A mixed-methods approach to repetitive formative assessment with timely feedback on instructional perception in doctor of pharmacy students.

INTRODUCTION: Formative assessment assists learning, but how Doctor of Pharmacy (PharmD) students perceive repeated formative assessment is unclear. METHODS AND MATERIALS: This study sought to determine perceptions of repeated formative assessment with timely feedback on student learning in third-year PharmD students. This mixed methods approach included four surveys and a qualitative interview. Five formative assessments were assigned to third-year PharmD students throughout a fall course, and then repeated in a spring course for the same cohort. Paired pre-and post-course surveys administered in both courses contained items corresponding to formative assessment perceptions. Survey items included domains of knowledge, engagement, feedback, and confidence, and effect size was determined using Cohen's d. Following the second course, students were invited to take part in a qualitative interview to further characterize perceptions. RESULTS: Overall, 19 and 18 students participated in paired fall and spring pre- and post-surveys, respectively. The standardized mean difference for 12 out of 24 total survey items (58.3%) indicated small to medium positive effect sizes following the intervention, two out of 24 (8.3%) with medium to strong positive effect sizes, and one out of 24 (4.17%) with a strong positive effect size. Eight students participated in a quantitative interview; response themes included "think," "critiquing," "helped," and "helpful." CONCLUSIONS: In two PharmD courses, students expressed a generally small to moderate perceived benefit on repeat formative assessment in domains representing knowledge, engagement, feedback, and confidence.

Effectiveness and Utility of Flowcharts on Learning in a Classroom Setting: A Mixed-Methods Study.

OBJECTIVE: Graphical representation of information organizes and promotes meaningful learning. As an example of graphical organizers, flowcharts can simplify and summarize complex information. The evidence of classroom use of flowcharts as an instructional tool is unclear. We investigated the effectiveness of flowcharts on student learning as an in-class instructional tool in a cardiovascular therapeutic course. Student experiences with the use and application of flowcharts were explored. METHODS: An explanatory sequential mixed-methods study was conducted with pharmacy students enrolled in an acute-care cardiovascular course from 2019-2021. The quantitative phase comprised a survey to determine flowchart effectiveness and a comparison of student performance in three content areas. The qualitative phase of the study used focused group interviews to understand student perceptions of flowchart use. RESULTS: Survey results indicated that using flowcharts improved understanding (110/128, 86%), integration of material (114/128, 89%), and overall knowledge (111/128, 87%). Student performance in the 3 content areas, shock, arrhythmia, and acute coronary syndrome were statistically significant with flowcharts implementation. Emerging themes from student interviews were (1) used as a medium for retention and recall, (2) used as a study tool, and (3) used as a decision-making framework. CONCLUSION: Flowcharts provide an alternative approach to teaching complex content, which allows students to organize and summarize information that promotes meaningful learning. The ease of implementation combined with the generalized nature of flowcharts makes it an effective graphical organizer that can be used across various disciplines.

Calcium handling genes are regulated by promoter DNA methylation in colorectal cancer cells.

Calcium signaling regulates various cellular processes, including proliferation and cell death. DNA methylation of gene promoters is an epigenetic modification that facilitates transcriptional suppression. Disruption of calcium homeostasis and DNA methylation in cancer are each linked to tumor development and progression. However, the possible connection between these two processes has not been thoroughly studied. Therefore, we measured the expression of six gene families involved in calcium regulation (ATP2A, ITPR, ORAI, RyR, STIM, and TRPC) in a colorectal cancer cell model, HCT116, with either genetic (Double Knock-out/DKO) or pharmacological (5-aza-2'-deoxycytidine/DAC) inhibition of DNA methyltransferases. Fourteen of the 20 examined calcium handling genes were expressed at higher levels in DKO cells as compared to HCT116. Expression of five genes was increased in HCT116 cells treated with DAC, three matching DKO. Due to a unique expression pattern of the three ATP2A genes in our model, encoding the Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase (SERCA) pumps, we chose to evaluate the methylation status of these genes, protein expression, and potential associated physiological effects, using the SERCA inhibitor thapsigarin (TG). We observed an expected pattern of promoter methylation coinciding with reduced expression and vice versa. This differential mRNA expression was associated with altered SERCA3 protein expression and cytosolic calcium levels with TG exposure. As a result, DKO cells displayed less TG-induced cytotoxicity, as compared to HCT116 cells. Overall, it is likely that at least several calcium regulatory genes are transcriptionally regulated by DNA methylation, and this may play a role in tumorigenesis through altering apoptosis in cancer.

The polyphenol ellagic acid exerts anti-inflammatory actions via disruption of store-operated calcium entry (SOCE) pathway activators and coupling mediators.

Ellagic acid, a naturally occurring phenol found in a variety of fruits and nuts has been shown to possess anti-inflammatory properties. However, the mechanism of action behind its anti-inflammatory action is unclear. Using human Jurkat T cells, our study examined the effects of ellagic acid (EA) on Ca2+ handling, in particular, store-operated Ca2+ entry (SOCE), a process critical to proper T cell function. We observed that the acute addition of EA-induced Ca2+ release with an EC50 of 63 µM. The Ca2+ release was significantly attenuated by Xestospongin C, a known inhibitor of the Inositol 1,4,5-trisphosphate receptor (IP3R) channel and was unaffected by the phospholipase C (PLC) inhibitor, U73122. Furthermore, chronic incubation of Jurkat T cells with EA not only decreased the ATP-induced Ca2+ release but also diminished the SOCE-mediated Ca2+ influx in a dose-dependent manner. This inhibition was confirmed by reduced Mn2+ entry rates in the EA-treated cells. The ATP-induced Ca2+ entry was also attenuated in EA-treated HEK293 cells transiently transfected with SOCE channel Orai1-myc and ER-sensor stromal interaction molecule (STIM1) (HEKSTIM/Orai). Moreover, EA treatment interfered with the Orai1 and STIM1 coupling by disrupting STIM1 puncta formation in the HEKSTIM/Orai cells. We observed that EA treatment reduced cytokine secretion and nuclear factor of activated T-cell transcriptional activity in stimulated T cells. Hence, by inhibiting SOCE mediated Ca2+ influx, EA decreased downstream activation of pro-inflammatory mediators. These results suggest a novel target for EA-mediated effects and provide insight into the mechanisms underlying EA-mediated anti-inflammatory effects.

The Na+/Ca2+ exchange inhibitor 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate (KB-R7943) also blocks ryanodine receptors type 1 (RyR1) and type 2 (RyR2) channels.

Na(+)/Ca(2+) exchanger (NCX) is a plasma membrane transporter that moves Ca(2+) in or out of the cell, depending on membrane potential and transmembrane ion gradients. NCX is the main pathway for Ca(2+) extrusion from excitable cells. NCX inhibitors can ameliorate cardiac ischemia-reperfusion injury and promote high-frequency fatigue of skeletal muscle, purportedly by inhibiting the Ca(2+) inward mode of NCX. Here we tested two known NCX inhibitors, 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)-isothiourea methanesulfonate (KB-R7943) and the structurally related 2-[[4-[(4-Nitrophenyl)methoxy]phenyl]methyl]-4-thiazoli dinecarboxylic acid ethyl ester (SN-6), for their influence on electrically or caffeine-evoked Ca(2+) transients in adult dissociated flexor digitorum brevis (FDB) skeletal muscle fibers and human embryonic kidney (HEK) 293 cells that have stable expression of type 1 ryanodine receptor (RyR1). KB-R7943 (< or = 10 microM) reversibly attenuates electrically evoked Ca(2+) transients in FDB and caffeine-induced Ca(2+) release in HEK 293, whereas the structurally related NCX inhibitor SN-6 does not, suggesting that KB-R7943 directly inhibits RyR1. In support of this interpretation, KB-R7943 inhibits high-affinity binding of [(3)H]ryanodine to RyR1 (IC(50) = 5.1 +/- 0.9 microM) and the cardiac isoform RyR2 (IC(50) = 13.4 +/- 1.8 microM). KB-R7943 interfered with the gating of reconstituted RyR1 and RyR2 channels, reducing open probability (P(o)), shortening mean open time, and prolonging mean closed time. KB-R7943 was more effective at blocking RyR1 with cytoplasmic conditions favoring high P(o) compared with those favoring low P(o). SN-6 has negligible activity toward altering [(3)H]ryanodine binding of RyR1 and RyR2. Our results identify that KB-R7943 is a reversible, activity-dependent blocker of the two most broadly expressed RyR channel isoforms and contributes to its pharmacological and therapeutic activities.

The actin cytoskeleton differentially regulates NG115-401L cell ryanodine receptor and inositol 1,4,5-trisphosphate receptor induced calcium signaling pathways.

Regulation of bi-directional communication between intracellular Ca(2+) pools and surface Ca(2+) channels remains incompletely characterized. We report Ca(2+) release mediated by inositol 1,4,5-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) pathways is diminished under actin cytoskeleton disruption in NG115-401L (401L) neuronal cells, yet despite truncated Ca(2+) release, Ca(2+) influx was not significantly altered in these experiments. However, disruption of cortical actin networks completely abolished IP(3)R induced Ca(2+) release, whereas RyR-mediated Ca(2+) release was preserved, albeit attenuated. Moreover, cortical actin disruption completely abolished IP(3)R and RyR linked Ca(2+) influx even though Ca(2+) pool sensitivities were different. These findings suggest discrete Ca(2+) store/Ca(2+) channel coupling mechanisms in the IP(3)R and RyR pathways as revealed by the differential sensitivity to actin perturbation.

Enantiomeric specificity of (-)-2,2',3,3',6,6'-hexachlorobiphenyl toward ryanodine receptor types 1 and 2.

Polychlorinated biphenyls (PCBs) with unsymmetrical chlorine substitutions and multiple orthosubstitutions that restrict rotation around the biphenyl bond may exist in two stable enantiomeric forms.Stereospecific binding and functional modification of specific biological signaling targets have not been previously described for PCB atropisomers. We report that (-)-2,2',3,3',6,6'-hexachlorobiphenyl [(-)-PCB 136] enhances the binding of [3H]ryanodine to high-affinity sites on ryanodine receptors type 1(RyR1) and type 2 (RyR2) (EC50 values ~0.95 microM), whereas (+)-PCB 136 is inactive at < or =10 microM.(-)-PCB 136 induces a rapid release of Ca2+ from microsomal vesicles by selective sensitization of RyRs, an effect not antagonized by (+)-PCB 136. (-)-PCB 136 (500nM) enhances the activity of reconstituted RyR1 channels 3-fold by stabilizing the open and destabilizing the closed conformational states. The enantiomeric specificity is also demonstrated in intact HEK 293 cells expressing RyR1 where exposure to (-)-PCB 136 (100 nM; 12 h) sensitizes responses to caffeine, whereas (+)-PCB 136 does not. These data show enantiomeric specificity of (-)-PCB 136 toward a broadly expressed family of microsomal Ca2+ channels that may extend to other chiral noncoplanar PCBs and related structures.Evidence for enantioselective enrichment of PCBs in biological tissues that express RyR1 and RyR2channels may provide new mechanistic leads about their toxicological impacts on human health

The size of zinc oxide nanoparticles controls its toxicity through impairing autophagic flux in A549 lung epithelial cells.

Zinc oxide nanoparticles (ZnONPs) widely used in various products, have been concerned with its impact on human health, in particular, on the risk of pulmonary toxicity. Our previous study indicated that ZnONPs could harness autophagy and impair the autophagic flux, which was positively linked to ZnONPs-induced toxicity. The objective of this study was to investigate whether ZnONPs-induced impairment of autophagic flux and cell death in lung epithelial cells is related to the size of ZnONPs. We demonstrate that ZnONPs with the average size of 50 nm could induce toxic effects in A549 lung epithelial cells, including accumulation of autophagosomes (the elevation of LC3B-II/LC3B-I ratio), impaired autophagic flux (the increase of p62 expression), the release of intracellular zinc ions (the increase of FluoZin-3 signal and ZnT1 mRNA expression), mitochondrial damage (the decrease of TMRE signal), lysosomal dysfunction (the aberrant expression of LAMP-2), oxidative stress (the increase of DCFH-DA signal and HO-1 expression) and cell death. Interestingly, ZnONPs with the average size of 200 nm failed to induce autophagy-mediated toxicity. Taken together, our results indicate that the size of ZnONPs is closely correlated with its toxicity, which is probably mediated by induction of impaired autophagic flux. This finding provides an insight into better understating of ZnONPs-associated toxicity, and mitigating the risk to humans and allowing the safer application.

Autophagy-dependent release of zinc ions is critical for acute lung injury triggered by zinc oxide nanoparticles.

Pulmonary exposure to zinc oxide nanoparticles (ZnONPs) could cause acute lung injury (ALI), but the underlying molecular mechanism remains unclear. Herein, we established a ZnONPs-induced ALI mouse model, characterized by the histopathological changes (edema and infiltration of inflammatory cells in lung tissues), and the elevation of total protein and cytokine interleukin-6 in bronchoalveolar lavage fluid in time- and dose-dependent manners. This model also exhibited features like the disturbance of redox-state (reduced of glutathione to glutathione disulfide ratio, elevation of heme oxygenase-1 and superoxide dismutase 2), the decrease of adenosine triphosphate synthesis and the release of zinc ions in the lung tissues. Interestingly, we found that ZnONPs exposure caused the accumulation of autophagic vacuoles and the elevation of microtubule-associated proteins 1A/1B light chain (LC)3B-II and p62, indicating the impairment of autophagic flux. Our data indicated that the above process might be regulated by the activation of AMP-activated protein kinase but not the mammalian target of rapamycin pathway. The association between ZnONPs-induced ALI and autophagy was further verified by a classical autophagy inhibitor, 3-methyladenine (3-MA). 3-MA administration reduced the accumulation of autophagic vacuoles, the expression of LC3B-II and p62, followed by a significant attenuation of histopathological changes, inflammation, and oxidative stress. More importantly, 3-MA could directly decrease the release of zinc ions in lung tissues. Taken together, our study provides the evidence that ZnONPs-induced pulmonary toxicity is autophagy-dependent, suggests that limiting the release of zinc ions by inhibiting autophagy could be a feasible strategy for the prevention of ZnONPs-associated pulmonary toxicity.

Zinc oxide nanoparticles harness autophagy to induce cell death in lung epithelial cells.

Although zinc oxide nanoparticles (ZnONPs) are widely used, they have raised concerns of toxicity in humans. Previous studies have indicated that reactive oxygen species (ROS) and autophagy are involved in the cytotoxicity of ZnONPs, but the regulatory mechanisms between autophagy and ROS remain to be elucidated. Herein, we comprehensively investigated the regulatory mechanism of autophagy and the link between autophagy and ROS in ZnONPs-treated lung epithelial cells. We demonstrated that ZnONPs could induce autophagy, and this process could enhance the dissolution of ZnONPs in lysosomes to release zinc ions. Sequentially, zinc ions released from ZnONPs were able to damage not only lysosomes, leading to impaired autophagic flux, but also mitochondria. Impaired autophagic flux resulted in the accumulation of damaged mitochondria, which could generate excessive ROS to cause cell death. We further demonstrated that the inhibition of autophagy by either pharmacological inhibitors or small interfering RNA (siRNA)-mediated knockdown of Beclin-1 and AMP-activated protein kinase could ameliorate ZnONPs-induced cell death. Moreover, we found that lysosomal-associated membrane protein 1/2 (LAMP-1/2), which were the most abundant highly glycosylated protein in late endosomes/lysosomes, exhibited aberrant expression pattern upon treatment with ZnONPs. Intriguingly, LAMP-2 knockdown, but not LAMP-1 knockdown, could exacerbate the ROS generation and cell death induced by ZnONPs treatment. Meanwhile, LAMP-2 overexpression alleviated ZnONPs-induced cell death, suggesting that LAMP-2 was linked to this toxic phenotype induced by ZnONPs. Our results indicate that autophagic dysfunction could contribute to excessive ROS generation upon treatment with ZnONPs in lung epithelial cells, suggesting that modulating the autophagy process would minimize ZnONPs-associated toxicity.