers of unique patients, an increase in management encounters for the DOAC group and decrease in the other anticoagulants group, and stable numbers of telephone encounters. Additionally, the number of new encounters for both groups increased. Throughout the study, pharmacy resources were reallocated within anticoagulation to adapt to the changing trends and patient satisfaction reached targets. Patients' characteristics remained stable, with the DOAC group having fewer comorbid conditions and concomitant medications that could increase bleed risk. This study showed that by reallocating resources within anticoagulation, AMS can maintain stable patient populations while continuing to expand access and satisfy patients following DOAC inclusion.

Background/aim Genetic variations of the non-coding RNA gene, ANRIL, have been associated with human diseases including cancer, type-2 diabetes, and atherosclerosis. In the present study, we investigated the potential associations of select ANRIL single nucleotide polymorphisms (SNPs) with overall survival and other clinical outcomes in adult patients with hematologic malignancies after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Patients and methods Genomic DNA was extracted from whole blood samples from 103 adult patients with hematologic malignancies who had received allo-HSCT followed by oral tacrolimus therapy. The genotypes of four select ANRIL SNPs, rs564398, rs1063192, rs2151280, and rs2157719 were determined using qRT-PCR-based genotyping assays. Results rs2151280 (C->T) in ANRIL was associated with worse overall survival in these patients (CT/CC vs. TT). Contrarily, rs2151280 and the other select ANRIL SNPs were not associated with death at Day-100 after transplantation, the incidence of graft-versus-host disease (GVHD), acute kidney injury (AKI), and neurotoxicity in the study cohort. Conclusion rs2151280 represents a potential prognostic biomarker for overall survival in adult patients with hematologic malignancies after allo-HSCT.

Purpose As health systems continue to expand pharmacy and clinical services, the ability to evaluate potential medication safety risks and mitigate errors remains a high priority. Workload and productivity monitoring tools for the assessment of operational and clinical pharmacy services exist. However, such tools are not currently available to justify medication safety pharmacy services. The purpose of this study is to determine methods used to assess, allocate, and justify medication safety resources in pediatric hospitals. Methods A 32-question survey was designed and distributed utilizing the Research Electronic Data Capture (REDCap) tool. The survey was disseminated to 46 pediatric hospitals affiliated with the Children’s Hospital Association (CHA). The survey was distributed in October 2018, and the respondents were given 3 weeks to submit responses. Data analysis includes the use of descriptive statistics. Categorical variables were summarized by frequencies and percentages to distinguish the differences between pediatric health systems. Results Of 26 respondents, 15.4% utilized metrics to justify medication safety resources. Metrics utilized were based on medication dispenses, projects, and error coding. Twenty-three percent of respondents were dissatisfied with current pharmacy-based medication safety resources within the organization. There was variability of medication safety resources within pediatric hospitals, including the number of dedicated full-time equivalents, time spent on tasks, and task prioritization. Conclusion Assessing medication safety resources at various pediatric hospitals highlights several potential barriers and opportunities. This information will serve as the foundation for the creation of a standardized workload assessment tool to assist pharmacy leaders with additional resource justification.

BACKGROUND Benign anastomotic esophageal stricture after surgical resection frequently occurs and requires endoscopic balloon dilation (EBD) or incision to maintain patency because of the significant recurrence rate. Our study was designed to evaluate the effectiveness and safety of endoscopic incision and selective cutting (EISC) as primary treatment on 5 patients for benign anastomotic esophageal stricture. METHODS Five patients with benign stricture of the esophageal anastomosis after radical resection for esophageal cancer underwent EISC in our center between April, 2018 and January, 2019. The effectiveness and safety of the procedure were observed during at least 12 months follow-up. RESULTS The EISC was successfully performed in all 5 patients. The diameters of the anastomoses were increased from 1-3 preoperatively to 14-18 mm (mean: 15.6 mm) intraoperatively (P

Background/aim Aberrant expression of the SEI1 oncogene has been prevalently found in a variety of human cancers, including oral squamous cell carcinoma (OSCC). Recent studies have shown that cisplatin up-regulates the expression of SEI1 in breast and bladder cancer cells, thus inhibiting apoptosis and cell death in these cells. In the present study, we investigated the impact of cisplatin on the expression of SEI1 in OSCC cells. Materials and methods Four OSCC cell lines, CAL27, SCC4, SCC15, and SCC22A were treated with cisplatin and 5-fluorouracil, and changes in SEI1 expression in these cells were evaluated using quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analyses. Results Cisplatin significantly induced SEI1 expression in the tested OSCC cells. Contrarily, cisplatin treatment did not affect the expression of gankyrin and BMI1, two oncogenes frequently overexpressed in a coordinate manner with SEI1 in OSCC. Additionally, 5-fluorouracil did not bring about any detectable changes in SEI1 expression in these cells. Conclusion Cisplatin-induced up-regulation of SEI1 expression in OSCC is specific, and such induction could underlie the development of resistance to cisplatin in OSCC.

BACKGROUND/AIM Aberrant expression of the BMI1 oncogene has been prevalently found in a variety of human cancers, including cervical cancer. Recent studies have shown that PTC209, a specific BMI1 inhibitor, exhibits high potency in inhibiting the growth of colon, breast, oral cancer cells and cancer-initiating cells, indicative of its chemotherapeutic potential. In the current study, we evaluated the inhibitory abilities of PTC209 in cervical cancer cells. MATERIALS AND METHODS Three cervical cell lines, C33A, HeLa, and SiHa were treated with PTC209. The impacts of PTC209 on BMI1 were investigated using quantitative reverse-transcription PCR assay (qRT-PCR) and western blotting; changes in cell viability, cell cycle distribution, and apoptosis were assessed using cell viability testing, colony formation assay and flow cytometry analyses, respectively. RESULTS PTC209 exhibited considerably high short-term and long-term cytotoxicities in all tested cervical cancer cell lines regardless of their HPV infection status, TP53 and pRb statuses. PTC209 significantly downregulated the expression of BMI1 in cervical cancer cell lines, and such downregulation led to G0/G1 arrest (p

Background For a vaccine to be successful, communities must perceive it as important, safe, effective, and necessary. However, there are many barriers and hesitancies to vaccination. Underserved patient populations may face additional challenges related to access and cost. Because community pharmacists improve vaccine access and increase vaccination rates, it is beneficial for pharmacists to understand perceptions and barriers to vaccinations in their community to increase vaccine confidence. Objectives This study aims to assess and compare barriers and perceptions of the annual influenza to the coronavirus disease 2019 (COVID-19) vaccine for underserved patients of a charitable pharmacy. Methods Patients who qualified to receive medications from an outpatient charitable pharmacy took an electronic survey when receiving medications. The survey incorporated questions developed by the World Health Organization’s Strategic Advisory Group of Experts on Vaccine Hesitancy on a 5-point Likert scale. Questions about the influenza and COVID-19 vaccines mirrored one another. Demographic data such as age, race, sex, and education level were also collected. Results Of the 189 patients surveyed at the charitable pharmacy, 71.7% were 55 years old and older and 58.9% were female. Of note, 78% and 77% of participants agreed or strongly agreed that the influenza and COVID-19 vaccines, respectively, were important for the health of others in their community. Adverse effects and the cost of the COVID-19 vaccine were noted to be statistically significantly more of a concern with the COVID-19 vaccine than that of the influenza vaccine (P Conclusion Ensuring equitable vaccine access, promoting the COVID-19 vaccine as free, and eliciting and addressing individual persons’ concerns related to vaccine safety and adverse effects are all important ways pharmacists and other health care providers and community stakeholders can help promote vaccine confidence within the populations they serve.

Objective. To determine the impact of the holistic redesign of top 200 medications learning activities within a Doctor of Pharmacy (PharmD) curriculum by comparing student performances on a comprehensive examination before and after the redesign. Methods. During a curricular revision at The Ohio State University College of Pharmacy that began with the class of 2020, learning activities involving the top 200 medications were implemented that involved repeated retrieval and mastery concepts, alignment with therapeutic coursework, and autonomous learning regarding the top 200 medications. A high-stakes comprehensive top 200 medications examination was administered to students at the end of their third professional year both before and after implementation of these activities. The difference in the percentage of students who achieved a satisfactory score on the comprehensive examination was compared between cohorts prior to and following the curricular redesign. Results. The study analyzed results from 134, 130, and 120 students from three PharmD classes (one before and two after the redesign of top 200 medications activities). Following the redesign, a higher percentage of students achieved a satisfactory score of 85% on the examination (class of 2020: 116/130, 89.2%; class of 2022: 107/120, 89.2%) compared to before the redesign (class of 2019: 88/134, 65.7%). Conclusion. The combination of repeated retrieval and mastery, alignment with therapeutic coursework, and development of autonomous learning can significantly increase student knowledge and retention of top 200 medications.

An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAAAC→GAG//GTAAGT) in the TOP2α gene’s suboptimal exon 19/intron 19 5′ splice site (E19/I19 5′ SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 mRNA/protein, and increased TOP2α/170 mRNA/protein. Sensitivity to etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition was restored to levels comparable to those in parental K562 cells. Together, the results indicate that our gene-editing strategy for optimizing the TOP2α E19/I19 5′ SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2α-targeted drugs. Significance Statement Results presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 5′ splice site in the DNA topoisomerase IIα (TOP2α) gene results in circumvention of acquired drug resistance to etoposide and other TOP2α-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2α 90 kDa isoform and increasing expression of full-length TOP2α 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2α-targeted drugs.

Triple-negative breast cancer (TNBC) is one type of the most aggressive breast cancers with poor prognosis. It is of great urgency to develop new therapeutics for treating TNBC. Based on current treatment guideline and genetic information of TNBC, a combinational therapy platform integrating chemotherapy drugs and mRNA encoding tumor suppressor proteins may become an efficacious strategy. In this study, we developed paclitaxel amino lipid (PAL) derived nanoparticles (NPs) to incorporate both chemotherapy drugs and P53 mRNA. The PAL P53 mRNA NPs showed superior properties compared to Abraxane(®) and Lipusu(®) used in the clinic including high paclitaxel loading capacity (24 wt.%, calculated by paclitaxel in PAL), PAL encapsulation efficiency (94.7% ± 6.8%) and mRNA encapsulation efficiency (88.7% ± 0.7%). Meanwhile, these NPs displayed synergetic cytotoxicity of paclitaxel and P53 mRNA in cultured TNBC cells. More importantly, we demonstrated in vivo anti-tumor efficacy of PAL P53 mRNA NPs in an orthotopic TNBC mouse model. Overall, these chemotherapy drugs derived mRNA NPs provide a new platform to integrate chemotherapy and personalized medicine using tumor genetic information, and therefore represent a promising approach for TNBC treatment.

Background: This study aimed to evaluate the conditional survival (CS) of appendiceal tumors (ATs) after surgery.Methods: A total of 3,031 patients with ATs who underwent surgery were included in the Surveillance Epidemiology and End Results (SEER) database from 2004 to 2016. A multivariate Cox regression model was used to analyze the prognostic factors affecting overall survival (OS) and cancer-specific survival (CSS). CS was used to calculate the probability of survival for another 3 years after the patient had survived x years. The formulas were COS3 = OS (x + 3) /OS (x), and CCS3 = CSS (x + 3)/CSS (x).Results: The 1-year, 3-year, and 5-year OSs for all patients were 95.6%, 83.3%, and 73.9%, respectively, while the 1-year, 3-year, and 5-year CSSs were 97.0%, 87.1%, and 79.9%, respectively. Age, grade, histology, N stage, carcinoembryonic antigen (CEA), and radiation were independent prognostic factors for OS and CSS. For patients that survived for 1 year, 3 years, and 5 years, their COS3s were 81.7%, 83.9%, and 87.0%, respectively. The CCS3s were 85.5%, 88.3%, and 92.0% respectively. In patients with poor clinicopathological factors, COS3 and CCS3 increased significantly, and the survival gap between OS and COS3, CSS and CCS3 was more obvious.Conclusions: CS for appendiceal tumors were dynamic and increased over time, especially in patients with poor prognosis.

Due to the limitations of existing medications, there is a critical need for new drugs to treat visceral leishmaniasis. Since arylimidamides and antifungal azoles both show oral activity in murine visceral leishmaniasis models, a molecular hybridization approach was employed where arylimidamide and azole groups were separated by phenoxyalkyl linkers in an attempt to capitalize on the favorable antileishmanial properties of both series. Among the target compounds synthesized, a greater antileishmanial potency against intracellular Leishmania donovani was observed as the linker length increased from two to eight carbons and when an imidazole ring was employed as the terminal group compared to a 1,2,4-triazole group. Compound 24c (N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)-2-isopropoxyphenyl) picolinimidamide) displayed activity against L. donovani intracellular amastigotes with an IC50 value of 0.53 μM. When tested in a murine visceral leishmaniasis model, compound 24c at a dose of 75 mg/kg/day p.o. for five consecutive days resulted in a modest 33% decrease in liver parasitemia compared to the control group, indicating that further optimization of these molecules is needed. While potent hybrid compounds bearing an imidazole terminal group were also strong inhibitors of recombinant CYP51 from L. donovani, as assessed by a fluorescence-based assay, additional targets are likely to play an important role in the antileishmanial action of these compounds.

Epithelial–mesenchymal transition (EMT) in cancer cells drives cancer chemoresistance, yet the molecular events of EMT that underpin the acquisition of chemoresistance are poorly understood. Here, we demonstrate a loss of gemcitabine chemosensitivity facilitated by human equilibrative nucleoside transporter 1 (ENT1) during EMT in pancreatic cancer and identify that cadherin switching from the epithelial (E) to neuronal (N) type, a hallmark of EMT, contributes to this loss. Our findings demonstrate that N-cadherin decreases ENT1 expression, membrane localization, and gemcitabine transport, while E-cadherin augments each of these. Besides E- and N-cadherin, another epithelial cell adhesion molecule, EpCAM, played a more prominent role in determining ENT1 membrane localization. Forced expression of EpCAM opposed cadherin switching with restored ENT1 expression, membrane localization, and gemcitabine transport in EMT-committed pancreatic cancer cells. In gemcitabine-treated mice, EpCAM-positive tumors had high ENT1 expression and reduced metastasis, whereas tumors with N-cadherin expression resisted gemcitabine treatment and formed extensive secondary metastatic nodules. Tissue microarray profiling and multiplexed IHC analysis of pancreatic cancer patient-derived primary tumors revealed EpCAM and ENT1 cell surface coexpression is favored, and ENT1 plasma membrane expression positively predicted median overall survival times in patients treated with adjuvant gemcitabine. Together, our findings identify ENT1 as an inadvertent target of EMT signaling mediated by cadherin switching and provide a mechanism by which mesenchymal pancreatic cancer cells evade gemcitabine therapy during EMT.

Objective. To assess the impact of the Patient Voices series on Doctor of Pharmacy (PharmD) students. Methods. A series of patient speakers and integrated simulation activities focused on underserved populations, otherwise known as the Patient Voices series, was embedded into a pharmacy skills laboratory curriculum. First-year PharmD students’ self-ratings of confidence were compared on pre- and post-course surveys. Using evaluations from first-year introductory pharmacy practice experiences (IPPEs), student self-evaluation data were compared to preceptor evaluations of student performance. Open-ended responses to course evaluations from first- and second-year PharmD students and student reflections from third-year PharmD students were assessed using conventional content analysis to identify and characterize student perceptions. Results. Significant increases were observed in first-year students’ confidence to show empathy (mean, 4.2 to 4.7) and to interact with patients from underserved communities (mean, 2.2 to 4.2). Preceptor ratings of students’ empathy were consistent with the students’ self-rated abilities, while students’ self-ratings on cultural sensitivity were higher than the preceptors’ ratings. Qualitative analysis of course evaluation surveys and reflections revealed common themes identified by students, such as understanding different perspectives, increased empathy for patients, and the value of including this content in the curriculum. Conclusion. Student confidence to interact with patients from a variety of underserved populations increased following introduction of the Patient Voices series into the PharmD curriculum. Students perceived the series to be a valuable learning experience.

An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAAAC→GAG//GTAAGT) in the TOP2α gene’s suboptimal exon 19/intron 19 5′ splice site (E19/I19 5′ SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 protein, and increased TOP2α/170 protein. Sensitivity to etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition was restored to levels comparable to those in parental K562 cells. Together, the results indicate that our gene-editing strategy for optimizing the TOP2α E19/I19 5′ SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2α-targeted drugs. SIGNIFICANCE STATEMENT: Results presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 5′ splice site in the DNA topoisomerase IIα (TOP2α) gene results in circumvention of acquired drug resistance to etoposide and other TOP2α-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2α 90 kDa isoform and increasing expression of full-length TOP2α 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2α-targeted drugs.

Purpose To assess pharmacy technician learning preferences using the VARK tool and through self-identification. Methods The VARK (visual, aural, read/write, kinesthetic) questionnaire was incorporated into a larger survey, which was distributed during live staff meetings and a continuing education session held by the Ohio Pharmacists Association attended by 204 pharmacy technicians across various practice settings. Results A 90% response rate was achieved. Most respondents (78.8%) self-identified a single predominant learning preference, with 60.3% indicating a preference for kinesthetic learning methods. In contrast, after assessment with the VARK questionnaire 37.9% of survey participants were categorized as having a quadmodal learning style incorporating all VARK modalities. With regard to the Pharmacy Technician Certification Exam, a large majority of participants (96.2% of those providing a response) indicated that they had taken the exam in the past, with 17 participants (9.3% of those providing a response) indicating more than 1 attempt to pass the exam. Furthermore, experiential (on-the-job) training was identified by a large majority of survey respondents (79.3%) as the preferred way to learn new information. Conclusion Learning preferences of pharmacy technicians vary amongst individuals, with many found to have multiple learning preferences through VARK questionnaire assessment. Incorporating experiential training and establishing learning preferences of pharmacy technicians may aid in development of accredited training programs that cater to the needs of pharmacy technicians.

Gankyrin, a newly defined oncoprotein also known as PSMD10 and P28, functions as a dual-negative regulator of the two most prominent tumor suppressor pathways, the CDK/pRb and HDM2/P53 pathways. Its aberrant expression has been prevalently found in human hepatocellular carcinomas (HCC) and esophagus squamous cell carcinomas (ESCC), indicative of the potential of gankyrin as a rational diagnostic and therapeutic target in cancers. Here, we review the unique structural features and functional diversity of gankyrin, and discuss its implication in cancer diagnostics and therapeutics from the perspective of chemical biology.

Genetic variations of the non-coding RNA gene, ANRIL, have been associated with human diseases including cancer, type-2 diabetes, and atherosclerosis. In the present study, we investigated the potential associations of select ANRIL single nucleotide polymorphisms (SNPs) with overall survival and other clinical outcomes in adult patients with hematologic malignancies after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Genomic DNA was extracted from whole blood samples from 103 adult patients with hematologic malignancies who had received allo-HSCT followed by oral tacrolimus therapy. The genotypes of four select ANRIL SNPs, rs564398, rs1063192, rs2151280, and rs2157719 were determined using qRT-PCR-based genotyping assays.rs2151280 (C-T) in ANRIL was associated with worse overall survival in these patients (CT/CC vs. TT). Contrarily, rs2151280 and the other select ANRIL SNPs were not associated with death at Day-100 after transplantation, the incidence of graft-versus-host disease (GVHD), acute kidney injury (AKI), and neurotoxicity in the study cohort.rs2151280 represents a potential prognostic biomarker for overall survival in adult patients with hematologic malignancies after allo-HSCT.

Nasopharyngeal carcinoma is one of the lethal cancers prevalent in Southeast Asia and Southern China. The frequent relapses, development of drug resistance, the adverse effects of chemotherapy and lack of therapeutic targets form the major hurdles in nasopharyngeal carcinoma treatment. This study was undertaken to investigate the role and therapeutic potential of miR-205 in human nasopharyngeal carcinoma cells.Expression analysis was performed by qRT-PCR. The WST-1 and colony formation assays were used for the assessment of the cell viability. Autophagy was detected by electron microscopy and apoptosis was detected by DAPI staining. Protein expression was determined by western blot analysis.The expression of miR-205 was significantly downregulated in human nasopharyngeal carcinoma cells. Overexpression of miR-205 caused significant inhibition in the proliferation of CNE1 nasopharyngeal carcinoma cells. The miR-205-triggered growth inhibition was found to be mainly due to the induction of autophagy which was associated with increase in LC3B II and decrease in p62 expression. The miR-205 overexpression also caused apoptotic cell death of CNE1 cells which was concomitant with increase in the Bax/Bcl-2 ratio. Additionally, miR-205 enhanced the chemosensitivity of the nasopharyngeal carcinoma cells to cisplatin and suppressed their migration and invasiveness. In silico analysis showed that miR-205 exerts its effects by inhibiting human epidermal growth factor receptor 3 (HER3). The expression of HER3 was found to be significantly upregulated in nasopharyngeal carcinoma cells and overexpression of HER3 could nullify the effects of miR-205 on the proliferation of nasopharyngeal carcinoma cells.miR-205 may exhibit therapeutic implications in the treatment of nasopharyngeal carcinoma.

Wearable devices play an important role in our daily life. We can use it in the sports area and the medical area. This review uses many articles to introduce how to use different sensors to analyze human kinetic characteristics. This review is divided into three parts. In the first sections, we will introduce the working principle and its calculation method of the different sensors such as IMUs sensors, pressure transducers, etc. The second part will show you some research about using wearable sensors to calculate the joint data and link the data with human movement. These researches can help us stay far away from joint diseases. In conclusion, we give some suggestions about the improvement of wearable devices.

High-dose melphalan (HDM) is part of the conditioning regimen in patients with multiple myeloma (MM) receiving autologous stem cell transplantation (ASCT). However, individual sensitivity to melphalan varies, and many patients experience severe toxicities. Prolonged severe neutropenia is one of the most severe toxicities and contributes to potentially life-threatening infections and failure of ASCT. Granulocyte-colony stimulating factor (G-CSF) is given to stimulate neutrophil proliferation after melphalan administration. The aim of this study was to develop a population pharmacokinetic/pharmacodynamic (PK/PD) model capable of predicting neutrophil kinetics in individual patients with MM undergoing ASCT with high-dose melphalan and G-CSF administration. The extended PK/PD model incorporated several covariates, including G-CSF regimen, stem cell dose, hematocrit, sex, creatinine clearance, p53 fold change, and race. The resulting model explained portions of interindividual variability in melphalan exposure, therapeutic effect, and feedback regulation of G-CSF on neutrophils, thus enabling simulation of various doses and prediction of neutropenia duration.

Aberrant expression of the SEI1 oncogene has been prevalently found in a variety of human cancers, including oral squamous cell carcinoma (OSCC). Recent studies have shown that cisplatin up-regulates the expression of SEI1 in breast and bladder cancer cells, thus inhibiting apoptosis and cell death in these cells. In the present study, we investigated the impact of cisplatin on the expression of SEI1 in OSCC cells.Four OSCC cell lines, CAL27, SCC4, SCC15, and SCC22A were treated with cisplatin and 5-fluorouracil, and changes in SEI1 expression in these cells were evaluated using quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analyses.Cisplatin significantly induced SEI1 expression in the tested OSCC cells. Contrarily, cisplatin treatment did not affect the expression of gankyrin and BMI1, two oncogenes frequently overexpressed in a coordinate manner with SEI1 in OSCC. Additionally, 5-fluorouracil did not bring about any detectable changes in SEI1 expression in these cells.Cisplatin-induced up-regulation of SEI1 expression in OSCC is specific, and such induction could underlie the development of resistance to cisplatin in OSCC.

Autologous hematopoietic stem cell transplant (autoHSCT) remains standard of care for fit patients with multiple myeloma [1]. The use of granulocyte colony-stimulating factor (G-CSF) after autoHSCT...

SLC7A5 is recognized as the major mediator of melphalan uptake into multiple myeloma (MM) cells; however, its contribution to the inter-patient variability of melphalan efficacy and toxicity is yet to be well elucidated. This study aimed to investigate the impact of a single nucleotide polymorphism (SNP) rs4240803 in SLC7A5 on the gene expression, ex vivo sensitivity to melphalan, and clinical outcomes in MM patients who were undergoing autologous stem cell transplantation with high-dose melphalan.Peripheral blood mononuclear cells (PBMC) were collected from 108 MM patients prior to melphalan therapy. Clinical data were also collected from these patients following melphalan therapy.rs4240803 was associated with elevated expression of SLC7A5 mRNA, higher ex vivo sensitivity to melphalan in PBMCs, and positive 90-day response in these patients (p=0.047, 0.10, 0.049, respectively).rs4240803 impacted the expression of SLC7A5, thus contributing to the clinical response of MM patients to melphalan therapy.

Epithelial-mesenchymal transition (EMT) in cancer cells drives cancer chemoresistance, yet the molecular events of EMT that underpin the acquisition of chemoresistance are poorly understood. Here, we demonstrate a significant loss of gemcitabine chemosensitivity facilitated by human equilibrative nucleoside transporter 1 (ENT1) during EMT in pancreatic cancer and identify that cadherin switching from the epithelial (E) to neuronal (N) type, a hallmark of EMT, contributes to this loss. Our findings demonstrate that N-cadherin decreases ENT1 expression, basolateral membrane localization and gemcitabine transport, while E-cadherin augments each of these. Besides E- and N-cadherin, another epithelial cell adhesion molecule, EpCAM, played a more prominent role in determining ENT1 membrane localization. Forced expression of EpCAM opposed cadherin switching with restored ENT1 expression, basolateral localization and gemcitabine transport in EMT-committed pancreatic cancer cells. In gemcitabine-treated mice, EpCAM positive tumors had high ENT1 expression and reduced metastasis, whereas tumors with N-cadherin expression resisted gemcitabine treatment and formed extensive secondary metastatic nodules. Tissue microarray profiling and multiplexed immunohistochemical analysis of pancreatic cancer patient-derived primary tumors revealed EpCAM and ENT1 cell surface co-expression is favored, and ENT1 plasma membrane expression positively predicted median overall survival times in patients treated with adjuvant gemcitabine. Together, our findings identify ENT1 as an inadvertent target of EMT signaling mediated by cadherin switching and provide a mechanism by which mesenchymal pancreatic cancer cells evade gemcitabine therapy during EMT. Citation Format: Brenna Weadick, Debasis Nayak, Avinash K. Persaud, Sau Wai Hung, Radhika Raj, Wei Chen, Junan Li, Terence M. Williams, Rajgopal Govindarajan. EMT-induced gemcitabine resistance in pancreatic cancer involves the functional loss of equilibrative nucleoside transporter 1 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-062.

Given the limitations of current antileishmanial drugs and the utility of oral combination therapy for other infections, developing an oral combination against visceral leishmaniasis should be a high priority. In vitro combination studies with DB766 and antifungal azoles against intracellular Leishmania donovani showed that posaconazole and ketoconazole, but not fluconazole, enhanced DB766 potency. Pharmacokinetic analysis of DB766-azole combinations in uninfected Swiss Webster mice revealed that DB766 exposure was increased by higher posaconazole and ketoconazole doses, while DB766 decreased ketoconazole exposure. In L. donovani -infected BALB/c mice, DB766-posaconazole combinations given orally for 5 days were more effective than DB766 or posaconazole alone. For example, 81% ± 1% (means ± standard errors) inhibition of liver parasite burden was observed for 37.5 mg/kg of body weight DB766 plus 15 mg/kg posaconazole, while 37.5 mg/kg DB766 and 15 mg/kg posaconazole administered as monotherapy gave 40% ± 5% and 21% ± 3% inhibition, respectively. Combination index (CI) analysis indicated that synergy or moderate synergy was observed in six of nine combined dose groups, while the other three were nearly additive. Liver concentrations of DB766 and posaconazole increased in almost all combination groups compared to monotherapy groups, although many increases were not statistically significant. For DB766-ketoconazole combinations evaluated in this model, two were antagonistic, one displayed synergy, and one was nearly additive. These data indicate that the efficacy of DB766-posaconazole and DB766-ketoconazole combinations in vivo is influenced in part by the pharmacokinetics of the combination, and that the former combination deserves further consideration in developing new treatment strategies against visceral leishmaniasis.

High dose melphalan followed by autologous stem cell transplantation remains standard of care for eligible patients with multiple myeloma, but disease response and toxicity, including severe mucositis, varies among patients. Our randomized trial investigated duration of cryotherapy (2 and 6 hours) for reduction of mucositis prevalence and severity and explored factors associated with variability in pharmacokinetics and outcomes from melphalan therapy. The results demonstrate 2-hour is at least as effective as 6-hour cryotherapy in decreasing severe mucositis. From a population pharmacokinetic model, we identified fat free mass, hematocrit, and creatinine clearance were significant covariates, as had been reported previously. Furthermore, we observed the rs4240803 SLC7A5 polymorphism was significantly associated with pharmacokinetic variability, and pharmacokinetics was associated with both mucositis and neutropenia. However, melphalan exposure was not associated with progression-free or overall survival in our dataset. These findings contribute to ongoing efforts to personalize melphalan dosing in transplant patients.

The presence of RD(INK4/ARF) (RD) enhancer in the INK4-ARF locus provides a novel mechanism to simultaneously increase the transcription of p15(INK4b) (p15), p14ARF (p14), and p16(INK4a) (p16). While such upregulation can be repressed through interactions between RD and oncoproteins CDC6 and BMI1, little is known about the involvement of RD in cancer. In this study we investigated RD deletions in 30 squamous cell carcinoma of the head and neck (SCCHN) and the patient-matched High At-Risk Mucosa specimens (HARM, "phenotypically normal" tissues neighboring SCCHN foci but beyond the surgical resection margin). RD was deleted (homozygously/heterozygously) in SCCHN and HARM at the incidence of 36.7% (11/30) and 13.3% (4/30), respectively. In comparison, no RD deletion was detected in 26 oral buccal brush biopsy specimens from healthy donors. Both p16 and p14 were lowly expressed in SCCHN and HARM, and their mRNA expression levels were positively associated with each other (P < 0.01). Moreover, BMI1 was highly expressed in both SCCHN and HARM, and BMI1 overexpression was associated with p16 downregulation in SCCHN (P < 0.05). These results indicate that RD deletion and BMI1 overexpression frequently occur in the early stage of oral carcinogenesis and BMI1 overexpression may downregulate the transcription of p16 and p14 through interfering with RD.